key: cord- -zupx qn authors: ni, yijun; ebido, chike chukwuenyem; odii, elijah chibueze; wang, jinhui; orakwelu, chinemerem hodges; abonyi, francis chukwuemeka; ngene, chinedu innocent; okoro, joseph onyekwere; ubachukwu, patience obiageli; hu, wei; yin, mingbo title: phylogeography and genetic diversity of the copepod family cyclopidae (crustacea: cyclopoida) from freshwater ecosystems of southeast nigeria date: - - journal: bmc evol biol doi: . /s - - - sha: doc_id: cord_uid: zupx qn background: copepods are key components of aquatic ecosystems and can help regulate the global carbon cycle. much attention has been paid to the species diversity of copepods worldwide, but the phylogeography and genetic diversity of copepods in nigeria is unexplored. results: using a mitochondrial cytochrome c oxidase subunit i marker, we preformed phylogenetic and phylogeographic analyses for cyclopidae copepods in southeast nigeria. a high species diversity of cyclopidae in nigeria: species of tropocyclops, species of mesocyclops and species of thermocyclops from cyclopidae were identified in populations. moreover, we detected unique haplotypes, which fell into two distinct clades. pairwise genetic distances (uncorrected p-distances) among the species of cyclopidae ranged from . to . . several species co-existed in the same lake, and some haplotypes were shared among different geographic populations, suggesting a dispersal of cyclopidae in our sampling region. finally, we found that the population genetic diversity for each species of cyclopidae was low in nigeria. conclusions: our findings explored the species diversity and distribution of copepods within the family cyclopidae for nigerian freshwater ecosystems: a high species diversity of cyclopidae copepods was detected over a small geographic sampling range. results from this study contribute to a better understanding of copepod diversity of nigerian freshwater ecosystems. co levels [ ] . thus, much attention has been paid to the bio-diversity of copepods in aquatic ecosystems [ , ] . copepods are the intermediate hosts of the parasitic nematode dracunculus medinensis, which causes a serious guinea-worm disease in nigeria and elsewhere [ ] . humans could become infected by drinking unfiltered water containing copepods which are infected with larvae of d. medinensis. therefore, most studies on copepods from nigeria have focused on their role in the dispersal of the pathogen [ , ] . only a few regional biogeographic studies have been conducted on copepods based on morphological species identification [ ] . for example, based on the morphology, a previous study showed the occurrence of the genera mesocyclops sars, and thermocyclops kiefer, in nigerian freshwater ecosystems: six mesocyclops species and three thermocyclops species were identified [ ] . moreover, it was believed that m. aspericornis was one of the most abundant species of mesocyclops in nigerian waterbodies, and t. decipiens was the most abundant species of thermocyclops from nigeria [ ] . however, the identification of different species of copepods solely based on morphology has technical limitations [ ] , as cryptic species are often detected. therefore, more discerning methods such as dna barcoding are needed to investigate copepod taxonomy, especially to recognize morphologically cryptic genetic lineages [ ] . dna barcoding has already been successfully applied to estimate the species/genetic diversity in many zooplankton taxa [ ] , as it can be used for rapid, accurate, reliable and remote identification of specimens of all metazoan [ ] . a fragment of the mitochondrial cytochrome c oxidase subunit i (coi) gene has proved to be a useful marker for many biodiversity studies [ , ] , as coi has advantages of being effective for species identification from a wider range of metazoan phyla and possessing a phylogenetic signal which can be used over a wider range of taxonomic levels [ ] . the coi marker has been successfully applied to species identification for cladocerans [ , ] . for example, dna barcoding was used to identify sibling cryptic species of the ceriodaphnia cornuta species complex from australia [ ] and to examine species of cladocera, such as daphnia, diaphanosoma, ceriodaphnia, moina and alona, from mexico and guatemala [ ] . this approach has also been applied to copepods [ ] . for instance, a study reported new sequences of marine copepod species by using a coi marker [ ] . elías-gutierrez et al. [ ] examined species of copepoda from mexico and guatemala by applying a coi marker. using coi is highly advantageous because it can also detect cryptic species, a phenomenon that is very common in copepod assemblages [ ] . for example, three genetically divergent but morphologically similar forms of hemidiaptomus (occidodiaptomus) ingens were detected throughout the distribution range of this species complex [ ] . moreover, oithona similis s.l. was found to be a complex of nine cryptic species instead of a single cosmopolitan species, according to a coi and a nuclear ribosomal s genetic marker [ ] . similarly, the nominal species "eudiaptomus hadzici" in the western balkans consists of four cryptic species according to a mitochondrial (coi) and a nuclear (nh ) marker [ ] . dna barcoding often reveals differences between allopatric populations. in that situation, it is difficult to decide whether this indicates different genetic lineages or simply geographical intraspecific variation. for instance, several different genetic lineages of moina which were allopatric in a phylogeny were assigned to a single species, because they had similar morphology [ ] . the phylogeny of copepods had been widely studied using molecular data. recently, a comprehensive study from asia showed a high species diversity of copepods in south korea [ ] . in that study, sequenced individuals represented species belonging to six different orders [ ] . another study has shown that sinocalanus tenellus consists of two very distinct clades in china, suggesting they are parts of a complex of cryptic species [ ] . moreover, karanovic [ ] detected a new species of schizopera from japan, which was the first member of its genus reported in japanese freshwater ecosystems, and it had no close relatives from elsewhere in the world. another study has revised the higher systematics of copepods and proposed the new taxa canuelloida ordo. nov., smirnovipinidae fam. nov. and cyclopicinidae fam. nov. [ ] . use of molecular data has not been restricted to species-level taxonomy [ ] , for example, the phylogeography of copepods has been also frequently investigated. they focused on the genealogical lineages of closely related species of copepods and their geographic distributions, by combining the information from phylogenetics, molecular genetics, population genetics, geology, paleontology, demography, ethology and historical biogeography [ ] . for instance, two species of copepods (i.e. neodiaptomus schmackeri and mongolodiaptomus birulai) occur in chinese taiwan: there was little gene flow among populations for both species [ ] . additionally, four populations of leptodiaptomus cf. sicilis in mexico were found to diverge into distinct phenotypes, and their specialization was further supported by molecular data which showed persistence of a founder effect, limited gene flow, and a pattern of allopatric speciation [ ] . there have been no studies on phylogeography and genetic diversity of copepods from nigeria. in this study, we analyzed copepod populations (out of pools/ lakes sampled) from nigeria. by analyzing sequence variation in the coi gene, we aimed to explore the species diversity and distribution of copepods among these populations. our expectation was to detect several members of the cyclopidae, as it is commonly observed worldwide [ , ] . we also investigated the phylogeography of cyclopidae in nigeria. one to specimens of cyclopidae were sequenced per location, and a total of cyclopidae coi sequences were successfully obtained from freshwater lakes around southeast nigeria, of which unique haplotypes were detected (tables and ). none of the coi sequences exhibited characteristics of nuclear pseudogenes (frame shifts or premature stop codons). two independent species-delimitation methods (i.e. gmyc and bptp) based on the coi bayesian tree consistently iden- (table ) . for each species, the population haplotype diversity (h d ) of coi ranged from to . , and the population nucleotide diversity (π) ranged from to . × − ( table ) . based on the haplotype network, seven out of species detected through analysis of the coi gene occurred at more than one locality in nigeria (fig. b) . the most frequently occurring species in this study was t. cf. prasinus, which had haplotypes and was distributed in lakes, including a g, aor, o m and u h, and one of the haplotypes was shared by lakes (a g, aor, o m). such a pattern was also observed in species m. cf. dussarti, which had haplotypes and one of them was shared by lakes (n o, n o and ubs) (fig. b) . different cyclopidae species co-existed in the same lake. for example, three species (i.e. t. cf. confinis, t. cf. prasinus and t. cf. crassus) co-existed in lake a g (fig. b) . similarly, t. cf. onabamiroi, m. cf. aequatorialis similis and m. cf. salinus co-existed in lake uii (fig. b) . moreover, five out of haplotypes were shared by different lakes (fig. b) . the most abundant haplotype was cth , including specimens shared by a g, a g and ihe. this was followed by cms , shared by n o, n o and ubs, and ctr , shared by a g, aor and o m (fig. b) . four species (i.e. t. cf. onabamiroi, t. cf. mellanbyi, m. cf. ogunnus and m. cf. aequatorialis similis) expressed only one haplotype with a single individual (fig. b) . through analysis of coi sequence variation, we explored the species diversity and distribution of copepods within the family cyclopidae for nigerian freshwater ecosystems, the first such study for west africa. our results suggested a high species diversity of cyclopidae copepods over a small geographic sampling range. high species diversity has already been reported in the copepods from nigeria [ , ] . forty species of cyclopidae copepods from nigeria were described based on morphological characteristics in the s [ ] . here, we did not detect any new species based on molecular data; all the species identified in the present study were described in [ ] . in agreement with a previous study based on morphology [ ] , we found that t. decipiens was the most abundant species of thermocyclops from nigeria. however, m. aspericornis was recorded as the most abundant species of mesocyclops in nigerian waterbodies [ ] , whereas we found that m. cf. dussarti is the most abundant species of the genus mesocyclops. this inconsistency might be explained by the relatively small sampling region in our study in southeast nigeria. globally, high levels of species diversity of copepods have also been reported [ , ] . for example, caligus species were present in chinese taiwan, and many more species remained to be discovered from this region [ ] . similarly, thirteen species of copepoda, including three members of calanoida (diaptomidae) and ten members of cyclopoida (eucyclopinae and cyclopinae), were recorded in chiapas, mexico [ ] . indeed, high species diversity, even in a relatively small area, has often been observed in copepods [ , , ] . for example, a study identified species that belonged to genera of copepods in sagami bay [ ] . another study identified species of copepods in tolo harbour, hong kong, and oithona rigida, o. simplex and paracalanus crassirostris were found to be the most abundant species [ ] . here, we detected species with several species and species complexes across three genera in nigeria; suggesting a high species diversity of cyclopidae in southeast nigeria. in agreement with a previous study of cyclopoida in nigeria [ ] , our results showed that the same species could be found in geographically separate populations, which also suggests that there are not extensive and common cryptic species in these sampled lakes. thermocyclops decipiens has also been detected in antilles, central america, columbia, venezuela, east of the andes, brazil [ ] and congo [ ] , indicating that this species has a wide distribution across continents. in contrast to several copepod species with high genetic divergence over short distances, e.g. tigriopus californicus [ ] , our data showed genetic similarity of the t. decipiens populations from different continents. a similar phenomenon has been detected in some open-ocean copepods which have more obvious dispersal routes. for example, it was found that several mtcoi haplotypes of calanus pacificus were distributed across multiple sampling location from the north pacific ocean [ ] . by using restriction site-associated dna sequencing, no significant genetic differentiation was found among centropages typicus samples collected from different nw atlantic regions with clear connectivity [ ] . zooplankton species often have vast ranges [ , ] . for example, daphnia galeata has been detected in both china and europe with some haplotypes shared across large distances [ ] . birds are often regarded as the key vectors for the dispersal of resting eggs of aquatic zooplankton [ ] , across geographical barriers. we found that different sibling species of cyclopidae co-existed in the same nigerian lake, a common finding in copepods [ ] . for example, a study of the genus mesocyclops conducted in africa reported that m. major and m. ogunnus often co-existed in the same waterbody [ ] . similarly, another study from nigeria reported that (see figure on previous page.) fig. a geographic locations of sampling sites for cyclopidae in nigeria. b haplotype network of cyclopidae from nigeria, based on the coi gene ( bp). each circle represents a unique haplotype and its size reflects the number of individuals expressing that haplotype. color codes denote geographic location of populations. portion of circles indicate distribution of haplotypes among different populations. the number of marks on connecting lines indicates the number of mutations between haplotypes. for lake abbreviations see table . the map was obtained from arcgis and edited in adobe illustrator fig. bayesian phylogenetic tree and species delimitation results for cyclopidae from nigeria, based on the coi gene ( bp). the ids for shared haplotypes are provided in table ; for origin of reference sequence ids see table s . only posterior probabilities > . are shown. species delimitation according to the gmyc and bptp methods are indicated. for the bptp method, the statistical support (pp) for species membership is also shown. paracalanus parvus was used as an outgroup it was common for up to mesocyclops species coexisted in a single locality [ ] . sympatry provides a possibility for interspecific hybridization, which is believed to be a common phenomenon in zooplankton [ ] . hybridization has often been observed in copepods. for example, hybrids between calanus glacialis and c. finmarchicus were detected along the atlantic and arctic canadian coast [ ] . another study also found that hybridization occurred between a female neocalanus cristatus and a male n. plumchrus, and was then followed by backcrossing to a n. plumchrus individual [ ] . our molecular data indicated paraphyly between thermocyclops and mesocyclops, which might reflect introgression resulting from hybridization [ ] . paraphyly has also been observed in other zooplankton, for example, in the daphnia pulex species complex [ ] and moina [ ] . future studies and nuclear markers are needed to investigate gene introgression among the copepod species from nigeria. here, cyclopidae in nigeria showed a high species diversity, but for each species, the haplotype diversity and nucleotide diversity were rather low. consistently, low haplotype and nucleotide diversities were observed in the copepods calanus finmarchicus [ ] , c. agulhensis and c. sinicus [ ] . patterns of population genetic diversity could be caused by different evolutionary forces, such as mutation, migration, genetic drift and natural selection. how these evolutionary forces affect the population genetic diversity depends on many factors, including species' response to environmental changes, and the past and present sizes of the population [ ] . another explanation might be that the structuring of a metapopulation together with founder effects resulted in low population genetic diversity [ , ] . however, the limited sampling of each species among populations cannot be ruled out as a cause for their low genetic diversity in this study. in conclusion, our data revealed a high species diversity of cyclopidae in southeast nigeria: twelve species were detected. our geographical sampling scale in this study was quite small, and therefore, further studies are called for a comprehensive understanding of species distribution and genetic diversity of cyclopidae in west africa. copepod specimens were collected from freshwater lakes around southeast nigeria (fig. a and table ). samples were collected using a -μm plankton net hauled vertically through the water column at three different sites per location. samples collected from different sites in the same location were pooled together and preserved in % ethanol. all specimens were identified morphologically according to the morphological description of copepods in nigeria [ ] [ ] [ ] ] , which also worked as taxonomic keys in this paper. copepods were processed for molecular analyses ( table ). the cephalosome portion of the prosome was obtained from each individual copepod to avoid dna contamination from prey items in the gut, using a microscopic tweezer and a sharp blade under the stereomicroscope. total genomic dna was extracted from the head using h buffer with proteinase k ( μl), containing mm tris-hcl, . m kcl, . % tween , . % np- and mg/ml proteinase k (merck, germany). samples were incubated overnight at °c in a water-bath with mild shaking. the proteinase k was irreversibly denatured after a -min incubation at °c. the homogenate was centrifuged briefly and stored at °c before use. a base-pair fragment of the coi gene was amplified using the consensus primer pair (forward: ′-ggt caa caa atc ata aag ata ttg g − ′; reverse: ′-taa act tca ggg tga cca aaa aat ca - ′ [ ] ;). polymerase chain reaction (pcr) was carried out in a total volume of μl, consisting of μl x pcr buffer ( mm tris-hcl, ph . , mm mgcl , mm kcl), μl . mm of each dntp, μl . μm of each primer, . μl water, units of taq dna polymerase (supertherm dna polymerase, taq hs from takara bio inc., california, usa) and μl of genomic dna. the pcr thermocycle protocol was as follows: denaturation at °c for min, then cycles of min at °c, . min at °c and . min at °c; followed by a final elongation at °c for min. the success of amplification was checked using agarose gel electrophoresis. afterwards, the pcr products were purified (high pure pcr product purification kit, roche diagnostics) and sequenced in the forward direction on an abi prism dna capillary sequencer by invitrogen trading co., ltd. (china). chromatograms were checked for ambiguous base calling and errors in base calling were corrected using mega [ ] , and the phred quality scores of the sequences were examined with chromas lite version . (technelysium pty. ltd., south brisbane, australia). sequences with double peaks or noise were re-sequenced in the reverse direction, and only chromatograms of high quality were applied to the following genetic analyses. all newly obtained sequences were submitted to genbank under accession numbers mn -mn . we identified unique haplotypes in dnasp . [ ] . muscle [ ] implemented in mega was used to align the sequences that were subsequently translated into amino acids to examine the presence of stop codons. afterwards, all haplotypes were aligned, together with reference sequences obtained from genbank (table s ), using the clustal w algorithm [ ] in mega . then, all the sequences were timed to a uniform length of bp in mega . for each species, the number of haplotypes (n ), haplotype diversity (h d ) and nucleotide diversity (π) per population (populations with sample size less than were excluded) were calculated in dnasp . . the test of xia et al. [ ] implemented in dambe [ ] was used to inspect potential loss of phylogenetic signal resulting from substitution saturation at the coi locus. a phylogenetic tree was then constructed using the bayesian method in beast [ ] , with a tree sampled every generations among , , , a burn-in of %, and the final , trees summarized using treeannotator. the best-fitting substitution model was gtr + g + i according to the corrected akaike information criterion in jmodeltest v. . . [ ] . we applied a strict molecular clock and set other tree priors to their default values. tracer v . [ ] was applied to ensure that enough generations were computed. paracalanus parvus, a member of the calanoida phylogenetically close to cyclopoida, was used as an outgroup. to test the hypothesis that the cyclopidae in nigeria contains high biodiversity, two independent species delimitation methods were applied: the general mixed yule coalescent model (gmyc [ ] ) and poisson tree processes methods (ptp [ ] ). the gmyc model is a likelihoodbased method using an ultrametric tree to delimit species by fitting within-and between-species branching models to reconstruct gene trees. we performed the gmyc modeling using the "splits" package [ ] in r . [ ] and conducted the ptp calculations on the bptp webserver (http://species.h-its.org/ptp/), with , markov chain monte carlo (mcmc) generations, thinning set to , burnin at % and a bayesian search performed. the input phylogenetic tree was generated using beast (see above). a network of coi haplotypes was then constructed to visualize genetic relationships among populations using haploviewer [ ] . the maximum likelihood tree inferred with mega using the best model gtr + g + i (by jmodeltest v. . . ) was applied as input. uncorrected pairwise genetic distances between species were calculated in mega based on coi. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : table s . list of reference coi sequences of cyclopidae (from south korea, brazil and china) and the outgroup used in this study. abbreviations coi: mitochondrial cytochrome c oxidase subunit i gene; pcr: polymerase chain reaction; bp: base pairs; gmyc: the general mixed yule coalescent model; ptp: poisson tree processes methods an introduction to copepod diversity. london: ray society copepod evolution. london: the ray society climatic warming and the decline of zooplankton in the california current entomology for the copepodologist high diversity of caligus species (copepoda: siphonostomatoida: caligidae) in taiwanese waters how many copepods mismatch between marine plankton range movements and the velocity of climate change reorganization of north atlantic marine copepod biodiversity and climate the value of the world's ecosystem services and natural capital phase transitions of wax esters adjust buoyancy in diapausing calanoides acutus climate change and marine plankton molecular phylogeny of the calanoida (crustacea: copepoda) cladistic analysis of the calanoid copepoda a survey of predilection sites and degree of disability associated with guineaworm (dracunculus medinensis) dracunculiasis in the north eastern border of ebonyi state, south eastern nigeria progress in dracunculiasis eradication in oyo state, south-west nigeria: a case study four new species of cyclops sensu lat. 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linguistic help, and the anonymous reviewer for useful comments on the earlier version of this manuscript. authors' contributions my designed the study, ce, eo, co, fa, cn, jo and pu collected samples, yn and jw carried out the molecular work. yn, wh and my contributed to data analyses. my wrote the manuscript with the help of yn. all authors read and approved the final version. this research was funded by the national natural science foundation of china ( ) and natural science foundation of shanghai ( zr ) to my. the funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. all the sequencing data are available via ncbi (under accession numbers mn -mn ). collection of zooplankton (copepods in this study) did not require specific permissions because these samples were obtained from unprotected lakes that are open for public activities. our study did not involve the use or collection of endangered or protected species. not applicable. the authors declare that they have no competing interests. key: cord- - j yic authors: donato, celeste; vijaykrishna, dhanasekaran title: the broad host range and genetic diversity of mammalian and avian astroviruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: j yic astroviruses are a diverse family of viruses that infect a wide range of mammalian and avian hosts. here we describe the phylogenetic diversity and current classification methodology of astroviruses based on the orf b and orf genes, highlighting the propensity of astroviruses to undergo interspecies transmission and genetic recombination which greatly increase diversity and complicate attempts at a unified and comprehensive classification strategy. astroviruses (astvs) were first described in as small viruses that are - nm in diameter with icosahedral morphology. astroviruses are named due to the distinct five-pointed or six-pointed star-like appearance of some virions when visualized under an electron microscope (em); astrovirus is derived from the greek word astron meaning star [ ] [ ] [ ] . astroviruses were first described from human infants with diarrhea and were subsequently identified in the young of numerous mammalian and avian species [ , ] . human astroviurses (hastvs) have been recognized as one of the major causes of acute gastroenteritis in children, associated with - % of infections [ ] . transmission of hastv occurs via the fecal-oral route, person-to-person contact, or contaminated food or water. following an incubation period of - days, symptoms including diarrhea, vomiting, abdominal pain, and fever are often reported [ , ] . whilst primarily associated with asymptomatic or diarrheal disease in humans, there are several reports of central nervous system (cns) complications such as acute flaccid paralysis [ ] , meningitis, and encephalitis [ , ] . animal astroviurses, have been isolated from numerous mammalian and avian species. in animals, astrovirus infection may be asymptomatic or associated with enteric disease and a range of other symptoms indicative of the involvement of other organ systems including hepatitis and nephritis in avian species [ , ] , and neurological symptoms in cattle [ ] [ ] [ ] and mink [ ] . astroviruses are classified within the unassigned astroviridae family and are non-enveloped viruses characterized by a positive sense, single-stranded rna (ssrna) genome . - . kb long comprised of a -untranslated region (utr), three open reading frames (orfs)-orf a, orf b, and orf , a -utr, and a poly a tail [ ] . the orf a region encodes a non-structural polyprotein (serine protease), orf b encodes a polyprotein including the rna-dependent rna polymerase (rdrp), and orf encodes the viral capsid protein [ ] . a further orf, termed orfx, has been observed in classic hastvs and some mammalian astroviurses, overlapping the end of orf which may be translated through a leaking scanning mechanism [ ] . astroviruses exhibit several distinctive features in addition to a distinctive morphology. the viruses lack a rna-helicase domain encoded within the genome and utilize a ribosomal frameshifting mechanism to translate the rdrp, which distinguishes the astroviridae family from other non-enveloped ssrna virus families such as picornaviridae and caliciviridae [ , ] . the greatest diversity in the genome is within the orf region, which is characterized by a highly-conserved n-terminal domain (amino acids (aa) - ), a hypervariable domain (aa - ) which is believed to form the capsid spike and contribute to receptor binding, and a highly acidic c-terminal domain [ , ] . the wide host range of astroviruses and the high degree of genetic diversity present within the astroviridae family have complicated attempts at a unified classification method. astroviruses are classified within the unassigned astroviridae family, which was initially comprised of a single genus (astrovirus) based on virion morphology [ ] . the classification of the astrovirus genus within the family astroviridae was recognized by the international committee for taxonomy of viruses (ictv) in and the classification scheme has been modified numerous times over the intervening years [ ] . in , two genera were recognized; mamastrovirus (mastv) and avastrovirus (aastv), that were known to infect mammalian and avian species, respectively, and viruses were classified solely on the species of origin [ ] . however, the advent of sequence based characterization rendered this approach inadequate, revealing that viruses isolated from different species can be genetically similar (reflecting prevalent interspecies transmission of viruses) and also revealing a large range of diversity of viruses within a single host species. with this in mind, the classification system proposed by the ictv astroviridae study group in recommended classification based on the amino acid sequence of the orf genome region, recommending that different strains of the same astrovirus species should share > % identity in the capsid proteins [ ] . additionally, there are proposals to define distinct variants within a recognized astrovirus species, with a variant defined as sharing < - % nucleotide similarity to the reference or prototype strain of each species or if phylogenetic analysis is used, a distance of > . based on analysis of the capsid protein [ , ] . astroviruses within the mamastrovirus genus are derived from numerous mammalian species in addition to humans (hastv), including farmed species such as pigs (pastv), sheep (oastv), cattle (boastv), domesticated animals including cats (fastv), and dogs (caastv), rodents and small mammals including mink (miastv), bats (bastv), rats (rastv), mice, rabbit (rabastv), fox, marmot (hhmastv), porcupine, shrew, vole, and larger species including deer (ccastv), monkeys, water buffalo (bufastv), yak, camel (dcastv), and cheetah (chastv) (figure a,b) . viruses from the mamastrovirus genus have also been characterized from marine mammals including stellar sea lion (sslastv) and california sea lions (cslastv), minke whale, orca whale, and bottlenose dolphins (bdastv) (figure a ,b) [ ] . the current ictv classification reveals recognized species of mamastrovirus (mastv- - ) within two genogroups gi and gii; mamastrovirus (gi.a-human); mamastrovirus (gi.b-feline); mamastrovirus (gi.c-porcine); mamastrovirus (gi.d-california sea lion); mamastrovirus (gi.e-canine); mamastrovirus (gi.f-human); mamastrovirus (gi.g-bottlenose dolphin); mamastrovirus (gii.a-human); mamastrovirus (gii.b-human); mamastrovirus (gii.c-mink); mamastrovirus (gii.d-california sea lion), mamastrovirus (gii.e-bat); mamastrovirus (gii.f-ovine); and mamastroviruses - (gii.g to gii.l-bat species), and numerous other strains awaiting classification, some of which are considered as tentative new species (designated by a ∧ symbol in the phylogenetic trees) (figure a ) [ , ] . viruses from the avastrovirus genus have been characterized from numerous farmed avian species including turkeys (tastv), ducks (dastv), chicken (castv), guineafowl (gfastv), pigeon (piastv), goose, as well as wild aquatic and terrestrial birds including heron, doves, penguins, and many other species (figure a) . the three species originally recognized within the genus were avastrovirus gi.a comprised of turkey astrovirus (tastv- ), avastrovirus gi.b comprised of avian nephritis virus (anv- ), avian nephritis virus (anv- ), and avastrovirus gii.a comprised of turkey astrovirus (tastv- ) and duck astrovirus dastv/c-ngb [ ] . avastrovirus gi.a, avastrovirus gi.b, and avastrovirus gii.a were renamed avastrovirus (aastv- ), avastrovirus (aastv- ), and avastrovirus (aastv- ), respectively [ ] . viruses from the avastrovirus genus have been characterized from numerous farmed avian species including turkeys (tastv), ducks (dastv), chicken (castv), guineafowl (gfastv), pigeon (piastv), goose, as well as wild aquatic and terrestrial birds including heron, doves, penguins, and many other species (figure a) . the three species originally recognized within the genus were avastrovirus gi.a comprised of turkey astrovirus (tastv- ), avastrovirus gi.b comprised of avian nephritis virus (anv- ), avian nephritis virus (anv- ), and avastrovirus gii.a comprised of turkey astrovirus (tastv- ) and duck astrovirus dastv/c-ngb [ ] . avastrovirus gi.a, avastrovirus gi.b, and avastrovirus gii.a were renamed avastrovirus (aastv- ), avastrovirus (aastv- ), and avastrovirus (aastv- ), respectively [ ] . currently, classification into species is based on the phylogenetic analysis of the amino acid sequence of the full length orf region of the genome that encodes the capsid. however, the limited number of capsid sequences available compared to rdrp sequences makes consistent classification difficult, especially with some novel viruses incompletely sequenced. there are numerous unclassified astroviruses, particularly isolated from aquatic and terrestrial wild birds which, according to the ictv, are "related viruses which may be members of the avastrovirus genus but have not been approved as species" [ ] . astrovirus infection in humans has been primarily associated with diarrhea and vomiting, accounting for up to % of sporadic gastroenteritis cases in some regions [ ] . cns complications associated with astrovirus infection have been reported in recent years, including acute flaccid paralysis, with some fatalities reported in children with underlying immune disorders [ , ] . historically human astroviruses (hastv) were classified into five serotypes in [ ] . subsequent molecular characterization based on viral reactivity to polyclonal antibodies and nucleotide sequence analysis led to the recognition of eight serotypes (hastv- - ), now termed "classic" hastv [ , , ] . the relatively recent advent of next generation sequencing (ngs) and metagenomic analysis has led to the identification of numerous novel strains considered "non-classic" hastv [ ] . currently, hastvs are classified within the species mastv- (hastv- - ), mastv- (mlb - ), mastv- (va /hmo-a, va , va , bf ), and mastv- (va /hmo-c, va /hmo-b) [ ] (figure a ). the mastv- species is comprised of hastv- - , and surveillance has revealed that hastv- is the most commonly detected type in children, followed by hastv- - , whereas hastv- - have been rarely detected [ ] . hastv- and hastv- have been associated with infection of older children and longer duration of diarrhea (> days) [ , ] . a hastv- strain was also isolated from an infant with fatal meningoencephalitis [ ] . based upon the phylogenetic analysis of the orf region, different lineages within each hastv type have been proposed; hastv- (hastv- a-d) and hastv- (hastv- a-d) have been divided into four lineages, whereas hastv- (hastv- a-b) and hastv- (hastv- a-c) have been classified into two and three lineages, respectively [ ] . the first "non-classic" hastv strain characterized was mlb , the virus was detected in a stool sample from a year old australian child with acute diarrhea in ; the child had previously received a liver transplant [ ] . the majority of mlb strains characterized to date have been detected in india, kenya, and japan with limited detected in the usa, china, bhutan, egypt, brazil, and italy and prevalence has been reported in the range of . % to % [ ] . however, a seroepidemiologic study in the usa revealed that primary exposure to mlb occurs in childhood and that seropositivity reached % by adulthood suggesting the widespread circulation of the virus in the human population [ ] . mlb viruses were first identified in vellore, india [ ] with the majority of strains subsequently identified in japan, the gambia, and switzerland with limited detection in turkey, usa, kenya, china, and thailand and prevalence reported in the range of . % to . % [ ] . mlb has been associated with meningitis and other cns complications and has been detected in immunocompromised children [ ] . mlb viruses were first detected in india in [ ] , with subsequent detection in kenya and the gambia and the prevalence in stools ranges from . % to . % [ , ] . there is a dual naming system for some hastv species due to the simultaneous characterization of these viruses by different researchers; these viruses are termed va/hmo named for va-virginia and hmo-human-mink-ovine-like viruses, due to their genetic relatedness to previously characterized mink and sheep viruses [ ] . in , va /hmo-a strains were detected in children with non-polio acute flaccid paralysis in nigeria, pakistan [ ] , and india [ ] . the prevalence of va /hmo-a viruses in stools ranges from . % to . %, with strains also detected in egypt, japan, usa, kenya, and china [ ] . va has only been detected in nepal [ ] and the bf strain has only detected in burkina faso [ ] . the va /hmo-b viruses were first identified in vellore, india [ ] with sporadic detection in nigeria, pakistan, and nepal with prevalence ranging from . % to . % [ ] . va /hmo-c viruses were first detected in during an outbreak of diarrhea in children in virginia, usa [ ] . va /hmo-c viruses have been detected and associated with encephalitis in immunocompromized children and adults [ , , ] and acute respiratory disease [ ] . the prevalence of va /hmo-c viruses in stools ranges from . % to . % in diarrheic and non-diarrheic subjects worldwide, with limited detection in nepal, japan, tanzania, the gambia, france, and the u.k. [ , , ] . seroprevalence of va /hmo-c has been reported at % in adults [ ] . the divergent human astrovirus-like virus tentatively named bastrovirus was isolated from patients in the netherlands. the capsid region is homologous to the capsid of hastv whilst the rdrp region is more closely related to members of the hepeviridae family. this virus remains to be classified by the icvt, however the capsid regions clusters closest to the mlb - viruses suggesting an evolutionary relationship to these divergent human astroviruses [ ] (figure a ). revealing the geographic and host diversity of this virus, divergent bastrovirus strains have also been isolated from bat, pig, and rat species in vietnam, forming a distinct cluster of strains that also await classification (figure a ). mamastroviruses are capable of infecting a wide range of mammalian species including companion animals (cats and dogs), intensively farmed species (pigs and cattle), as well as terrestrial and aquatic wild mammalian species. unexpectedly there is no clear clustering of viruses separated by hosts of terrestrial or aquatic origins (figure a,b) . in addition to the classified mastv species, there are numerous divergent viruses isolated from diverse species which likely represent new species awaiting formal classification (figure a ) [ ] . not surprisingly, domesticated animals such as cats and dogs harbor astrovirus strains more closely related to hastv than the viruses harboured by many other animal species. feline astrovirus was first identified in , and feline strains form a small discrete cluster defined as the species mastv- , closely related to the human strains comprising the species mastv- [ ] (figure a ). based on evolutionary analysis, an interspecies transmission pathway has been hypothesised whereby porcine strains may have been transmitted to cats and subsequently to humans, possibly involving other intermediary species suggesting sustained interspecies transmission events [ ] . characterization of an outbreak of diarrhea in a group of captive cheetahs in a breeding facility identified an astrovirus strain most closely related to feline strains (figure a ), however it is not clear if this is a recent transmission from domesticated cats or if the virus is circulating independently in cheetahs [ ] . despite dogs also being a companion animal, canine astrovirus, first described in the s, are not as closely related to human strains as feline strains appear to be, based on phylogenetic analysis (figure a,b) . canine strains form a small, discrete cluster comprising species mastv- , within a lineage comprised of dolphin and sea lion clades representing species mastv- and - [ , ] , and feline, porcine, and human strains are more distantly related within the same lineage (figure a) . a large diverse lineage, largely comprised of unclassified viruses or tentatively classified viruses awaiting approval encompasses divergent marmot and rabbit strains (mastv- ), small, discrete clusters of porcine/ovine/bovine strains (mastv- ), divergent rat strains (mastv- ), and unclassified mouse and mink strains forming a small discrete cluster (figure a) . numerous porcine strains also cluster within this lineage; pastv comprise mamastrovirus and were first detected by em in pigs in the u.k. and the usa [ ] , and are now recognized to have worldwide distribution [ ] . there is a high prevalence of astrovirus detection in pigs, up to % in some studies, suggesting that pigs may be persistently infected with various strains of pastv [ ] . there are five recognized lineages (pastv - ) reflecting the diversity of strains, suggesting swine are highly permissive to astrovirus infection often without symptoms of disease (figure a,b) . this diversity likely reflects the varying origins of these viruses, in particular highlighting frequent interspecies transmission and recombination events [ ] [ ] [ ] . a study in the usa revealed the high level of co-infections in pigs ( . %), revealing the frequent opportunity for recombination, especially between viruses of different lineages [ ] . the close clustering between some porcine and humans strains, in particular mastv- and mastv- viruses, reflects the close contact between these species with the close sharing of environments and co-housing documented in some countries facilitating frequent interspecies transmission [ ] . wild boars showing no symptoms of enteric disease, housed in a captive breeding park with no contact with domesticated pigs, were found to harbor astrovirus strains with a high degree of genetic similarity to porcine strains comprising mastv- and suggests that the virus may be derived from commonly circulating porcine strains [ ] (figure a,b) . porcupine strains isolated in china also cluster with unclassified pastv- strains suggesting further interspecies transmission of these viruses (figure a ) [ ] . unexpectedly, limited astrovirus strains have been isolated from sheep. ovine astrovirus was first identified by em in [ ] , and oastv- clusters with bovine strains comprising mastv- and a second strain from a healthy sheep characterized in , oastv- , clusters with porcine and bovine strains comprising mastv- [ ] (figure a) . similarly, astroviruses are not associated with a significant burden of diarrheal disease in bovine species. the first bovine astrovirus was detected in england in [ ] and bovine astrovirus strains have been detection in association with neurological disease, including encephalitis (boastv-ch /neuros ) [ ] [ ] [ ] [ ] [ ] [ ] and diarrheal disease in calves in south korea [ ] and cattle and buffalo calves in china [ ] . two serotypes were previously recognized, boastv- and boastv- [ ] , however based on phylogenetic analysis there are multiple lineages of boastv strains circulating in farmed bovine populations, and the close clustering of bovine, porcine, and ovine strains in multiple lineages reflects the common interspecies transmission events that occur between farmed animals (figure a,b) . phylogenetic analysis also reveals that bovine-like astrovirus strains have been isolated from numerous wild species including water buffalo, yak, and european roe deer (ccastv- and ccastv- ) suffering from gastroenteritis [ ] . unclassified astroviruses from dromedary camels (dcastv) [ ] also cluster in a lineage comprised of porcine and bovine strains, further suggesting multiple interspecies transmission events (figure a) . substantial astrovirus strain diversity has been observed in small mammals, primarily rodents and bats, forming both species-specific clusters reflecting endemic transmission and co-clustering of strains with other host species reflecting widespread interspecies transmission (figure a,b) . novel murine astrovirus strains have been isolated from laboratory mice in the usa and japan [ , ] . divergent viruses have also been detected, such as those detected in rats (mastv- ), highlighting the need for more detailed detection and characterization of these viruses to better understand the role these animals play in astrovirus transmission between varied species. one of the mammalian species with the highest burden of symptomatic astrovirus infection is mink; infection is associated with pre-weaning diarrhea syndrome and the neurological condition "shaking mink syndrome" [ , ] . mink viruses cluster within multiple lineages suggesting that the species is permissive to infection with multiple, diverse lineages of viruses (figure a) . bat astroviruses were first detected in in hong kong [ ] and subsequently detected in bats in china [ , ] , north america [ ] , germany [ ] , hungary [ ] , and the czech republic [ ] from numerous bat species, with detection rates ranging from % to % in miniopterus magnater bats and from % to % of miniopterus pusillus bats sampled in hong kong [ ] . a diverse population of viruses appears to be highly prevalent in bats without causing disease [ ] (figures and ) . the majority of bat astroviruses are divergent from other characterized mammalian astroviruses, and display a high degree of genetic diversity forming numerous recognized and proposed species (figure a) . some bat sequences clustered with strains from other species including fox, cattle, and mice, suggesting that bats are highly permissive to infection with diverse astrovirus strains from multiple hosts and play a key role in astrovirus diversity and interspecies transmission (figure a,b) . astroviruses have been detected in aquatic mammalian species including californian sea lions, steller sea lion, bottlenose dolphin, killer whale, and minke whale [ ] . the strains from these aquatic species do not cluster together, instead forming multiple discrete clusters, suggesting several transmission events from terrestrial mammals to aquatic mammals (figure a) . minke whale and bdastv- strains are divergent to characterized astrovirus strains, with bdastv- strains comprising mastv- and a diverse group of sea lion viruses comprising the mastv- cluster with porcine and canine strains (figure a ). there are numerous divergent strains that are yet to be classified that may reflect interspecies transmission events. a single divergent feline strain along with a fox strain cluster within a diverse lineage comprised of human hmo strains (mastv- and - ), sea lion (mastv- ), unclassified sea lion, mink (mastv- ), and bat (mastv- ) strains. a himalayan marmot strain clusters with mastv- strains (figure a,b ). there appears to be a large diversity of mastv strains not captured by the currently available sequences, highlighting the need for more detailed sampling and characterization of animal strains. the isolation of astroviruses from avian species predates their isolation in humans, with disease in ducklings described in , however the virus was not formally recognized as an astrovirus until [ , ] . avian astroviruses have been documented to cause infection in poultry leading to economic losses in farms and affecting food production worldwide [ ] . avian astroviruses have been associated with a spectrum of disease ranging from subclinical infection in seemingly healthy adult birds to heavy flock losses. pleomorphic symptoms include enteritis in turkeys, chickens, and guineafowl, mild growth depression and nephritis in chickens, and hepatitis in ducklings [ ] . astrovirus infection has been implicated in pre-hatching mortality in ducklings and goslings [ ] . in addition to aastv- comprised of tastv- , aastv- comprised of anv and anv , and aastv- comprised of tastv- and duck astrovirus (dastv- ), there are numerous yet-to-be classified viruses, including turkey astrovirus (tastv- ), chicken astrovirus a (castv-a), chicken astrovirus b (castv-b) [ , ] , gfastv, and multiple viruses isolated from ducks including duck hepatitis virus (dhv- /dastv- ) [ ] , dhv- /dastv- -like viruses, dastv-cph (dastv- ) [ ] , dastv- , dastv-yp/dastv- -like, and diverse viruses isolated from wild birds [ ] . avian astrovirus have been detected in outbreaks of enteric disease in turkey poults, and avian astrovirus were first reported as an agent of gastroenteritis and mortality in young turkeys in , associated with a condition known as poult enteritis mortality syndrome (pems) [ , ] . subsequently there have been sporadic reports of astrovirus outbreaks in turkeys, associated with enteritis and growth depression [ ] . based on serological and genetic analysis, two types of tastv have been recognized (tastv- and tastv- ). tastv- comprises the avastrovirus species and was first described in the u.k. [ ] . tastv- has limited detection in other avian species with sporadic detection in chicken and ducks and forms a discrete cluster in both the capsid and rdrp phylogenetic analysis (figure a,b) . tastv- was identified in [ ] , and is likely to be classified within the species avastrovirus and is primarily associated with pems [ ] . tastv- is the predominant tastv lineage, with a wider global circulation and greater genetic diversity compared to tastv- , reflected by the co-circulation of multiple sub-lineages (figure a,b) . based on phylogenetic analysis of the rdrp gene there appears to be limited interspecies transmission of tastv- -like viruses detected in chicken and duck (figure b) . astroviruses infecting guineafowl are closely related to tastv- strains based on analysis of the rdrp region [ ] , however the capsid region is distinct to tastv- strains, forming a discrete cluster in a lineage of unassigned viruses suggesting they are closely related to castv-b strains (figure a) . these gfastvs were possibly derived from recombination and interspecies events followed by sustained transmission in the guineafowl population. this highlights the limitation of classifying viruses based on a single region of the genome. chicken astrovirus has been associated with runting-stunting syndrome (rss) in chickens characterized by poor weight gain, lower feed conversion, and mortality resulting in economic losses [ ] , and "white chicks" disease associated with increased mortality of embryos and chicks, weakness, and white plumage [ ] . currently two serotypes of castv have been described [ ] , and both serotypes form discrete clusters in the phylogenetic analysis of the capsid region within a lineage of unclassified viruses (likely to be classified within aastv- ). the castv-a viruses form a smaller, discrete lineage, clustering closest to dastv- strains (figure a) . castv-b strains form a large lineage clustering closest to gfastv strains. based on phylogenetic analysis of the small region of the rdrp gene commonly sequenced, it does not allow castv-a and -b strains to form discrete clusters as seen in the capsid analysis, possibly reflecting multiple recombination events in castv strains (figure b ). castv strains have limited detection in other avian species with sporadic detection in pigeon and duck (figure a,b) . avian nephritis virus was identified in association with intestinal nephritis in chickens and growth retardation. the first serotype of anv (anv- ) was isolated from a healthy broiler chick in [ ] and was initially regarded as a picornavirus, subsequently reclassified within the astroviridae family in [ ] . based on capsid phylogenetic analysis, anv- strains cluster within the aastv- lineage forming a small, discrete, relatively conserved cluster (figure a) . the second serotype (anv- ) was later described from chicks with stunted growth [ ] . based on capsid phylogenetic analysis, anv- strains cluster within the aastv- lineage, forming a larger, more diverse sub-lineage compared to anv- . anv- and anv- viruses have also been sporadically detected in ducks and turkeys (figure a) . a third serotype (anv- ) detected in chickens and turkeys with rss and locomotion impairment has been proposed based on sequencing of the orf a region, however complete genome sequences are unavailable for adequate comparisons to the recognized serotypes [ ] . the virus designated pigeon anv (p-anv) was detected during an outbreak of gastrointestinal illness in young pigeons in china [ ] . p-anv represents an interspecies transmission event from chickens to pigeon; based on phylogenetic analysis, p-anv strains share a high degree of genetic similarity to anv- strains and cluster closely with strains also circulating in china suggesting a localized transmission event, and these viruses should be considered as anv- viruses and do not require a distinct designation (figure a ) [ ] . based on phylogenetic analysis of the small region of the rdrp gene commonly sequenced, it does not allow anv and strains to form discrete clusters as characterized in the capsid analysis (figure b ). whilst this may reflect common recombination events between anv strains, the analysis of a small region does not adequately allow for distinct clustering. astrovirus infection in ducks has been associated with a highly contagious and fatal hepatitis, historically known as duck hepatitis virus type (dhv- ), which was described in the u.k. and subsequently serotype dhv- was isolated in the usa [ , , , ] . the th ictv report classified dhv- and dhv- as dastv- and dastv- , respectively [ ] . dastv- strains form a small, discrete cluster within the aastv- lineage based on phylogenetic analysis of the capsid region, clustering closest to tastv- strains (figure a) . dastv- viruses, currently unclassified, form a discrete, highly conserved cluster closest to castv-a strains (figure a) . based on phylogenetic analysis of the rdrp region, dastv- is closely related to other unclassified duck strains whilst dastv- strains are more closely related to tastv- strains (figure b ). further unclassified serotypes have been described; dastv- -cph, dastv/c-ngb, and dastv- [ ] . duck astrovirus dastv- /cph has been suggested to transmit horizontally and vertically [ ] and dastv- /cph viruses have also been detected in goslings [ ] . phylogenetic analysis of the rdrp region highlights the diversity of aastv strains circulating in duck species (figure b) . strains endemic to ducks have limited detection in other avian species including geese (figure a,b) . astroviruses have been detected in numerous wild aquatic species including teals, pintails, and shovelers (belonging to the order anseriformes), sanderlings (order charadriiformes), and herons and spoonbills (order pelecaniformes) [ , ] . fewer land dwelling wild birds have been found to harbor avian astroviruses including doves and pigeons (order colombiformes), european roller (order coraciiformes), and black-naped monarch (order passeriformes) [ , , , ] . these viruses are highly divergent and largely unclassified, suggesting these viruses are endemic to the wild bird population (figure a,b) . the migratory behavior of some of these species provides ideal conditions for virus dissemination and diversification as during migration there is a high degree of co-mingling and increased density of birds of different species that may originate from varied geographic regions [ ] . phylogenies indicate that the genetic diversity of mamastroviruses has been shaped by extensive interspecies transmission events that have occurred in the past between wild and domestic species and humans. however, the inference of astrovirus interspecies transmission events is hampered due to (a) sequencing of only a small portion of the genome; (b) inadequate sampling; or (c) that the event occurred early during the divergence of astrovirus lineages. a few exceptions where host-jumps are apparent have been in livestock where a greater level of sampling has occurred. this includes hosts with a greater level of interaction (e.g., mastv- in ovine and bovine) or host genetic similarly (e.g., mastv- in wild boars and domestic pigs). with the exception of hastv- strains associated with fatal meningoencephalitis, it is interesting to note that viruses from multiple species, all recognized to cause neurological symptoms, are closely related including human va /hmo-c viruses and mink and bovine viruses also associated with neurological symptoms, suggesting that these related viruses may have a distinct phenotype compared to other mastv strains (figure a,b) . astrovirus strains identified from fecal samples of multiple non-human primate species from wild, captive, and peri-urban environments in bangladesh and cambodia reveal multiple interspecies transmission events, with viruses closely related to the va/hmo lineage of human viruses, and non-human mammalian and avian astroviruses (figure a,b) [ ] . similarly, there appears to be evidence for a high degree of cross species transmission of avian astroviruses between farmed poultry species as described in the above sections. there also appears to be transmission between avian and mammalian species. the highly divergent strains isolated from european roller (er/szal /hun/ and er/bmtk /hun/ ) exhibited low identity to avian and mammalian astroviruses, cluster within the mastv lineage, and were likely derived from multiple recombination and interspecies transmission events [ ] . the carnivorous diet of this avian species may have facilitated the interspecies transmission event between a rodent or small mammal species. this is the only report of a mamastrovirus strain in an avian species; in contrast, there have been more reports of avastrovirus strains detected in mammalian species which may reflect greater sampling density of the mammalian population. a highly divergent group of mink strains detected in china represent a novel clade of astroviruses that were distantly related to previously described mink astrovirus and were closely related to chicken and turkey astroviruses (figure a,b) [ ] . these viruses are recombinant strains with the capsid region clustering with castv-b strains and the rdrp region clustering with human mastv strains and castv strains (figure a,b) . there have been two avastrovirus strains detected in humans; a strain clustering close to anv- strains detected in turkey and chicken was isolated from a child in the gambia, and a strain clustering with anv- strains detected in chicken was isolated from a child in kenya [ ] . serological studies have been used to screen human sera from poultry workers for antibodies to tastv- , with up to % of participants positive with the highest detection in abattoir workers and turkey growers [ ] , suggesting avian strains may be readily transmitted to humans under prolonged close contact. the important role that ecotones play in astrovirus cross-species transmission has been proposed [ ] . ecotones are ecological transition areas such as small and medium sized farms which rear multiple species. the co-rearing of poultry such as domestic ducks, chickens, turkey, and guineafowl can facilitate transmission between these species but also transmission to wild birds [ ] . farms and abattoirs have also been recognized as environments facilitating transmission between livestock species and to farm and abattoir workers [ ] . many other species have contact with livestock in a farming environment; in addition to wild species, companion animals such as cats and dogs and other peri-domestic animals have contact with livestock and their biological waste providing substantial opportunities for cross-species transmission. astroviruses also persist in bodies of water making the aquatic environment ideal for the transmission of viruses infecting avian species, aquatic mammalian species, and possible transmission between terrestrial and aquatic species. untreated or inadequately treated sewage and waste water from domestic and farmed areas can reach fresh and marine bodies of water transmitting human and animal viruses. astroviruses have been detected in the environment and the durability of the virus in this environment may greatly contribute to cross-species transmission within and between terrestrial and aquatic species, generating significant diversity [ ] . in addition to interspecies transmission which generates significant diversity in astrovirus species, both intra-species and inter-species recombination can rapidly generate novel, divergent viruses. full-and partial-genome sequence analysis has identified multiple strains that have undergone recombination events, which are predominately located within the orf b/orf junction region of the genome, which is a region with an rna secondary structure predicted to contain a stable hairpin structure [ , , [ ] [ ] [ ] [ ] . a virus with a recombination event within the orf a region has been identified [ ] . some recombinants appear to be highly stable and show widespread detection [ ] , whilst others are detected sporadically as single strains. numerous human recombinant strains have been reported between hastv strains, including strains with hastv- (orf b) and hastv- (orf ) parental viruses, hastv- (orf b) and hastv- (orf ) parental viruses, hastv- (orf b) and hastv- (orf ), and va (orf b) and mlb (orf ) parental viruses [ , [ ] [ ] [ ] . divergent species often represent recombination events between strains of different species. in , the study conducted by rivera et al. suggested the possibility of a recombination event between human and california sea lion astrovirus strains [ ] . a recombinant strain derived from porcine astrovirus and human hastv- strains was reported from piglets and children from various regions of colombia [ ] . anv strains that appear to be derived from recombination events between anv- (orf b) and anv- (orf ) have been described in the usa [ ] . the increased sampling density of numerous host species combined with the more prevalent use of ngs technologies and viral metagenomic studies will increase the detection of novel strains, further driving the need for a unified, complex, and encompassing classification system. the previously common practice of sequencing a relatively conserved rdrp amplicon of - bp renders many sequences available in genbank of little use in detailed phylogenetic analysis, as does the frequent missing metadata regarding species of isolation, country, and date of collection. sequencing small regions is not adequate to determine if a virus strain is a novel, divergent strain or a recombinant virus. recommendations should be made to encourage full genome sequencing where possible and the deposition of associated host and demographic information. although analysis of amino acid sequences of the capsid region is required for classification, it leads to confusion regarding appropriate phylogenetic analysis, with highly inconsistent publication of nucleotide and amino acid trees further complicating attempts to clarify diversity and classification. the topologies of amino acid trees differ to those of nucleotide trees, particularly for the analysis of mastv, and whilst amino acid trees are required for classification, nucleotide trees may be more appropriate for describing within species diversity (figures s and s ) . incorporating a standardized nomenclature to aid in classification has proven invaluable in the classification of numerous viruses, including rotavirus and influenza [ ] . adopting a nomenclature that records the appropriate metadata associated with sample collection including host, location, date of collection, and determined species and serotype as proposed by martella and colleagues would vastly improve the usability of strains for more complex analyses [ ] . the following are available online at www.mdpi.com/ - / / / /s , 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and sewage treatment plants, evaluated by a competitive reverse transcription-pcr high evolutionary rate of human astrovirus nationwide surveillance study of human astrovirus infections in an italian paediatric population detection of all serotypes of human astrovirus by the polymerase chain reaction evidence of a recombinant wild-type human astrovirus strain from a kenyan child with gastroenteritis lineage diversification and recombination in type- human astroviruses novel astroviruses in children uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) key: cord- -oxahu nz authors: lawes, roger a.; murphy, helen t.; grice, anthony c. title: comparing agglomerative clustering and three weed classification frameworks to assess the invasiveness of alien species across spatial scales date: - - journal: divers distrib doi: . /j. - . . .x sha: doc_id: cord_uid: oxahu nz to prioritize weed management at the catchment scale, information is required on the species present, their relatively frequency, abundance, and likely spread and impact. the objective of this study was to classify the invasiveness of alien species that have invaded the upper burdekin catchment in queensland, australia, at three spatial scales. a combination of three published weed classification frameworks and multivariate techniques were employed to classify species based on their frequency and cover at a range of spatial scales. we surveyed the upper burdekin catchment for alien species, and for each species determined the following distribution indices — site frequency, total cover, transect frequency per site frequency and quadrat frequency per site frequency, cover per quadrat when present, cover per transect when present, and cover per site when present. these indices capture the effect of species abundance and frequency between sites (site frequency and total cover), within sites (transect frequency per site and cover per transect when present), and within transects (quadrat frequency per site frequency and cover per site). they were used to classify the species into seven groups using a hierarchical cluster analysis. the relationship between the indices was explored to determine how effective the small scale, site‐specific indices were at predicting the broader, landscape‐scale patterns. strong correlations were observed between transect frequency per site and frequency (r ( ) = . ) and cover per transect when present and total cover (r ( ) = . ). this suggests that if a weed is abundant at the site level, it has the potential to occupy large areas of the catchment. the species groupings derived from the application of the three published weed classification frameworks were compared graphically to the groupings derived from the cluster analysis. one of the frameworks classified species into three groups. the other two frameworks classified species into four groups. there was a high degree of subjectivity in applying the frameworks to the survey data. some of the data were of no relevance to the classification frameworks and were therefore ignored. we suggest that the weed classification frameworks should be used in conjunction with existing multivariate techniques to ensure that classifications capture important natural variations in observed data that may reflect invasion processes. the combined use of the frameworks and multivariate techniques enabled us to aggregate species into categories appropriate for management. new species entering a landscape are subject to community-level processes and may or may not become invasive. this fate is governed by the characteristics of the species and habitat it has entered (williamson & fitter, ; richardson & py s ek, ) . the ecological definition of invasiveness has been discussed at length by williamson ( ) , lonsdale ( ) , davis & thompson ( ) , rejmánek et al . ( ) , and colautti & macisaac ( ) . despite these discussions, ecologists have struggled to classify successful invasions, partly because successful invasions are quite rare. according to the 'tens rules' (williamson & fitter, ) , a species has approximately a − probability of becoming a pest once introduced to a novel ecosystem. initially, ecologists focused on classifying species based on important plant characteristics (e.g. mack, ) culminating in models of plant invasiveness with a view to predicting which species are likely to become invasive (e.g. perrins et al ., ; rejmánek, ) . biosecurity agencies adopted key concepts from these studies to develop weed risk assessment protocols (pheloung et al ., ) , although recent reviews of these protocols found intentional human dispersal of propagules to be more important than plant species' ecological traits such as dispersal mechanism or seed size (caley & kuhnert, ) . richardson et al . ( ) , davis & thompson ( ) , and colautti & macisaac ( ) all developed frameworks to enable researchers to either describe a species as a 'type' of invader (davis & thompson, ; richardson et al ., ) or classify the invasion itself as a series of stages (colautti & macisaac, ) (table ). the ability to classify an invasion is necessary to prioritize weed management. the framework of richardson et al . ( ) comprised three components: introduction, naturalization, and invasion. introduction implies that the species has been transported by humans across major geographical boundaries. naturalization occurs when the species has overcome abiotic and biotic barriers to survival and can cope with environmental stochasticity. invasion occurs when the species produces reproductive offspring distant from the parent and culminates in new self-perpetuating populations. six key barriers that limit the spread of introduced plants were defined to enable researchers to classify species into one of the three categories (table ) . py s ek et al . ( ) recently discussed further details on the use and application of richardson et al . ( ) 's framework. davis & thompson ( ) proposed a scheme modeled after rabinowitz's ( ) classification of rarity, to distinguish 'invaders' as distinct from 'successional colonizers' and 'non-invasive colonizers' . the authors defined eight different types of 'colonizers' table data requirements to implement the weed classification framework devised by richardson et al. ( ) ; colautti & macisaac ( ) ; davis & thompson ( ) introduction barrier based on three distinct characteristics: dispersal distance (short/ long), uniqueness (novel/common to the region colonized), and impact on the new environment (small/great) ( table ). the interpretation of dispersal distance and the origin of the colonizer are scale dependent, while impact may be assessed from either a productivity or an ecosystem function. colautti & macisaac ( ) recently proposed a framework that attempted to eliminate the need for universal definitions of current terms by using operational terms with no a priori meaning (i.e. as one of five possible stages) in a process based model. the first stage ( ) begins when a propagule enters the habitat of interest. a species proceeds through stage , dispersal and survival in the habitat, and stage , survival and reproduction. if the weed satisfies these criteria, it may then be classified into separate stages that describe the frequency and abundance of a species in an environment. stage species are considered localized and numerically rare. stage a species are widespread but rare, stage b species are localized but dominant in those localities, and stage species are considered widespread and dominant (table ) . again, the environment can be any region of interest. the definitions of widespread and abundant are also open to interpretation. in essence, all three frameworks put forward a series of hypotheses that can be applied to data to classify the invasive characteristics of a species. the data requirements necessary to implement the frameworks vary, although each draws heavily on frequency and abundance information to classify the invasive characteristics of a species. the strengths and weaknesses of these frameworks were recently evaluated by murphy et al . ( ) who used existing data on invasive species in canada, and tested whether the frameworks were operational given a relatively rich species data set, and whether the different frameworks identified the same sets of species. these authors had difficulty defining the rate of spread, required by richardson et al . ( ) 's scheme, and impact as required by davis & thompson ( ) 's, and concluded that both schemes had limited potential in classifying regional alien floras. in this study, we surveyed the riparian zone of the burdekin river system for alien species. this river system, the third largest in australia, extends for over km and connects an array of pastoral properties. alien plant species such as cryptostegia grandiflora and ziziphus mauritiana are present in the catchment and affect the productive capacity of pastoral properties in this catchment (grice et al ., ) . however, the extent of their geographical distribution and possible impact along the riparian zone remains unknown. other alien grass species, such as andropogon gayanus , have dramatically altered ecosystem function in other australian rangeland systems and may be present in this catchment (rossiter et al ., ) . it was therefore necessary to document the weed status of this ecosystem to assist managers and state agencies in formulating a regional weed management strategy. the frequency and abundance of each species were recorded at course and fine scales. we apply a series of multivariate techniques and the frameworks devised by richardson et al . ( ) , davis & thompson ( ) , and colautti & macisaac ( ) to each species objectively, using the data derived from the survey. the frameworks present a series of hypotheses about invasion and apply these to data. we propose evaluating weed invasions as part of a larger, existing plant community where invasive species are classified based on their relative abundance, frequency, and cover at local and landscape scales. this approach differs from those proposed by the authors of previous frameworks as species are classified using all available data acquired from an extensive, multiscale landscape survey of species frequency and cover. the patterns derived from multivariate analyses may help formulate hypotheses about invasion processes in the landscape of interest and can be applied to any data set. once critical invasive species have been identified, management strategies can be formulated to target important species in the ecosystem. surveys were conducted on the upper burdekin catchment, situated in north queensland, australia (fig. ). the burdekin river and associated tributaries extend for km. eighty sites were surveyed at intervals of approximately km in the riparian zone of this catchment. at each site, a transect consisting of contiguous m × m quadrats was surveyed on the riverbank, on the mid-slope of the riparian zone and on the upper bank of the riparian zone. within each site, this design was replicated three times, with each replicate separated by m. in all, quadrats were surveyed throughout a site. at each quadrat, trained observers visually estimated and recorded the percentage canopy cover of each alien species. from these observations, the presence or absence of each species was determined at the quadrat, transect, and site levels. as part of the survey, canopy cover, c , in the i th site, j th transect, and k th quadrat was recorded as a percentage of the quadrat. these were transformed into a portion by dividing by to allow the portions in individual quadrats to be summed. seven indices were derived from the surveys to determine what portion of area the weed occupies, when present, at the quadrat level, transect level, and site level. if cover in the quadrat was greater than zero, then the presence, p , of a weed , in the i th site, j th transect, and k th quadrat was recorded as one, otherwise it was zero. for each species, four measures of cover were used: total cover, c ; cover per site, c s ; cover per transect, c t ; and cover per quadrat, c q (table ) . total cover was the cumulative sum of the proportion of cover measured in each quadrat. cover per quadrat, cover per transect, and cover per site provided a measure of cover occupied by the weed at each scale. as total cover was the cumulative sum of the portion of cover in all quadrats, it was necessary to scale lower level frequency measures to match this estimate of total cover. therefore, transect frequency was multiplied by , as there are quadrats total cover (portion) cumulative sum of portion of cover in each quadrat cover per quadrat, c q c q = total cover/frequency of quadrats where weed was present cover per transect, c t c t = total cover/frequency of transects where weed was present cover per site, c s c s = total cover/frequency of sites where weed was present in each transect. site frequency was multiplied by and as there were nine transects in each site and quadrats in each transect. these indices differ from estimates of mean cover because absences, at different scales, are deliberately ignored in order to give an indication of the proportion of land a weed occupies once it has invaded. three measures of frequency were also defined: site frequency, transect frequency per site frequency, and quadrat frequency per site frequency. these were simply derived from the three frequency indices presented in table . site frequency was the number of sites where the species was present. transect frequency per site frequency provides a measure of the mean number of transects occupied by the species when present at the site level. nine transects were surveyed at each site and this was the maximum possible value. quadrat frequency per site frequency provides an indication of the mean number of quadrats occupied by the weed when present. the maximum possible value was . the two relative frequencies indicate the extent to which a species occupies a given site. for each species, these indices collectively capture the variation in frequency and cover at three spatial scales. site frequency and total cover provide information on these effects at the landscape scale. transect frequency per site and cover per transect explain the effects of a species along a m transect within a site. quadrat frequency per site and quadrat cover per site capture details of species effects at a quadrat level within the site. prior to analysis, data were centred by subtracting the attribute mean, and standardized by dividing by the centred attribute's root mean square, enabling all attributes to be considered with equal weight. data were analyzed using non-metric multidimensional scaling to summarize the relationship between the seven indices for the species identified in the survey. the dissimilarities between species cover values were calculated using a euclidean distance measure. data were furthered summarized using an agglomerative hierarchical cluster analysis using the same distance matrix as the non-metric multidimensional scaling (gordon, ) . species were agglomerated using ward's minimum variance method that seeks to minimize the within group variation (see gordon, for discussion). small groups of species were agglomerated into larger groups using the same method and this iterative process continued until all species formed a single group. in this way, group membership was optimized at each stage of the agglomeration, and was essentially a local, or single stage optimization (gordon, ) . results of this process were displayed in a dendrogram, arbitrarily cut at a height of seven. it is realized that trade-offs exist between a parsimonious interpretation of data and the need to preserve and identify the inherent variation that exists between species groups. weed classification frameworks were applied to every species. the criteria used for each of the three frameworks are shown in table . davis & thompson ( ) required information on dispersal distance, uniqueness to the region, and impact on the new environment. dispersal distance (short or long) was derived from site frequency, not from an understanding of the species dispersal mechanism. plants with high site frequency (> sites out of surveyed, or %) were considered long-distance dispersers, whereas those occupying fewer sites were considered short-distance dispersers. uniqueness to the region, whether common or novel, was difficult to classify from the data alone. davis & thompson ( ) argue that species introduced into north america during colonization should now be considered common as they are unlikely to ever be eradicated. nearly all the species surveyed here satisfy that criterion, with most present in australia for over years. therefore, all cluster groups were considered common. impact, either little or great, was determined as cover per quadrat. species occupying more than % of a quadrat when present were considered to have a great impact, and those that occupied less than this were considered to have a small impact. impact was assessed at a very local scale. site and transect level impacts were excluded from the assessment for parsimony, although the three measures were strongly correlated (fig. b ). colautti & macisaac ( )'s classification system is largely devised around concepts of frequency and abundance and it was a relatively simple process to classify species based on the survey data, where cover provided a surrogate measure of abundance. species were all able to reproduce and survive, and by default, had reached stage . species with low cover per quadrat (< %) and very low site frequency (< sites) were considered stage species (localized and rare). species with high cover per quadrat (> %) and low to moderate site frequency (< sites) were considered stage b species (localized but dominant). species present at more than sites but with low quadrat cover were considered stage a species (widespread but rare). all other species, with moderate to high quadrat cover and a high site frequency were considered stage species (widespread and dominant). it was more difficult to apply the survey data to richardson et al . ( ) 's framework. all species had overcome major geographical barriers, environmental barriers at introduction, reproduction barriers, and local/regional dispersal barriers. therefore, all species were naturalized. we decided that a species should be present at more than sites before being considered invasive and those that occupied more than sites and had moderate or high quadrat cover were considered transformer species. data were not available to assess whether high levels of quadrat cover actually transformed the ecosystem, as suggested by richardson et al . ( ) . we adopted a threshold approach to determine when a species moved from one category to the next. the application of frequency and cover data to the frameworks was complicated by the subjectivity associated with determining when this transition should occur. this is an inherent weakness of all three classification systems. as such, many of the thresholds chosen were arbitrary. it was arguably easier to apply data to davis & thompson ( ) 's and colautti & macisaac ( ) 's frameworks. nevertheless, subjective elements of the classifications varied between the frameworks and this complicated our ability to arrive at seemingly analogous classifications across the three frameworks. this may be because none of the frameworks was designed to be used with frequency and cover data, such as those collected here. it was therefore difficult to implement the frameworks as their authors intended. processes such as impact and dispersal were not explicitly captured and therefore inferred from the available data. the ecological impact of invasive species in the burdekin catchment has not been quantified, further limiting our ability to implement the frameworks. groupings derived from cluster analysis and the three frameworks were evaluated graphically by superimposing all four groupings onto the ordination to assess the performance of each framework in reduced multivariate space. species were classified into three of the four possible categories using richardson et al . ( ) 's framework, where were 'naturalized' , were 'invasive' , and were 'transformer' species (table ) . species were classified into four of the six groups using davis & thompson ( ) 's framework (table ) . five were considered long-distance dispersal, common with great impact. twelve were evaluated as long-distance dispersal, common with small impact, and nine were short-distance dispersal, common with great impact (table ). the remaining species were short dispersers, common, with small impact. similarly, colautti & macisaac ( ) 's framework also classified the species into four groups. forty-five species were classified as stage , localized, but numerically rare, as stage a, widespread but numerically rare, four as stage b, localized and abundant. five species were classified as stage , widespread and abundant (table ) . analogous classifications were obtained for the most abundant and widespread species, bothriochloa pertusa, cenchrus ciliaris, c. grandiflora, parthenium hysterophorous , and urochloa mosambicensis . these were classified as transformer (richardson et al ., ) , long-distance/common/great (davis & thompson, (colautti & macisaac, ) . similarly, minor species were all classified as naturalized (richardson et al., ) , short-distance/common/great (davis & thompson, ) , or stage species (colautti & macisaac, (richardson et al., ) , a short-disperser, common with great impact (scg) (davis & thompson, ) and at stage b (colautti & macisaac, ) . in contrast, jatropha gossypifolia was classified as naturalized (richardson et al., ) , while classified as a short-disperser, common with great impact scg (davis & thompson, ) and at stage b (colautti & macisaac, ) (table ) . the first two dimensions accounted for . % of the variation with a stress of . . the first dimension primarily separated species on total cover, where the score was negatively correlated with total cover, cover per site, cover per transect and cover per quadrat (r = - . , − . , − . and − . , respectively). urochloa mosambicensis was the most abundant species. other species with negative scores for the first dimension included the woody species, ziziphus mauritiana, c. grandiflora and lantana camara. the grasses panicum maximum, c. ciliaris and b. pertusa all had negative scores for the first dimension, as did the forb parthenium hysterophorus. the small forbs, sida cordifolia, alternanthera bettzikiana, xanthium strumarium and a native perennial shrub carissa ovata were the only other species with negative scores for the first dimension. the second dimension separated species on site frequency. ziziphus mauritiana and panicum maximum had low scores for the second component, suggesting they occupied relatively few sites. conversely, s. cordifolia, c. grandiflora and c. ovata had high scores on the second dimension, corresponding to a high site frequency (fig. a,b) . the first and second dimensions captured the main effects present in the data. minor species had small positive scores on the first axis and small negative scores on the second. it is apparent from the correlations between the attributes and the axis scores that these species had low total cover and low site frequency. seven groups were identified by the agglomerative hierarchical cluster analysis (table ). the groups agglomerated species based on their total cover and site frequency and the groupings concur with the output derived from the non-metric multidimensional scaling (figs c & ) . detailed characteristics and species membership of each group are shown in table . the first three groups, with , , and species had high total cover and were present in , , and sites, respectively. all three groups were widely dispersed throughout the site. group species or u. mosambicensis, occupied quadrats per site when present, while groups and species occupied quadrats per site when present. groups and also occupied approximately % of the quadrat when present, while species in group occupied % of the quadrat. the remaining four groups, with , , , and species, respectively, occupied negligible proportions of the catchment, although group species were widespread and present in sites. all four groups were rare, even when present at the site, as species were found in few quadrats per site, and groups and species were generally found in just one of the nine transects surveyed per site. two species, u. mosambicensis and chloris babata, were outliers in this survey, because of their very high site and quadrat per site frequency (u. mosambicensis) and very high cover per quadrat but low site frequency (c. babata). as a result, they were excluded from all other groups. categorical data, such as life form, were ignored in this study and grouped species often had different ecological characteristics. for example, grasses, forbs, vines, and trees were all found in cluster groups , , , and (table ) . other information, such as time since introduction, was either unavailable or not applicable to this catchment as species were usually first introduced elsewhere in australia. we did not consider it necessary to further separate the few species present at a large number of sites and therefore discounted information on the spatial aggregation of individual species. the two site level measures of frequency, transect frequency per site and quadrat frequency per site, were moderately correlated with the landscape scale measure, site frequency (r = . and . , respectively). similarly, two of the three site-level measures of cover, cover per transect and cover per site, were highly correlated with total cover (r = . and . , respectively). cover per quadrat was only moderately correlated with total cover (r = . ). the relationship between attributes and the dimensions from the multivariate analysis are presented in fig. (b) . in general, there was an orthogonal relationship between site frequency and cover per quadrat, cover per transect, and cover per site. this suggests that the area occupied by a species, captured by the various measures of cover, was unrelated to the processes governing its dispersal at the catchment scale, as represented by site frequency. however, dispersal within site, measured by quadrat per site and transect per site, was correlated with cover per site and cover per transect. collectively, these relationships suggest a dichotomy between the processes involved in long-distance dispersal between sites and shorter dispersal distances for within site measures. weed classification schemes are designed to provide clarity and consistency to the field of invasion ecology through the use of broad, generally applicable terminology and concepts for species invasions. these frameworks put forward a series of hypotheses about the process of invasion and are intended to enable ecologists to qualitatively assess characteristics of invasive species and group them concordantly. the three frameworks require different criteria to perform the classifications. application of the frameworks is further complicated by the subjective interpretation of classification criteria and implementation of the classification systems. taxonomic classification systems, such as soil classifications (e.g. isbell, ) or land systems (e.g. tongway & hindley, ) , traditionally use a key that can be repeatedly applied to the soil or to a landscape. the classification systems employed in the present study have not been developed to the extent of soil or landscape classification systems, possibly because the data necessary to evaluate their performance was unavailable. their application is invariably subjective. for example, if we had considered species occupying more than sites to be 'widespread' (colautti & macisaac, ) and 'long-distance dispersers' (davis & thompson, ) , then the number of species categorized as stage widespread and abundant (colautti & macisaac, ) and longdistance, common with great impact, would have increased from five to eight species. every application of these frameworks is likely to be different and arguably site and scale specific, with different interpretations of what is frequent, abundant, local, common, high-impact, or long-distance dispersal. the frameworks were also unable to make use of additional available data, such as the cross-scale indices used in this study. given these limitations, it seems unlikely that widespread application of these frameworks, in their current form, can advance the field of invasion ecology. this may be because each framework has a discrete set of hypotheses about what constitutes a serious invader. the suitability and application of these hypotheses to a localized problem, with unique or inappropriate data, may not always be relevant for managerial purposes. there are elements of subjectivity in application of the clustering algorithms to the frequency and abundance data. this subjectivity exists at two levels. first, the decision to cut the dendrogram at a height of seven is arbitrary. cutting at a height of would have resulted in just three groups and yielded groupings synonymous with those derived from the classification frameworks. for example, groups and would have been amalgamated, even though group species were present, on average in more sites but occupied just % of the quadrat when present. in contrast, group species occupied % of the quadrat. we consider it productive to preserve this variation in the data since it potentially reflects key differences in modes of invasive spread that should be considered in management. thus, group species, while having the capability to become regionally common, probably through a greater capacity for long-distance spread, rarely become locally dominant. in contrast, group species appear to spread well locally and become dominant where they occur but at present have not dispersed as widely. in practical terms, management strategies could target group species because they have yet to disperse widely. they may also pose a greater threat to the ecosystem simply because they occupy a larger portion of the quadrat and transect when present. alternatively, group species have demonstrated proficiency for dispersal. if this trend continued, these species could threaten a much larger area, but with slightly lower levels of cover than group species. group species displayed a similar penchant for dispersal, but occupied very minor portions of the transect or quadrat. these are arguably the hardest species to classify with confidence. because they are successful dispersers, they have the potential to threaten the ecosystem, although at this point in time their effect is minor. our interpretations of the effect of species from group and group are also based on the frequency and abundance data at this point in time. their presence and overall effect in the catchment are minor. however, our analyses and interpretations are limited by the data available. additional information, such as time since introduction into the catchment may alter the assessment, but these data were unavailable for most species. some species may still be adapting, or may not have had the opportunity to establish a sufficiently large population to create a problem. individual species in groups , , and may indeed be sleeper weeds and become a greater problem than they are. this concept was recently reviewed by grice & ainsworth ( ) . in addition, the cluster analysis, detected two extreme outlier species, group , u. mosambicensis, and group , c. babata. urochloa mosambicensis was introduced as a pasture grass in the s and the observed frequency and abundance reflect its importance to the grazing industry as it would have been deliberately planted by graziers since its introduction. chloris babata was only found in three sites, but, on average occupied % of the quadrat and % of the transect, suggesting that this species could have a dramatic impact on the landscape. the other element of subjectivity lies with the algorithms used to assess the dissimilarity between species attributes and the algorithms used to combine species based on their dissimilarities (e.g. pielou, ) . in this study, euclidean metric scaling was applied to normalized ordinal data, ensuring that each attribute would contribute equally to the group structure. alternative distance measures would be required if data were categorical. clustering algorithms have been widely used and although elements of subjectivity remain, they can be applied to a weed data set and discriminate between species at some level. they use whatever data are available and help formulate hypotheses about the invasion in the landscape of interest. we used the classification frameworks in the first instance to provide baseline parameters for a mixture method of clustering using the software developed by fraley & raferty ( ) . data were not multivariate normal and the resulting classifications were influenced by the initial group structure and often identified local optima (fraley & raferty, ) . this approach may be adopted in the future if the methods are modified to cope with poisson distributions, such as those encountered here. the extensive survey data used to derive the landscape indices may be time consuming to collect in some landscapes. the similarities between the seven variables imply that meaningful classifications of invasive species can be achieved with a reduced sampling effort. the simple site level indices derived in this study also provide insights into how invasive a species may become at a larger scale. the particularly strong relationship between cover per transect and total cover in the landscape suggests that, if a species consistently occupies more than % of a transect in a specific habitat, then it has the potential to impact and invade that habitat. the link between these scales is worthy of further study to determine the relationship between local and regional impact by species at a certain stage in the invasion process. it may be argued that a species that occupied even a small portion of the landscape in one location has overcome many of the barriers to colonization identified in richardson et al., ' s framework and has the potential to invade similar habitats, providing a dispersal mechanism exists. both quantitative and qualitative forms of classification are developed to enable researchers and managers to classify an invasion and determine which species have dispersed widely and had the greatest impact on the ecosystem. both have limitations. the classification techniques provide a means for classifying an invasion based on a series of hypotheses that describe an invasion. in contrast, the multivariate techniques enabled us to partition species into meaningful groups, based on observed patterns within the data set. this data set contained information on species frequency and cover at a range of spatial scales. we suggest that the subjective, qualitative approaches developed by richardson et al., , davis & thompson, , and colautti & macisaac, be used in conjunction with multivariate techniques developed here to derive classifications that are data driven, but spatially and temporally specific. a classification scheme should ensure that species within a group have attributes that are more similar than those in an adjacent group. this concept may be extended further, where the species in the same group should be subjected to similar levels of management. the subjective use of thresholds that often separate species using a single attribute, arrived at non-sensical classifications when all the data are considered and viewed in multivariate space ( fig. c-f ). this may be because the thresholds chosen were inappropriate, or the frameworks lacked the capacity to make the best use of the data available. the frameworks had a tendency to under-classify the data. the other purpose of these analyses was to assess the characteristics of the data to determine which species have similar levels of frequency and abundance across a range of spatial scales. from this information, management strategies could be developed for as few or many groups as necessary. outliers may emerge in this analysis and warrant preferential treatment. our use of a simple cluster analysis to objectively group species with similar population-level distributional characteristics has several advantages over classification using any of the three individual frameworks. most importantly, it is repeatable, using data alone to drive the classification and assigning species to like groups independently of preconceived notions of their relative invasiveness. furthermore, it is regionally and temporally specific, lending itself well to a management application. a strong advantage also lies in the potential for integration of the outcomes of our analyses with current frameworks for assessing regional-scale dynamics of native species. application and evaluation of classification trees for screening unwanted plants a neutral terminology to define invasive species eight ways to be a colonizer, two ways to be an invader: a proposed nomenclature scheme for invasion ecology mclust: software for modelbased clustering, density estimation and discriminant analysis sleeper weeds -a useful concept regional and landscape-scale patterns of shrub invasion in tropical savannas the australian soil classification global patterns of plant invasions and the concept of invasibility predicting the identity and fate of plant invaders: emergent and emerging approaches. biological conservation invasiveness in exotic plants: immigration and naturalization in an ecological continuum do annual weeds have predictable characteristics? acta oecologia weed risk assessment. a weed risk assessment model for use as a biosecurity tool evaluating plant introductions the interpretation of ecological data alien plants in checklists and floras: towards better communication between taxonomists and ecologists the biology aspects of rare plant conservation what makes a species invasive? plant invasions -general aspects and special problems biological invasions: politics and the discontinuity of ecological terminology plant invasions: merging the concepts of species invasiveness and community invasibility naturalization and invasion of alien plants: concepts and definitions testing the grass-fire cycle: alien grass invasion in the tropical savannas of northern australia manual for assessment of soil condition of tropical grasslands. csiro division of wildlife and ecology the characters of successful invaders we thank lindsay whiteman, mike nicholas and brett abbott for assisting with fieldwork and professor iain gordon for constructive comments on earlier versions of the manuscript. we also thank the crc for australian weed management and crc for tropical savannas for funding various components of the study. key: cord- - khcmta authors: delaney, martha a.; treuting, piper m.; rothenburger, jamie l. title: rodentia date: - - journal: pathology of wildlife and zoo animals doi: . /b - - - - . - sha: doc_id: cord_uid: khcmta this chapter includes diseases of animals in the order rodentia, in which there are over species representing % of all mammals. this incredibly diverse order includes members inhabiting every continent, either naturally or in human-made environments. while rodents have been the cause or implicated in disease transmission that has lead to human pandemics, such as the black death, and the decimation of certain animal species, like island-dwelling birds; genetically modified rodents have contributed significantly to the advancement of biomedical research and human health. there are more than species of endangered rats, mice, voles, squirrels, and marmots. the recent extinction of the bramble cay melomys represents the first human-induced rodent extinction linked to climate change. rodents are the reservoir host of several human and domestic pathogens of concern listed by oie. herein, we highlight those diseases of rodents that lead to clinically important gross and microscopic lesions. the order rodentia includes families, genera, and over species. two separate systems exist to classify rodents (carleton and musser, ) . the first classification scheme by j.f. brandt ( ) is based on morphology of the jaw and skull together with masticatory muscle position and has three suborders: sciuromorpha (squirrellike); myomorpha (mouse-like); and hystricomorpha (porcupine-like). the second separates rodents into two suborders, sciurognathi and hysticognathi, based on the position of incisors and the angle of the jaw (carleton and musser, ) . molecular studies yield seven clades containing three phylogenetic lineages: squirrel-related (sciuriodea and gliridae); mouse-related (anomaluromorpha, castoridae, geomyoidae, and myodonta); and ctenohystrica. with diverse ecologic strategies, rodents are found naturally in a variety of habitats globally except antarctica. the natural history and biology of members of this extensive order exhibit substantial variation. nevertheless, similarities exist among rodents including dentition, anatomy, and disease susceptibility. the unifying characteristic of all rodents is their dentition, which includes two pairs of continuously growing (elondont) incisors. brachydonts have elondont incisors with brachydont molars; in hystricomorphs, all of the teeth are elondont. dentition is important when considering the diet and husbandry needs of these groups. brachydonts, which include mice, rats, hamsters, and gerbils, need high calorie diets with low amounts of fiber, whereas the hystricomorphs (porcupine-like; guinea pigs, chinchillas, agoutis) are strictly herbivores and require ample abrasive roughage. dental formulae vary but in general, rodents have four incisors, no canines, - premolars, and numerous (up to ) molars. as most rodents are omnivorous, their digestive system typically consists of a simple stomach (monogastric) and a variably adapted intestinal tract depending on the degree of hind gut fermentation (stevens and hume, ) . most practice coprophagy or cecotrophy to maintain normal microflora and absorb microbially derived amino acids and vitamins (e.g., b and k). as such, herbivorous, omnivorous, insectivorous, and carnivorous rodents have different gastric and large intestinal anatomy. some rodents lack gall bladders (e.g., rats and pocket gophers). rats and hamsters are unable to vomit due to a muscular sphincter at the esophageal-gastric junction. members of caviidae lack the enzyme l-gulonolactone oxidase, making vitamin c (ascorbic acid) a necessary dietary requirement for this group (see scurvy in section, nutritional diseases). rodents are typically small (as small as g) and rotund with short legs, though the largest rodents are almost kg (e.g., capybaras). rodents are nocturnal and though some species hibernate (e.g., woodchucks) but most of the species do not. there are numerous specializations among rodents including cheek pouches (food transport), extremity and claw development (e.g., burrowing, jumping, climbing, flying), sensory organ variations (e.g., vibrissae and large ears or eyes), special integument and pelage (e.g., porcupine quills and chinchilla fur), and tail adaptations (e.g., prehensile, fat storage). rodents have a strong sense of smell and a well-developed vomeronasal organ, which is important for reproduction and social behavior through the sensing of pheromones. since rodents lack sweat glands, they are susceptible to overheating. reproductive anatomy varies though most female rodents ovulate spontaneously and are polyestrous. like rabbits, chinchillas have two cervices that communicate with individual uterine horns. vaginas are imperforate prior to sexual maturing and during nonbreeding and/or postpartum intervals. placentation is discoid and hemochorial. mammary gland anatomy varies among species. the suborders pathology of wildlife and zoo animals scuirognatha and stricognatha have altricial and precocial young, respectively. some male rodents have testes within a scrotum, though the testes of porcupines, agoutis, chinchillas, cavies, and capybaras are located within the inguinal canal, which remains open in most male rodents. male rodents also have well developed accessory sex glands and an os penis. prairie dogs have perianal sacs similar to canids and felids, which are important for sexual communication. male beavers have persistent paramesonephric ducts that resemble a uterus and are referred to as masculine uterus (doboszynska and zurowski, ; meier et al., ) . south american rodents including guinea pigs and related species have unique pulmonary anatomy as do burrow-dwelling, subterranean mole-rats that live in low oxygen tension environments, for example, large and dilated, primitive bronchi and bronchioles (ilgun et al., ; widmer et al., ) . lung morphology of different rodent species representing families is well described (wallau et al., ) . common microscopic findings in rodents that may be misinterpreted as lesions include: multinucleated, karyomegalic, and cytomegalic hepatocytes are common in several rodent species and can increase with age ( fig. . ); hepatocellular intranuclear cytoplasmic invaginations (pseudoinclusions) (fig. . ); eosinophilic cytoplasmic spherical inclusions in renal tubular epithelial cells and hepatocytes seen predominantly male mice, rats, and hamsters; splenic extramedullary hematopoiesis, which is very common in healthy rodents of all ages (fig. . ); hemosiderin, lipofuscin, ceroid, and melanin (in dark or black coated animals) are commonly detected in various tissues, such as spleen, liver, kidney, and adrenal glands; cardiac muscle in the tunica of pulmonary veins in the lung is a normal finding in mice; male rodents may have refluxed seminal coagula in the urinary bladder and urethra that is thought to occur peri mortem; and adrenal x-zone vacuolation in female mice. furthermore, there are evident sexual dimorphisms of some organs and glands, such as the cuboidal parietal epithelium of the glomeruli in male rodents. hematological reference ranges have been established for some common domestic, zoo-housed, and ubiquitous free-ranging species. in general, the lifespan of rodent erythrocytes is shorter than other mammals, thus, higher levels of circulating reticulocytes ( %- %), seen as polychromasia and anisocytosis, is considered normal. howell-jolly bodies may also be present. in all age groups, lymphocytes are more numerous than neutrophils. in fact, lymphocytes are the predominant white blood cells in guinea pigs ( %- %). in addition, guinea pigs and capybaras have kurloff cells (fig. . ), which are lymphocytes containing a single, large intracytoplasmic mucopolysaccharide inclusion body, known as a kurloff body (jara et al., ) . though the actual function of these cells is unknown, they are presumed to be natural killer cells. the number of kurloff cells in females fluctuate with the reproductive cycle stage, thus they are considered to be estrogen-dependent. pregnant females may have %- % kurloff cells in circulation while males typically have fewer. guinea pigs have heterophils similar to rabbits. rats, mice, and hamsters commonly have neutrophils with ring-form nuclei. chinchillas and porcupines have welldefined and segmented neutrophil nuclei. band neutrophils may be a normal finding in some species including new world porcupines, coypu, and agoutis. serum biochemical parameters have been compiled for some rodent species (kurtz et al., ; . of note is that serum levels of alanine aminotransferase (alt) are not sensitive biomarkers of liver health in guinea pigs and figure . normal liver in an aged mouse. common findings inlcude anisocytosis, anisokaryosis, and eosinophilic intranuclear pseudoinclusions of invaginated cytoplasm. within the red pulp are hematopoietic elements including erythroid and myeloid lineages. note the large multinucleated megakaryocytes, which are readily visible at this magnification. some other rodents as they normally have low hepatic levels of this enzyme. in contrast, gamma glutamyl transferase is present in the liver of all rodents and can be evaluated to determine the hepatic injury or disease. hamsters and other desert species may have high levels of serum calcium. rodents like mice and rats, and specifically those evolved in arid regions, have a profound ability to concentrate their urine, thus it is often turbid and contain calcium crystals. the following list of diseases of rodents is not exhaustive but instead highlights the more common findings in species of which there is published literature or knowledge. a plethora of information exists regarding the anatomy, physiology, and diseases of rodents used as animal models in biomedical research (i.e., laboratory animals) and is a valuable resource when examining lesser-studied rodent species using a comparative approach greaves, ; suckow et al., ; treuting and dintzis, ) . as many rodents are important prey species, diseased or dead rodents are exceedingly difficult to detect in nature using the passive surveillance techniques of traditional wildlife disease monitoring. thus, less is known about the diseases and pathology of free-ranging rodents as compared to larger and charismatic mammals. one of the most well described nutritional diseases in rodents is hypovitaminosis c or scurvy. guinea pigs and a number of other species including capybaras and humans, lack the enzyme l-gulonolactone oxidase which is required for l-ascorbic acid (vitamin c) production therefore, these species require dietary vitamin c (cueto et al., ) . vitamin c is necessary for the formation of collagen via hydroxylation of proline/lysine for cross linking of fibrillar collagen. hypovitaminosis c results in decreased collagen fibril cross linking, leading to the increased fragility of blood vessels, cartilage, and osteoid. in affected individuals, gross and histologic lesions are characteristic and include capillary hemorrhage and lack of bone deposition and remodeling as a result of poorly formed and weak collagen. other gross lesions include evidence of anemia (e.g., pallor, reactive bone marrow), periarticular hemorrhage, and swollen joints, specifically at the costochondral junctions (scorbutic lattice). characteristic oral lesions include gingival swelling, erythema, hemorrhage, erosion, ulceration, and tooth loss. histological examination is necessary to confirm and define bone lesions, which include: dilation of metaphyseal blood vessels, irregular columnization and hypertrophy of physeal cartilage, calcification of metaphyseal cartilage with reduced or absent osteoid, microfractures with surrounding and subperiosteal hemorrhage, medullary fibrosis, and decreased osteoclastic activity and remodeling. tooth loss may result from a combination of abnormal odontoblasts, dentin resorption, pulp fibrosis, and a lack of/ abnormal alveolar bone remodeling. like lagomorphs, some hindgut-fermenting rodents are susceptible to gut stasis and related diseases if they are not able to obtain enough fiber from the diet. obesity can be common in rodent species if fed a high caloric diet and can predispose to diabetes mellitus, cardiac disease, pregnancy toxemia, and hepatic lipidosis in females. dystrophic mineralization occurs in guinea pigs, hamsters, and free-ranging rats (rothenburger et al., a) . mineralization most often affects the myocardium but can also occur in other tissues, such as the diaphragm, tongue, liver, kidney, lungs, cornea, and aorta (see section congenital/ genetic). though the pathogenesis is not fully elucidated, mitochondria appear to be preferentially mineralized based on ultrastructural studies. gross lesions may include cardiomegaly with chalky to gritty white material and streaking within the myocardium of the atria and ventricles. histologically, affected cardiac myocytes are degenerative and/ or necrotic with rupture of the sarcolemma. damaged myocytes will have variable and progressive mineralization until the entire myofiber is densely mineralized. variably mature fibrosis surrounds individual and clusters of dead and mineralized myocytes. satellite cells are frequently hypertrophic. pregnancy toxemia occurs in overweight, pregnant, and lactating female guinea pigs and hamsters. there are two forms in guinea pigs, a fasting type that results from decreased carbohydrate intake and subsequent mobilization of fat stores and a circulatory type that occurs when the gravid uterus compresses the aorta and causes reduced blood flow to abdominal organs. characteristic gross and histologic lesions include hepatic lipidosis and fat deposition in renal tubular epithelial cells. similarly, hepatic lipidosis and associated metabolic effects occasionally affect overweight nongravid sows and boars. urolithiasis is common in guinea pigs, rats, and mice due to high urine ph and calcium concentrations. obstructive urolithiasis has also been reported in captive male agoutis resulting in bilateral hydronephrosis, urinary bladder rupture, and peritonitis (batista et al., ) . a number of other endocrine diseases have rarely been documented in various rodent species. they have typically been associated with hormone-producing neoplasms (adrenal, islet, thyroid, parathyroid, and pituitary tumors). fluorosis is well studied in rats as they are used as models in toxicology studies. guinea pigs and rabbits are also susceptible and fluorosis has occurred in free-ranging cotton rats in sites contaminated by petrochemical waste (rafferty et al., ) . pathognomonic lesions occur in teeth and bones. periosteal hyperostosis occurs first along the medial surface of the metatarsals, then progresses to affect the mandible, metacarpals, and ribs though all bones can be affected in severe, chronic disease. bones are brittle and chalkywhite. pathological fractures are common but joints are typically spared. although characteristic, enamel hypoplasia only occurs when fluorosis is present during tooth development. tooth lesions include dull, dry, and chalky hypomineralized or hypoplastic enamel with pitting, grooves, and discoloration (due to oxidation); excessive attrition affects the incisors first. irregular wear, malocclusion, and fractures may be present. histologically, ameloblasts are small while osteoblasts are vacuolated and disorganized. there is abundant globular dentin and hypomineralization of the outer enamel layer. a differential for enamel hypoplasia in young syrian hamsters is hamster parvovirus (hapv); concurrent testicular hypoplasia, and cerebral malacia is present with this viral infection. cholecalciferol (vitamin d) is used as a rodenticide. the deliberate poisoning of free-ranging rats and mice results in inadvertent toxicity of nontarget animals including other rodents, such as squirrels or carnivorous predators that consume affected carcasses. multisystemic mineralization occurs prior to death in affected individuals. unlike other rodents, naked mole-rats, beavers, and woodchucks do not require dietary vitamin d and toxicity can develop if they are fed rodent chow or diets supplemented with vitamin d. lesions consistent with calcinosis cutis and circumscripta can develop in naked mole-rats fed such diets. soft tis-sue calcification is not limited to pressure points and can be found throughout the body in severe cases. resolution occurs with correction of the diet (delaney et al., ) . anticoagulant rodenticides including warfarin and its derivatives are frequently used to control free-ranging mice and rat populations. these compounds antagonize vitamin k and thus inhibit production of vitamin k-dependent clotting factors (i, ii, vii, ix, x). gross lesions include multisystemic hemorrhage, particularly subcutaneous and periarticular hematomas. "bait," some of which are brightly colored (e.g., teal-green), may be present within the intestines. inadvertent consumption by nontarget wildlife may have fatal consequences. hindgut fermenting rodents are susceptible to similar antibiotic-related toxicities as rabbits (see chapter ). nephrotoxicosis can be the result of exposure to numerous agents and has been extensively described in laboratory animals. for example, swainsonine toxicity occurs following the ingestion of plants from the genus swainsona and results in acquired alpha-mannosidosis in rats. histologically, renal tubular epithelial cells are severely and diffusely vacuolated. dystrophic mineralization, also known as dystrophic cardiac calcinosis is considered as a genetic disease in inbred mouse strains, particularly in aged individuals (eaton et al., ) . cardiac mineralization is detected grossly as pinpoint foci to larger irregular crusts composed of white, gritty material within and overlying the epicardium. histologic lesions vary depending on severity and chronicity with degeneration and necrosis of individual myocytes to extensive regions of myocyte loss and replacement by deeply basophilic granular material. in severe cases, mineralization extends into the myocardium resulting in scar formation and can also be seen in the kidneys and lungs. the prevalence of this disease in wild mice from which these strains originated is unknown. diabetes mellitus (dm) in chinese hamsters is a heritable, autosomal recessive disease (green et al., ) . symptomology and lesions are similar to those found in humans and include hydropic degeneration and degranulation of pancreatic islet β-cells with secondary vascular lesions, such as arteriosclerosis and glomerulosclerosis. in addition, affected hamsters are predisposed to pancreatic adenocarcinomas. serum levels of α- globulins are a useful indicator of risk for diabetes mellitus development in these populations. polycystic kidney disease has been diagnosed in captive populations of brazilian agoutis (müller et al., ) . most affected individuals have no clinical signs and are potentially related genetically. grossly, affected agoutis have a range of bilateral renal changes including atrophy, roughened granular cortical surfaces, and multiple cysts. histologically, there is cystic dilatation of renal tubules and bowman's capsules with mesangial and capsular thickening accompanied by mononuclear interstitial nephritis and fibrosis. many degenerative and age-related diseases are described in rodents used in toxicology studies pettan-brewer and treuting, ) . a significant disease confounding these studies is chronic progressive nephropathy, which is best described in rats though mice, naked mole-rats, and australian rodents, particularly the sticknest rat, are also affected (delaney et al., a) . the exact pathogenesis is unknown; however genetic factors may play a role. grossly, kidneys are bilaterally pale and either enlarged or shrunken with numerous pinpoint to large, mm cortical microcysts creating a nodular surface. histologically, renal tubules are markedly ectatic and tortuous and many contain brightly eosinophilic fluid (proteinosis) ( fig. . ). tubules have a range of degeneration, necrosis, and regeneration with occasional tubular hyperplasia and adenoma formation (predominantly in rats). glomeruli are variably affected by membranous and/or proliferative changes with thickened basement membranes, sclerosis, and obsolescence. synechiae, dilated bowman's capsules, and hypertrophied parietal epithelium are common. the interstitium is expanded by lymphoplasmacytic infiltrates with pigment-laden macrophages, mild hemorrhage, and edema. there is often substantial interstitial fibrosis and in severe cases, infarction. diagnosis is based on histologic evaluation though in some species, clinical pathology may be preferentially used to determine renal function. cardiomyopathy is common in aged mice, rats and hamsters; it is particularly common in syrian hamsters and is reported in woodchucks (chanut et al., ; rothenburger et al., a; roth and king, ) . lesion severity varies though it appears to increase with age and is more common in males. though considered strain and thus possibly genetically related in laboratory rats, free-ranging rats develop similar disease (rothenburger et al., a) . grossly, there may be cardiomegaly but macroscopic changes are not always apparent. histologically, myocardial lesions consist of multifocal and perivascular lymphoplasmacytic infiltrates accompanied by fibrosis, myocyte degeneration, and drop out ( fig. . a,b). trichrome, movat pentachrome, or other stains that highlight the fibrosis are useful to evaluate severity and chronicity. syrian hamsters develop cardiomyopathy associated spontaneous atrial thrombosis and subsequent coagulopathy that is often fatal (fig. . ). females appear to be affected earlier in the life than males. like other species, rodents are susceptible to skeletal degeneration and related diseases. guinea pigs, specifically the duncan-hartley breed and syrian hamsters, frequently develop degenerative joint disease. gross lesions include thickened joint capsules, variable osteophyte formation, roughened cartilage surfaces, eburnation, joint effusion, and synovial proliferation. depending on which joints are affected and chronicity, crepitus may be present when joints are manipulated. microscopic examination of joints reveals degeneration, necrosis, and ulceration of the articular cartilage with proliferation and fibrosis of the synovium and joint capsule, periarticular and periosteal bone formation, and remodeling. there may also be variable inflammation and hemorrhage within joint spaces. amyloidosis is typically seen in aged animals. amyloid is an extracellular accumulation of insoluble protein. excessive amyloid deposits can lead to tissue atrophy and dysfunction. amyloidosis is most common in myomorphs (mice, rats, hamsters, gerbils) and appears in the kidneys, spleen, adrenals, and liver as well as the gastrointestinal lamina propria (mouse) (fig. . ). there is a higher prevalence in females. of note is the eosinophilic substance within the nasal planum of aged mice that was once thought to be amyloid, through histochemical and ultrastructural studies, has instead been shown to consist of collagen and complex carbohydrates produced by the nasal gland epithelial cells (doi et al., ) . hemosiderosis is the accumulation of iron within various tissues, most commonly the liver. it is described in numerous species including primates, birds, rhinoceros, hyraces, and rodents. in one zoological institution, hemosiderosis was present in approximately % of adult naked mole rats (delaney et al., ) . grossly, livers appear bronze. iron accumulation appears histologically as zonal to diffuse hepatocellular accumulation of brown granular to figure . chronic progressive nephropathy in a naked molerat. renal tubules are variably ectatic, atrophied, degenerate, necrotic, and replaced by collagenous fibrous tissue. within the interstitium are accumulations of mononuclear cells (primarily lymphocytes and fewer macrophages). there is also sclerosis of glomeruli and periglomerular fibrosis. globular pigment. pigment is most intense in the periportal hepatocytes. the pigment stains positively with prussian blue and is not refractile with polarized light. aged mice can develop intrapulpal denticles, which are dysplastic tooth-like growths arising in the pulp of incisors. these may result in abnormal wear and or breakage of the incisors contributing to periodontitis and decreased ability to effectively gnaw (pettan-brewer and treuting, ). despite their protective quills, porcupines are particularly susceptible to cutaneous trauma and tears due to an exquisitely fragile integument. secondary infections resulting in pyoderma and persistent ulceration may occur. stress dermatosis (or dermatitis) is a condition of several south american rodents including, the agouti, acouchis, and capybaras. this syndrome is seen in captive and free-ranging individuals in densely populated groups. it presents as alopecic skin lesions and lacerations along the lumbosacral spine. stress is thought to increase skin fragility and overcrowding with intraspecific aggression contributes to lesion development. sun exposure, pruritus, and self-mutilation result in large patches of alopecia with erythema and excoriated skin with loss of the long hairs along the dorsum and tail. microscopic lesions correlate to severity and chronicity and include variable epidermal degeneration, necrosis, erosion/ulceration, and associated dermatitis with hemorrhage and edema. diagnosis is based on clinical history, demonstrated absence of a parasitic etiology in skin scrapings and histopathology. malocclusion is common especially in hysticomorphs, though it is also seen in brachydonts. it is a significant disease in captivity and may be related to genetics, nutrition and diet, and husbandry practices (losco, ) . ovarian cysts are common in rodents. cystic rete ovarii are prevalent in female guinea pigs (veiga-parga et al., ) and can be distinguished from other ovarian cysts as they arise from the rete and compress adjacent ovarian tissue ( fig. . ). other ovarian cystic structures reported in female rodents include: bursal cysts, epithelial cysts, follicular cysts, luteal cysts, and paraovarian cysts . the hemochorial placentation in rodents predisposes them to trophoblast emboli. this incidental lesion is best described in chinchillas and is not associated with clinical ramifications or gross lesions. histologically, intravascular trophoblasts are present in the lung, uterus, spleen, liver, and other sites, such as the adrenal glands with no associated inflammation. trophoblasts are large (> µm) with abundant amphophilic granular cytoplasm and large nuclei with prominent nucleoli. many neoplasms of inbred mice and rats appear to be strain related. nevertheless, certain neoplasms occur more frequently among free-ranging and domesticated outbred rodents than in other mammalian orders. several of them are described below. a list of other common neoplasms in rodent species is listed in the supplemental materials (table e ) . naked mole-rats are a popular zoo species and are heralded as animal models for cancer and aging due to their extreme longevity and purported cancer resistance. rare cases of neoplasia have been reported in naked mole-rats and in the related damaraland mole-rat, which is also longlived and appears to be relatively resistant to cancer and agerelated diseases (delaney et al., b; taylor et al., ,) . other subterranean rodents used as animal models for carcinogenesis research include some members of spalacidae (mole-rats, blind mole-rats, zokors, and bamboo rats). there are currently no reported spontaneous tumors in this group; however, subcutaneous tumors (fibromas and fibrosarcomas) may be induced with application of carcinogenic compounds. odontoma, or more aptly named odontogenic dysplasia, is described in several rodent species (rats, mice, guinea pig, prairie dogs, degus, chinchilla, chipmunk, squirrels; . most odontomas are not true neoplasms and instead represent dysplastic changes resulting from chronic inflammation or trauma and associated regional tissue response. as there is no demonstrated metastatic potential or criteria for malignancy, these lesions are increasingly identified as hamartomas versus neoplasms (and also referred to as pseudo-odontomas). nevertheless, these toothy masses are regionally destructive and almost exclusively affect the incisors. grossly, odontogenic dysplasia presents as nodular swellings around the maxillary or mandibular incisors. microscopically, these lesions are composed of disorganized and dysplastic proliferations of odontogenic epithelium, enamel matrix, and mineralized enamel, dentin, pulp, and cementum. in rodents, odontomas have been described as complex because they contain fully differentiated tooth elements but do not form the tooth-like structures grossly present in compound odontomas seen in other species (e.g., horse). histologically, rodent odontomas are composed of welldifferentiated odontogenic epithelium among an enamel organ with dental pulp mesenchyme and a variably mineralized enamel matrix. several important infectious and zoonotic diseases of rodents described are later with a focus on those that cause significant lesions in rodent species. additional pathogens, including zoonotic pathogens that occur in rodent species are listed in the supplemental materials (table e ). several references exist pertaining to rodent reservoirs of emerging and reemerging zoonotic pathogens ). poxviral diseases in rodents are caused by members of orthopoxviridae, which includes mousepox and cowpox (oie listed reportable zoonotic diseases). several species of rodents serve as the reservoir for cowpox virus, which results in cutaneous lesions in humans. in laboratory mice, mousepox, more commonly known as ectromelia, is an important disease. this infection can be acutely fatal with minimal lesions. in the chronic form, gross lesions include crusting lesions of face, legs, and tail, conjunctivitis, an enlarged, swollen and friable liver and spleen, and in cases of recovery, splenic fibrosis. necrosis of the kidney, thymus, and lymph nodes may also be grossly evident. the small intestine, urinary bladder, and vagina can have mucosal erosions and hemorrhage. histologically, skin and mucosal lesions include epithelial ballooning degeneration, hyperplasia, and erosions with characteristic intraepithelial, intracytoplasmic inclusion bodies. within the parenchymal organs, splenic and hepatocellular necrosis is typically coagulative and multifocal to coalescing. diagnosis is based on characteristic lesions with viral inclusions. electron microscopy, pcr, and virus isolation are useful for confirmation of the viral strain. in , an outbreak of monkeypox (orthopoxviridae), a zoonotic virus, occurred in prairie dogs sold as companion animals in the united states (gaurner et al., ) . fatal infections in infected prairie dogs were characterized by necrotizing bronchopneumonia, conjunctivitis, and glossal ulcers. viruses and viral antigens were detected in multiple tissues and cell types via immunohistochemical staining, pcr, and electron microscopy. infected humans developed fever and vascular rashes. forty seven confirmed and probable cases of monkeypox were reported from six states. the prairie dogs contracted the virus following exposure to infected african rodents. other rodents that also developed disease and died included: rope squirrels, tree squirrels, gambian giant rats, brush-tailed porcupine, dormice, and striped mice. fibromas and fibromatosis of gray squirrels are potentially fatal conditions caused by squirrel fibroma virus (leporipoxviridae). disease is mainly reported in the eastern north america (terrell et al., ) but also occurs in red squirrels and has been reported in a fox squirrel (wilcoxen et al., ) . transmission is likely through biting arthropods (fleas, ticks, and mosquitoes). gross and histologic lesions are similar to shope fibromas in rabbits (both are leporipoxviruses). multifocal to coalescing tan, firm nodules may cover all parts of the body and begin as alopecic nodules with progression to plaque-like to pedunculated masses ( fig. . a ). systemic disease manifests as renomegaly and multiple pulmonary nodules . histologically, the skin nodules exhibit marked epidermal hyperplasia with ballooning degeneration of keratinocytes, spongiosis, and intracytoplasmic eosinophilic viral inclusions (fig. . b,c) . pulmonary nodules correlate histologically to adenomatous hyperplasia with similar viral inclusions. other microscopic lesions may include atypical mesencyhmal proliferation in the liver and seminal vesicles, and renal tubular epithelial hyperplasia with viral inclusions. diagnosis is based on characteristic lesions and detection of virus by pcr or isolation. parapoxvirus is an important cause of disease in red squirrels in the united kingdom and ireland where it is a suspected factor in severe population declines tompkins et al., ) . the causative agent is referred to as red squirrel parapoxvirus. grey squirrels are the likely maintenance hosts since a large percentage of healthy gray squirrels are positive for the virus and their range overlaps with that of red squirrels. lesions in red squirrels include exudative dermatitis with crusting that is similar to other poxviral diseases. viral inclusions can be detected histologically within the cytoplasm of infected cells. electron microscopy and pcr are used to detect the virus. woodchuck hepatitis virus (whv; hepadnaviridae) is a disease in captive and free-ranging animals . although not zoonotic, this disease is a valuable animal model for viral hepatitis and hepatocarcinogenicity associated with hepatitis b in humans. it affects both sexes and is typically found in animals greater than years of age. activated t cells (cd +) cause cellular and dna damage leading to persistent hepatitis. simultaneously, viral dna integrates into the host genome at loci containing oncogenes, creating the potential for carcinogenic transformation. hepatocellular carcinomas are reported in % of experimentally infected woodchucks. grossly, most carcinomas are solitary and involve one hepatic lobe. histologically, neoplastic masses have differing growth patterns: trabecular, adenoid, or solid with and without cystic spaces, hemorrhage, and necrosis. an interesting feature of these tumors is the presence of atypical, bizarre, and giant neoplastic cells. adenomas may be present and are considered a precursor to carcinomas. mitotic index and cellular morphology are used to classify tumors as adenomas versus carcinomas. the remaining hepatic parenchyma often contains acute and/or chronic hepatitis with neutrophilic to mononuclear inflammation, variable hepatocellular degeneration, necrosis, fibrosis, and biliary changes. electron microscopic identification of the virus is definitive. beechey ground squirrels have a similar virus with variable hepatic lesions (cullen and marion, ) . hamster polyomavirus (hapyv), a papovavirus, causes cutaneous trichoepitheliomas in syrian hamsters . the virus is transmitted via urine and is highly contagious, with reported % morbidity in affected breeding colonies. early lesions consist of wartlike growths and alopecic nodules of the face and perineum with progression to severe, multifocal to coalescing nodules throughout the body. histologically, these characteristic skin tumors contain multiple islands of basilar to variably differentiated follicular epithelium and abrupt central keratinization with faded anuclear keratinocytes (ghost cells). rudimentary hairs may be present and follicular rupture may be associated with inflammation. other polyomaviruses-associated neoplasms include other cutaneous tumors, lymphomas and salivary gland tumors. in virally induced lymphomas, affected tissues are effaced by diffuse sheets of neoplastic lymphocytes among scant stroma. viral inclusions are not present. in a case of polyomavirus-induced, multicentric lymphoma in an -week old syrian hamster, the virus was detected in tumor cells, renal tubular epithelial cells, and enterocytes via in situ pcr indicating some degree of tissue tropism . diagnosis is based on clinical history, gross and histologic lesions, and detection of the virus via pcr and/or electron microscopy. mouse hepatitis caused by mhv (coronaviridae) is enzootic in some colonies of inbred laboratory mice . mouse hepatitis may also occur in nonlaboratory strains of mice in zoo settings. mhv causes polytropic and enterotropic disease syndromes, both of which occur following highly contagious viral infection by the oronasal route. grossly, there is hepatomegaly, splenomegaly, lymphadenomegaly, and lymphadenopathy with jaundice and ascites. there may be gross evidence of intestinal (ileal and cecal) disease with thickened and necrotic mucosa. within the liver, spleen, and lymph nodes, there are multiple coalescing and random white necrotic foci. histologically, polytropic disease includes necrotizing hepatitis with syncytial cells within necrotic foci, and demyelination in cns in immunodeficient mice. the enterotropic disease lesions are age-dependent and may include villous attenuation and syncytial cell formation with eosinophilic intracytoplasmic inclusion bodies in enterocytes, mesenteric lymph nodes, and endothelial cells; granulomatous serositis may also be present. diagnosis is based on histologic features and immunohistochemical demonstration of viral antigens within lesions. virus isolation, pcr, and serology are used to confirm infection and exposure, respectively. sendai virus infection (parainfluenza- ; paramyxoviridae) is common in wild and pet rodents and is actively excluded from laboratory populations of mice, rats, and gerbils (easterbrook et al., ) . disease manifestations occur quickly following the aerosol inhalation, direct contact, or in utero exposure. the virus targets respiratory epithelium and type ii pneumocytes, which may impair ciliary activity and predispose affected individuals to secondary mycoplasma pulmonis and other bacterial infections. gross lesions include dark purple discoloration, atelectasis, and consolidation of the cranioventral lung lobes and diffuse pulmonary edema. splenomegaly and lymphadenomegaly are common and if infection occurs in a gravid female, fetal death may occur. histologically, lesions include necrotizing rhinitis, laryngotracheitis, bronchitis, and bronchointerstitial pneumonia. in immunocompromised animals (e.g., athymic and scid mice), intracytoplasmic and intranuclear inclusion bodies may be present. diagnosis is based on supportive clinical history, histologic findings, and pcr detection of the virus. sialodacryoadenitis is a disease specific to rats and is caused by sialodacryoadenitis virus (sdav; coronaviridae). the virus may be enzootic in some research colonies and in pet and free-ranging rats (easterbrook et al., ) . virus is shed in nasal secretions and/or saliva. sdav infects and replicates in the respiratory tract and spreads to salivary and lacrimal glands resulting in secondary lesions of the nasopharynx, respiratory tract, and eyes. grossly, the parotid and submandibular salivary glands, harderian glands, exorbital glands, and cervical lymph nodes are enlarged, pale, and edematous. the harderian glands may exhibit brown pigmentation, which correlates to accumulations of porphyrin within acini. histologically, there is acute necrotizing adenitis. in chronic cases, there is fibrosis and extensive squamous metaplasia of glandular tissue. there may be rhinitis, tracheitis, bronchitis, and bronchiolitis in severe and chronic cases. histologic lesions are characteristic and supportive of the diagnosis. within this section are several zoonotic and notable nonzoonotic bacteria that infect rodents. several others that are notable but not associated with disease in their rodent host are listed in the supplemental materials (table e ). plague is an oie-listed and who-reportable zoonotic disease that is important to consider when working with rodents. fleas spread the causative agent, yersinia pestis, between hosts. cats and other rodent-eating predators can also contract infection through ingestion and rarely via inhalation. historically, plague has been implicated in the deaths of millions of humans, most notably in th century europe during the black death. there are two main host types: enzootic reservoirs (voles and deer mice) and epizootic amplifiers (prairie dogs, rats, and squirrels). with close to % mortality, plague outbreaks in prairie dog colonies are a devastating and significant threat not only this species but to endangered black-footed ferrets, who are susceptible to disease because prairie dogs are their primary food source (cully et al., ) . there are three disease forms: bubonic (lymphadenomegaly with abscessation); pneumonic ( necrotizing pneumonia with fibrinous pleuritis); and septicemic (multifocal necrosis of liver, lung, spleen, kidney, eye, brain). gross lesions include multifocal pulmonary hemorrhage (fig. . ) , hemorrhagic lymph nodes, erythema of axillary and inguinal skin, splenomegaly and localized hemorrhage and necrosis in the dermis and subutis (suspected site of flea bite). histologic lesions of bubonic disease include necrosuppurative lymphadenitis; in the pneumonic form, necrotizing pneumonia; and in the septicemic form, multifocal abscessation. all lesions contain gram and giemsa positive coccobacilli with characteristic bipolar morphology resembling a safety pin. bacterial culture must be completed at a certified, biosecure laboratory; identification using immunohistochemistry or immunofluorescence is a beneficial diagnostic aid. yersiniosis (pseudotuberculosis) is caused by y. pseudotuberculosis and y. entercolitica. rodents may serve as carriers of this zoonotic bacterial agent, though some species, including mice, voles, beavers, and muskrats may succumb to acutely fatal disease or develop more characteristic lesions and symptoms. gross and histologic lesions include multifocal necrotizing hepatitis and splenitis and/or fibrinonecrotizing and ulcerative enterocolitis (fig. . ). agoutis appear to be uniquely susceptible in zoo-settings (k. terio, personal communication). tularemia caused by francisella tularensis is an important zoonotic and oie listed reportable disease that is maintained by wild rodents and lagomorphs. disease occurs in many rodents including mice, beavers, and muskrats, while voles appear to be subclinical carriers and serve as a reservoir (nelson et al., ; rossow et al., ) . lesions of affected species are similar to those described in rabbits (see chapter ). miliary to multifocal and coalescing hepatic necrosis with variable inflammation (depending on chronicity) characterize the disease (fig. . ) . unlike yersiniosis and similar to listeriosis, bacteria are not readily detected but can be better visualized with special stains. listeriosis is a common zoonotic bacterium in rodent species. it can cause variable, yet similar lesions as for rabbits (see chapter ) . the systemic form is most common. during an outbreak in bushy-tailed jirds, death occurred without clinical signs (tappe et al., ) . salmonellosis is reported in guinea pigs, rats, and mice, though any rodent species can be infected and develop disease. salmonella enterica enteritidis or typhimurium are the most common zoonotic serovars. guinea pigs become septicemic and often die acutely. other rodents may develop gastrointestinal disease that includes diarrhea and abdominal pain prior to systemic spread. in hamsters, salmonellosis must be considered and ruled out in cases of "wet tail," which is typically associated with infection by lawsonia intracellularis. gross lesions of salmonellosis include cyanosis of mucous membranes and polyserositis with splenomegaly. hepatic, splenic, and lymph node necrosis are typical, as are multifocal necrosis and button ulcers in the colon with segmental intestinal infarction. histologic lesions correlate to gross findings and include small foci of hepatocellular necrosis containing necrotic cell debris and variable numbers of macrophages, lymphocytes, and fewer neutrophils (paratyphoid nodules) and kupffer cell hyperplasia; granulomatous hepatitis, splenitis, and lymphadenitis; and fibrinonecrotic ileotyphlocolitis. intestinal infarction can be attributed to vasculitis and thrombosis of the mesenteric or mesocolic vessels. culture of the causative agent and histologic lesions confirm the diagnosis. mycobacteriosis is a zoonotic disease that is rarely reported in rodents, though some species may serve as reservoirs. there are reports of mycobacteriosis (mycobacterium microti) in british voles and wood mice (cavanagh et al., ) , hamsters, and korean and richardson's ground squirrels in spain. chlamydophila caviae and less frequently c. psittaci cause disease in guinea pigs, primarily juveniles. conjunctivitis, bronchitis, and pneumonia may occur and result in debilitation and death. histologically, there is heterophilic keratoconjunctivitis and uveitis. pneumonia has a cranioventral pattern and is heterophilic. some animals may be asymptomatic. diagnosis is achieved through cytology of ocular discharge and/or pcr of affected tissues. immunohistochemistry of tissue sections is also available. bordetella bronchiseptica causes bordetellosis (epizootic pneumonia), a common respiratory disease in guinea pigs though is also reported in free-ranging squirrel populations . similar disease can occur in other rodents, particularly in the context of immunosuppression or coinfection. grossly, there is cranioventral pneumonia with multifocal to coalescing, discrete, reddish-gray consolidated regions affecting multiple lobes, and pleuritis. mucopurulent exudate may be present in the nares, nasal passages, trachea, and tympanic bullae. there is often crusting conjunctivitis. females may have metritis and pyosalpinx. histologically, lung lesions consist of necrotizing and heterophilic bronchopneumonia with obliteration of the airways. diagnosis is confirmed with bacterial culture or pcr. other predisposing pathogens (e.g., adenovirus, mycoplasma spp.) must be ruled out. ciliated associated respiratory (car) bacillus is an unclassified bacterium that colonizes the ciliated epithelium of the respiratory tract of rats, mice, rabbits, and a variety of other species. this agent acts synergistically with other respiratory pathogens including mycoplasma pulmonis, sendai virus, coronavirus, streptococcus pneumoniae, corynebacterium kutscheri, bordetella bronchiseptica, klebsiella pneumoniae, or pasteurella spp. lesions include suppurative to mucopurulent bronchopneumonia, perivascular and bronchiolar lymphoid hyperplasia, rhinitis and lymphoplasmacytic tracheitis in free-ranging, pet and laboratory rats rothenburger et al., b) . coinfection of car bacillus and mycoplasmas has been reported in rats and spinifex hopping mice with pneumonia (mackie et al., ) . murine respiratory mycoplasmosis is caused by mycoplasma pulmonis . gross lesions include catarrhal exudate in the upper respiratory tract with cranioventral bronchopneumonia and mucopurulent exudate in airways with atelectasis. histologic lesions include peribronchial lymphoid cuffing with balt hyperplasia; neutrophilic bronchopneumonia; type ii pneumocyte hyperplasia of alveolar epithelium; and emphysema; bronchiectasis is a characteristic finding. histology alone cannot differentiate m. pulmonis and car bacillus respiratory infections (rothenburger et al., b) . other lesions associated with m. pulmonis include endometritis, salpingitis, and perioophoritis. diagnosis is based on histologic features, culture or pcr detection, and elisa. pasteurellosis caused by p. multocida affects young and immunocompromised rodents, most frequently following direct transmission from the dam. strain virulence and species determine lesions, which in general, are similar to those reported in other species, including rabbits (see chapter ) . prairie dogs develop upper respiratory disease and pneumonia, while conjunctivitis is more common in rats and mice. hamsters have variable susceptibility. cervical lymphadenitis, a disease classically described in guinea pigs, is caused by streptococcus equi subsp. zooepidemicus. infection is associated with coarse foods that cause traumatic lesions of the oral mucosa; bite wounds may serve as another route of entry. grossly, the cervical and submandibular lymph nodes are markedly enlarged and contain thick purulent yellow-white to red-gray exudate. lymph nodes in severely affected and chronic cases may rupture. histologically, affected lymph nodes are effaced by heterophilic lymphadenitis with central necrosis and peripheral fibrosis. thoracic lesions include fibrinous pleural adhesions, fibrinosuppurative bronchopneumonia, pleuritis, and pericarditis. chains of gram-positive cocci are present within lesions. diagnosis is based on gross and histologic lesions and culture of causative agent. tyzzer's disease, caused by clostridium piliforme, is a potentially fatal disease of many rodent species. it has been reported in captive gerbils, hamsters, and spinifex hopping mice (stannard et al., ) . infection results in a triad of gross lesions including icterus and hepatomegaly with military gray foci, congestion, and edema of the intestine, and lymphadenomegaly with edema and hemorrhage. sudden death without clinical disease or lesions is possible in peracute cases. histologically, multifocal random to coalescing hepatic necrosis is surrounded by hemorrhage, heterophils, and macrophages (fig. . a ). at the necrotic margins, hepatocytes may accumulate characteristic criss-crossed bundles of faintly staining bacilli that are best visualized with silver stains (fig. . b ). proliferative ileitis (wet tail) due to lawsonia intracellularis is most commonly reported in hamsters. similar to this infection in other species, there is segmental thickening of ileum with serosal nodules. histologically, there is marked crypt hyperplasia and herniation and villous elongation, hyperplasia, and fusion with variable necrosis and hemorrhage. silver stains and pcr are useful to confirm infection. a diverse array of pneumocystis spp. are carried without clinical signs by a variety of wild rodents; these organisms are thought to be highly host-specific. pneumocystis spp. are found within alveoli and alveolar macrophages and appear as small, eosinophilic round organisms with a small central body. in severe cases, organisms within alveolar spaces and alveolar macrophages can obliterate alveoli; in some cases they can be associated with interstitial pneumonia (fig. . a,b) . this fungus can cause significant opportunistic infections in immunocomromised individuals. cryptococcosis (cryptococcus neoformans) can be a significant pathogen in some rodent species. for example, approximately % of slender-tailed cloud rats at one zoological institution died with or as a result of cryptococcal pneumonia over a -year period (berliner et al., ) . these large, arboreal rodents were likely infected by contaminated substrates and may be a highly susceptible species, similar to some species of nonhuman primates and felids. lesions included none or mild to severe, chronic granulomatous pneumonia with or without necrosis and regional lymphadenitis. disseminated disease can produce subcutaneous nodules ( cryptococcoma) and meningoencephalitis. microscopically, characteristic fungal yeasts with narrow-based budding are present in cytologic preparations and histologic sections. gms staining can enhance detection of fungal yeasts, which are - µm diameter with a distinct, thick ( - µm), nonstaining mucopolysaccharide capsule, the latter of which is best visualized using the pas reaction and mucin stains. culture can confirm infection ( fig. . ) adiaspiromycosis is a systemic fungal disease reported in several captive and free-ranging rodent species. it is caused by the saphrophytic fungi chrysosporium parum and c. crescens. once inhaled, these fungi cause pulmonary granulomas with fungal spherules and conidia. additional diagnostic tests may be useful, including immunodiffusion and complement fixation. these fungi are zoonotic. other systemic mycoses of rodents include infections caused by the endemic (and zoonotic) fungi blastomcyes dermatitides and histoplasma capsulatum. reported cases include juvenile mice and chinchillas. immunity to fungal pathogens appears limited in mice. dermatophytosis is a cutaneous fungal disease that can be found in any rodent species and is similar to that of other mammalian species. causative agents include trichophyton mentagrophytes, microsporum canis, m. gypseum, and epidermophyton. of note, these fungi are zoonotic and can cause skin lesions in other animals. additionally, sporothrix schneckii associated cutaneous disease is reported in several rodent species and can be zoonotic. dermatitis lesions vary from suppurative and necrotizing to granulomatous and fibrosing depending on chronicity. paspositive yeast are present within macrophages. character-istic organisms on cytologic preparations of skin scrapes prepared with potassium hydroxide support the diagnosis. capillaria hepatica (synonymn calodium hepaticum) affects a broad host range worldwide, including rodents and humans. it is a ubiquitous parasite of globally invasive norway and black rat populations . gross lesions consist of multifocal to coalescing, tortuous, white tracks in the liver parenchyma ( fig. . a ). histologically, varying combinations of lymphoplasmocytic and granulomatous inflammatory infiltrates, fibrosis, and bioperculate eggs and occasionally adult parasites replace hepatic parenchyma (fig. . b ). cross sections of viable or mineralized capillarid nematodes may be present. eggs are only released from the liver following carcass decomposition or ingestion by a predator/cannibalistic conspecific. therefore, diagnosis is based on the presence of the nematode and/or eggs in the liver and not fecal parasitology. many species of free-ranging rodents are susceptible to fatal visceral larval migrans associated with the raccoon nematode baylisascaris procyonis (fig. . ). significant clinical disease is often related to parasite migration in the brain, where they may be accompanied by low numbers of eosinophils, gliosis, spheroids, and rarefaction or necrosis of the neuropil. rodents, particularly squirrels are prone to notoedric mange cornish et al., ; . mites burrow into the stratum corneum inducing marked hypekeratosis, which results in patchy alopecia, and crusting and thickening of the skin (lichenification), which can appear yellow to gray. gross lesions are predominantly found on the ears, nose, tail, external genitalia, inguinal and perianal regions, and feet though they can cover the entire body in severe cases. peripheral lymphadenopathy may also be seen. skin scrapings and fecal floats are beneficial to best examine mite features and presence of eggs, respectively. histologically, there is marked epidermial hyperplasia with parakeratotic hyperkeratosis forming caps over tunnels containing mites with superficial, perivascular eosinophilic dermatitis ( fig. . a,b) . secondary bacterial infections are common. additional notable parasites of rodents are listed in the supplemental materials (table e ) . chronic progressive nephropathy (cpn), naked mole-rat, kidney. in the kidney, there is marked ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. . ). hepatic hemosiderosis is seen in sections of the liver. eslide: vm .e chronic progressive nephropathy (cpn), naked mole-rat, kidney. in the kidney, there is marked ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. . ). hepatic hemosiderosis is seen in sections of the liver. eslide: vm .e chronic progressive nephropathy (cpn), naked mole-rat, kidney. there is mild to moderate ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. . ). eslide: vm .e hepatic hemosiderosis, naked mole-rat, liver. prussian blue staining highlights bright blue granular intracytoplasmic (iron) pigments throughout the liver. the kidney has changes consistent with chronic progressive nephropathy. eslide: vm .e yersiniosis, beaver, liver and diaphragm. yersiniosis in the liver and diaphragm of a free-ranging north american beaver. bacterial infection causes multifocal random,necrosis throughout the hepatic parenchyma. large, central colonies of the causative agent, yersinia pseudotuberculosis are surrounded by mild neutrophilic inflammation. (see fig. . ). eslide: vm .e tularemia, beaver, liver and spleen. tularemia in the liver and spleen of a free-ranging north american beaver. large, multifocal random areas of coagulative necrosis are present throughout the hepatic and splenic parenchyma. note the relative lack of inflammation. unlike yersiniosis, bacterial colonies are not readily detected with routine hematoxylin and eosin staining. (see fig. . ). eslide: vm .e pneumocystis murina pneumonia, mouse, lung. multifocally, alveoli are partially to completely filled with large macrophages containing small round yeasts.(see fig. . ). eslide: vm .e pneumocystis murina pneumonia, mouse, lung, gms. gomori methenamine silver (gms) staining highlights the intrahistiocytic yeasts within the alveoli. (see fig. . ). eslide: vm .e pulmonary cryptococcosis, slender-tailed cloud rat, lung. the interstitium and air spaces are moderately to markedly expanded and distorted by myriad fungal yeasts that are associated with histiocytic to granulomatous inflammation. aggregates of lymphoplasmacytic inflammation are present but infrequent. (see fig. . ). eslide: vm .e pulmonary cryptococcosis, slender-tailed cloud rat, lung. gms. intralesional yeasts are hhighlighed gray/ blue with silver staining. gms (see fig. . ). eslide: vm .e pulmonary cryptococcosis, slender-tailed cloud rat, lung. mucicarmine. mucicarmine staining highlights the thick mucopolysaccharide capsule of the crytpococcal yeast and stains it bright pink. mucicarmine (see fig. . ). eslide: vm .e capillaria hepatica (calodium hepaticum), norway rat, liver. capillaria hepatica in the liver of a freeranging norway rat. adult nematodes and eggs invade and efface the liver parenchyma. there are examples of viable and degenerative adults. the eggs are characteristically bioperculate with a thick shell. inflammation is lymphoplasmacytic to granulomatous and is associated with fibrosis indicating chronic infection. in some areas, there is multifocal mineralization adjacent to eggs. (see fig. . ). eslide: vm .e baylisacaris visceral larval migrans, groundhog, brain. multifocally, multiple sections of larval nematodes with prominent lateral alae and lateral cords, a thick cuticle, and coelomyarian musculature, features consistent with larval ascarids, are present in the brain. inflammation, including eosinophils, gliosis, and spheroids and rarefaction of the neuropil due to parasite migration, are multifocal and occasionally associated with intralesional nematodes. (see fig. . ). eslide: vm ( ); krinke ( ) , krinke ( ) mice, rats mammary fibroadenoma rudman ( ); krinke ( ) , krinke ( ) gray squirrel mammary (mixed malignant) williams ( ) mice, rats zymbal gland adenoma/carcinoma rudman ( ); krinke ( ) , krinke ( ) mice, rats preputial/clitoral adenoma/carcinoma rudman ( ), , krinke ( ) , krinke ( ) female reproductive ovarian adenocarcinoma , krinke ( ) , krinke ( ) mice teratoma dixon ( ), mice, rats uterine decidual reaction/deciduoma dixon ( ) woodchuck, mice, rats uterine leiomyoma foley ( ) , mice, rats uterine leiomyosarcoma dixon ( ) woodchuck adenoma of the rete testis foley ( ) rat, woodchuck interstitial cells tumors foley ( ) , , creasy ( ) woodchuck sertoli cell tumors foley ( ) woodchuck seminoma foley ( ) woodchuck, mice teratoma foley ( ) , , creasy ( ) hepatobilliary foci of cellular alteration thoolen ( ) woodchuck, rat, mice hepatocellular adenoma roth ( ) , thoolen ( ) , krinke ( ) , krinke ( ) woodchuck, rat, mice hepatocellular carcinoma , thoolen ( ) , krinke ( ) , krinke ( ) hemolymphatic mice, rats histiocytic sarcoma krinke ( ) , krinke ( ) , kogan ( ) , guinea pig, rat lymphocytic leukemia yarto-jaramillo ( ), guinea pig, hamster, mice, woodchuck, rat lymphoma nagy ( ) , , yarto-jaramillo ( ), kogan ( ) , woodchuck, mice, rats myeloproliferative disease roth ( ) , kogan ( ) , ( ), , krinke ( ) , krinke ( ) ground squirrel integumentary gland adenocarcinoma carminato ( ) damaraland mole rat melanomas sura ( ) yellow cheeked vole sebacious gland adenoma williams ( ) capybara, tundra vole, arctic ground squirrel,woodchuck squamous cell carcinoma hamanno ( ) , takahisa ( ), anderson ( ) beaver, insular vole, porcupine squamous papilloma williams ( ) hamsters, red squirrels trichoepithelioma , guinea pig trichofolliculoma ( ) beaver rhabdomyoma williams ( ) red backed vole rhabdomyosarcoma williams ( ) tundra vole osteoma williams ( ) guinea pig alveologenic carcinoma yarto-jaramillo ( ) guinea pig bronchogenic carcinoma yarto-jaramillo ( ) guinea pig papillary adenoma yarto-jaramillo ( ) gray squirrels pulmonary adenomatosis red squirrels, mice pulmonary carcinoma , , renne mice, rats pulomnary adenoma , renne ( ) red and gray squirrels, mice, rats renal adenoma , williams ( ) , frazier spontaneous neoplastic and hyperplastic skin lesions of the woodchuck cutaneous and systemic poxviral disease in red (tamiasciurus hudsonicus) and gray (sciurus carolinensis) squirrels pathology of laboratory rodents and rabbits non-proliverative and proliferative lesions of the cardiovascular system of the rat and mouse endocrine system odontoma-like tumours of squirrel elodont incisors -elodontomas spontaneous hibernomas in sprague-dawley rats adenocarcinoma of the dorsal glands in european ground squirrels (spermophilus citellus) synhimantus (nematoda) associated with gastric squamous tumors in muskrats proliferative and nonproliferative lesions of the rat and mouse male reproductive system nonproliferative and proliferative lesions of the rat and mouse female reproductive system neoplastic and nonneoplastic lesions of the reproductive tract of the woodchuck (marmota monax) polyomavirus infection in hamsters and trichoepitheliomas/ cutaneous adnexal tumors proliferative and nonproliferative lesions of the rat and mouse urinary system proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium squamous cell carcinoma in a capybara (hydrochoerus hydrochaeris) malignant pleural mesothelioma in a woodchuck (marmota monax) oral leiomyosarcoma in a woodchuck (marmota monax) changes in the major ocular glands nonneoplastic and neoplastic changes in the harderian and lacrimal glands pulmonary lesions produced by fibromas viruses in squirrels and rabbits bethesda proposal for classification of nonlymphoind hematopoietic neoplasms in mice lymphosarcoma in the laboratory woodchuck obstructive respiratory disease in prairie dogs with odontomas proliferative and nonproliferative lesions of the rat and mouse respiratory tract harderian gland neoplasms in captive, wild-caught beechey ground squirrels (spermophilus beecheyi) chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection hepatic lesions in woodchucks (marmota monax) serongative for woodchuck hepatitis virus lesions associated with eucoleus sp. in the non-glandular stomach of wild urban rats (rattus norvegicus) proliferative and nonproliferative lesions of the rat and mouse mammary, zymbal's preputial and clitoral glands hamster polyomavirus infection in a pet syrian hamster (mesocricetus auratus) causes of mortality and pathological lesions observed post-mortem in red squirrels (sciurus vulgaris) in great britain endocrine system. endocrine system neoplasia and granulomas surrounding microchip transponders in damaraland mole rats (cryptomys damarensis) squamous cell carcinoma in a capybara (hydrochoerus hydrochaeris) comparative histomorphological review of rat and human hepatocellular proliferative lesions spontaneous tumors of free-ranging terrestrial mammals of north america fowler's zoo and wild animal medicine leptospira and leptospirosis natural history of plague: perspectives from more than a century of research plague and yersiniosis bartonella infection in rodents and their flea ectoparasites: an overview. vector borne zoonotic dis rodent reservoirs of future zoonotic diseases a review of listeria monocytogenes and listeriosis yersiniosis in wildlife and its public health implications rodent-borne diseases and their risks for public health the ecology of tularaemia tularemia in deer mice (peromyscus maniculatus) during a population irruption in saskatchewan *this list includes the zoonotic pathogens carried by rodents that cause disease in wild animals and those deemed most relevent to wildlife and zoo animal specialists ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver nathural pathogens of laboratory mice, rats, and rabbits and their effects on research toxoplasmosis in a woodchuck (marmota monax) and two american red squirrels (tamiasciurus hudsonicus) baylisascariosis-infections of animals and humans with 'unusual'roundworms echinococcus multilocularis detection in live eurasian beavers (castor fiber) using a combination of laparoscopy and abdominal ultrasound under field conditions synhimantus (nematoda) associated with gastric squamous tumors in muskrats besnoitia jellisoni (sporozoa: toxoplasmea) in rodents from utah and california a survey of hemoparasite infections in free-ranging mammals and reptiles in french guiana systemic toxoplasmosis in a five month old beaver, (castor canadensis) echinococcus vogeli rausch and bernstein, , from the paca, cuniculus paca l.(rodentia: dasyproctidae), in the departamento de santa cruz pathomorphologic findings in short-tailed voles (microtus agrestis) experimentallyinfected with frenkelia microti pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus more than a rabbit's tale-encephalitozoon spp. in wild mammals and birds studies on the nematode parasite, gongylonema neoplasticum (spiroptera neoplasticum) and avitaminosis a in the forestomach of rats: comparison with fibiger's results echinococcus multilocularis in a european beaver from switzerland frenkelia sp. from the brain of a porcupine (erethizon dorsatum) from alberta the role of wildlife rehabilitation as sentinels for one health issues at the wildlife and public health interface: reports of taenia crassiceps cysticercosis in woodchucks (marmota monax) and squirrels (sciurus carolinensis) in maryland and virginia cysticerci of taenia mustelae in the fox squirrel characteristics of natural infections of the stomach worm, obeliscoides cuniculi (graybill), in lagomorphs and woodchucks in canada first identification of echinococcus multilocularis in rodent intermediate hosts in sweden parasites of ferrets, rabbits, and rodents klossiella infection of the guinea pig the taxonomic status of echinococcus cruzi brumpt and joyeux, (cestoda: taeniidae) from an agouti (rodentia: dasyproctidae) in brazil capillaria hepatica in wild norway rats (rattus norvegicus) from vancouver lesions associated with eucoleus sp. in the non-glandular stomach of wild urban rats (rattus norvegicus) cutaneous leishmaniasis in the amazon: isolation of leishmania (v.) lainsoni rodent agouti paca (rodentia: dasyproctidae) in the state of para causes of mortality and pathological lesions observed post-mortem in red squirrels (sciurus vulgaris) in great britain pathologic findings in western gray squirrels (sciurus griseus) from a notoedric mange epidemic in san bernardino moutnains, california natural infection of the ground squirrel (spermophilus spp.) with echinococcus granulosus in china ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver. can nathural pathogens of laboratory mice, rats, and rabbits and their effects on research cutaneous and systemic poxviral disease in red (tamiasciurus hudsonicus) and gray (sciurus carolinensis) squirrels pathology of laboratory rodents and rabbits diseases of agouti (dasyprocta agouti) rained in captivity diagnosed by pathological examination cryptococcus neoformans pneumonia in slender tailed cloud rats (phloeomys pallidus): a review of seven cases odontoma-like tumors of squirrel elodont incisors -elondontomas order rodentia mycobacterium microti infection (vole tuberculosis) in wild rodent populations spontaneous cardiomyopathy in young sprague-dawley rats: evaluation of biological and environmental variability notoedric mange in western gray squirrels from washington scurvy in capybaras bred in captivity argentine non-neoplastic liver disease associated with chronic ground squirrel hepatitis virus infection disease limits populations: plague and black-tailed prairie dogs. vector-borne zoonotic dis spontaneous histologic lesions of the adult naked mole-rat (heterocephalus glaber): a retrospective survey of lesions in a zoo population renal pathology in a non-traditional aging model: the naked mole-rat (heterocephalus glaber) initial case reports of cancer in naked mole-rats (heterocephalus glaber) nonproliferative and proliferative lesions of the rat and mouse female reproductive system anatomical studies of the male genital organs of the european beaver eosinophilic substanceis "not amyloid" in the mouse nasal septum a survey of rodent-borne pathogens carried by wild-caught norway rats: a potential threat to laboratory rodent colonies dystrophic cardiac calcinosis in mice polyomavirus infection in hamsters and trichoepitheliomas/cutaneous adnexal tumors monkeypox transmission and pathogenesis in prairie dogs histopathology of preclinical toxicity studies prediction of spontaneous hereditary diabetes mellitus in chinese hamsters by means of elevated alpha- serum levels rodent reservoirs of future zoonotic diseases macroscopic anatomy of the lower respiratory system in mole rats (spalax leucodon) kurloff cells in peripheral blood and organs of wild capybaras pulmonary lesions produced by fibromas viruses in squirrels and rabbits the clinical chemistry of laboratory animals dental dysplasia in rats and mice concurrent infection with cilia-associated respiratory bacillus and mycoplasmas in spinifex hopping-mice (notomys alexis) with pneumonia rodent-borne diseases and their risks for public health persistent paramesonpehric ducts (masculine uterus) in the male north american beaver (castor candadensis) polycystic kidney disease in adult brazilian agoutis (dasyprocta leporina) francisella tularensis infection rodentia chapter | without lesions in gray tree squirrels (sciurus griseus): a diagnostic challenge practical pathology of aging mice fluorosis risks to resident hispid cotton rats on land-treatment facilities for petrochemical wastes detection of francisella tularensis in voles in finland. vector borne zoonotic dis congestive cardiomyopathy in the woodchuck, marmota monax chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection capillaria hepatica in wild norway rats (rattus norvegicus) from vancouver survey of cardiovascular pathology in wild urban rattus norvegicus and rattus rattus respiratory pathology and pathogens in wild urban rats (rattus norvegicus and rattus rattus) hamster polyomavirus infection in a pet syrian hamster (mesocricetus auratus) causes of mortality and pathological lesions observed postmortem in red squirrels (sciurus vulgaris) in great britain pathologic findings in western gray squirrels (sciurus griseus) from a notoedric mange epidemic in san bernardino moutnains, california contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients the laboratory rabbit, guinea pig, hamster, and other rodents neoplasia and granulomas surrounding microchip transponders in damaraland mole rats (cryptomys damarensis) four cases of spontaneous neoplasia in the naked mole-rat (heterocephalus glaber), a putative cancer-resistant species an epizootic of fibromatosis in gray squirrels (sciurus carolinensis) in florida parapoxvirus causes a deleterious disease in red squirrels associated with uk population declines listeriosis in seven bushy-tailed jirds spontaneous reproductive pathology in female guinea pigs lung morphology in rodents (mammalia rodentia) and its implication for systematics working underground: respiratory adaptations in the blind mole rat fibroma virus infection in an eastern fox squirrel (sciurus niger) from sangamon county illinois fowler's zoo and wild animal medicine chirodiscoides caviae skin none guinea pigs morrisey ( ) demodex skin alopecia, dermatitis, pruritis gerbil, hamster morrisey ( ) gliricola porcelli skin alopecia, crusting dermatitis, pruritis guinea pigs morrisey ( ) gyropus ovalis skin alopecia, crusting dermatitis, pruritis guinea pigs morrisey ( ) myobia musculi skin alopecia, pruritis, selfmutilation mice myocoptes musculinis skin alopecia, pruritis, selfmutilation mice morrisey ( ) notoedres sp. skin alopecia, crusting dermatitishamster, grey squirrel, ratsanholt ( ), , morrisey ( ) , ornithonyssus bacoti skin anemia rats morrisey ( ) polyplax serrata skin anemia, pruritis, dermatitis mice morrisey ( ) polyplax spinulosa skin anemia, pruritis rats morrisey ( ) psoregates simplex skin follicular nodules mice morrisey ( ) radfordia affinis skin alopecia, pruritis, selfmutilation mice morrisey ( ) trixacarus caviae skin alopecia, crusting dermatitis guinea pigs morrisey ( ) key: cord- -b r cyp authors: maritz, julia m.; land, kirkwood m.; carlton, jane m.; hirt, robert p. title: what is the importance of zoonotic trichomonads for human health? date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: b r cyp trichomonads are common parasites of many vertebrate and invertebrate species, with four species classically recognized as human parasites: dientamoeba fragilis, pentatrichomonas hominis, trichomonas vaginalis, and trichomonas tenax. the latter two species are considered human-specific; by contrast, d. fragilis and p. hominis have been isolated from domestic and farm mammals, demonstrating a wide host range and potential zoonotic origin. several new studies have highlighted the zoonotic dimension of trichomonads. first, species typically known to infect birds and domestic mammals have been identified in human clinical samples. second, several phylogenetic analyses have identified animal-derived trichomonads as close sister taxa of the two human-specific species. it is our opinion, therefore, that these observations prompt further investigation into the importance of zoonotic trichomonads for human health. the trichomonad lineage in phylum parabasalia trichomonads are anaerobic, flagellated protists belonging to the large and diverse groups trichomonadea and tritrichomonadea of phylum parabasalia [ ] . they are characterized by the presence of three to five anterior flagella, hydrogenosomes -hydrogen-producing organelles corresponding to anaerobic versions of mitochondria [ ] , a parabasal body (a large golgi), and a complex cytoskeleton. a few species have been isolated from environmental samples and may represent free-living species; however, the majority of species form symbiotic interactions (see glossary) with various animal hosts. among the parasitic trichomonads, several species inhabit the oral, digestive, and urogenital tracts of invertebrate and vertebrate hosts, including livestock, pets, and humans. historically, phylum parabasalia was divided into two groups based on morphological characteristics; however, the recent inclusion of molecular data recovered six groups: trichomonadea, tritrichomonadea, hypotrichomonadea, cristamonadea, spirotrichonymphea, and trichonymphea [ ] . the trichomonadea, tritrichomonadea, and hypotrichomonadea are of primary concern to parasitologists; however, the evolutionary relationships within and between these groups are unclear [ ] . several molecular phylogenies have attempted to resolve these evolutionary relationships using phylogenetic markers such as ribosomal rna (rrna) and protein coding genes (figure ), which give inconsistent phylogenies [ , ] . four species of trichomonad are considered human parasites: trichomonas vaginalis (found in the urogenital tract) [ ] , trichomonas tenax (localized to the oral cavity) [ ] , and pentatrichomonas hominis and dientamoeba fragilis (located in the digestive tract) [ , ] . only one species has wellestablished pathogenic potential: t. vaginalis, the cause of the most prevalent non-viral sexually transmitted infection in humans, trichomoniasis [ ] . only t. vaginalis and t. tenax are considered human-specific, with the former characterized by the richest, although still limited, epidemiology data [ ] , but very little is known about the latter. p. hominis and d. fragilis can cause gastrointestinal symptoms in some patients, such as abdominal pain and diarrhea [ , ] , d. fragilis has also been proffered as a potential causative agent of irritable bowel syndrome (ibs) [ , ] , but debate surrounds its pathogenicity, infection route, and epidemiology [ ] . in addition, several trichomonad species are of veterinary importance, such as the avian pathogens trichomonas gallinae, tetratrichomonas gallinarum, and histomonas meleagridis [ ] [ ] [ ] [ ] , and tritrichomonas foetus, the causative agent of a venereal disease in cattle [ ] . this extensive host range, along with the isolation of d. fragilis [ ] and p. hominis [ ] from various animal hosts, suggests that certain species of trichomonads may exhibit the characteristics of zoonoses. although the question of zoonotic trichomonads has been considered for some years (e.g., [ , ] ), recent results from several different sources have highlighted this potential. here we summarize the clinical and phylogenetic studies that suggest a zoonotic potential for trichomonads, discuss their implications for human health, and the next steps required for investigation into their epidemiology, pathobiology and evolution. new evidence supports the zoonotic potential of trichomonads human trichomonad infections are not body site-specific the four trichomonad species recognized as human parasites were initially thought to be site-specific [ ] (table ) . however, various clinical studies have shown that they can also be found in atypical locations. for example, t. tenax, a commensal of the human mouth found in patients with poor oral hygiene [ ] , has been identified by microscopic and molecular methods in the upper and lower respiratory tracts [ , ] . one possibility that could account from this 'aberrant' location is inhalation of the parasite from the oral cavity into the respiratory tract. however, in some cases where t. tenax was identified in the respiratory tract, no parasites were found in the mouth [ ] . other human trichomonad species have also been identified in the respiratory tract including the sexually transmitted species t. vaginalis [ , ] and the gut parasite p. hominis [ ] , which suggests that these species too can proliferate outside their usual body sites. at least five species of trichomonad, including p. hominis, t. tenax, t. vaginalis, t. foetus, and t. gallinarum, have been identified in the human respiratory tract and as causative agents of pulmonary trichomoniasis (table ) . they have been found in up to % of patients with pneumocystis pneumonia (pcp) and in up to % of patients with acute respiratory distress syndrome (ards) [ ] . because trichomonads are microaerophilic it is unlikely that they initiate and cause these diseases themselves, but may represent secondary and opportunistic infections that could exacerbate symptoms and prolong illness [ ] . these trichomonad respiratory infections seem to depend upon: (i) the presence of bacteria on which to feed and (ii) local anaerobic conditions caused by pcp or ardsassociated infections [ ] but not necessarily upon immunosuppression, because drugs against pcp consistently cure patients of pulmonary trichomonosis and, in one study, treated ards patients were not found to be immunocompromised [ ] . thus, the presence of an increasing number of distinct trichomonads in a broader range of clinical samples from patients with diverse diseases, such as aids, rheumatoid arthritis, prostate cancer, pulmonary infections (empyema and pneumonia in addition to pcp and ards), and digestive conditions such as diarrhea and ibs [ ] [ ] [ ] , is becoming increasingly apparent. indeed, the frequency of pulmonary trichomonosis infections may be higher than reported because transformation of parasites from the motile, pear-shaped stage to the amoeboid stage renders microscopic identification in clinical samples difficult [ ] , highlighting the importance of molecular data to identify such infections [ ] . non-human species of trichomonad have been isolated from clinical samples trichomonads were thought to have strict host specificity [ ] ; however, trichomonad parasites not previously reported as infecting humans have recently been found in human clinical samples (table ) . for example, parasites belonging to the genus tritrichomonas can be isolated from the reproductive tract of cattle (tritrichomonas foetus), the nasal mucosa and intestine of pigs (tritrichomonas suis), and the intestine of non-human primates (tritrichomonas mobilensis) [ ] . another example is t. foetus, historically considered specific to cattle [ , ] . nonetheless, experimental cross-infections of the parasites between pigs and cattle in addition to analysis of molecular data suggest that these three species should be considered glossary commensal: : a form of symbiosis between two organisms where one derives benefit, whereas the other is unaffected. some gut trichomonads are thought to represent commensals. disease incidence: : the number of new disease cases that occur in a population for a given time period (typically per year). dysbiosis: : an imbalance of the microbiota (the microbial populations at a particular body site of an animal host) that leads, or predisposes, the host to disease conditions [ ] . emerging infectious disease: : outbreaks of previously unknown diseases or known diseases that show an increase in incidence, expansion in geographical range, or spread to a new population. emerging infections can be caused by previously unknown or undetected infectious agents, newly evolved strains, environmental changes, and changes in human demography [ ] . a recent review found that over % of human emerging infectious diseases are zoonotic in origin [ ] . examples include influenza, hiv/aids, and severe acute respiratory syndrome (sars) coronavirus. intermediate host: : a host in or on which a pathogen spends a part of its life, usually a transition period, but does not reach sexual maturity. mutualism (mutualist): : a form of symbiosis between two organisms in which both benefit from the relationship. some gut parabasalids from termites are thought to represent mutualists [ ] . opportunistic: : a potential pathogen that typically does not cause disease in a healthy host, but can in particular situations cause disease, for example, owing to the compromised immune system (e.g., attributable to aids, chemotherapy, or malnutrition) of the host. lung trichomonads represent probable opportunistic infections -see main text. parasitic (parasite): : a non-mutual symbiosis between two species where one, the parasite, benefits at the expense of the other, the host. parasites typically do not kill their hosts but exploit them for resources necessary for their survival. obligate parasites cannot complete their life cycles and reproduce without a suitable host. pathogenic (pathogen): : a broad term that refers to the ability of an organism to cause disease. it is typically used to describe an infectious agent or microorganism, such as a bacterium, protist, virus, etc., that causes disease in its host. some pathogens, for example, protists acanthamoeba spp. and naegleria fowleri and fungi aspergillus spp. are free-living species thriving in the environment and occasionally infect humans, often in an opportunistic manner in compromised hosts [ ] . pathogenicity: : the ability of a pathogen to overcome host defenses and cause disease. re-emerging infectious disease: : the reappearance of a historically known infectious disease after a significant decline in incidence. acquired resistance of pathogens to antimicrobial medications is an important factor in the reemergence of many diseases. examples include west nile virus, cholera, mrsa (methicillin-resistant staphylococcus aureus). reservoir: : the habitat or host that harbors an infectious agent, where it can live, grow, and multiply. reservoirs can include humans, animals, and serve as a source of potential disease outbreaks. symbiosis: : a close and often long-term relationship between two or more different biological species. these relationships can be obligate or facultative, mutualistic, commensalistic, or parasitic. transmission: : the passing of an infectious agent from one host to another host. direct transmission routes include: physical contact, contact with a contaminated environment or surface, airborne transmission, and fecal-oral transmission. indirect transmission routes involve another organism such as an insect vector or intermediate host. vector: : an organism that carries and transmits a pathogen from an infected individual to another individual. virulence: : a property of a pathogen, such as specific structural elements or biochemical compounds commonly called virulence factors that cause a reduction in host fitness or damage to the host. it is now recognized that virulence is multifactorial and involves characteristics of both the pathogen and its host, which influence the outcome of their interaction and hence the observed virulence (e.g., an opportunistic pathogen in immunocompromised patients) [ ] . zoonosis: : an infectious organism, such as a bacterium, virus, parasite, or fungus, transmissible between wildlife or domesticated animals and humans. examples include: (i) the lyme disease bacterium borrelia transmitted to humans by ticks from a natural reservoir in rodents; (ii) the malaria parasite plasmodium knowlesi transmitted by anopheles vectors that causes malaria in monkeys and humans; and (iii) cryptosporidium parvum, a parasite found in cats, dogs, and farmed animals and transmitted as a cyst in contaminated water, food, or through the fecal-oral route. zoonoses are the leading cause of emerging infectious diseases worldwide, responsible for devastating disease outbreaks, mortality, and serious socioeconomic consequences [ , , ] . zoonotic potential: : the potential for infectious diseases of wildlife or domestic animals to be transmitted to humans. trends in parasitology july , vol. , no. strains of the same species [ , ] . in addition, several different genotypes of t. foetus have been identified as causing diarrhea in cats in countries [ , ] and have also been isolated from dogs with diarrhea [ ] . moreover, in several new clinical cases, t. foetus or t. foetus-like organisms have unexpectedly been identified in the lungs of human patients [ ] . such findings suggest that t. foetus is a zoonotic parasite capable of colonizing an extensive range of hosts and body sites. other examples of species of non-human trichomonads recently found to infect humans are members of the genus tetratrichomonas, currently the largest genus in phylum parabasalia. tetratrichomonas species are found in the small intestine of a wide spectrum of invertebrate and vertebrate hosts, such as leeches, birds, and rodents [ ] . indeed, some species of tetratrichomonad are known to infect a wide range of unrelated hosts, such as tetratrichomonas prowazeki, which has been found in species of amphibians and reptiles [ ] . another example, tetratrichomonas gallinarum, is primarily thought of as an avian parasite of the digestive tract in domestic and wild birds [ ] , although its pathogenicity is not well established [ ] . however, several recent studies have identified tetratrichomonas strains isolated from human lungs or the human the primary host may not represent the true natural history of the species, which may have a broader host range. c pulmonary infections include pcp, ards-associated infections, pneumonia, and can lead to empyema. trends in parasitology july , vol. , no. oral cavity as t. gallinarum or t. gallinarum-like organisms [ , , ] . studies have also shown that genus tetratrichomonas is much more diverse than previously thought and that t. gallinarum comprises at least three cryptic species with variable host specificity, some that represent human isolates [ , ] . notably, experiments failed to transmit two tetratrichomonas of human origin to birds, although the authors suggest this result could be explained by either adaptation of the t. gallinarum-like trichomonads to the human host or extensive in vitro culturing, so that infection of birds was no longer biologically achievable [ ] . molecular phylogenies reveal close relationships between human and avian trichomonads recent molecular phylogenetic analysis of trichomonads using rrna and protein coding genes (e.g., rpb ) has begun to answer important questions regarding trichomonad phylogeny. rpb is a ubiquitous eukaryotic gene coding for the largest subunit of rna polymerase ii and is present as a single copy in many eukaryotes. a recent analysis of rpb generated a fully resolved phylogeny of trichomonadea, tritrichomonadea, and hypotrichomonadea, and species and isolates within these groups (figure ) [ ] . interestingly, the phylogeny recovered some avian isolates of trichomonas spp. as sister taxa to t. vaginalis, and t. tenax as closely related to t. gallinae; these findings are consistent with previous phylogenies based upon rrna and other protein coding genes [ , [ ] [ ] [ ] . the common ancestor to this complex is also related to the avian t. gallinarum (e.g., [ ] ). these new phylogenies complicate the inferred relationship between t. vaginalis and t. tenax, which on the basis of host specificity might be expected to be sister taxa [ ] . in this scenario, an ancestor of both species infected humans and subsequently differentiated into two distinct species with different body site preferences. however, the lg+g+i model, bootstrap replicates, based on unambiguously aligned sites (the alignment is available upon request)], based on rpb illustrating the relationship between human-and animal-specific trichomonad species and one isolated from the environment (pseudotrichomonas keilini). note the high similarity of sequences derived from the human trichomonas vaginalis isolates and trichomonas sp. (hmo ) isolated from a dove. this sequence and other trichomonas gallinae sequences are clearly distinct from the recently defined trichomonas stableri (kf ) species isolated from band-tailed pigeons [ ] . taxa in red were isolated from humans and those in blue isolated from birds; accession numbers of each sequence are shown. adapted from [ ] . trends in parasitology july , vol. , no. phylogenetic data suggest the zoonotic transfer of trichomonad parasites from humans to birds, and/or vice versa, on at least two occasions, or a combination of these events. other studies investigating outbreaks of avian trichomonosis in wild birds, primarily attributed to t. gallinae infections, have also confirmed the close relationship between avian and human trichomonads [ ] . epidemic infections by t. gallinae of passerine species in europe have recently been described and associated with high mortality and population decline [ ] . this dramatic case of disease emergence demonstrates the potential for a trichomonad to jump host species (columbiform to passerine) and spread rapidly through populations. the disease is thought to have been initiated and spread through transfer of the parasite via contaminated water and bird feeders, as well as through direct contact between passerines [ ] . furthermore, the recent isolation and preliminary characterization of trichomonas stableri associated with epidemic mortality in california band-tail pigeons suggest that avian trichomonosis may be caused by pathogens other than t. gallinae and h. meleagridis [ , ] . genetic analysis at multiple loci indicated t. stableri to be more closely related to t. vaginalis than to the bird-associated t. gallinae [ ] (figure ), further supporting the hypothesis that trichomonads are crossing host boundaries. generating genetic markers and whole genome sequences of t. stableri and other t. vaginalis-like isolates derived from cases of epidemic avian trichomonosis will provide important insight into the evolution and origins of these pathogens. for example, does the trichomonas sp. isolate from a whitewinged pigeon extremely closely related to isolates of t. vaginalis represent a case of human to bird transfer (accession hm in figure )? the implications of zoonotic trichomonads for human health according to current disease dynamic models, the zoonotic emergence of parasitic diseases in humans is typically associated with several characteristics, including broad host range, genetic variability, presence of genotypes better suited to the parasitism of humans, and modified pathogenic potential [ ] . emergent zoonoses are thought to appear through a number of different stages, for example, some develop as animal 'parasitoses' that are newly transmissible to humans, although the source of the disease remains the animal reservoir [ , ] . in other cases, parasites are able to cross the species barrier, modify their specificity, and become sustainably transmissible from human to human [ , , ] . the evolution of these emergent parasitoses is not linear, and an explanation for such a complex process requires consideration of the multi-host ecology and complex dynamics of zoonotic infections [ ] . based on these models and the clinical and molecular evidence discussed previously, it may be that several trichomonads are at different stages of zoonotic emergence ( figure ). these observations raise important questions regarding the implications of the potential widening pathological spectrum of trichomonads in humans and suggest that owing to links with other diseases these parasites may be of greater medical importance than previously thought. historically, trichomonads have not been considered as emerging infections because of their site-and host-specific occurrence. nonetheless, the presence of trichomonads in a diverse array of clinical disorders suggests that they may exhibit a form of opportunism and multiply when local conditions are favorable. for example, the diseases in which trichomonads are found as co-infecting agents in respiratory infections are probably not limited to pcp and ards-associated infections, but may include other pulmonary diseases such as cystic fibrosis [ , ] . overall, the high prevalence of pulmonary diseases globally [ ] combined with the higher burden of both lung conditions and zoonotic diseases among people in resource-limited settings [ , ] suggest that only the 'tip of the iceberg' of pulmonary trichomoniasis may currently be known. digestive tract infections by trichomonads are also increasingly recognized as being common. although the exact clinical profile of d. fragilis is still poorly understood some consider this species to have pathogenic capabilities [ ] , and recent studies have associated the rise of ibs with a high prevalence ( %) of d. fragilis in europe [ ] . however, the pathogenicity of d. fragilis has been questioned owing to the asymptomatic nature of many infections, and it is considered by some as a commensal of the intestinal flora [ ] . indeed, treatment of d. fragilis-infected children with metronidazole was not associated with better clinical outcomes [ ] . because association is not evidence for causality, additional data are required to establish the pathogenicity of trichomonads in the digestive tract and 'aberrant' body sites such as the lungs. in this context, it is important to consider characteristics of both host and parasite with regard to the outcome of their interactions. for example, one extreme is represented by severely immune-compromised patients that are more susceptible to a wider range of microbial infections compared with immunocompetent hosts [ ] . when studying the outcome of human-microbe interactions, a complex interplay among viruses, bacteria and archaea, microbial eukaryotes, and animal parasites influence the health status of the human host, with mucosal microbiota playing a key role influencing health and disease status [ , ] . based on these considerations and examples, trichomonads may be more prevalent and have a wider pathological spectrum in humans than currently recognized, influencing human health through direct pathologies but also indirectly through dysbiosis of the mucosal microbiota and local inflammation, facilitating transmission of pathogens -a prime example being t. vaginalis infection and bacterial vaginosis contributing to hiv transmission [ , ] . the potential influence of gut trichomonads to human health will also have to consider their potential impact on the gut microbiota, which might explain observations of the association between d. fragilis and ibs through inducing gut dysbiosis [ ] . indeed, the ability of trichomonads to live on a variety of mucosal tissues may be the key to their wide host range and ability to develop infections at different body sites, as well as contribute directly or indirectly to pathologies. once the capacity to thrive on vertebrate mucosal surfaces has developed, there may be less of a barrier to cross both species and mucosal sites. for example, in the case of review trends in parasitology july , vol. , no. t. foetus, this sexually transmitted species may represent a recent transfer from the digestive to the urogenital tract, with a capacity of the parasite to thrive in the gut in different species (e.g., pigs, cats, and dogs). trichomonads provide a unique system for the study of the origins and pathobiology of zoonotic and emerging infectious diseases. in addition, they have attracted interest as model systems for evolutionary biology and comparative genomics [ , ] , and for biochemical, molecular, and cell biology investigations [ , , ] . the t. vaginalis genome sequence published in was the first species of trichomonad to be sequenced [ ] , and others are currently underway including isolates of t. foetus, p. hominis, t. gallinae, and t. tenax. these sequences will enable comparative analysis of common and unique parasitic modes of life cycle, and possible adaptive mechanisms. for example, t. vaginalis and t. foetus appear to have evolved independently to colonize the urogenital tracts of different mammalian hosts [ ] . in addition, t. foetus has been isolated from the digestive tract of cats and dogs, indicating that it is capable of colonizing a broad range of hosts and environments [ ] . these two species of sexually transmitted trichomonad probably represent cases of convergent evolution and provide an opportunity to compare the derived similarities and the origins of these traits that coincide with a shared niche. species of trichomonad exhibit a range of genome sizes, from mb for the p. hominis genome to mb for the t. foetus genome [ ] . the t. vaginalis genome sequence revealed the -mb genome to be a result of expanded transposable elements and protein coding gene families, . speculative models of zoonoses caused by trichomonads.trichomonads are listed on the right and colored according to primary hosts assigned historically in the literature. unbroken lines represent known infections or transmission routes, and broken lines represent speculative infections or transmission routes for which data are lacking. the relationships are represented as follows: (blue box) trichomonads identified in wild bird species (e.g., green finch [ ] and toucan [ ] ) in partially domesticated species (rock dove) and in fully domesticated species (chicken) circulate within these populations with variable host specificity [ ] (blue unbroken circle with arrow). two of the four avian trichomonads listed (tetratrichomonas sp. and tetratrichomonas gallinarum) have been identified in human lungs [ ] , and trichomonas gallinae and trichomonas stableri are also included owing to their close relationship to trichomonas tenax and trichomonas vaginalis [ , ] . (red box) t. vaginalis and t. tenax are the two species considered human-specific, with known human-to-human infections (unbroken red circle). the close genetic relationship of the human and avian trichomonads ( figure ) suggests either independent zoonotic acquisitions from avian sources (broken blue arrow) or transfer of the parasites from humans to birds through environmental contamination (broken red arrow). (green box) tritrichomonas foetus has been isolated from a variety of pets and farm animals, with the same strain known to infect cattle and pigs (unbroken green arrow) [ ] , but different genotypes infecting cattle and cats [ , ] ; the origins of dog infections remain unclear [ ] . thus, there are at least two t. foetus genotypes capable of colonizing an extensive range of hosts, including humans [ ] (broken green circle and arrow). the lack of precise epidemiological data is indicated by '?'. (purple box) pentatrichomonas hominis has been isolated from a variety of pets and farm animals [ ] , but little is known about its infection route and epidemiology; the same strain could be circulating between all identified hosts (broken purple circle). (orange box) dientamoeba fragilis has been isolated from farm animals (pigs) and non-human primates (gorillas), with the same strain known to infect pigs and humans [ ] (unbroken orange arrow). recent evidence suggests that household pets do not play a role in transmission [ ] ; however, the origins remain unclear and multiple strains could be circulating in animal hosts (broken orange circle and arrow). additionally, given recent prevalence and transmission data it seems unlikely that transmission from non-human hosts represents a significant proportion of infections. contaminated surfaces and water [ ] , uncooked meat, or direct contact with pets and farm animals could lead to animal-to-human transmissions of trichomonads. initial infections were presumably through the digestive tract (via oral ingestion) with further progression to the lungs for some (various) species or the urogenital tract (t. vaginalis). trends in parasitology july , vol. , no. including those responsible for interaction of the parasite with its immediate environment [ ] [ ] [ ] . it has been hypothesized that the large genome size may be a recent event that occurred when the most recent common ancestor of t. vaginalis underwent a population bottleneck during its transition from the digestive tract to the urogenital tract. because genome size is positively associated with cell volume, an increase in genome size and concomitant increase in cell size might have increased its phagocytic potential as well as surface area for the interaction of the parasite with host cell tissue [ , ] . in this context, it will be particularly interesting to compare the genomes of t. vaginalis with its closely related isolates and species derived from birds ( figure ) to gain detailed insight into correlations between genome evolution and how this might relate to parasite pathobiology in humans and birds. lateral gene transfers from bacterial donors sometime in the evolutionary past have also importantly influenced the evolution of t. vaginalis protein coding genes [ , ] . for example, the parasite has gained an almost complete pathway for the degradation of complex glycans present in host mucosal secretions, factors which may contribute to its adaptive potential and pathogenicity [ , ] . comparison of a broad range of trichomonad genomes will help test this hypothesis and may pinpoint gene families whose acquisition and/or expansions correlate with pathogenicity and facilitated or mediated transitions from: (i) an ancestral animal-only stage to human-inclusive infections or (ii) from the digestive to urogenital tracts. trichomonads also provide a unique system to study the features of a zoonotic lifestyle via comparative examination of molecular and cellular characteristics. for example, successful t. vaginalis infections are probably favored by virulence mechanisms such as cytoadherence and phagocytosis [ , ] . the predicted protein coding genes of t. vaginalis [ ] includes a plethora of candidate genes for surface molecules mediating interaction with host tissues and membrane trafficking and signaling, important processes involved in parasite pathobiology [ , ] . cysteine proteases in particular have been identified as virulence factors central to the host-pathogen interface in t. vaginalis [ , [ ] [ ] [ ] [ ] . transcriptomic studies have shown upregulation of some of these t. vaginalis virulence factors in response to contact with host cells in vitro [ ] and have also documented their expression under in vitro growth conditions in t. foetus [ ] . similar to t. vaginalis, recent studies have shown the presence of cysteine proteases in the cell-free filtrate of t. gallinae and demonstrated their involvement in its in vitro cytopathogenic effects [ ] . mining other trichomonad genome data to identify important virulence proteins will improve our understanding of the molecular and cellular basis of infections and can be used to test hypotheses, such as whether zoonotic organisms show greater diversity in key virulence proteins underlying their capacity to parasitize a variety of host species and mucosal sites [ ] . concluding remarks and future perspectives although we have discussed several recent studies that provide strong evidence for the zoonotic origin and potential of trichomonads, regular and sustained zoonotic transmission of these microbes has yet to be definitively established. to improve our knowledge of the zoonotic origins of trichomonads, detailed investigations including systematic surveys of trichomonads in humans and animals will be required. molecular methods have been instrumental in our understanding of the biology and complexity of trichomonads so far; however, more data and novel approaches are needed to resolve evolutionary relationships and to improve diagnostic tools. wide sampling and whole genome sequencing of trichomonads, with subsequent comparative genomic investigations, will facilitate identifying the closest relatives of human trichomonad pathogens, providing a solid evolutionary framework for how these diseases have emerged and forming a basis for epidemiological studies across both animal (wild and domestic) and human hosts. environmental studies such as the 'microbes, sewage, health and disease' metagenomics project in new york city (http://www.nyu.edu/ about/news-publications/nyu-stories/video-mapping-nycs-metagenome.html) will provide important information on potential transmission routes and the patterns, nature, and occurrence of trichomonad infections in humans, animals, and birds; and establish the importance of trichomonads zoonotic transmissions -as has been established for species of trypanosoma, cryptosporidium, and toxoplasma [ ] . furthermore, studies investigating the potential pathogenicity of these parasites in various mucosae (respiratory, digestive, and urogenital) are needed to determine the clinical significance and public health implications of trichomonads. 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early infection of vaginal epithelial cells and amoeboid transition cysteine peptidases, secreted by trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture data-mining approaches reveal hidden families of proteases in the genome of malaria parasite oxford textbook of zoonoses symbioses of flagellates and prokaryotes in the gut of lower termites molecular confirmation of trichomonas gallinae and other parabasalids from brazil using the . s and its- rrna regions evaluation of the easyscreen enteric parasite detection kit for the detection of blastocystis spp., cryptosporidium spp., dientamoeba fragilis, entamoeba complex, and giardia intestinalis from clinical stool samples persistence of two trichomonas gallinae isolates in chlorinated and distilled water with or without organic material we thank four anonymous referees for their constructive comments that allowed us to improve the manuscript. j.m.m. is supported by the maccracken program in the graduate school of arts and science, and by a new york university grand challenge project. r.p.h. acknowledges past wellcome trust funding for his work on trichomonas vaginalis. key: cord- - ghzpqct authors: odasz, ann marie title: nitrate reductase activity in vegetation below an arctic bird cliff, svalbard, norway date: - - journal: j veg sci doi: . / sha: doc_id: cord_uid: ghzpqct abstract. vegetated sites below bird‐nesting cliffs are uniquely nutrient‐rich habitats in the otherwise nutrient‐poor arctic environment. plants from six distinct vegetation zones below such a cliff at ° n, svalbard, norway, were collected for analysis under greenhouse conditions. leaf nitrate reductase activity (nra) was analysed in species representing % of the svalbard vascular flora. the species mean nra values ranged from . to . μmols of nitrite ions formed per gram of plant fresh weight per hour. species in the vegetated zone growing closest to recent guano deposits had the highest nra values, (mean = . ) whereas plants growing farther below the cliff had significantly lower values (mean = . ). a similar pattern was detected in a duplicate set of plants induced with mm kno( ); vegetation zone means for nra ranged from . to . μmols of nitrite ions formed per gram of plant fresh weight per hour. maximally induced species nra values were highest in the first zones below the cliff and decreased downslope. this gradient paralleled the steep soil nitrate gradient, which decreased from . mg/l at the cliffbase to . mg/l downslope. correspondingly, soil ammonium ions in the vegetation zones ranged between . mg/l at the cliff‐base to . mg/l downslope. correlations between nra and soil nitrate provide a systematic basis for assigning scalar ‘nitrogen figures’ as indicators of habitat preference, here for the first time applied to arctic species. nitrogen, the element taken up in greatest amounts by plants from soil, is of major significance in plant ecology because of its involvement in plant growth and metabolism. nitrogen constitutes as much as % of the total dry weight in some plants (lee & stewart ) and is essential for all activities of living cells, including the synthesis of organic compounds such as aminoacids, proteins, and nucleic acids. although in a few cases nh + provides the exclusive form of available soil n , in some soils no is the predominant form available for uptake. the capacity for no reduction in plants is therefore a prerequisite for, and an indication of, the optimal utilization of the natural n supply. of the enzymes participating in reduction, nitrate reductase (nr) determines the rate of the reaction as a whole (beevers & hageman ) . occurrence and activity of nr are therefore of considerable ecological interest. low nitrogen levels limit plant growth over much of the biosphere and especially at high latitudes (shaver & chapin ; maessen et al. ) . moreover, in the arctic, nitrogen availability is limited by abiotic factors, e.g., low temperatures, little precipitation, slow chemical weathering of bedrock, poor soil aeration, low evapotranspiration, and impeded drainage of soils underlain by permafrost. biotic processes such as slow natural decomposition rates, aggravated by the dominance of wet, snowy, and ice-covered surfaces, severely hinder nutrient cycling during the short to week growing season in the arctic. exceptional nitrogen-rich 'oases' occasionally exist in the generally nitrogen-poor landscape of the arctic. large colonies of migratory birds transport nitrogen from sea to land during cliff-nesting activities in the spring and summer. the ornithogenic soils associated with these nesting sites receive seasonal flushes of nutrients during snowmelt-runoff. later in the season rainfall redistributes these deposits of rich organic material to maintain a typical steep nitrogen gradient characterized by high levels at the cliff-base and lower concentrations downslope. distinct identifiable zones of lush vegetation are present along this rich nitrogen gradient, and are best developed on the south-facing slopes where net solar radiation is highest. a bird cliff habitat at ° n on svalbard provides a unique 'natural experiment' where the competitive advantage of effective nitrate assimilation can be investigated in plants growing along a natural nitrate gradient. the aim of this study was to investigate the relationship between the distribution of plant species in bird cliff vegetation and the amount of soil nitrate available in the region of sea-bird guano deposition. the species' capacity to synthesize nr may be a key factor for plant survival and distribution along the vegetation zonation. to test this hypothesis, constitutive nra analyses were conducted on plants collected in their natural soils and transported to a controlled greenhouse environment. at the same time, induction capacity of the nitrate assimilating enzyme was investigated in a nitrate applicationfertilization experiment conducted on a subset of the collected plants. field investigations of nr activity have been conducted on temperate and boreal species from various environments (gigon & rorison ; havill et al. ; lee & stewart ; gebauer et al. ; högbom & ohlson ) . this study is the first field investigation of nr activity in arctic plants. the investigation focused on the vegetation zones under the casimir perierkammen bird cliff, ( ° ' n ° ' e) in the krosfjorden region on northwest svalbard (fig. ) . summers are cool with a june to august mean temperature of °c and midday temperatures reaching °c . annual precipitation of mm has been recorded in ny-Ålesund, km distant (aune ) . ca. pairs of kittiwakes (rissa tridactyla) and brunnich's guillemots (uria lomvia) nest on a vertical south-facing cliff and deposit about kg of guano per day on the vegetated slopes below the cliffs. zones of low-growing herbaceous plants cover a vertical drop of more than m below the cliffs. mats of accumulated organic material are greater than m thick in the lower zones. bedrock consists of gneisses and magmatites, with some calcareous rocks in the lower slopes. there are h of sun from april to august with the bird cliff receiving ca. h of potential direct sun during this period. immediately below the bird cliff is a -m wide 'sterile' zone ( ° slope). guano and other organic matter (e.g. nesting material) accumulate in this area; seedling establishment is restricted, mainly due to the lack of soil, repeated local mechanical disturbance caused by litter and rock fall, and a high osmotic gradient in the guano. below this zone a series of vegetated zones fan out to more than m at the base (fig. ) . cochlearia groenlandica dominates the first vegetated zone of ca. m . fine moist litter material on the ° slope is stabilized by a network of shallow c. groenlandica roots (fig. ) . a -m strip of vegetatively reproducing puccinellia angustata is located at the top of the zone and a few individuals of draba arctica and d. norvegica are located close to the cliff side-walls. see table for species composition of each zone. oxyria digyna dominates the next zone, characterized by a moderate slope ( - °) and undulating topography due to the accumulated rockfall. the substrate is moist and emits a pungent urea odour. oxyria plants are robust with thick tuberous roots and completely cover the m of this zone. cerastium arcticum dominates an area of m . the zone has a slight slope ( - °) and interdigitates with the lower boundary of the oxyria zone. species diversity increases in the cerastium zone and is at a maximum in two saxifraga zones with s. hieracifolia and s. cespitosa farther downslope. the combined saxifraga zones are m wide and support a continuous mat of the moss, aulacomnium palustre, which is present to a depth of over m and extends into the permafrost. the tundra area has rolling topography and abutts this lower edge of the bird cliff site. samples (n = ) were collected from the top cm of soil in each of the vegetation zones. nitrate, ammonium, phosphate (mg/l), and ph were determined on fresh soil samples immediately after collection (from odasz ), and on dry samples from tundra sites (odasz ) . nitrate and ammonium were extracted in calcium chloride and phosphate was extracted in ammonium lactate. plants of species were collected from the bird cliff vegetation following a random sampling design within each zone. replicates of all species were collected in their original soil during the peak growing season ( - july). the number of replicates varied because some species were small and more abundant allowing for easy collection, resulting in many replicates while plants showing signs of stress after field collection were excluded from analyses. each individual plant was placed in a -mm diameter, -mm tall plastic pot and transported by boat for h ( km) to a greenhouse located in ny-Ålesund (fig. ). plants of each species were randomly separated into two groups both of which were analysed for nra. one group represents constitutive enzyme activity values and these plants were kept in natural soil conditions. the second group was fertilized regularly with mm kno to test if species had induction capacity of nitrate reductase activity above the field, or constitutive, levels. plants were maintained at - °c to simulate field conditions. nra was determined during - august, a period with minimal spectral variation (hisdal ) . analyses were standardized using fresh leaf material, in mid-day sunlight at constant temperature, to minimize potential variation in enzyme activity associated with different plant organs (lee & stewart ; gebauer et al. ) , circadian rhythms and leaf-age (hipkin et al. ; lillo ) , or the influence of light and temperature (nicholas et al. ; margolis et al. ). nra was estimated by measuring the amount of nitrite reduced from nitrate following the methods of guerrero ( ) . the bioassay was conducted on leaves because root reduction is a minimal portion of total plant nra when large amounts of nitrate are available (gebauer et al. ; muller & garnier ) . within each species, care was taken to use . - . g young leaf material. because of betweenspecies differences in leaf size, the sampled mass corresponds to different numbers of leaves. these were cut into ca. mm × mm pieces which were placed in test tubes with phosphate buffer containing propanol- and mm kno and incubated in the dark for h at °c in a water bath. activity was stopped by immersion in boiling water for min. the product, no -, was dyed with n( -naphtyl) ethylene diamine dichloride, sulphamide, and m hcl, and measured at nm with a spectrophotometer (schimadzu, model uv- ). nra was calculated and reported as µmol of nitrite ions formed per gram of fresh weight of the plant per hour (µmol no -/g fw /h). the standard assay was conducted on all species. analysis of variance was used for statistical comparisons among vegetation zones and treatments. correlation analyses were conducted on constitutive and induced nra, and on nra and soil nitrate values (statview plus, machintosh). the difference between the nra values measured in the fertilization treatments and those in natural soil conditions indicates increase in enzyme activity due to induction. in this study i used the maximally induced nitrate reductase activities of species to establish nitrogen indicator figures for arctic plants, thus extending the system of ellenberg ( ) for central-european plants for different levels of nitrogen, using a scalar system from , environments poor in n, through , rich in n. soil nutrient values decrease downslope with the sole exception of a relatively high phosphate level in the oxyria zone ( table ). the nitrate gradient, decreases downslope from . mg/l to . mg/l. correspondingly, the soil ammonium ranged from . mg/l at the top to . mg/l downslope. the ratio no -: nh + decreased downslope; the high value for saxifraga cespitosa zone is accounted for by extremely low soil nh + ( table ) . the soil ph, on the other hand, increases downslope. sea-bird guano contains a wide range of phosphate minerals (tatur & barczuk ) and the concentrations found in this site are unlikely to be limiting to plant growth (table ) . nra was detectable in all species tested in natural soil. mean nra ranged from . µmol no -/g fw /h in the saxifraga zone to . µmol no -/g fw /h in the cochlearia zone (table ) . thus a decrease in values of over an order of magnitude was detected for species along a gradient downslope from the guano deposition site. only nine species (mean nra= . µmol no -/ g fw /h) grow in the two richest vegetation zones, while a total of species (mean nra= . µmol no -/g fw /h) thrive in the lower zones where nutrients are less concentrated. in table the significant differences between mean nra values for each zone are indicated by different letters, i.e. 'a' is significantly different from the table . mean nra values for constitutive field nra and mm kno treatment for all individuals (n) of the species sampled in each vegetation zone below the casimir perierkammen bird cliff. means (x) and one standard error (se) for soil nutrients in mg/l, and mean, minimum and maximum ph, (data from odasz ) and the no -: nh + ratios are from fresh soil samples from each vegetation zone. tundra data are from dry soil samples, mg/ g (odasz ) . letters indicate significant differences (p < . ) for parameters between vegetation zones (see text for details). two saxifraga zones and in the tundra zone had lowest nra. draba norvegica, in the cochlearia zone, showed the greatest induced increase in nra amounting to . µmol µmol no -/g fw /h above the constitutive nra value (table ). in the oxyria zone, poa pratensis ssp. alpigea increased . µmol no -/g fw /h and draba daurica increased . µmol no -/g fw /h above the constitutive values. six species of the cerastium zone showed nra increases of over µmol no -/g fw /h whereas in the saxifraga hieracifolia zone three species were µmol over the constitutive value (table ) . the mean induced nra for the vegetation zones ranged from . µmol no -/g fw /h in the saxifraga cespitosa zone to . µmol no -/g fw /h in the cochlearia zone. induced nra values for the dominant species in each zone decreased significantly downslope: cochlearia groenlandica: . ; oxyria digyna: . ; cerastium arcticum: . ; saxifraga hieracifolia: . ; saxifraga cespitosa: . ; and dryas octopetala representative of the tundra sites: . (table ) . accordingly, constitutive and induced nra for all samples and for all species decreased significantly downslope (table ) . species with high constitutive enzyme activity also had additional capacity for considerable induction of the enzyme (fig. a) . all correlations between nra and soil no -, for both mean nra values zones indicated by 'b', but is not different from zones indicated by 'a', etc. the nra of most plants induced with mm kno increased above the constitutive values and ranged from . to . µmol no -/g fw /h (table ) . species in the may be genetically constrained to a small size and must compete for sunlight through the dense stands of the dominating c. groenlandica and o. digyna plants. fieldbased habitat studies such as these are prerequisites for understanding ecological behavior which is not necessarily identical with physiological demands measured in isolation under laboratory conditions. the r-vs. k-selection (stearns ) may be useful in describing growth strategies which relate to species differences in nra. c. groenlandica is short-lived, has a fast growth rate, high productivity, and high reproductive achievement (fig. , group ) , high capacity for photosynthesis and nutrient absorption, and the ability to exploit nutrient-rich environments. the rapid assimilation of absorbed nitrate into tissues and the capacity to store large concentrations of nitrate in stems and leaves allows concordant high nra in nitrophilous ruderal species (lee & stewart ) . on the other hand, smaller, slow-growing species such as cassiope tetragona and silene acaulis are more typical of k-strategists. these tend to live longer and allocate less biomass to reproduction (fig. , group ) . a combination of low capacity for nra and the small size of these k-strategists makes sunlight competition with the fast growing species difficult. the k-strategists in group (fig. ) are known to be long-lived, dryas: yr, silene: yr, salix: yr (callaghan & emanuelsson ) , and these show low nra values both before and after fertilization. in group , the longevity of luzula exceeds yr, whereas in group , cerastium may reach yr, oxyria, yr, and in group , cochlearia lives to a maximum of yr. group species all have relatively high nra. thus, life expectancy is inversely related to nra and reproductive biomass. in the upper vegetation zones, however, the position of the two species, cochlearia and oxyria, contradicts the otherwise direct correlation between plant nra and soil nitrate. the mean induced nra for oxyria, . µmol no -/g fw /h, is greater than the mean of . µmol no -/g fw /h for cochlearia, which is growing on more concentrated soil nitrate (table ) . species establishment is influenced by substrate composition, and cochlearia roots form a shallow network which stabilizes the dry unconsolidated nesting litter fall. in contrast, the thick long-lived tuberous oxyria roots act to stabilize the undulating topography of the rockfall prevalent in the next zone. oxyria produces greater root biomass in arctic than alpine stands (mooney & billings ) , and, once established, oxyria prevents most other species from germinating in its understory where limited light also restricts potential nra. furthermore, the relationship between root morphology and the ability to establish and stabilize in the contrasting substrate types may also be regulated by for the entire data set and maximum nra values for each species were significant (p < . ) (fig. b) . the correlations of zone mean for all mean nra values and maximum nra values of the dominant species of each zone were not significant; correlation coefficients were similar here, but there were fewer degrees of freedom. plants from specific taxonomic groups showed similarities in habitat preference along the guano gradient. species from the brassicaceae, poaceae and polygonaceae families were growing high on the slope, nearest the cliff, on soils with the most concentrated nitrate levels, and had capacity for the greatest nra (fig. c) . the capacity for enzyme activity is also related to the growth strategy of plants and the amount of biomass allocated to reproductive tissues (fig. ) . the distribution of the species in the vegetation and their capacity for maximally induced nra was the basis for assigning n-figures (referring to ellenberg et al. ) to species of this study, values ranged from . for salix reticulata to a maximum of . for oxyria digyna. values are highest in the top zones and decrease downslope. species dominating the vegetation zones with concentrated soil nitrate showed greater capacity to synthesize nr than species growing in areas of lower nitrate (tables and ). such evidence supports the suggestion that the differential capacity for nra in vascular species results in the development and maintenance of distinct vegetation zones in the bird cliff meadow. effective assimilation of nitrate may provide a competitive edge in interspecific establishment and growth of arctic plants in ornithogenic soils. c. groenlandica and o. digyna appear to out-compete most other species in their respective zones. genetic differentiation may have resulted in specially adapted populations of these species. by means of effective nitrate assimilation (mean induced nra = . , and . µmol no -/g fw /h, respectively) and probable tolerance to concentrated ammonia and other nutrients in the sites, these robust species thrive in a dense, high biomass vegetation (plant cover - %) and grow rapidly to heights of over cm in the bird cliff vegetation. the large individuals of these two species may be a result of great growth response due to abundant useable nutrients in the mesic bird cliff meadow. draba species, however, have high nra, but they root size and ion flux at root surfaces. small roots, like those of cochlearia, have higher demand per unit root surface area (clarkson et al. ) , which may explain the dominance of this species at high soil nitrate levels. water availability and limitation of micronutrients at different ph levels are potential limitations depending on root and substrate conditions. the 'natural experiment' of this bird cliff provides an example of how extremes in abiotic conditions limit species diversity. in nutrient-rich habitats, inorganic n nutrition usually prevails, no is the dominant form and nra is of ultimate importance (lee & stewart ) . however, in poor sites species have other strategies for improving nutrient status such as nitrogenfixing mutualists, ericoid, or ectomycorrhizal symbioses which utilize complex organic sources of n. conversely some species have preference for ammonium. often species commonly mycorrhizal in nutrient-poor sites do not develop the association when growing in rich habitats. cyanobacteria, for example, were found associated with moss in the bird cliff vegetation but the high nutrient levels inhibited nitrogen-fixation (b. solheim pers. comm.). väre et al. (in press) found that although polygonum viviparum, silene acaulis and saxifraga oppositifolia are ectomycorrhizal in other areas, they did not have this association in svalbard habitats. some species can increase the amount of nitrate uptake per unit root as nitrate becomes limiting. another adaptive strategy is to reduce growth rather than produce nutrient-deficient tissue. among families, assimilation of n is most pronounced in the brassicaceae, dominating the top vegetation zone and represented by draba species in the next zone (fig. c) . high levels of nra may be the most important explanation for the successful ruderal habit of this phylogenetic group. poaceae (puccinellia angustata, poa pratensis), and polygonaceae (oxyria digyna) dominate the richer soils, whereas salicaceae, ericaceae and scrophulariaceae have a significantly lower capacity to induce nra and dominate lower zones (fig. c) . the two scrophulariaceae species in this study, pedicularis dasyantha and p. hirsuta, are hemiparasitic plants with low nra. hemiparasitic plants have access to nitrate in host plant xylem sap and are not solely dependent on nitrate assimilation from the soil (lee et al. ). constitutive nra values in the bird cliff, ranging from . to . no -/g fw /h for the species, are within reported mean values for other fertilized sites such as areas where cattle congregate, which reach values as high as . µmol no -/g fw /h (gebauer et al. ) . gradients in nra have been recognized along successional series, ruderal to climax species (lee et al. ) and from old field ruderals to forest climax species (franz & haines ) . the importance of nitrogen as an ecological factor is emphasized by comparing nitrate reductase activity values for diverse habitats, fellfield = . , bog = . , woodland = . , and ruderal = . (mean constitutive nra in no -/g fw /h) (lee et al. ). other habitats influenced by ornithogenic soils have been investigated in penguin rookeries in antarctica (speir & cowling ; tartur & barczuk ) , in an estuary in canada (bedard et al. ) , and on the coast of south africa (bosman et al. ). surface soil horizons from sites receiving continuous additions of fresh penguin guano had high soil enzyme activity. on the coast of south africa guano deposition by colonial sea birds was found to enhance intertidal algal growth. nra and soil nitrate status of the species analysed in the present study provide a basis for assigning scalar n-figures to arctic plants for the first time. in a similar way, gebauer et al. ( ) related nitrate and nitrate reduction levels of some central-european plants to the n-figures of ellenberg ( ; ellenberg et al. ). in the arctic bird cliff vegetation, values of maximally induced nra reach . in the top two zones and decrease to . in lower zones; they provide appropriate scalar n-figures for the species. ellenberg and gebauer et al. did not use figures above , and in order to relate to their system the n-figures would have to be converted. it is not yet clear how this should be done. a check on the ellenberg-values for some species included both in his list (as alpine and montane species) and in mine gave confusing results; oxyria digyna, the relatively most nitrophilous species on the arctic cliff, has an ellenberg value of only . the n-figures as based on the maximally induced nra figures are included in table . n-figures can be used to predict changes in species composition and distribution following changes in nitrogen supply. nitrogenous fertilizers are known to produce marked changes in vegetation species composition (e.g. thurston ) , thus leading us to predict changes in bird cliff vegetation if nitrogen supply (e.g. decline or increase in seabird populations). in addition, plants with high n figures may be quick to respond to managed fertilization and be useful in revegetating disturbed areas in the arctic. klima; 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bourhy, hervé; lavenir, rachel; serra-cobo, jordi title: seroprevalence dynamics of european bat lyssavirus type in a multispecies bat colony date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: me p we report an active surveillance study of the occurrence of specific antibodies to european bat lyssavirus type (eblv- ) in bat species, scarcely studied hitherto, that share the same refuge. from to , sera were obtained from nine bat species. blood samples were subjected to a modified fluorescent antibody virus neutralization test to determine the antibody titer. eblv- -neutralizing antibodies were detected in six of the nine species analyzed (pipistrellus pipistrellus, p. kuhlii, hypsugo savii, plecotus austriacus, eptesicus serotinus and tadarida teniotis). among all bats sampled, female seroprevalence ( . %, % ci: . %– . %) was not significantly higher than the seroprevalence in males ( . %, % ci: . %– . %). the results showed that the inter-annual variation in the number of seropositive bats in t. teniotis and p. austriacus showed a peak in (> % of eblv- prevalence). however, significant differences were observed in the temporal patterns of the seroprevalence modeling of t. teniotis and p. austriacus. the behavioral ecology of these species involved could explain the different annual fluctuations in eblv- seroprevalence. wildlife plays a key role in emerging infectious diseases by providing a -zoonotic pool‖ from which pathogens may emerge [ ] . zoonotic pathogens represent approximately % of all pathogens able to infect humans [ ] . in recent years, bats have been implicated in numerous emerging infectious disease events and have been recognized as important reservoir hosts for viruses that can cross the species barrier to infect humans and other domestic and wild mammals [ ] . the role of bats in viral diseases is well established, particularly their role as hosts for lyssaviruses, coronaviruses, flaviviruses, astroviruses and adenoviruses [ ] [ ] [ ] . bats have several unique features that may maximize their effectiveness as reservoir hosts for viruses. bats are the second largest order of mammals. currently, there are about recognized bat species worldwide, accounting for approximately % of all mammalian species. bats have the potential to rapidly and widely spread viruses (having a high mobility, they are the only mammals capable of flight). they have a long lifespan and a high survival rate, and many bat species have a gregarious behavior. bats can fly long distances between their summer and overwintering sites, permitting the exchange of viruses between conspecifics or bats of other species, i.e., in france, rabies virus infections have been associated with the migratory routes of nathusius' pipistrelle (pipistrellus nathusii) bats [ ] . persistent viral infections occurring among long-lived bats, coupled with their often gregarious roosting behavior, could greatly increase the potential for intra-and inter-species transmission of viruses [ ] , especially in summer and winter periods. seasonality in temperate zone bats includes birthing periods, migration, gregarious behavior and torpor. each of these strategies may affect population density, contact rates and immune response, thus leading to spatiotemporal variation in infection dynamics [ , ] . numerous bat species have been found to be infected by lyssaviruses [ ] . bats serve as reservoirs of of the lyssavirus species described (the only lyssavirus species that have not been isolated from bats, to date, are mokola virus and ikoma virus). furthermore, recently described lyssavirus species enlarged the genetic diversity of lyssaviruses found in bats [ ] [ ] [ ] , suggesting that the lyssaviruses originated in these mammals and progressively diverged from a common ancestor [ , ] . in europe, four of the lyssavirus species recognized, european bat lyssavirus types and (eblv- and eblv- , respectively), bokeloh bat lyssavirus (bblv), the west caucasian bat virus (wcbv) and one tentative species, lleida bat lyssavirus, circulate among several bat species [ , , ] . eblv- is widely distributed throughout europe, and two variants have distinct distributions and evolution histories: one is eblv- a, which has an east-west distribution from russia to france, with very little genetic variation; and the other is eblv- b, which exhibits a south-north distribution and far more genetic diversity [ ] . different studies showed that lyssavirus dynamics exhibits a strong seasonal pattern [ ] and that the breeding period could favor the infection of bats [ ] [ ] [ ] . many bat species roost in very large and dense maternity colonies. this dense clustering of individuals can provide large opportunities for viral exchange in bat colonies [ ] . previous studies have observed a higher seroprevalence in multispecies colonies compared to monospecific colonies, suggesting that interspecific virus transmission plays an important role in eblv- dynamics [ ] . however, in some cases, infection cycles may be maintained among specific host species and transmission may be minimal among sympatric bats [ ] . furthermore, differences in the ecological behavior of species (e.g., migration, torpor) can drive different bat infection dynamics. in this sense, a higher number of species might not only increase the rates of contact between bat groups, but could also facilitate virus entry or spread through the higher mobility of individuals among colonies, especially if there are migratory species involved [ ] . few studies have addressed the inter-annual dynamics of lyssavirus among bat multispecies that are roosting in the same refuge, despite these studies giving a better understanding of the dynamics of bat lyssaviruses. our previous investigations have analyzed the temporal dynamics of lyssavirus in one bat species (myotis myotis) roosting in two colonies [ , ] . the present report is based on a long-term (nine years) longitudinal study of the prevalence of eblv- neutralizing antibodies and provides the first report on the inter-annual dynamics of eblv- in p. austriacus and t. teniotis, both being bat species scarcely studied hitherto. we chose this locality, because we found three species (p. pipistrellus, p. austriacus and t. teniotis) that were eblv- rna-positive by nested reverse transcriptase-polymerase chain reaction in the first year of study [ ] . our specific goals were: (i) to provide information about eblv- seroprevalence in the wild bat community where several european bat species share the same refuge; and (ii) to compare the temporal patterns of seroprevalence mainly in two less-studied bat species that, moreover, exhibit different ecological strategies. this study was carried out at the san pedro de los griegos pothole ( ° ' n, ° ' e; elevation: m), situated km from oliete village (teruel province). the cavity is an enormous hole with an entrance of × m and a -m maximum depth. crevices in the walls are optimal roost sites for many birds and bat species. however, the pothole is totally illuminated and shows a large lagoon inside ( figure ). around the cavity, the vegetation is dominated by a mix of low growing stipa sp., brachypodium retusum, rosmarinus officinalis and thymus vulgaris. local weather is characterized by continental climate with a mean annual temperature of . °c and a mean annual precipitation of mm (mainly in spring). however, mean daily temperature is over °c between june and august (with . °c and . °c as the mean minimum and maximum temperatures, respectively). the permanent availability of water and nutrients, the dampening of hard external climatic conditions and the suitability of the habitat for the reproduction of various vertebrate species make the san pedro pothole a site of unprecedented high biodiversity in europe [ ] . bats were captured in summer (from june to july) over a -year period ( - ). mist nets were employed to capture bats at sunset when emerging from the pothole to forage. all bats were identified to species based on published identification keys of the bats of europe [ ] . individuals were sexed, and the reproductive status of adult females was classified as pregnant or lactating, based on palpation of the abdomen and nipple condition [ ] . blood samples were obtained by a small puncture made in the median artery. the amount of blood sampled varied from . ml to . ml, according to the size of the animal. pressure with a sterilized absorbent hemostatic sponge impregnated with gelatin was applied to prevent bleeding and facilitate healing. the bats were given % glucose water to drink to prevent dehydration and to provide rapidly assimilated compounds for energy. once bleeding ceased, the bat was released. vials containing blood were stored at °c for a few hours. samples were centrifuged for minutes at × g, and the serum was extracted with a micropipette. serum samples and blood pellets were stored at - °c before analysis. all animals were handled in strict accordance with good animal practices, as defined by current european legislation. bat capture and blood sampling were authorized by permit from the spanish regional committee for scientific capture. the technique used to detect eblv- neutralizing antibodies is an adaptation of the rapid fluorescent focus inhibition test (rffit) [ , ] . a constant dose of a previously titrated (calibrated to give % fluorescent foci-infected cells), cell culture-adapted eblv- challenge virus ( fra) was incubated with -fold dilutions of the sera to be labelled. after incubation of the serum-virus mixtures, a suspension of bsr cells (a clone of bhk cells) was added. after hours incubation, the cell monolayer was acetone-fixed and labelled with a fluoresceinated anti-nucleocapsid antibody (bio-rad, marnes-la-coquette, france). the optimal challenge dose (the dilution giving % infected cells for each virus production) was calculated. titers are presented as an arithmetic mean of two independent repetitions. serum samples with antibody titers < are considered negative for eblv- neutralizing antibodies. this cut-off value is similar to that applied in other studies [ , , , ] . to study the variation in eblv- -antibody prevalence, we conducted two analyses: first, three explanatory variables (sex, species and year) were first screened using a univariate analysis and a chi-square test to check for statistically significant associations with serological status ( : negative; : positive). in the second analysis, we used a generalized additive model (gam) to study the temporal patterns of eblv- -antibody prevalence in only two species (p. austriacus and t. teniotis). more specifically, we used a generalized additive model with the binomial error distribution, where the seroprevalence was the response variable and sex, species and year ( - ) were the explanatory variables. the -year‖ variable was modeled as a covariate fitted with penalized cubic regression splines and sex and species as a fixed categorical factor. to avoid over-fitting and to retain more easily interpretable relationships in the gam smoothing function, an upper limit of degrees of freedom was set for the year variable when fitting the models. we used an information-theoretic procedure and the akaike information criterion corrected for small sample sizes (aicc) to compare models [ ] . modeling was performed using the -lme ‖ and ‗‗mgcv'' packages in the r program v. . [ ] . we report the results of the prevalence of specific eblv- neutralizing antibody analysis from the - period in nine bat species roosting in the same refuge. five of these species (eptesicus serotinus, p. kuhlii, p. pygmaeus, myotis myotis and m. daubentonii) were captured sporadically (sample size < individuals during the whole study period), while the rest of the species sampled (p. pipistrellus, hypsugo savii, plecotus austriacus and tadarida teniotis) were captured often. the larger samples (> individuals) were obtained in p. austriacus and t. teniotis, because they form large colonies in this cavity. t. teniotis form a colony of several hundred individuals. the colony of p. austriacus is smaller and consists of individuals, approximately [ ] . we observed pregnant females in all bat species, except in e. serotinus, p. pygmaeus and m. myotis, where females were never captured, indicating that this cavity is a breeding roost for the rest of the species found. males were also captured during the breeding period, indicating that males, either as solitary individuals or forming part of the maternity colonies (e.g., p. austriacus), are present during the breeding period in the cave. among the sera obtained, ( . %) were positive for eblv- -neutralizing antibodies. eblv- antibodies were detected in ( . %) of the nine species analyzed (p. pipistrellus, p. kuhlii, h. savii, p. austriacus, e. serotinus and t. teniotis) ( table ) . no significant differences in eblv- seroprevalence were detected among seropositive bat species (χ = . , df = , p = . ). the highest seroprevalence was observed in h. savii. we did not find any difference in eblv- seroprevalence between females ( . %, % ci: . %- . %) and males ( . %, % ci: . %- . %) (χ = . , df = , p = . ) when all species were analyzed together and when only bat species with a large sample size-p. austriacus and t. teniotis-were considered (table ) . capture-mark-recapture of some bats during the study period allowed the tracking of temporal changes in eblv- seroneutralization titers. seven p. austriacus were captured and analyzed almost two times at intervals of one or several years. four of these seven bats showed positive antibody titers, becoming negative in the following recapture sessions after some years, indicating that these bats survive at least several years after their seroconversion (table ) . the models that incorporate sex and species variables were not significantly different from the model without these variables (Δaicc < ) ( table ). the best model showed a significant different nonlinear pattern in the eblv- seroprevalence along p. austriacus and t. teniotis. the effect of year fitted with the spline was highly significant for two species (p. austriacus: df = . , p < . and t. teniotis: df = . , p = . ), suggesting a different inter-annual pattern among these species (figure , table ). although no positive sera were detected in three bat species (m. myotis, m. daubentonii and p. pygmaeus), this result is probably due to the very low sample size. the high percentage ( %) of seropositive species found and the lack of significant differences in eblv- seroprevalence among seropositive species suggest that most of the bat species can be exposed to eblv- in this pothole although most of these species are not considered as lyssavirus reservoirs by previous studies [ , , , ] . previous studies have shown higher prevalence in females than in males [ , ] . this difference may be due to the gregarious behavior of female bats in summer (nursing colonies are composed almost exclusively of adult females). in these colonies, virus transmission may be favored by high contact rates during social grooming, nursing or olfactory or lingual contact with body fluids. reproductive activity may also play a role in virus transmission [ ] , because an increased susceptibility to infectious disease during pregnancy and lactation has been demonstrated in bats [ ] and other mammals [ ] . however, we report in this study no sex differences of eblv- seroprevalence. the presence of males in this cavity during summer could indicate that males also are present in maternity colonies, as observed in p. austriacus colonies, or roost near these colonies. significant fluctuations in the percentage of seropositive bats are indicative of several different episodes of eblv- infection occurring in p. austriacus and t. teniotis colonies during the period of study. a quick increase and a high seropositive percentage after a lyssavirus episode are not unusual in a gregarious behavior species and could explain the sudden increase in the percentage of seropositive bats in t. teniotis and p. austriacus colonies. a similar quick increase with seropositive peaks of %- % was observed in different colonies of m. myotis in mallorca [ , ] . however, in m. myotis colonies, the evolution of seroprevalence after infection peaks follows a more gradual decline over subsequent years, until a new episode takes place, very different from what is observed here. the delay between the waves is then dependent on the rate of inflow of susceptible bats into the colonies as a consequence of new births, bat immigration from neighboring colonies and the expiration of eblv- specific immunity in previously infected animals [ ] . when a sufficient fraction of susceptible bats in the colony is reached, the virus spreads again if infected individuals join the colony. in the t. teniotis and p. austriacus colonies, the increase of seroprevalence is followed by a rapid decline until seropositive bats are not detected. the difference in the seropositive percentage evolution can be due to a higher rate of inflow of individuals in colonies of t. teniotis and p. austriacus. no data of inflow are available on t. teniotis, but very few recaptures were obtained during the study, indicating probably a high inflow rate in this colony. however, recapture rates in the p. austriacus colony were higher, suggesting a lower inflow in this species. another hypothesis could be a different lifespan of immunity in these species. recent studies estimated the lifespan of the m. myotis immunity from eblv- to be around two years [ ] . in this respect, it is possible that the immunity lifespan would be shorter in p. austriacus and t. teniotis than in m. myotis. the best model obtained by gam analysis indicated that inter-annual patterns of seroprevalence evolution were significantly different for t. teniotis and p. austriacus. annual fluctuations could result from the behavioral ecology of the species involved [ ] . t. teniotis and p. austriacus are two species with a different social organization and behavior. while t. teniotis forms large maternity colonies and can make long seasonal movements, p. austriacus forms smaller maternity colonies constituted by both sexes and makes shorter seasonal movements [ ] . different host ecology, behavior and movement could explain the different temporal variations in seroprevalence in these two species. changes in density during migration or colony formation may affect contact rates and, thus, disease dynamics [ , ] . differences in eblv- exposure dynamics could also be related to host community composition and inter-species interaction. higher eblv- seroprevalence was observed in large and multispecies colonies compared to smaller and monospecific colonies, suggesting that interspecific virus transmission plays an important role in dynamics. a higher number of species might not only increase the rates of contact between bat groups, but could also facilitate virus entry or spread through the higher mobility of individuals among colonies, especially if there are migratory species [ ] . in this sense, m. schreibersii (a species that often shares roost with m. myotis) has been considered as a regional reservoir and an essential species for eblv- persistence in the balearic islands [ ] . other bat species present in the san pedro pothole, such as p. pipistrellus and p. kuhlii, showed lower eblv- seroprevalence than p. austriacus and t. teniotis. however, previous studies of bat rabies surveillance in europe did not find eblv- -neutralizing antibodies in both species of pipistrellus (for review see [ , ] ). these results could be indicative of a low public health risk associated with these synanthropic species. furthermore, the lack of a standardized serological test procedure, including arbitrary cut-off values, makes the comparison between previous european studies difficult. however, the higher values of eblv- seroprevalence in our study could be due to differences in virus circulation and dynamics resulting from regional differences or selection of different types of colony (large multispecies maternity colonies in this case) [ , ] . research programs that focus mainly on multi-host systems will help advance our understanding of the ecology of bat diseases. this research addresses the role of multiple hosts in the infection dynamics of lyssavirus. to advance our understanding of the ecology of bat lyssavirus, we report the results of specific eblv- neutralizing antibody analysis in nine bat species roosting in the san pedro de los griegos pothole. these results suggest that most bats species roosted in this cave were exposed to the eblv- lyssavirus. the evolution of seroprevalence in t. teniotis and p. austriacus colonies after infection peaks is different from that observed in m. myotis colonies. differences in behavior ecology and population dynamics among bat species could explain the differences in the inter-annual variability of eblv- seroprevalence. emerging infectious diseases of wildlife -threats of biodiversity and human health ecology of zoonoses: natural and unnatural histories bats: important reservoir hosts of emerging viruses bats as a continuing source of emerging infections in humans a preliminary study of viral metagenomics of french bat species in contact with humans: identification of new mammalian viruses the migrations of pipistrellus nathusii in france-possible implication on the spreading of rabies (in french). mammal concepts of infectious disease epidemiology host and viral ecology determine bat rabies seasonality and maintenance ecology of zoonotic infectious diseases in bats: current knowledge and future directions bats, emerging infectious diseases, and the rabies paradigm revisited shimoni bat virus, a new representative of the lyssavirus genus ikoma lyssavirus, highly divergent novel lyssavirus in an african civet. emerg. infect. dis host switching in lyssavirus history from the chiroptera to the carnivora orders genomic diversity and evolution of the lyssaviruses phylogenetic relationships of irkut and west caucasian bat viruses within the lyssavirus genus and suggested quantitative criteria based on the n gene sequence for lyssavirus genotype definition phylogeography, population dynamics, and molecular evolution of european bat lyssaviruses ecology of rabies virus exposure in colonies of brazilian free-tailed bats (tadarida brasiliensis) at natural and man-made roosts in texas. vector borne zoonotic dis amplification of emerging viruses in a bat colony urban habituation, ecological connectivity and epidemic dampening: the emergence of hendra virus from flying foxes (pteropus spp.) ecological factors associated with european bat lyssavirus seroprevalence in spanish bats european bat lyssavirus infection in spanish bat populations temporal dynamics of european bat lyssavirus type and survival of myotis myotis bats in natural colonies espagne): un refuge de biodiversité sans é quivalent en europe (in french) illustrated identification key to the bats of europe ageing and assessment of reproductive status of pipistrelle bats, pipistrellus pipistrellus in commission des laboratoires de référence et d'expertise, editors. méthodes de laboratoire pour le diagnostique de la rage model selection and multimodal inference: a practical information-theoretic approach r: a language and environment for statistical computing. r foundation for statistical computing aná lisis demográ ficos y sanitarios en las colonias de plecotus austriacus y tadarida teniotis de la sima de san pedro (oliete, parque cultural del rí o martí n) (in spanish) active surveillance of bat rabies in france: a -year study reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes (pteropus scapulatus) immunosuppression during pregnancy and lactation insights into persistence mechanisms of a zoonotic virus in bat colonies using a multispecies metapopulation model bat migrations in europe. a review of banding data and literature; federal agency for nature conservation animal migration and infectious disease risk bat rabies surveillance in europe twenty years of active bat rabies surveillance in germany: a detailed analysis and future perspectives the authors wish to thank sergi vives, departament d'estadística de la facultat de biologia, university of barcelona, for his mathematical support. we thank pepe royo of the centro de arte rupestre -antonio beltrán‖ del parque cultural del río martín of ariño (teruel, spain) for providing access to installations and for support during sample collection. we thank xavier bayer and cisco guasch for sharing his team's fieldwork.the research leading to these results has received funding from ministerio de sanidad y servicios sociales e igualdad, dirección general de salud pública y sanidad exterior. the authors declare no conflict of interest. key: cord- -gs oo no authors: kaandorp, jacques title: veterinary challenges of mixed species exhibits date: - - journal: fowler's zoo and wild animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: gs oo no nan with acknowledgement of the original source. these permissions are granted for free by elsevier for as long as the covid- resource centre remains active. modern zoos like to show larger groups of animals, preferably in natural habitat-like mixed species exhibits, but it is not always easy to combine different species in one exhibit. the size of an exhibit is essential when mixing animals, especially when mixing larger mammals. aviaries and aquaria are examples with a long-standing experience of combining various species, but in mammals this experience is often poor. most often, zoos still show single species exhibits because of lack of space or simply to prevent problems associated with mixing different species. safari parks in europe were very popular in the s, showing more natural displays of animals. however, because of the difficulties of handling animals in mixed exhibits, many of these parks later closed their gates. the parks that remained and still exist gained experience regarding which species may be kept together with others and which species shouldn't be mixed. the main advantage of mixed species enclosures is behavioral enrichment (fig. - ) and the obvious educational value. there are even mixed species exhibits of carnivores. for example, dierenrijk in nuenen, the netherlands, combines european grey wolves (canis lupus) with european brown bears (ursus arctos; fig. - ) and gelsenkirchen zoo, germany, combines arctic foxes (vulpes lagopus) with kodiak bears (ursus arctos middendorffi; fig. - ) . in this chapter, an incomplete listing of diseases and problems is presented to make the reader aware of the broad variety of veterinary challenges of mixed species exhibits. it is meant to encourage ideas and suggest further reading in veterinary literature about specific diseases and problems when mixing different species of animals. veterinary problems arising because of keeping different species together may be categorized as trauma, nutrition-related problems, infectious diseases, and parasitic diseases. in mixed species exhibits, trauma is the most frequent and serious cause of health problems ( fig. - ) . competition for nesting sites in birds, establishment of territories, and competition for food and watering stations in all taxa may provoke fighting and trauma in mixed exhibits such as aviaries and large exhibits of mammals. for example, young antelopes born outside will be chased in the beginning of their lives by curious zebras, leading to death or a fatal myopathy, as has also been seen in young or newborn giraffes and antelopes. play of young animals may not be understood by other species (fig. - ). pinioned birds fly in unrecognizable ways in the eyes of other animals and may become victims of other birds or mammals. another factor is that when animals are frightened because of thunder or other events, or when animals are chased by other animals because of unexpected or differing circumstances, fleeing against fences or walls may cause fatal trauma. seasonal aggression, especially in deer (rut), may lead to interspecies conflicts, but different males of the artiodactylae family will fight intraspecifically over their territory or interspecifically with other animals to protect their herd (fig. - ). antlers and horns are weapons capable of causing stab wounds, fractures, or even immediate death. capping horns and cutting of antlers may limit the severity of trauma. after traumatic injuries, pathologic studies should always be carried out. for example, when birds kill one another, pathology often reveals underlying disease and explains the noticed aggression. to prevent trauma, next to appropriate size of the exhibit, pole gates (creeps), where small animals can flee from larger animals, creation of large obstacles in phillipsi) and sable antelope (hypotrachus niger), or toxicities such as vitamin e toxicity in pelicans or iron storage disease in birds and some primate species, should be avoided when developing feeding protocols for mixed species exhibits. these should incorporate the specific needs and required feeding supplementations of the various species in a mixed exhibit. various herpesviruses are known to be responsible for disease outbreaks in mixed species exhibits. other viruses such as rabies or bacteria (e.g., mycobacterium tuberculosis complex) or a variety of endoparasite, ectoparasite, or fungal infections (e.g., aspergillosis) may each be detrimental in mixed exhibits, because not only one species will be infected, as in single-species exhibits. measures to control these diseases may have an enormous impact on a collection and demands for an effective preventative veterinary protocol. decisions about which species are to be housed together should be made based on this information. , and banteng (bos javanicus) are especially susceptible to these diseases. , in white-tailed deer (odocoileus virginianus), a new mcf virus has been recognized that causes classic mcf. do not mix wildebeest with giraffes and preferably get rid of all sheep and goats in a zoo collection. carrier species should at least not be in breeding situations in direct contact or close to susceptible species. there are examples of zookeepers owning sheep at home that transmitted the virus to giraffes, resulting in high mortality. it is questionable whether zookeepers should be allowed to take care of household sheep and goats at home. exhibits so animals may circle around these when chased, hiding places, and provision of multiple feeding and watering sources are workable preventive measures when planning mixed exhibits. there is no definition of an appropriate size, but for animal welfare reasons and to avoid trauma, exhibits should be as large as possible. adequate nutrition is vital for every living being. in mixed exhibits, a sufficient number of feeding stations is essential to ensure that all animals may eat and at the same time prevent that some don't overeat. also, to prevent interspecies aggression, a sufficient number of feeding stations is necessary. spreading food over larger areas in aviaries or among hoofstock prevents aggressiveness, and is even more effective when food is provided several times a day. free-flying wild birds may be a nuisance when feeding, such as kangaroos and birds (e.g., storks and cranes). a special configuration of feeding places may be helpful to prevent this. precautions should be taken to prevent animals from not being able to eat enough and losing too much weight. requirements of trace minerals and other nutrients such as vitamins differ among species. deficiencies such as copper deficiency in blesbok (damaliscus pygargus species. there are many carriers of these viruses. coronaviruses are also known to cause winter dysentery in adult ruminants. spider monkeys (subfamily ateles) and squirrel monkeys (saimiri sciureus) should not be placed in mixed exhibits with other primates. squirrel monkeys are hosts of two herpesviruses (herpesvirus tamarinus, h. saimiri) and spider monkeys may transfer h. ateles to callitrichids, aotids, marmosets, and tamarins, causing fatal disease in these species. rhesus macaques (macaca mulatta) and other macaques may host h. simiae (hvb or, most recently, cercopithecine hv- ) and is transmitted by biting and scratching and by dried secretions-for example, to colobus monkeys (colobus guereza). do not mix african and asian monkeys. at least, macaques should be seronegative for hvs when housed in mixed exhibits. another herpesvirus, the simian varicella group (svv), is hosted by macaques; it produces mild, self-limiting signs in the host species but may be fatal in patas monkeys (erythrocebus patas) and other african cercopithecines. simian hemorrhagic fever (shf) and simian immunodeficiency virus (siv) are other reasons not to mix african and asian monkeys, because they will spread among these primates. in mixed primate exhibits, salmonellosis, campylobacteriasis, bordetellosis, and shigellosis should be monitored as preventive measures. in marine mammals (cetaceans, pinnipedia), morbillivirus, orthopoxvirus, and parapoxvirus infections may occur when water systems are connected between basins. the morbillivirus outbreak in the north of the netherlands, germany, and denmark has shown that vaccination using canine distemper virus (cdv-iscom) (immunestimulating complex) vaccine is effective in protecting harbor seals (phoca vitulina) from phocid distemper in . this vaccine from erasmus university, rotterdam, the netherlands, halted the spread of the disease. using inactivated canine distemper virus will do the same, but is not allowed for use in the european union (eu). avian herpesviruses such as the pacheco disease virus may be carried by conures and should be taken into equine herpesvirus has led to problems with bactrian camel (camelus bactrianus), llama (lama glama), and a thompson's gazelle (gazella thomsoni). the virus is shed by infected horses (equus caballus), zebra (e. grevyi, e. zebra, e. quagga), and onager ( e. hemionus) during respiratory infection, parturition, and abortion. vaccination is no guarantee for preventing an outbreak. when introducing equids into mixed exhibits, it is advisable to use only seronegative equids. mixed exhibits with ruminants should be monitored serologically for diseases such as leptospirosis, brucellosis, infectious bovine rhinotracheitis (ibr; bovine herpesvirus [bhv- ]), bovine virus diarrhea (bvd), tuberculosis (mycobacterium tuberculosis, m. bovis), paratuberculosis (m. avium subsp. paratuberculosis), leucosis (enzootic bovine leucosis, bovine leukemia virus), neosporosis (neospora caninum), bovine respiratory syncytial virus (brsv; e.g., ovine lentivirus, maedi-visna)because most of these diseases spread between different ruminant species. brucellosis, leptospirosis, and tuberculosis will also affect numerous other mammalian species. the following are other mammalian diseases mycobacteriosis in the form of paratuberculosis or the mycobacteriaceae responsible for the tuberculosis complex are not easy to control in mixed species exhibits. bacterial infections such as leptospirosis, erysipelas, listeriosis, pseudomoniasis, and infections caused by enterobacteria and clostridia are found in many species. they are responsible for an enormous variety of disease problems, especially in mixed species exhibits. almost all notifiable diseases as listed in many parasites have a broad host range and are a threat in mixed species exhibits. one animal imported into a collection may be hazardous not only to its own species, but also other species in mixed species exhibits. protozoal parasites easily contaminate exhibit substrates. preventive protocols should be taken to avoid serious problems. in mixed primate exhibits, various protozoal infections may be seen and may cause problems such as gastric amoebiasis, giardiosis, hexamitiasis, trichomoniasis, and cryptosporidiosis. it is popular to mix gorillas in exhibits with other african species such as colobus monkeys (c. guereza) and mangabeys ( fig. - ) . however, be careful when mixing them, because cercopithecine monkeys are often carriers of balantidium coli. all great apes, especially gorillas, may become very ill from these infections. , toxoplasmosis may be found in all vertebrates and may be spread by all felid species. in mixed aviaries, trichomonas spp. are common in columbiforms but may spread to passeriforms or psittacines, who may become seriously ill. puffins and penguins are very susceptible to plasmodium infections causing avian malaria. the infection is endemic in many continental birds in europe and north america and, from these carrier birds, the disease is spread to the susceptible penguins and puffins by mosquito vectors. antimalarial drugs as a preventive measure are widely used in these birds. another protozoal parasite, neospora caninum, causes abortions in some herbivores. endoparasites such as nematodes, trematodes, and cestodes should also be monitored in mixed species account when mixing birds; they may cross to different species and cause devastating outbreaks in other species of psittacine birds. avipoxvirus infections are seen in mixed aviaries because a variety of birds are susceptible to the virus. tortoises are also known for herpesviruses that will spread among different species. some species act as reservoirs, whereas other species show high mortality. another herpesvirus causing fibropapillomatosis is seen in various marine turtles. gray patch disease in marine turtles is probably also caused by a herpesvirus. it requires the same strict regimen of hygienic measures and quarantine as the other herpesviruses when mixing these animals. ophidian paramyxovirus may be transmitted between snakes. viperids show a variety of susceptibility to this virus and may infect other groups of snakes, such as boids, elaphids and colubrids. mixed exhibits of amphibian species may have an extra chance of outbreaks of chytridiomycosis caused by batrachochytrium dentrobatidis. frogs, toads, salamanders, and others are susceptible. in amphibians, the spread of adenovirus infections among lizards, snakes, and crocodiles may only be prevented by in-house biosecurity measures. west nile virus (wnv) is a vector-borne disease noted in almost species of birds and a variety of domestic and exotic mammals. using the u.s. data acquired after the wnv outbreak in recent years, vaccination protocols and vector control should be proactively discussed before wnv becomes endemic in europe. salmonella spp. (especially s. typhimurium and s. enteridius) in mixed bird exhibits and in reptile departments in zoos are difficult to control. also, among mammals, different salmonella spp. may result in high morbidity and mortality. yersinia pseudotuberculosis and y. enterocolitica are responsible for mortality in various species of birds, rodents, and primates (e.g., squirrel monkeys, saimirinae). often, transmission occurs through uninvited vector species (e.g., rats, mice, wild birds) who share a mixed exhibit with collection species. chlamydophila psittaci affects psittacines, passerines, and columbiformes. this well-known zoonosis may also cause significant infections in other nonavian species. fungal diseases are seen in all taxa. trichophyton spp., microsporum spp., aspergillosis, candidiasis, malassezia, exhibits because many of them cross species lines. every large animal practitioner knows not to keep horses and donkeys together-donkeys are carriers of lungworms, without clinical problems, but horses are vulnerable to these parasites. mixing a variety of artiodactylids will lead to a burden of possible parasitical infections, especially enteric nematode infections. in mixed aviaries, helminths are potentially lethal among birds (especially capillaria spp. and syngamus trachea) and preventive measures are a necessity. ectoparasites such as sarcoptes and chorioptes spp. are capable of affecting different, often related species and are not always easy to control in larger mixed species exhibits. transmissible diseases handbook: european association of zoo and wildlife veterinarians gvp (good veterinary practice) regarding (emerging) infectious diseases-a political issue? presented at the th scientific meeting of the european association of zoo and wildlife veterinarians european association of zoo and wildlife veterinarians-infectious diseases working group malignant catarrhal fever in two closely related zoos in the netherlands health problems in mixed-species exhibits key: cord- - j euvc authors: williams, ernest h.; bunkley-williams, lucy title: life cycle and life history strategies of parasitic crustacea date: - - journal: parasitic crustacea doi: . / - - - - _ sha: doc_id: cord_uid: j euvc different parasitic life strategies are described including four new life cycles: complex rebrooding, micro-male, mesoparasite and prey-predator transfer. four new life cycle behaviours are named: nursery hiding, mid-moult stage, positive precursor (intraspecific antagonism) and negative precursor (ambush strategy). further strategies discussed are opossum attack, double parasitism (doubling of the normal reproductive set), duplex arrangement (separated male-female pairs), simple rebrooding, and describing how displaced parasites and superinfections may partly elucidate life cycles. proportional stunting masks life history effects of parasitism; cuckoo copepods are true parasites and not just associates; burrowing barnacles (acrothoracicans) are not parasites. further findings based on life cycle information: branchiurans and pentastomes are possibly not related; firefly seed shrimp are not parasites; copepod pre-adult life cycle stages are common in the western pacific but rare in caribbean; harpacticoids on vertebrates are not parasites; cuckoo copepods are true parasites; explained the importance of pennellid intermediate hosts. crustacean parasite life cycles are largely unknown ( % of species). most crustacean life cycles represent minor modifications from the ancestral free-living mode. crustacean parasites have less complex and less modified life cycles than other major parasite groups. this limits their exploitation of, and effectiveness, in parasitism. however, these life cycles will be an advantage in global change. most metazoan parasites will be eliminated while crustaceans (and nematodes) will inherit the new world of parasites. life cycles and life histories are arguably the most significant functional traits of all organisms (e.g. stearns ; roff ; mcgill et al. ) , and understanding those life cycles and life histories is essential to understanding the autecology and evolution of each organism, as well as its impact on community and ecosystem processes (e.g. heppell et al. ; mcgill et al. ) . parasitism is the most common consumer strategy, and parasites are estimated to account for approximately half of all biodiversity (reviewed by hatcher and dunn ) . carlson et al. ( and references) considered that conservation of parasites is essential to maintain the diversity in major ecosystems, particularly during global climate change. because parasites are typically small and cryptic and often infect multiple hosts and/or host species during their life cycle, unravelling the life cycles of even a small percentage of parasite species has proven to be challenging. indeed, while life cycles and life histories have been extensively investigated for a small number of model species (mostly species that impact the health of humans and/or plants and animals of economic importance to humans), we know virtually nothing about the vast majority of species. the phylum arthropoda is the most diverse animal phylum, with more than , , described species. while insects are the most diverse class within this phylum, the class crustacea has more than , described species (zhang ) . the crustacea also includes the greatest diversity of parasitic forms, with over one quarter of the described species. as with parasites generally, details of the life histories for most of these remain unknown. the goal of this chapter is to provide an overview of the current state of knowledge regarding life cycles of parasitic crustacea and offer insights and suggestions for future research; our suggestions and analyses are based on the authors' combined years of experience working with this group. we consider each parasite group separately in phylogenetic order, giving a brief summary of the life cycle(s) with recent discoveries and new details regarding life history strategies and concluding with some new information. we only present a brief overview of the impacts on hosts and on broader ecological aspects as these are reviewed in chaps. and , respectively. general larval descriptions (unless new) have been kept to a minimum because martin et al. ( ) presented an extensive description and collection of drawings and photographs of larval forms. the classification given in chaps. and has been followed. to improve readability and referencing, an annotated glossary has been included with less familiar terms in italics in the text. common names, with accompanying scientific names, are at the end (sect. . ). this is a small group of~ (worms ) species in four genera that largely ectoparasitise freshwater fishes (poly ) , with a few taxa on coastal marine fishes (< ; møller ) , and some attach to tadpoles, salamanders, and even alligators (møller ). most non-fish infections appear to be incidental or accidental, but poly ( ) described a species from a mexican salamander. because of their temporary association with hosts, they may more appropriately be referred to as 'micropredators' (e.g. lafferty and kuris ) ; however, we feel that serial parasites * is more appropriate. much of the interest in this group stems from their negative effects on aquaculture and aquarium fishes (lafferty et al. ) and vector fish viral diseases (møller ) . the japanese fish louse, argulus japonicus thiele, , is probably the most famous and has been spread around the world with cyprinids (bunkley- williams and williams ) . however, the common fish louse, argulus foliaceus (linnaeus, ), is also widespread, and occurs from fresh to marine waters (møller ) . in two cases, these parasites have infected humans. hargis ( ) found argulus laticauda smith, , in the eye orbit of a child in virginia; and an argulid has also been observed in the orbit of a tilapia aquaculturalist in venezuela (williams and bunkley-williams, personal observation) . the life cycles of only~ branchiuran species have been examined, mostly in argulus müller, , and a few species of dolops audouin, . most males transfer sperm directly to the females using a variety of modified structures on the third and fourth thoracic legs; however, in dolops, sperm are transferred in chitinous spermatophores. sperm morphology originally linked fish lice with tongue worms (see sect. . ) . molecular evidence also supports their similarities. only the life cycle of argulus is well known (poly ; neethling and avenant-oldewage ) , and no life cycles of marine species are known. a mature female argulus leaves its host and lays eggs in rows on a hard, submerged surface ( fig. . ). as many as eggs are laid at any one time and are cemented to the substrate. the eggs hatch - days later, varying by species and water temperature. eggs hatch into ( ) free-swimming metanauplius-like larvae ( species of argulus), ( ) freeswimming juvenile-like larvae ( argulus, dolops) or ( ) non-swimming larvae ( chonopeltis thiele, ). the three larval stages (above) moult into secondstage juveniles, which are parasitic and repeatedly change hosts (serial parasite). the - stages before the adult are also parasitic and host-hopping*. the maxillule undergoes a profound metamorphosis around the fifth stage, changing from a long limb bearing a powerful distal claw, into a short but powerful circular sucker (martin et al. ). this is a remarkable transformation. møller et al. ( ) described swimming and self-cleaning in the hatching, free-swimming stage and the subsequent juvenile stages of argulus foliaceus. wilson ( ) and reproduced with permission from benz and bullard ( ) . mature adults copulate on or off the host, and gravid females will then swim and find hard inanimate objects (such as stones, walls, equipment, etc.) on which to deposit eggs. hatching times vary according to temperatures and can take from weeks to months to occur. once hatched, infective larvae will search for a host, attach to the suitable host, and undergo multiple moults before reaching maturation. image modified from benz and bullard ( ) neither larvae, nor juveniles, nor adult chonopeltis can swim and are therefore referred to here as 'non-swimming fish lice*'. adults leave the host, mate, and deposit eggs, and how, or if, they return to a host is unknown. they have seven to eight free-living, developmental stages and are said to have intermediate hosts, but, as in other fish lice, these are really transfer hosts*. all host infection and reinfection are through host contact with the bottom. intermediate hosts are small, bottomdwelling fishes, and the definitive hosts are larger, bottom-dwelling fishes (grundlingh ) . how they infect or attach to hosts is still unknown, possibly only floating to a new host (fryer ; piasecki and avenant-oldewage ) . few parasite embryos have ever had mutualistic symbionts, but banerjee et al. ( ) have found one. a rotifer, philodina roseola ehrenberg, , feeds off the jelly coat of the egg strips of argulus bengalensis ramakrishna, . this makes the coating thin enough for larvae to break out. if the rotifer does not feed, the larvae cannot emerge. they suggested this knowledge of the life cycle could afford parasite control. van as and van as ( ) found adult and larvae chonopeltis lisikili van as & van as, , on the same host specimens and suggested host-change does not occur in c. lisikili, as was reported for other chonopeltis spp. these stages, and freeswimming adults, are attracted by bright objects, light, and motion, as mikheev et al. ( ) demonstrated in aquarium studies. in daylight, the parasite employed hoverand-wait tactics with low swimming speed and an inclined position of the body. in the dark, cruising tactics were employed, characterised by a much higher swimming speed and a horizontal position of the body. vision, olfaction and mechanoreception are used in daylight, whereas only the latter two are used at night. swimming speed was - times greater at night than in the daylight. host-induced cues increased mean swimming speed by a factor of . - . in adults starved for - days, the swimming speed was - times greater than those freshly detached from the host. a longer starvation caused a decrease in swimming activity (mikheev et al. ) . mikheev et al. ( ) found receptive females largely rested on hosts and attracted free-swimming males with pheromones. they also found females deposit eggs in fish spawning or nursery areas, where their offspring will have access to many fish. mikheev et al. ( ) suggested fish lice manipulate the behaviour of hosts for their benefit: ( ) when one attaches to a fish, the host reaction attracts other lice; ( ) injuring a host by attacking it may cause a predator-attack tightening of a school, favouring more parasite attachment. however, these effects seem too inadvertent, reactive, and temporal, to be called parasite-induced host behavioural changes. they do not help the individual parasite causing the reaction, possibly even harming it, similar to a positive precursor. they are certainly nothing like the host behaviour changes such as found in parasitic barnacles. mikheev et al. ( ) also suggested microbial pathogens were changing the behaviour of hosts and fish lice to spread their infection. they found 'sick' fish had more fish lice and stated that the parasites vector these diseases (see chap. ). banerjee's et al. ( ) mutualistic rotifer has obviously co-evolved with argulus bengalensis. we suggest that at one point, the rotifer appears to have been a hyperparasite (many in its genus are parasites) or predator harming the egg strip. the parasite gradually thickened the coating on its egg strip, protecting it from the rotifer. finally, the embryos were sufficiently protected, to turn the rotifer into a mutualist, and the organisms became inexorably linked. our suggestion is that this is first evidence of a hyperparasite evolving into a mutualist. while fish lice metamorphosis of suckers is an interesting change from the freeliving forms, they show no other major morphological developments towards parasitism. thus, while unusual, their larvae are strange, but not necessarily modified well for parasitism. there are~ extant species mostly parasitising the respiratory tracks of terrestrial vertebrates (christoffersen and de assis ; siveter et al. ) . they are of little commercial importance even though they may be found on crocodile and alligator farms, and the eggs of ten species can infect humans with nymphs (li et al. ). their phylogenetic relationship was once mysterious because they only have reduced parasitic morphologies. until recently, no fossil forms (~ . billion years ago) were known. molecular work has suggested a relation to fish lice (e.g. li et al. and references) , although this is still debated. williams ( ) suggested that these parasites were important and were once parasites of dinosaurs since the remaining species parasitise many extant close relatives of dinosaurs (e.g. crocodilians and birds). bunkley- williams and williams ( ) found sebekid nymphs in freshwater largemouth bass and peacock bass in puerto rico and speculated that spectacled caiman was the final host (williams and britton ) . we now identify these nymphs as sebekia oxycephalum (diesing, ) , and they are common in puerto rico (williams and bunkley-williams unpublished data) . reported nymphs in coral reef fishes in okinawa and suggested the final hosts were sea snakes. males fertilize females soon after they mature in the respiratory tract. males do not live long, and often only females are found in the definitive host. stored sperm fertilize ova released continuously from the ovaries of mature females. fertilized eggs mature as they descend the uterus of porocephalids. gravid females of armillifer sambon, , and linguatula frölich, , species may contain millions of eggs. the eggs of cephalobaenids are stored in a saccate uterus until they contain - % fully mature primary larva and are infectious; then egg deposition begins. the vagina is equipped with a sieve-like mechanism only allowing mature eggs to escape. they lay eggs in the respiratory track of vertebrates, which are either coughed or sneezed out by the host or leave the host body through the digestive system. usually, an insect or vertebrate ingests the eggs. the larva hatches into a nymph, penetrates the intestinal wall, and forms a cyst in the intermediate host's body. the nymph is rounded in form, with - short legs. the final host is infected when it eats the intermediate host, and the nymph crawls into the respiratory tract from the oesoph agus or stomach. it moults several times to become a post-larval juvenile and finally an adult. a few species, mostly in birds, have direct life cycles. subtriquetra subtriquetra (diesing, ) , in south american crocodiles, is the only tongue worm known to have a free-swimming larva. it searches for fishes as its intermediate hosts (winch and riley ) . they occur worldwide but mostly in the tropics and subtropics. very few reliable taxonomic characters exist, even in adults, and these few characters change in different adult stages (supra-adults). the long cherished hope of fossil forms revealing clues to relations with other groups has not been realised. siveter et al. ( and references) found these forms as nearly characterless and enigmatic as the extant species. the few fossil forms known are isolated larvae, which appear to have been freeliving. siveter et al. ( ) found adults ectoparasitic on a marine ostracod. these life cycle forms are completely different from the present-day endoparasites of terrestrial, semiterrestrial, and vertebrates. there has been either a monumental and complete change between the fossil and extant life cycles, or, which is more likely, the fossil forms are not in the same lineage as extant tongue worms. the fossils may be related to extant tongue worms, but do not represent their ancestors. furthermore, without any interconnecting forms over half a billion years, it is difficult to try to join these fossils in a lineage with the extant tongue worms. more likely, the fossil and extant forms represent parallel evolution. chapter does not recognise siveter et al. ( ) fossil as a tongue worm. if correct, this leaves tongue worm fossil forms without an adult and without a host. these apparently free-swimming and unattached forms do have a modern equivalent in the larvae of subtriquetra subtriquetra. sanders and lee ( ) suggested that these larval forms parasitised conodonts (early, eel-like organisms, famous for first teeth in the fossil record). this would agree with this only modern analogue, which parasitises fishes as intermediate hosts. however, they considered the small fossil forms adults, not larvae, with direct life cycles. large forms with indirect life cycles only developed after the air-breathing tetropods were available~ million years ago. while these are interesting life strategy hypotheses, they lack any supporting evidence. tongue worm life cycles are like those of any other crustacean parasite. in their life evolution, they have invaded the land and colonised all four classes of terrestrial vertebrates. they are completely endoparasitic, with the exception of a free-living stage in one species. no other crustacean parasite is even similar. their drastically different life cycles suggest they may not be crustaceans. many other analyses agree (e.g. de assis ( , ) , place them in their own phylum), but most place them with the fish lice (branchiura). sebekia oxycephalum is a generalist having little specificity in fish intermediate, and crocodilian and snake definitive, hosts (silva et al. ) . vague reports of nymphs in lizards and reports in snakes probably represent paratenic hosts. it has the greatest range (southeastern usa to southern south america) of any tongue worm and the greatest host diversity. seed shrimp are a large group of largely free-living, marine species. a few are commensal on invertebrates, and extremely few are apparently parasitic on a shark, a ray, pacific sea urchins, one polychaete, groundwater isopods, and gammaridean amphipods (smith ) . many host records are based on few observations and specimens, and some relationships are unclear. their shells in sediments and extensive fossils are very useful indicators of past conditions, climate changes, oil deposits, and crustacean sexual development. they are well known to scuba divers for their painful bites at night, for their bioluminescent glow and for their nocturnal attacks on injured fishes (stepien and brusca ) . mating typically occurs in swarms with large numbers of females swimming to join the males. however, some are partially or wholly partheno-genetic. all seed shrimp, except punciids (no shell), brood their eggs between the upper (dorsal) part of the body and the shell. most ostracods shed eggs directly into the water as plankton or attach them to vegetation or the substratum. in some groups, one or two larval moults occur before the larvae are shed. eggs hatch into nauplius larvae with a hard, bivalve shell, except punciids that have a single headshield. a nauplius stage is usually followed by - metanaupliar moults. kretzler ( ) described the seven instars in the life cycle of echinophilus xiphidion kretzler, , in pacific sea urchins. he also found intense wave action inhibited the infection of sea urchins. males and females occur together on hosts. most adults do not moult. often, only a few specimens of parasitic seed shrimp are reported, although they can be very abundant. kretzler ( ) found specimens in host specimens of four species of sea urchins. he reported no damage; therefore, even heavy infections do not obviously affect hosts. bennett et al. ( ) found of epaulette sharks examined had sheina orri harding, , ostracods attached in the gills. light and scanning electron microscopy showed ostracods were anchored to gill tissues with their mandibular and maxillular claws. they damaged host tissues and were often located in distinct pockets, formed by local distortion of shark respiratory lamellae, strongly suggesting that they had been attached to the gills for considerable time. these details were presented because of some controversy whether sheina orri was parasitic. it has also been found in the bluespotted ribbontail ray, skogsbergia squamosa (mueller, ), and may be a bony fish parasite (monod ) , but this was not clear. wilson ( ) found , , and photeros parasitica (wilson ) in the gills and nasal tubes of three smooth hammerheads, one on the gills of a rock hind, and three on a blue runner in jamaica. hypothesised that p. parasitica was specific to sharks and rays and only accidental on bony fishes in the caribbean. cohen and morin ( ) reported that p. parasitica is a luciferin bioluminescent carrion feeder, not a parasite. brian ( ) found cypridina sp. on the gills of dolphinfish (coryphaena linnaeus, ) and called them parasites. however, this form is another luciferin bioluminescent seed shrimp, like p. parasitica, and is unlikely to be a parasite . it is related to the famous sea firefly. thus, 'firefly seed shrimp' do not appear to be parasites. bioluminescent seed shrimp are sometimes reported as gill parasites because they feed on detritus, and the gills of an organism are the first part to deteriorate. most of the~ , described copepod species (worms ) are free-living, some are commensal of invertebrates, and many parasitise invertebrates and fishes (~ described species,~ species in fishes alone). some parasites are little changed from the free-living form and even capable of free swimming between hosts (serial parasites). at the other end of the copepod, parasite spectrums are highly modified forms, which are fully embedded inside their hosts and can only be recognised as copepods by their larval forms. the basic life cycle of copepods has two phases (naupliar and copepodid) (fig. . ). the egg usually hatches into a nauplius larva with a small, unsegmented body, and three pairs of functional appendages (antennules, antennae and mandibles). a maximum of six naupliar stages can occur, and all six are found in most freeliving copepods and in some parasites. nauplii may be planktotrophic (feed on plankton) or rely on its yolk (lecithotrophic). parasitic nauplii are usually lecithotrophic, have reduced setation on the three limb pairs, and no naupliar feeding process on the coxae of the antenna. in many parasites, the naupliar phase is abbreviated or occasionally lost. the final nauplius stage moults to become the first copepodid with a segmented body, a full adult set of cephalic appendages, and the first and second swimming legs. free-living copepods have a maximum of five copepodid stages with one body somite added at each moult. in almost all copepod parasites, copepodid i is a free-swimming stage. only parachordeumium aphiurae (hérouard, ) and internal sea cucumber copepods have copepodid ii hatching from their eggs (martin et al. ). copepodid i is in the typical crustacean form with two pairs of biramous swimming legs, each with -segmented rami. it begins free-living but is usually the infective stage. the copepodid stages provide a gradual transition from the copepodid body form to adult morphology, however transformed. in the more derived families, successive copepdid stages have increasing modifications in body form and limb structures. the fifth copepodid stage moults into an adult male or female. following this moult, the female becomes sexually receptive. adult males may conduct precopulatory mate guarding and holding pre-adult females until the final moult. males use an array of chemosensory aesthetics on their antennules to detect pheromones produced by females. mating takes place soon fig. . the generalised life cycle of an ergasilus von nordmann, , species showing the freeliving naupliar and copepodid stages as well as the parasitic adult female. image from smit and hadfield ( ) after the female becomes sexually receptive and consists of mate detection, mate recognition, and mate capture and culminates in copulation. sperm-containing spermatophore(s) are transferred to the female and usually discharged via copulatory pores, into seminal receptacle(s) within the genital region of the female. sperm are stored for fertilization, which occurs as egg batches are laid. females may produce several batches of eggs during her life. most parasitic copepods extrude their eggs into paired egg sacs or uniseriate egg strings, although some are stored internally. copepods have a great diversity of invertebrate and fish hosts but are remarkably limited among other vertebrate groups with a single species on whales and dolphins, none on reptiles or birds, and only a very few, almost accidentally, on amphibians. their simple life cycles may inhibit them from colonising more diverse vertebrates since their only mammal parasite has their most complicated life cycle. the life strategies of copepods suggest the simpler the host, the easier it is to parasitise. reported, based on decades of research, that copepodids, chalimus, and immature adult copepods were very rare on caribbean coral reef fishes but rather common on western pacific coral reef fishes (a -year study). one possible explanation for this difference is that caribbean, small cleaner gobies (elacatinus jordan, ) , are much more efficient in locating and removing these small, life cycle stages than are the larger indo-pacific cleaner wrasses (labroides bleeker, ). thus, life strategies of caribbean and indo-pacific parasitic crustaceans may operate under quite different selective pressures. brusca ( ) found adult cymothoid isopods of the genus nerocila leach, , with damaged pleotelsons and uropods and speculated these injuries might represent predation by cleaner fishes. williams and williams (unpublished data) have found numerous injuries and missing parts of fish lice, fish-parasitic copepods, fish isopods, and gill worms (monogenea) on, and in the gills, or mouths of fishes, and have observed copepods on fishes scurrying away from cleaner fishes, even though they were obviously too large to be removed. cleaners may bite and injure crustacean parasites that are too large for them to remove (cleaner nipping*). cleaner nipping is a widespread, important, but hitherto unrecognised, life history peril for ectoparasites. mahmoud et al. ( ) experimentally induced nipping and removal of fish parasitic isopods by portunid crabs. cleaner shrimp similarly snip off the legs of small crustacean parasites to remove and eat them (williams and bunkley-williams b, unpublished data many copepod parasites of invertebrates also have direct life cycles, but some have endoparasitic larvae and free-swimming adults, mesoparasitic larvae and ectoparasitic adults, and abbreviated or no larval stages. some endo-and mesoparasitic forms can be quite modified. most are small and free-living, but rarely planktonic, in marine and freshwaters. some are commensal with invertebrates and only rarely damage their hosts (williams and wolfe-walters ) . many parasitise molluscs, sea anemones, sea squirts, fishes, and a caridean shrimp (conradi et al. ) . the most well known is the anchor worm-an economically important fish pest, which was globally spread on goldfish and asian carp and is now common worldwide. species of the family ergasilidae cause the most important problems in aquaculture and are distributed globally (garcia and williams ; williams et al. a williams et al. , b, thatcher and williams ; . eggs are usually carried in paired or single sacs attached to first abdominal somite. however, some notodelphyids and pectinophilus nagasawa, bresciani, & lutzen, , store eggs internally. the full life cycle occurs in many copepods parasitising invertebrates and in ergasilid fish parasites. ergasilids are also unusual in having naupliar stages feeding on unicellular algae. thaumatopsyllids have a life cycle similar to that of the monstrilloids with parasitic nauplii inhabiting the gut of brittle stars and nonfeeding adults living in the plankton. the copepodid phase in thaumatopsyllus paradoxus sars, comprises the full five stages preceding the adult, and the entire phase from final nauplius to adult is completed without further food intake. parachordeumium amphiurae (hérouard, ) hatches directly as an infective copepodid ii, having passed through the first within the egg. in the tunicate parasite, gonophysema bresciani & lützen, , the infective copepodid larva settles on the host and moults into an onychopodid larva, which is reduced to a simple elongate sac-like body provided with grasping antennae used for attachment. the onychopodid penetrates the skin of the tunicate and transforms into an amorphous, lobate adult (rohde ) . the life cycle of anchor worms (lernaeids) has been described as direct with only one host, indirect with an intermediate host, or with a transfer host. the confusion lies in the apparent occurrence of all three cycles in the same species of anchor worm. the first copepodid usually attaches and develops through copepodid stages on the gills of a fish host. this may occur on the definitive host specimen, on a different specimen of the same species, or on a different species of fish. in the final copepodid stage, the female usually leaves the gills and attaches on the body of the same fish specimen (direct) or on a different one (indirect). thus, a real intermediate host can occur, but this is not obligatory. some of these species may be evolving towards an obligate intermediate host. this would represent a third method of developing, through an intermediate host, for which we propose the term parallel incorporation*. deep-sea copepods have been found resting and feeding on the mucus of gelatinous plankton (humes ) . gasca et al. ( ) found mating males and females and early to late copepodid stages of pseudolubbockia dilatata sars, , in the subumbrella cavity of deep-sea hydromedusae. we suggest this copepod is another protelean parasite in the short-antennae copepods. recent reports and descriptions of additional copepodids or of pre-adults in shortantenna copepods were probably growth stages, not moult stages (martin et al. ). the free-living stages in the life cycles of ergasilids and many of the copepod species parasitising invertebrates suggest that they have more recently evolved a parasitic lifestyle. the most modified female of any of the fish-parasitic copepods are species of sarcotaces olsson, . osman et al. ( ) described and pictured the naupli of possibly a new species of sarcotaces apparently host specific to a brownspotted grouper in the arabian gulf. surprisingly, they found no females in six gall cysts, even though nauplii and males were present. eggs were attached to the inner wall of the galls. nagasawa et al. ( ) found eight cysts with females, males, nauplii, and eggs in a blacktip grouper (fig. . a, b) in the ryukyu islands. some short-antenna copepods occur in the musculature and sinus canals of fishes. rosim et al. ( ) reported a new genus of ergasilid in the urinary bladder of fishes and considered the process of becoming an endoparasite. the muscle parasites are mesoparasites; however, their host positions do complicate their life cycles. forsskal, ) , collected in okinawa. (a) embedded (skin has been removed to expose large, sack-like cysts), (b) removed large, sack-like cyst. nagasawa et al. ( ) found eight cysts with females, males, naupli, and eggs in this host specimen. images © kazuya nagasawa . . harpacticoida: wormlike copepods* these are a very large group of mostly benthic copepods. very few associate with other organisms, and almost none are known to be parasitic. in the family tisbidae, species parasitise deep-sea octopuses. red bug, an aquarium pest, has often been called a parasite of corals and is caused by the copepod tegastes acroporanus humes, . neoscutellidium yeatmani (zwerner ) was also said to be a parasite of fish, and other wormlike copepods were noted to be parasites of whales, sea turtles, and manatees (e.g. aznar et al. ) . however, others have disagreed with these statements (e.g. suárez-morales et al. a; domènech et al. ). an apparent commensal species, the ochre copepod*, balaenophilus manatorum (ortíz, lalana & torrez, ) , has been observed in caribbean manatees and sea turtles (badillo et al. ; williams and bunkley-williams unpublished data) . haracticoids probably follow the usual copepod life cycle of six naupliar stages and five copepodids found in their family. dahms et al. ( ) discussed all that is known about the naupliar stages in tisbidae. no development stages of parasitic forms had ever been found until lópez- gonzález et al. ( ) described copepodids iii and v. they suggested similar copepodid stages occurred internally (actually mesoparasitic) for the other adult forms found in other octopuses. therefore, their discovery completed the life cycle of these parasites (see additional information below). they hypothesised copepodid i was the infective stage and all these occurred internally. they did not discuss the naupliar stages, but these are expected to be the usual, six, free-swimming forms. adult males have only been found in four of the known haracticoid species. they may not live very long, not stay on the host very long, or even move to new hosts to copulate with other females. the more complete life cycle suggested by lópez- gonzález et al. ( ) is interesting and possibly correct. the only problem is that it is in disjunct halves. until the external portion is matched with the internal portion in a single species, ockham's razor would suggest these are portions of two different life cycles of tisbids on octopuses. the complete life cycle is thus still unresolved. ogawa et al. ( ) suggested balaenophilus aurivillius, , species on sea turtles spend their entire life cycle on one host and cannot swim, like whale lice. domènech et al. ( ) experimentally found nauplii can only crawl, but copepodids and adults can swim, albeit only for short distances. zwerner's ( ) discovery of neoscutellidium yeatmani in the gills of antarctic eelpout has been uncritically repeated so many times that it appears to be widely believed. however, this form has never been reported again. he found a mere seven specimens in the gills of fish. these numbers are too low to sustain a viable life cycle for a parasite. this was very likely an accidental infection. this deep-water fish is known to eat molluscs, and species of parasites in this family infect the gills of deep-water octopuses. these copepods could have spilled from an octopus to the fish consumed (see prey predator transfer). more specimens with this association, with higher numbers per host, would need to be observed before this species could be confidently regarded as a fish parasite. many authors, even very recently (e.g. aznar et al. ) , have called the ochre copepod on sea turtles and manatees an ectoparasite, but it appears to be only a commensal. suárez-morales et al. ( a) found that it was a harmless epibiont. this copepod formed ochre-coloured patches on the skin with no positive precursor relationship with barnacles or algae. badillo et al. ( ) explored the possibility of parasitism of wormlike copepods on whales, sea turtles, and manatees in detail and believed that they ate keratin. they therefore claim this makes them commensals of whales, yet ectoparasites of turtles. this interpretation does not appear convincing, and we assert that they are all commensals. suárez-morales et al. ( a) also dismissed, another harpacticoid, harpacticus pulex humes, , associate. it had been reported on a manatee only once, in captivity, and appears to be a non-associated predacious species. thus, we surmise wormlike copepods do not parasitise vertebrates or corals (red bugs). some may be obligate commensals. some may also harm vertebrates in captivity but are not parasites. larval parasitic copepods are wholly parasitic and occur worldwide (tropical, temperate, polar) in marine waters and infect benthic gastropod and bivalve molluscs, polychaete worms, and sponges (martin et al. ) . approximately species are known in five genera in a single family. monstrilla dana, (latin for monster) is the best-known genus. they are not abundant anywhere but more often found in coastal and coral reef areas. their biology and ecology are poorly known. the only mortalities attributed to larval parasitic copepods was a partial die-off of cultured brown mussels (suárez-morales et al. b) caused by copepodids, the most damaging stage. suárez-morales ( ) reviewed the diversity, as well as the life cycles of larval parasitic copepods. they have a protelean life history unique among metazoan parasites (martin et al. ). the first naupliar stage is free-living, but the rest are endoparasitic. all the copepodid stages are parasitic. copepodid v, called subimago by suárez-morales et al. ( ) , is the emergent stage. it moults rather quickly into an adult after it leaves the host. the adults are nonfeeding, free-swimming, reproductive, and pelagic. most copepods produce egg sacs or spawn freely in the water column, but larval parasitic copepod females attach their eggs on their long, ovigerous spines with mucous secreted by the terminal part of the oviduct. egg masses are produced iteratively corresponding to when the ovigerous spines grow. eggs hatch into lecithotrophic nauplii that locate a mollusc or polychaete host and burrow into its tissues. they metamorphose into sac-like naupliar stage in the host's blood system. two antero-ventral root-like processes absorb nourishment from the host. this stage is like no other crustacean larvae. development continues endoparasitically until the last copepodid escapes from the host and undertakes a single moult into a reproductive adult. suárez-morales et al. ( ) described the first copepodids (iii, iv, v) in detail, finding copepodid v to be pre-emergent and emergent. adults spend very little time in the plankton; therefore, they are rarely found and usually in low numbers. suárez-morales ( ) described one mass aggregation in the caribbean sea off mexico. pelagic adults lack all cephalic appendages except antennules. the known hosts include pyramidellid and vermetid prosobranch gastropods, bivalves, and polychaete worms. pairing the same species of free-swimming males and females morphologically has been difficult. the few morphological characters have also made taxonomic work difficult and often inconclusive, and descriptive standards have only relatively recently been upgraded (grygier and ohtsuka ) . suárez-morales ( ) summarised the morphologies of the group to aid in identifying adults and life cycle stages. suárez-morales et al. ( b) were the first to find a monstrilloid in a commercial bivalve mollusc and to document the consequent harm and mortalities. a radical placement of the larval parasitic copepods within the sea lice, based on sem data, and antenna and caudal rami morphology, was proposed by huys et al. ( ) . the differences suárez-morales et al. ( ) found between these groups' copepodids suggested such a combination would be incorrect. their life histories are also completely different in almost every respect. a subimago refers to a pre-adult mayfly with wings, but no functional genitalia. it can fly, but cannot mate, and can be morphologically very similar to the adult. it moults into an adult. the copepodid v from suárez-morales et al. ( ) does not appear sufficiently different to warrant the use of this borrowed term. kuris et al. ( ) suggests larval parasitic copepods are parasitoids. they do have a life cycle similar to parasitoids; however, we believe they are parasites because they do not kill their hosts. only species (~ %) descriptions are based on both sexes, on females only, and on males. molecular studies might help pair females and males of the species (suárez-morales ). siphon-mouth copepods have siphon-like mandibles and a frontal filament that attaches to the hosts. these attributes have contributed to their great diversity (~ species in families) and success. sea lice (caligoids) are well known fish copepods and are very damaging to fishes in cage culture, salmon being particularly damaged by the salmon louse. cuckoo copepods*, or nicothoids (fig. . ) , are the most famous of this group of parasites of invertebrates that harm commercially important lobsters and spider crabs. this order holds % of the known copepod parasites of fishes ( species in families). most species are marine, but a few are freshwater (garcia and williams ; williams et al. a williams et al. , b, ). the full copepod life cycle occurs in many families of siphon-mouth copepods, especially those utilising invertebrates as hosts, such as asterocherids and cancerillids. in parasitic copepods, the infective larva is, with rare exceptions, the first copepodid, and life cycles are direct, involving only a single host. in fish parasites, the nauplius phase is reduced to two lecithotrophic stages and has uniseriate egg strings in which disc-shaped eggs are closely packed into one row extending the length of the string; and most nauplii have a single pair of modified caudal setae known as balancers (function unknown). related families with fig. . cuckoo copepods (nicothoids) parasitise the eggs of lobsters and spider crabs in asia and brazil. they mimic the eggs of their host and are not removed from the host's eggs, which they eat. the copepod (arrow) has egg strings. see life cycle in otake et al. ( ) . image © kaori wakabayashi multiseriate egg strings, such as lernaeopodids, sphyriids, and two genera of hatschekiids, have nauplii lacking balancers. in some lernaeopodids (allela leigh-sharpe, ; clavella oken, ; and nectobranchia hesse, ) , the nauplius phase is reduced to one stage. in other lernaeopodids and some pennellids (salmincola wilson, ; cardiodectes wilson, ; and peroderma heller, ) , it is lost completely, and eggs hatch directly into the infective first copepodid. life cycle abbreviation also occurs in some parasites of invertebrates. the herpyllobiidae and the genus trochicola dollfus, , have only two naupliar stages, and only one nauplius is known for gonophysema bresciani & lützen, , and for some genera of nicothoids. in other genera of nicothoids, some chordeumiids, and cucumaricolids, there is no nauplius stage. izawa ( ) experimentally showed that there could be five naupliar stages in gangliopus pyriformis gerstaecker, . in most fish parasites, the first copepodid secrets a chitinous frontal filament from an anteriorly located gland, soon after it settles on the host. this filament anchors the developing chalimus larva securely to its host (rohde ) . the life cycles of sea lice have been the topic of much research and debate of late. this attention is due to the damage that they cause cage cultured fishes, particularly of salmonids (lafferty et al. ) . knowing the correct life cycle is critical in determining when to treat for the damaging stages. caligid sea lice were thought to have four chalimus stages and one or two pre-adult stages. several recent papers have challenged this scenario (e.g. hamre et al. ; venmathi maran et al. ) . they found that the typical caligid life cycle comprised eight stages: two naupliar, one copepodid, and four chalimus stages preceding the adult in caligus müller, , but with the four chalimus stages represented by two chalimus and two pre-adult stages in lepeophtheirus heegaard, . this is a profound change with significant implications for the aquaculture industry. as the typical caligid life cycle may not exist, it may be necessary to determine the life cycle of every species of damaging sea louse. again, only . % ( ) of life cycles are known for caligid species (venmathi maran et al. ) . the new stingray laser gun has only been used to shoot adult sea lice (bevanger ) . considering the flexibility and accuracy described for the gun, it could probably be used to shoot multiple life cycle stages. pre-adults also secrete a frontal filament during moulting but soon detach and become motile. frontal filaments and chalimus larvae occur in most fish parasites for which the larvae are known, but none occur in the lernanthropids. nicothoids use a similar filament to attach their developing larva to the exoskeleton of a crustacean host. the basic copepodid stages, as primitively retained in cancerilla dalyel, , comprise five stages plus the adult. one pre-adult stage in caligus clemensi parker & margolis, , or two pre-adult stages in sea lice, as true moult stages, have been added to the basic life cycle. the general trend in parasites is to simplify or reduce ancestral free-living life cycles. these additions are quite unusual and have only been found in this order. pennellids (marine anchor worms*) differ from all other copepods by needing (obligate) intermediate hosts (fish, squid, pelagic gastropod) in order to develop. the copepodid larva becomes a chalimus larva stage on the intermediate host. some male and female chalimus individuals mate on this host. others leave the host and mate in the water column. males soon die; females have a short planktonic period, find a final host, and metamorphose into an adult (poulin a) . brooker et al. ( ) reviewed the literature and thoroughly described the life cycle and life history of the famous pennellid copepod lernaeocera branchialis (linnaeus, ) . not all marine anchor worms have intermediate hosts. okawachi et al. ( ) suggest peniculus minuticaudae shiino, , has a direct life cycle, unlike most pennellids, because copepodids, chalimi, adult males, premetamorphic adult females, and post-metamorphic adult females of the parasite were all found on a single fish. they also describe adult male, copepodid i, and late chalimus stages and redescribe post-metamorphic and premetamorphic adult females. ismail et al. ( ) described a complete, direct life cycle of a pennellid, peniculus minuticaudae shiino, . the hatching stage was an infective copepodid followed by four chalimi and adult instars. males associated with various pre-adult females, but copulation only occurred between adults. fertilised premetamorphic adult females carrying spermatophores may detach from the host and settle again before undergoing massive differential growth into the post-metamorphic adult female. many marine anchor worms (pennellids) have intermediate hosts. this is the first life cycle of the group in which the female remains in the same position on the same host specimen (ismail et al. ) . otake et al. ( ) had named a new cucukoo copepod* in and described its abbreviated nicothoid life cycle of free-living nauplius i (ni) observed hatching from female egg sac, copepodid i (ci) found on body of host, and copepodid ii+ (cii+) and adults found on host eggs. they surmised ni develops into infective ci in the water column, ci settles on the body of host, and ci moults to cii, migrates to host egg masses, and develops into cii+ and then adults. adults mate on host egg masses. brazenor and hutson ( ) examined the effects of temperature and salinity on the life cycle of lernanthropus latis yamaguti, , on the euryhaline barramundi in australia. nauplii hatched best at - c and ‰. none hatched in freshwater and only a few in brackish water. lernanthropus latis is euryhaline, but freshwater can be used to break its life cycle. Økland et al. ( ) described two new rhabdovirid viruses, which occurred in all life cycle stages of the salmon sea louse in norway. the viruses caused tissue necrosis in adult copepods but did not infect fish. they speculate the copepod injects the virus in the fish to confuse its immune system as part of its purposeful life cycle strategy. we find their suggestion interesting but rather astonishing. copepods do not purposely use viruses; theirs was not the first report of copepod viruses but the fifth; many other fish-parasitic viral vector mutualists* exist to the benefit of crustacean infective stages; and we designated the first viral crustacean mutualists*. sea lice are notorious for causing problems in marine aquaculture, particularly of salmonids (e.g. gonzález and carvajal ; lafferty et al. ) . the contamination of the environment by salmon sea lice from fish farms is a politically and economically important question that has received recent attention. serra-llinares et al. ( ) found that farms increase the infection of local, wild salmonids. they also noted thorstad et al. ( ) , and others strongly suggested the transmission of lice from farm salmon to wild salmonids in systems where the fish occur in close vicinity. these adult caligids can freely swim between hosts (host hop*) and build up in fish cages. few parasitic copepods have this life-history advantage (serial parasites). the filtering effect of cages tends to concentrate sea lice. adult male and female caligids are frequently found in plankton samples (venmathi maran and ohtsuka ) . they must spend considerable periods free-swimming off hosts. some species have even been described only from the plankton, and their hosts remain unknown. ohtsuka et al. ( ) previously described a dajid isopod and a nicothoid copepod parasitising the marsupial lumen of a mysid in japan. the adults eat mycid eggs and drastically reduce the population of mysids. infective stages of the copepod penetrate host body tissues, feed, and grow. infective isopods penetrate the space between the carapace and the dorsal tergites. remarkably, isopods and copepods rarely occur together, but alternately, albeit continuously, parasitised the same host at different times of the year. this life history association is unique in parasitology. we will call it alternate host sharing*. this allows both parasite species to use all available resources of the host. three species of copepods are known to fully encyst in intermediate host fish tissues. only one has been named. lewis ( ) found the first pandarids to encyst in the fins of bony (teleost) fishes in hawai'i. lewis only found male copepodids and immature males, which he tentatively identified as nesippus cf. costatus wilson, . amaterasia amanoiwatoi izawa, , was described from female copepodids i, iii, iv, and v and a female escaping from a copepodid v. izawa found , , , and copepodids in fin galls on a single striped triggerfish from the eastern pacific (izawa ) . a new species of amaterasia izawa, , was found in individual cysts, as lewis ( ) had found, not galls, on the body and dorsal fin of species of fishes in puerto rico (williams and bunkley-williams unpublished data). tang et al. ( ) found that lewis' ( ) younger early encysted stage was a copepodid iv, the older early encysted stage was a copepodid v, and the lateencysted and recently excysted stage was an immature adult male. izawa ( ) and tang et al. ( ) speculated about the life cycle of their species but made no descriptions. tang et al. ( ) suggested encystment could be protection from coral reef cleaners. we propose a new life cycle for these encysted copepods*: there are five planktonic nauplii (izawa ) and a first copepodid stage. the ci is infective (found in galls; izawa ) and settles from the plankton onto a host and forms a cyst or gall under the skin on the fins or body of a variety of different bony, coral reef fishes. lewis ( ) found surgeonfishes (acanthurids) were preferred in hawai'i. however, parrotfishes were preferred in puerto rico (williams and bunkley-williams unpublished data) . the cysts are open posteriorly for respiration (mesoparasite). after feeding, developing, and moulting through ci-civ, the fifth copepodid emerges from the cyst leaving a moulted exoskeleton behind. lewis ( ) found these forms were soft and pliant, which he interpreted to be of value in leaving their cysts. it was actually because they had recently moulted. the cv then swims out and searches for a shark definitive host. there could be some predator transfer involved since the copepodids are in hosts preyed upon by sharks. the encysted copepod life cycle* is the only mesoparasitic life cycle known in parasitology and only the second obligate intermediate host life cycle discovered in copepod parasites of fishes. many tapeworm (cestode) shark parasites have bony fish intermediate hosts, but this is the first crustacean one ever discovered. the evolutionary usefulness or necessity of pennellid intermediate hosts has never been explained (poulin a; martin et al. ) . we can discern at least seven, nonmutually exclusive explanations: . . faster attachment-when the female settles on the final host, water currents, host movements, and sometimes host cleaning or cleaner organisms make her stay perilous (loose on the host). nutrients from the intermediate host can be used to permanently attach without waiting to feed on the final host and digest. . widen host range-the only crustacean parasite to infect mammals cannot do so without an intermediate host. kik et al. ( ) found lepeophtheirus acutus heegaard, , was a potentially dangerous sea louse of elasmobranchs in captivity. not only did it damage sharks and rays but could complete its life cycle in an aquarium. muñoz et al. ( ) found early and late copepodids of two species of caligus, two of trifur wilson, , and two of unknown families, on juvenile fishes. they examined thousands of nearshore, planktonic fishes, found % infected, and % infected by multiple species. they thus opened a completely new dimension into parasitic life strategies, which we have termed planktonic juvenile fish infection*. juveniles of a single host species have sometimes been examined for parasites (e.g. nielson et al. ; herrera ), but masses of juveniles have seldom been studied (e.g. herrera ; felley et al. ; cribb et al. ) . muñoz et al. ( ) concluded these juvenile fishes were intermediate hosts for these copepods. however, caligids are not known to have intermediate hosts, and pennellid intermediate hosts are adult fish or squids. they had also concluded the copepods would mature too soon to develop with the host fishes. however, we believe the hosts and the parasites will grow up together in these cases. alternatively, these may be small predators* feeding on juvenile fishes as a part of their life cycles (table . ). whichever is the case, this extends and prolongs life cycles further than we had imagined. interestingly, only copepods are taking advantage of this resource, not only among crustacean parasites but also among all parasites. muñoz et al. ( ) also searched for internal parasites and found none. venmathi maran et al. ( ) cuckoo copepods are named after the nest-parasitic birds by that name (cuculidae). these copepods parasitise the eggs of lobsters and spider crabs in asia and brazil. they mimic the eggs of their host ( fig. . ) and are thus not removed by the host, as they resemble the real eggs, which they eat. otake et al. ( ) called these copepods 'associates'. kuris et al. ( ) calls them 'symbiotic egg predators'. however, we consider them to be true parasites, damaging and feeding off their hosts, with adults that never leave the host. flyingfishes are food for many large offshore predators, which host pennella spp. høeg et al. ( ) found that cypris larva morphologies of the barnacles reinforced the concept that this larva was a prerequisite to the tremendous success of that taxon. the evolution of parasitism, obligatory in three major taxa, was the result of convergent evolution. thecostraca was distinct from tantulocarida (sect. . ) because they differed in the life cycle stages that penetrated their hosts (høeg et al. ). these very small, naked barnacles bore into calcareous material such as animal shells and inanimate hardgrounds. burrowing barnacles produce a slit-like hole in the surface known by the trace fossil name rogerella saint-seine, . they feed on plankton ). they do not feed on their associates. burrowing barnacles are only found in their hosts' shells and never touch their flesh, except possibly in the case of corals. they are not obligates, perfectly happy living on inanimate hard ground, and do not harm their hosts. we do not understand how they can be called parasites? williams et al. ( ) called a burrowing barnacle a parasite and showed that it fed on its host hermit crab's eggs. however, this was predation, not parasitism. murphy and williams ( ) suggested burrowing barnacles in hermit crab shells were 'transient parasites' because they somehow consumed hermit crab eggs and preferred female hermit crab shells. in our opinion, burrowing barnacles are not parasites, as least when considering the current available information. the parasitic barnacle is the 'poster child' for gross modification of parasitic forms. adults are unrecognisable as crustaceans, let alone barnacles. only their larval forms resemble those of normal barnacles (see sect. . . ). they are also famous for controlling the behaviour and morphology of their hosts. they damage commercially important crustaceans. about species are known, about a quarter of all barnacle species. they infect crustaceans, mostly true crabs (brachyuran) and anomuran crabs (hermit crabs, squat lobsters, etc.). a few parasitise caridean shrimp, mantis shrimp, peracarids, and even other barnacles. we worked on the button-crab parasite* on the blue crab in the gulf of mexico but have not found such obvious parasitic barnacles in the caribbean. unlike most barnacles, parasitic barnacles have separate sexes. adults are sessile, with females consisting of a sac attached to the crab host (externa) (fig. . ) with rootlets of tissue flowing cancer-like through the host's body (interna) and dwarf males inside the female. some females sequentially only produce male larvae from large eggs, female larvae from small eggs or mixed sexes. they have the usual naupliar instars and infective cyprid; however, in some species, embryos develop directly into cypris larvae before adult females release them. the nauplii are smaller than those found in other barnacles, which may be necessary to produce them in much greater numbers. the larvae are lecithotrophic. the cypris are usually at least days old before they settle on a host. in kentrogonids, male cyprids are larger than female ones. some have a naupliar eye, and others have compound eyes. injection of the vermigon (the migratory internal stage) happens within - days after settlement. when many male larvae exist, only the fastest and strongest will succeed. when there are few males, the female remains receptive longer. when a virgin is found, the male cyrid must settle close to the orifice, enter the brood chamber, and inject a trichogon stage. the trichogon looks like a verogon, except with a spiny collar. it becomes a dwarf male and reaches one of two male receptacles. once established, the dwarf male undergoes spermatogenesis and is nourished by the female parasite for the duration of its life (cryptogonochorism). the male cypris of akentrogonids penetrate the host or the virgin female with their antennules and without a kentrogon or trichogon. a single male can fertilise all the broods of the female. a female externa produces several batches of larvae and drops off the host just before the host moults. a new, young externa is produced from the interna and emerges from the host body (waiho et al. ) . , ("greater than" symbol-shaped mass-red arrow), externa of this rizocephalan parasitic barnacle, which, in turn, was hyperparasitised and rendered sterile by four liriopsis pygmaea (rathke, ) (pearl shapes-black arrow) epicarid parasitic isopods (cryptoniscidae). a different black-and-white photograph of these associations was in lovrich et al. ( ) . associates in this present colour photograph have been misidentified in several popular sites online. image © gustavo a. lovrich the externa takes the place of the crab egg sac. the host's behaviour is chemically altered causing sterilisation and only moults when the aged externa drops off. the host treats the externa as if it were its egg sac. male crabs, which would never have carried eggs, care for the externa. they are even more affected since their tail shape changes to the female configuration to better protect the externa ( feminization). an externa may last for several years. these life cycles show parasitic barnacles are the most parasitic of the crustaceans. they totally penetrate all the tissues of their host, control the behaviour of the host, and are drastically modified for the parasitic existence. glenner and hebsgaard ( ) made a comprehensive phylogenetic analysis of the evolution of life history strategies in parasitic barnacles. they found they were monophyletic with a filter-feeding barnacle-like ancestor. the host-infective kentrogon larva, inserted in the life cycle of kentrogonida, was ancestral, and a homologue of the juvenile thoracican barnacle. the host inoculation in akentrogonida (last pelagic larval stage directly injects into haemolymph) is derived and has evolved only once within the parasitic barnacles. the ancestral host is anomuran (hermit crabs, squat lobsters, etc.). alverez et al. ( ) described the externae of the button-crab parasite in detail. unlike most parasitic barnacles, they found only a single male receptacle but two implanted males. they questioned what sorts of male-male competition occurs when they are not separated. glenner et al. ( ) used light and sem microscopy of cypris larvae to supplement molecular data showing that parasitic barnacles, thought to be the most primitive, were actually the most advanced, along with many evolutionary extrapolations. these parasitic barnacles are sometimes hyperparasitised by cryptic isopods (fig. . ). just as the barnacle sterilises its crab host, the hyperparasite sterilises its fellow parasite. there are some indications that swimming decapods, which must remove epibionts, are more resistant to the attachment of settling crustacean parasites; however, no experimental evidence exists (boyko and williams ). in the reverse, li et al. ( ) found a species of crab parasitised by a rhizocephalan had many more barnacles, and other epibionts, than those not parasitised. this is an example of our positive precursor*. several authors have recently suggested parasitic barnacles could be host-specific control agents for nonindigenous crabs, such as the problematic green crab. however, parasites seldom make effective controls, and well-intended introductions have often been disastrous. this group containing the normal acorn and gooseneck barnacles has four parasitic species in three families and three genera. the shark barnacle* is little modified for parasitic existence except for the loss of its shell and adding a tough tegument. polychaete barnacles* are moderately modified. the jellyfish barnacle* is the least modified with small, thin plates. the cirri (feeding legs) are still used for obtaining food. these free-living-to-parasitic transitionals are, of course, of great scientific interest (rees et al. ) . the shark barnacle is famous for promoting charles darwin's interest in barnacles. they are hermaphroditic with all individuals possessing a penis, and no dwarf males are known in shark barnacles and polychaete barnacles. adults are receptive as females for fertilisation after moulting. they use their penis to copulate with an adjacent individual as other hermaphroditic free-living barnacles. barnacles have the longest penises, relative to body size, in the animal kingdom. oddly, dwarf males have been found attached to larger hermaphrodites in some species (androdioecy; sawada et al. ) . the fertilised egg hatches into a standard, barnacle nauplius, a one-eyed, pearshaped larva with a head, a naupliar eye, a pair of horns, and a telson. nauplii are usually brooded by the parent and released after the first moult swimming freely with setae. towards the end of the sixth instar, they begin to develop compound eyes and a globular shape. they undergo months of growth, passing through five instars, before transforming into the cyprid stage, which has a carapace, is torpedo-shaped and is the stage before adulthood. it does not feed and only searches for a host. this may last for a period of days to weeks. it explores potential hosts with modified sensory antennules. once it finds a host, it undergoes metamorphosis into a juvenile barnacle. shark barnacles are usually found in pairs near the dorsal fin of their shark host; therefore, the cypris larvae must not only find a host but also a partner (see founder pair*). shark barnacles breed through the year and live on their host for at least a year. both genera feed on the host by roots formed from their peduncles. they mostly infect small specimens of sharks ( %), and incidence is reduced ( %) in large ones. gonads of infected sharks never develop (nutritional sterilisation*). reported a prey-predator transferred isopod in a lantern shark but found no barnacles. some epiphytic normal barnacles attach to hosts or parasites (e.g. williams ; williams and williams b; mignucci-giannoni et al. ) , and the sea turtle barnacle has been found on the carapace of a speckled crab at dauphin island, alabama (williams and bunkley-williams unpublished data). shark barnacles parasitise at least seven deep-sea lantern sharks and dogfish (ommundsen et al. ). furthermore, two species of polychaete barnacles and a jellyfish barnacle are known . the jellyfish barnacle is an obligate associate of jellyfishes, and pagès ( ) found it attached in an area with few nematocysts near the gonads of the jellyfish host. we believe the lack of stinging cells may have originally attracted the barnacle infective stage to this area, and the gonads became a convenient food source later. a simple development of parasitism is still in progress. the species in the larval genus hansenocaris Îto, of y-parasites remain a mystery in parasitology. y-larvae have been known for years, but their assumed parasite adult forms and hosts have never been found. these parasites are both everywhere and nowhere. they must be both parasitologically and ecologically very important, but how remains a mystery. life cycle y-nauplii are egg-shaped, with a faceted cephalic shield and carapace (reticulated cuticular ridges, forming plates), from which the group derives its name, and a relatively long, ornamented abdomen. they have a characteristic bobbing motion when swimming that makes them easy to distinguish in plankton samples. the function and homology of the naupliar horn pores and dorsocaudal organs have been much debated but remain uncertain. they are either planktotrophic or lecithotrophic. only lecithotrophic nauplii have been raised through all five instars. planktotrophic nauplii have food visible in their stomachs. unlike barnacle larvae, the y-cyprid is constantly swimming. this larva is distinctively the costracan. the y-cyprid has a univalved carapace that only partially covers the larval body and resembles an inverted boat but with elongated sharp posterior ends (fig. . ). paired compound eyes lie anteriorly in the body with antennules, labrum, paraocular processes, postocular filamentary tufts, and two pairs of rudiments of antennae and mandibles underneath (ventrally). the antennules have four segments. all larval stages are free-living and semitransparent. the cyprid does not feed. a number of species have been described based only on a y-cyprid (n ¼ ) or even a y-nauplii (n ¼ ). as with barnacles, the cyprid seeks a host to infect. it has compound eyes, is ambulatory with its antennae, and can produce an adhesive glue. recently, possible juvenile forms have been produced by treating y-cyprid with the hormone -hydroxyecdysone to stimulate ecdysis and the transition to the next life cycle phase. the resulting slug-like, unsegmented, and limbless form was called 'ypsigon' (fig. . ). it is formed in the cypris and escapes from its body (glenner et al. ). this may be a juvenile. see høeg et al. ( ) for more detailed descriptions, illustrations, and photographs. molecular studies support the present phylogeny of the y-parasites . they remain mysterious parasites with unknown adults and hosts. glenner et al. ( ) suggest the ypsigon is the 'vermigon of the y-parasites'. just like the barnacle vermigon, it is the injected form that will become the parasitic adult. this may be true, but just because formless structures are produced by similar methods and appear similar does not make them the same. their notion is courageous, albeit premature. glenner et al. ( ) found that + morphological types of y-larvae occurred very abundantly around sesoko island in okinawa. this unusual situation may indicate a centre of origin or, possibly, a diversity hot spot. yet, the adults remain unknown. the 'phantom' adults are neither too rare to discover, nor in some obscure unexamined hosts, nor in some restricted localities. the larvae are just too abundant, widespread, and diverse for such restrictions. this is an adult that parasitologists may be encountering all the time but just cannot recognise. the adult y-parasites may be morphologically similar to another parasite, with which they are confused, or so morphologically indistinct as to be unrecognisable as a parasite. glenner et al. ( ) came to a similar 'highly simplified structure' conclusion. showing the carapace, the thorax with six pairs of natatory legs and the segmented abdomen, (b) an ypsigon (red arrow) within minutes of leaving the empty cuticle of the spent y-cyprid (black arrow). the cuticles of the carapace, thorax, appendages and abdomen are clearly visible, but no tissues remain in the spent y-cyprid. the worm-shaped ypsigon exits by amoeboid bending and peristalsis movement of the body and is believed to be the initial parasitic stage that enters into the tissue or body cavities of a still unknown host. details in glenner et al. ( ) . images © jt hoeg, m grygier, y fujita, h glenner, j olesen . ascothoracida: copebarnacles* these are uncommon parasites that cause little damage and infect non-commercially important hosts. copebarnacles are a small group of~ species in genera, families, and orders, which are ecto-, meso-, and endoparasites. they occur from the intertidal to the deep sea around the world. coral copebarnacles are ecto-, meso-, and endoparasites of corals (scleractinia, zoantharia, antipatharia and alcyonacea) and ectoparasites of crinoids (waginella). the suggested common name is based on their bodies that are similar to copepods but are related to barnacles. echinoderm copebarnacles are meso-and endoparasites of echinoderms (asteroidea, echinoidea and ophiuroidea). sexes are separate except for the hermaphroditic petrarcids. sex determination is genetic, and some male and female larvae differ in the armature of chemosensory aesthetascs (as in most parasitic barnacles). a-nauplii have an oval, bowl-shaped head shield, which is broader anteriorly, and setiform frontal filaments. a-cypris have a bivalve carapace and antennules with hooked claws. echinoderm copebarnacles brood their larvae and only release a-cypris, while coral copebarnacles release nauplii. usually, six naupliar instars (sometimes two brooded) are followed by one to two cypris-like stages (a-cypris, ascothoracid larvae) (similar to the cirripede single cypris and the y-cypris of y-parasites). many are lecithotrophic, but some species are planktotrophic. coral copebarnacles a-cypris occur in the plankton. the a-cypris (ascothoracid-larva) attaches by grasping antennules rather than by glandular secretions as in cirripede cyprids. when two a-cyprid instars occur, the second is the settlement stage. many species have an abbreviated ontogeny, and the entire naupliar phase is sometimes brooded or embryonised. no complete life cycle is known for any copebarnacle. neither host infection, nor copulation has ever been observed. most females have seminal receptacles on their legs, but not in dendrogastrids. some male a-cyprids have testes with mature sperm and possibly fertilise the females through the pore in the host. some dwarf males sit close to the aperture, and others live in the mantle cavity of the female. the transition from a bivalved stage to a sac-like carapace is suspected to occur in one moult, but this remains unresolved. a 'post-larval' stage of females and males has been discovered, and second-stage a-cyprids, ready to moult to the male stage, have been observed. the cypris for the modified, enlarged female, and dwarf males of gorgonian copebarnacles (gorgonolaureus utinomi, ) are not known. the complete life cycle is equally not known (kolbasov et al. ) . threefive naupliar instars occur. gorgonolaureus muzikae grygier, has been noted to be frequently infected by hyperparasitic cryptoniscoid isopods (kolbasov et al. ) . some ectoparasitic males and females can swim from host to host. others are semipermanently glued in place. some are endoparasites in galls in corals and sea stars. others even start as an endoparasite but eventually create an opening to the outside and become mesoparasitic. parasites of cnidarians occupy individual polyps or nodules formed from several polyps. many of those in echinoderms sterilize their hosts. feeding methods are uncertain. many have piercing-sucking mouthparts, and the cuticle of at least one species seems capable of absorptive feeding (as in parasitic barnacles). hyperparasitism of ascothoracidans by cryptoniscid isopods is not uncommon, occurring in four of the six families. some sterilise their copebarnacle hosts. in a spectacular case, a copebarnacle, which had sterilised its host, was, in turn sterilised by an isopod. their body is enclosed by a bivalve carapace often modified and enlarged for brooding and possibly food absorption in females. minute-crustacean parasites are a highly specialised, small ( species in genera and families) group of minute (< . mm) ectoparasites on small benthic crustaceans (copepods, isopods, tanaids, amphipods and ostracods). they occur from the subtidal to the abyssal, widespread in the southern and northern hemispheres, and in both cold and warm waters. they claim the fame of the world's smallest arthropod ( μm). minute-crustacean parasites have asexual and sexual life cycles. the first is when a parthenogenetic female develops from a tantulus larva and remains permanently attached to its larva, and the host, as sort of a 'frankenstein' female. it feeds off the host and produces numerous eggs. this adult female has a large, sac-like trunk attached by the larval head. the larval trunk is sloughed leaving a scar, but no complete moult occurs. eggs develop within the trunk sac and hatch directly into the infective tantulus larval stage. the second life cycle occurs when a free-living semelparous female is produced by an attached tantulus larva, escapes from the larva, and swims away to copulate with a similarly produced free-swimming male. these minute-crustacean parasite life cycles are unique. instead of the standard moulting cycle of all other crustaceans, the mature adults develop in the attached parasitic tantulus larva. the extreme brevity of early ontogeny seems to be an adaptation to parasitism in situations where a high dispersal ability is not advantageous. minute-crustacean parasites have a dramatic reduction in body form compared to other crustaceans, with an unsegmented sac-like thorax and a much reduced abdo men. the attached larvae and parthenogenetic females are permanently attached to their host by the oral disc with an adhesive. in the centre of the disc, they make a minute puncture, through the host integument, with their cephalic stylet. this is their only access to the body fluids of the host. the free-swimming, nonfeeding adults lack cephalic appendages but possess two clusters of aesthetascs on its anterior margin. they are free swimming and have six pairs of large thoracopods without endites. the first two thoracic somites are incorporated into the cephalothorax. the male abdomen bears a posteriorly directed, median stylet and intermittent organ. it originates on the first abdominal somite. the parthenogenetic females live longer than their sexual doppelganger and are responsible for the majority of the reproduction of their species. knudsen et al. ( ) described tantulus larvae, developing males, parthenogenetic females, and only the third developing sexual female ever found. they noted that the taxonomy of the group is based mainly on the tantulus larvae, which is quite an unusual role for a larval life cycle stage. some suggest these minute crustacean parasites, with no larval stages, are the adults of the orphan mysterious y-parasites (facetotectans), which have no known adults, only larvae. there are myriad reasons why these life cycles are, unfortunately, unlikely to fit together. scuds are a minor group of known parasites, but they have many species commensal on ascidian, bryozoan, cnidarian, echinoderm, mollusc, sponge, and crab hosts. many of these may be parasitic or at least well on their way to parasitism. they are called commensals because we just do not know enough about most of them to make a proper categorisation. whale lice and jelly parasitoids are known to be parasitic, and we believe a bivalve scud is also an unrecognised parasite. there are species in genera and one family (cyamidae) of whale lice. they have no carapace, and their bodies are dorsoventrally flattened instead of laterally compressed as in other amphipods. they are one of the few aquatic crustaceans that cannot swim in any part of their life history. molecular studies of whale lice have determined the evolution of their host whales (kaliszewska et al. ) . infections can be heavy on whales and other marine mammals (mignucci-giannoni et al. ; colón-llavina et al. ), and superinfections can harm whales. whale lice will also attach to humans handling whales. there is an easily discernible life cycle. eggs are held in the marsupium of the female. females produce more eggs in each brood as they age. a quarter to half of the eggs die in the marsupium. the eggs hatch directly into a juvenile form with no larval stages, similar to fish isopods and all peracarida. they have clawed pereopods and immediately attach to the cetacean skin. sexual maturity is usually reached after six moults. some eat their exuvia (exoskeleton remains) after moulting. the complete life cycle may take or months. whale lice parasitise cetaceans (whales, dolphins, porpoises). since whale lice cannot swim, they can only be spread by direct contact among hosts such as during mating, nursing or care giving. they feed on dead cetacean skin and algae and are attracted to wounds on hosts but more for attachment than actual feeding. they may even clean up wounds and so speed up healing. whale lice also like creases, crevices, and barnacles for attachment. by eating algae, they control its growth on their host. in general, slow swimming whales have more whale lice, than fast swimming whales. some suggest that host jumping or breaching is done in order to knock off whale lice. some online videos show human divers easily brushing whale lice off whales with only their fingers. heavy infections reportedly harm humpback and gray whales. we found a heavy infection on a sperm whale that may have contributed to its death (mignucci-giannoni et al. ). melita anmyeonensis shin, coleman, & kim, , is found between the gills and mantle cavity of the broad angel wing bivalve, barnea dilatata (soulelet, ), in south korea. scuds in six families associate with bivalve mollusc hosts but never in high numbers of adults. shin et al. ( ) found no damage to the host. kretzler ( ) could not detect obvious physical damage to sea urchins even by very heavy infections of scuds. they could not determine if the association was obligate. they called it commensalism. we feel the high prevalence ( %) and intensity [ - (av. . )] in the broad angel wing, and the lack of free-living collections suggests that it is an obligate parasite. this bivalve is no stranger to crustacean parasitism having two species of parasitic copepods. shin et al. ( ) noted host abandonment* of m. anmyeonensis from damaged and captured broad angel wings. host abandonment is a frequent strategy of crustacean parasites for finding another host. they are often called parasites or parasitoids (lafferty and kuris ; kuris et al. ) but are not exactly parasites either. we term them serial parasitoids*. in various parts of their life histories, they may be free-living, kleptoparasites, minipredators*, parasitoids, serial parasitoids, or a mix. they have an obligatory association with jellies (salps and jellyfishes) as a nursery for their young (parasitoidism), often rest on jellies (phoresis), but spend most all of their lives as minipredators (see table . ). jelly parasitoids have~ species in families. they damage and kill some gelatinous zooplankton but have no known ecological or commercial importance. now that jellyfish are held and reared in major aquaria, jelly parasitoids have become a problem. effective chemical treatments have been developed (e.g. boonstra et al. ) . mating occurs on a host jelly, males depart, and ovigerous females remain on the host while brooding the eggs. brood sizes range from to several hundred eggs. the eggs are relatively small for amphipods. larval stages are in the female marsupium. the first stage is the pantochelis larva with four cheliform pereopods and unsegmented and limbless metasoma and urosome. the pantochelis stage metamorphoses into a 'protopleon' larva (often divided into three sub-stages), having a segmented metasome and imperfect pleopods. in many species, there is no pantochelis stage, and the egg hatches directly into a first protopleon stage. the last (or only) protopleon stage gives rise to the first juvenile stage (a miniature adult) and marks the demarsupiation or the deposition by the female of the larvae (in rare cases the juveniles) into a host. during demarsupiation, the gravid female swims out to find a host and deposits one or a few larvae upon the host specimen (depending on host size and capacity). she continues infecting one host after another. some females penetrate a host, split a gonad with its mouthparts, and inserts the larvae deeply into the organ. as they grow, the juveniles leave the gonad and start feeding on the prey trapped by the host. some females deposit pantochelis larvae on the surface of salps with their specialised seventh pereopods. when the larvae moult a few hours later, the ensuing protopleon larvae enter the branchial cavity and eat its wall or feeds on the collected suspended matter. once the host can no longer support the young as they mature, they leave it for another salp in the chain. species of phronimids excavate solitary salps or pyrosomes into 'barrels' open at both ends and in which they hide and use as a nursery. the larvae are demarsupiated into the barrel where they soon bunch together into a tight cluster that slowly moves around on the inner barrel wall. the female stays with the barrel and prevents the young from passing to the outer surface. at intervals, she makes short excursions into the water and returns with prey to feed her brood. the pereopods are used to maintain the position of the animal within the barrel, and beating of the pleopods propels the combined barreljelly parasitoids through the water (bio-jet ski). some adults feed on small plankton, at least part of the time; others steal small plankton out of jellies; and some eat jelly tissues as minipredators. they certainly use jellies as places to rest. jelly parasitoids seem immune to the stings of medusa. males are better swimmers than females. gasca and browne ( ) found ten hyperiid amphipods, a copepod, and a pycnogonid in jelatinous plankton in the gulf of california. they also named a new species, megalanceoloides aequanime gasca, , based on a redescription of m. remipes (barnard ) in gasca and haddock ( ) , distributions, sizes, and a few morphological differences. we name megalanceoloides gascae n. sp. based on the descriptions by vinogradov ( ) and vinogradov et al. ( ) of 'm. remipes'. we also distinguish m. gascae from m. remipes on the basis of geographical distributions in the northern indian ocean (vinogradov ) vs southwest atlantic (barnard ; usnm , ) , south-east pacific (usnm , ), and antarctic ocean (usnm ). the former three records represent two new locality records. the two species are further distinguished based on the sizes of females, mm female (vinogradov ) vs mm male (barnard ) , and morphological differences discussed by gasca and haddock ( ) . the holotype is the specimen collected by vinogradov ( : - , by monotypy iczn : art . . ) and also in illustrated ions by vinogradov ( : figs. , ) and vinogradov et al. ( : fig. ) (iczn : art . . , . . ) . the new species is the third in the megalanceoloides remipes species complex and in genus megalanceoloides zeidler, . . isopoda: isopods . . anuropidea: jelly isopods* jelly isopods are giant, blind isopods that occur in all oceans except the indian ocean (to date) and parasitise large scyphozoans in the deep sea of the eastern pacific and japan (ohtsuka et al. ). ten species in one genus, anuropus beddard, , are known, but only two have been associated with scyphozoans. they live in and feed on their host, but very little else is known (ohtsuka et al. ). our lack of knowledge may be an artefact of destructive net collections (gasca and browne ). fish-associated isopods are relatively large as adult parasites, in comparison with most other parasitic crustacea, and are often seen by divers on the outside of marine-reef fishes (figs. . and . ) and by fishermen in the mouths and gill chambers. fish gnats* (see below) are much smaller ( - mm) and highly mobile ectoparasites on marine and estuarine fishes (smit and davies ; tanaka ) . fish gnats are not true parasites but serial parasites. four other families have been variously stated to have parasitic species. no cirolanids are parasites, some corallanids may be parasites (gentil-vasconcelos and tavares-dias ) , and, very likely, some salve bugs (aegidae) and nasal isopods* (tridentellidae) are parasites (e.g. bruce and wong ) . the life cycles of fish-associated isopods and fish gnats are so completely different that their sharing a superfamily seems incongruous. fish-associated isopods are permanently parasitic as adults and fish gnats only as juveniles. fishassociated isopods hold their eggs in a marsupium and fish gnats in pouches in the female body. fish-associated isopods attach with their pereopods and fish gnats by their mouthparts. we have collected these isopods on fishes but have not considered them to be true parasites, because so little is known about their associations. some species do feed on fish blood. gentil-vasconcelos and tavares-dias ( ) considered excorallana berbicensis boone, , to be a parasite of south america freshwater fishes and e. tricornis (hansen, ) a facultative parasite of many marine fishes. they may be correct about e. berbicensis, but we are not ready to accept e. tricornis as a true parasite (bunkley-williams and williams a). we would call it a serial parasite. very little is known about any life cycles of excorallana stebbing, . the most famous cymothoid is the so-called tongue-replacement isopod (brusca and gilligan ) , which was also featured as the monsters in the horror movie 'the bay' ( ). fish isopods may cause some problems in aquaculture (williams ; woo ) . they drastically affected fisheries in a large lake in egypt (mahmoud et al. ) . juveniles may kill juvenile fishes, and adults may seriously stunt and slow the growth of hosts (bunkley- williams and williams a; mladineo ) . fish isopods have also been shown to increase swimming drag and metabolic demand of their hosts (Östlund-nilsson et al. ). more than species of fish isopods exist in genera (see chap. ). they occur in and on fishes around the world, but mostly in the tropics and subtropics, in coastal waters, with some in freshwaters largely in south america with a few species in africa and asia (bunkley- williams and williams a; smit et al. ) , and even the deep ocean (quattrini and demopoulos ; williams and bunkley-williams ) . some almost complete life cycles are known (e.g. williams and bunkley-williams ; aneesh et al. ) . klompmaker and boxshall ( ) listed many fossil fish parasitic isopods but dismissed all due to insufficient evidence. nagler et al. ( ) claimed the oldest fossil parasitic isopod based on sucking mouthparts and legs suited for attachment, but these could be just as indicative of a minipredator. we are also in the process of describing a parasitic fossil isopod (williams and bunkley-williams , unpublished data) , which may prove equally disputable. fish isopods may go through four parasitological life cycle stages: free-living, smaller*, serial parasite, and true parasite (see table . ). brood pouch development may include egg, oblong embryo predator, curled embryo with enlarged cephalic end and tapered posterior, uneyed embryo, eyed embryo, pre-manca, and manca juvenile (williams and williams a, b) . embryology is often neglected in species descriptions and even in life cycle studies. embryos in brood pouches number from to (adlard and lester ) but are usually in the low to mid-hundreds. larger and older supra-females have more offspring. although some brood mortality is expected, bakenhaster et al. ( ) found none in glossobius hemiramphi williams & williams, . pre-manca in the marsupium of some species are larvae. mancae in, and escaping from, the marsupium are juveniles. to avoid confusion, we here refer to this as a manca juvenile*. once the manca juvenile begins to form the seventh pair of legs in - moults, we consider these instars juveniles. the few juvenile stages that we know spend the daytime in the surface plankton. they descend at night, finding transfer or definitive hosts. the first to settle on a definitive host develops through the male stage and directly into a female. the second becomes a male. this was the traditional infective assumption based on little data. however, they may actually be infected by founder pairs. mladineo and valic ( ) and mladineo ( ) found only two ceratothoa oestroides (risso, ) manca infected each annular sea bream even when more mancae were available. they attached to the fish body and migrated under the operculum, rather than being swallowed as previously suspected (e.g. bunkley-williams and williams a). supposedly, a few complete life cycles of fish isopod are known, but none are complete. first, a natural release of mancae has only been closely observed once (williams and williams c) . a female on a brown chromis in a coral reef reared her posterior from the surface of the host and released a juvenile from the rear of her marsupium, slowly lowered back down, and reared up again to release another. this process was slowly and methodically repeated, although the complete release was not observed. adlard and lester ( ) found it took - h in the lab. the juveniles swam upwards towards the surface. some authors have mistaken our burst release (williams and williams c) with the normal release process. burst release occurs when a host is caught or struck. all juveniles are released at once even if they are not quite mature (pre-manca and manca; williams and williams c) . adlard and lester ( ) caused this response in the lab with pressure on the dorsal surface of a female and found it took - min. the second problem with most 'complete' life cycles is they do not include the free-swimming juvenile stages. we have found up to six stages ; and see below). the fish isopod manca juvenile has six pairs of legs. most manca juveniles are much more setose than later juvenile stages. the final juvenile has seven pairs of legs. intermediate juveniles may have . -, . -, and/or . -leg pairs. some suggest escaping mancae require a free-swimming period before they can attach to hosts; however, thatcher ( ) found they were ready to attach to fishes as soon as they left the brood pouch, and williams and williams ( c) reported that anilocra chromis williams & williams, , and cymothoa oestrum (linnaeus, ) could attach immediately. large samples of all post-manca juvenile stages (c. oestrum) have only been examined by williams and bunkley-williams ( ) . juveniles are positively phototaxic and can be captured in light traps at night. they swim to the surface light when released from the female. they can also be captured with surface plankton nets during the day. they thus appear to avoid the diurnal planktivores on the reef. adlard and lester ( ) found they rested at the surface of the water with their hooks (dactyls) through the day, but this was demonstrated in aquaria and not in the field. they descend back to the reef at night and can be caught near the bottom with diver-towed plankton nets. some have suggested juveniles must feed within - days to survive (lester ) . however, manca juveniles, unfed, for more than a week, are still capable of infecting hosts. adlard and lester ( ) reported that only half of the mancae were infective after days. juveniles of glossobius hemiramphi and livoneca ovalis (say, ) use resting hosts before locating their final hosts. cook and munguia ( ) found mancae of cymothoa excisa perty, had a window of days to infect hosts. juveniles of l. ovalis are micro-males since an adult male has never been found. resting hosts are commonly used by many species (thatcher ) and may be a part of the normal life cycle. this predisposes them to becoming micro-males. thatcher ( ) described an interesting life cycle strategy. a manca juvenile swimming right-side up (dorsal up), stops swimming, falls to the bottom landing upside-down (ventral up), and does not move. when a small fish comes near to investigate or eat it, the manca springs to life and attaches to the hapless fish. the isopod now has either a resting host to feed on or a final host on which to mature. this 'dead bug' or 'playing opossum' behaviour, we will call the opossum attack*. thatcher ( ) found mancae could feed on and kill up to four small fishes in h. this behaviour was in freshwater isopods. mancae attach all over the body of the host and move to the normal attachment site (adlard and lester ) as we have seen (williams and bunkley-williams unpublished data). legrand ( ) suggested mancae of anilocra physoides (linnaeus, ) were attracted to the motion of fish fins. some mancae fall off when they attempt to move to the normal attachment site (williams and bunkley-williams unpublished data). cook and munguia ( ) found that manca of cymothoa excisa located hosts by visual and chemical clues. the number of juvenile moults have not been determined for most species; however, williams and bunkley-williams ( ) reported finding six post-manca juveniles in cymothoa oestrum (fig. . ) . a juvenile attaches to a host and begins to develop through a series of instars: juvenile-male transitional, immature male, male, male-female transitional, immature female, virgin female (no oostegites), and ovigerous female. the second juvenile that arrives will cease developing and remain as a male, with development hormonally controlled by the female. if the female dies, the associated male resumes developing into a female. anilocra leach, , juveniles replace solitary dead females. williams and bunkley-williams (unpublished data) have often seen a juvenile attached in the attachment scar where a female was formerly located. micro-males may be attracted to dying females and begin to develop into females protected underneath the 'cougar' (old female). williams and bunkley-williams (unpublished data) have reared juvenile anilocra haemuli williams and williams, , to females and a. chromis to male-female transitionals, on their natural hosts, in the lab. adlard and lester ( ) found anilocra pomacentri bruce, , recruited from july to december, with a peak in september to october, at heron island, great barrier reef. they never saw males with females despite intense efforts in the field and laboratory. one of their hypothetical scenarios is essentially our micro-male life cycle. aneesh et al. ( ) gave the 'complete' life cycle for cymothoa frontalis milne edwards, . however, they have the same problems discussed above: ( ) omitting how the manca are naturally released (demarsupiation) and ( ) omitting the number of free-swimming juvenile stages. they did recognise six female stages. however, their stages fs- to fs- appear to be within the first female instar, which is confusing. furthermore, size alone does not determine supra-stages or instars (see size discussion below). their fs- would be our vegetative* supra-female (sf- ). their fs- to fs- would be the second marsupial stage (sf- ). overall, this does agree with our assertation that females have more than one brood with feeding vegetative stages in between. however, we think most fish isopods have more than two broods. when all brown chromis hosts with anilocra chromis on seven m segments of a linear coral reef were eliminated, and recovery followed for a year, the hosts recruited evenly from the plankton, but the isopod recruitment was significantly higher on the ends of the linear reef adjacent to areas still populated with isopods. the same result was obtained in a subsequent year (williams and bunkley-williams unpublished data). thus, the swimming juvenile stages only appear to disperse over very short distances. several studies on cymothoid-host associations have examined impacts of anilocra on components of host fitness (reviewed in chap. ). adlard and lester ( ) found that anilocra pomacentri reproductively compromised its female host. (meadows and meadows ; welicky and sikkel ) . brown chromis males infected with anilocra chromis appear unable to maintain a spawning site (williams and bunkley-williams unpublished data). contrastingly, meadows and meadows ( ) and robinson ( ) found little to no effect on host mating success or fecundity. a tenet in the strategy of fish isopod infection has always been that the first manca juvenile to arrive becomes a female and the second remains a male (epigametic sex determination). mladineo and valic ( ) and mladineo ( ) found a pair of ceratothoa oestroides manca juveniles became established simultaneously together in the mouth of a host and excluded all other mancae. aneesh et al. ( ) found something similar with cymothoa frontalis. possibly almost all fish isopod juveniles infect hosts in juvenile pairs, which we will call founder pairs*. this may change all cymothoid infection methods. life strategies sometimes consistently 'deposit' the male and the female of a pair on different sides of a host. have described these duplex arrangements for males and females in opposite gill chambers and on different sides of a fish caudal peduncle (williams and williams ) . this allows space for larger females and larger brood pouches, allows smaller fishes to be parasitised , prevents double parasitism, and separates areas of isopod feeding and damage. female hormonal fixing of their male appears to remain intact over these distances. thatcher ( ) found this arrangement with anphira xinguensis thatcher, , in the gill chambers of the beaked pacu. occasionally, site-specific isopods are found in a different location on their hosts, and they do not migrate to their normal site. when these parasites are kept from their site because of preoccupation, we call them displaced parasites*; when their normal sites are available, we say they are accidental attachments*. for example, we have seen normally under-eye anilocra attach above the eye or on top of the head when both of their normal sites were occupied. these are the normal attachment sites for other species of isopods and may help to explain how utilisation of different sites evolved. we found longsnout bullhead sculpins*, most with female elthusa sp. isopods in their gill chambers and males underneath the females. however, we also found three males, of the same size and species, attached in two of the same hosts on the body near the opercular openings (fig. . ) . the evolution of gilldwelling isopods into external attaching isopods has been previously hypothesised (brusca ) but never demonstrated. these normal female-male gill-chamberdwelling isopods with externally attaching cohorts represent the first ever observation of an example of this event (fig. . ). when two reproductive sets of the same species occupy the same host, when normally only a single set occurs, we call it double parasitism. this often occurs with the micro-male life cycle. it is common in anilocra chaetodontis williams & williams, , but less so in other anilocra spp. we found it was detrimental with a. brillae welicky, hadfield, sikkel & smit, on red hinds and coneys, and with livoneca redmanii leach, in cero (williams and bunkley-williams unpublished data). it is rare in gill-chamber isopods, and, of course cannot occur in oral cavity ones. the duplex arrangement also prevents this situation. as is typical of isopods generally, adult females continue to grow and moult on the host. older, larger females can be quite different morphologically from males and immature females (supra-females, bunkley-williams and williams ). these differences have caused many taxonomic problems. adult females moult on the host in two parts. this is probably necessary to have some hardening of the pereopods to maintain attachment on the host. we have seen this many times on anilocra spp. where the posterior part is shed usually posterior to the rd pereon, sometimes the th. there must be a considerable delay between moult halves, or we would have never seen so many. we call these mid-moult stages. the delay allows the new hooks (dactyls) to harden before the last anterior segments are shed. we have seen mid-moult stages with a large posterior body and a much smaller anterior body (williams and bunkley-williams unpublished data). we have also seen mid-moult stages in four wild cymothoa oestrum juveniles . discerning the different juvenile stages of cymothoids is most difficult. mid-moult specimens clearly mark the lower size range of each juvenile stage. when enough specimens exist to find mid-moults, the complete juvenile life cycle can be discerned. four mid-moults in c. oestrum allowed us to discern six postmanca juvenile stages ; williams and bunkley-williams unpublished data). as far as we are aware, this is first time all juvenile stages have been identified in the wild. post-juvenile mid-moults allow the elucidation of what changed in the moult and to positively identify instars. some supra-females regain their marsupium in one moult. spectacular, mid-moult stage half-female/half-male forms have been observed (williams and bunkley-williams unpublished data), as have supra-females with half formed marsupiums, indicating two moults are needed to form a marsupium (williams and bunkley-williams unpublished data). as noted above, isopod effects on hosts often cannot be measured with fish condition factors. we studied specimens ( infected) of doctorfish with - specimens of two species of adult isopods and noted there was no difference in condition factors (williams and bunkley-williams unpublished data). the isopods cause the host to grow more slowly, but they remain proportional (proportional stunting). Östlund-nilsson et al. ( ) also found no conditional factor difference and summarised the literature on this topic. a malefemale pair of livoneca redmanii in each gill chamber of mackerel will cause a decline in condition factor and often kills the host . lanzing and o'connor ( ) also found host condition was only reduced in multiple isopod infections. however, robinson ( ) found a reduced condition in bicolor damselfish infected with single anilocra partiti williams, females, and sala-bonzano et al. ( ) found no condition factor effect of ceratothoa italica schioedte & meinert, , on sand steenbras in a protected area, but severe effects in a similar overfished (unprotected) area. the prevalence of infection was also different ( vs %, respectively). obviously, the life cycle success rate was improved by stresses on the host, particularly when the lower abundance of host specimens should have had the opposite effect. the sizes of gill-and mouth-dwelling cymothoids are closely correlated with their host size due to space constraints; externally attaching ones are slightly less coordinated. however, isopod and host sizes are related suggesting they grow simultaneously. this also suggests most hosts are infected young, by young parasites, and the host and parasite grow up together. some have suggested that females only have one brood and others that they have one brood immediately after another. in many cases, the so-called virgin female (no oostegites) was the largest of the female specimens collected williams , a) . obviously, these females are in a feeding (vegetative) stage between broods. the number of adult moults and their morphological stages are not known for any cymothoid. however, aneesh et al. ( ) found six adult female stages corresponding to two of our supra-females, including a vegetative one (see above). adlard and lester ( ) found spent (demarsupiated) females could moult, feed, rejuvenate, and lay new eggs, all in one instar. no intervening vegetative instar was necessary. evidence exists to support two life cycle portions in these fish isopods: simple and complex rebrooding. simple rebrooding* with relatively small brood sizes, where the female internal organs are only flattened by the marsupium, not atrophied, and no vegetative moult is necessary to re-establish feeding. complex rebrooding* with large brood sizes, where female internal organs are atrophied, a moult to a vegetative supra-female is necessary to re-establish feeding, and a second moult is necessary to re-establish a supra-female with a marsupium. simple rebrooding is more economical and faster but limited numerically in offspring. complex rebrooding is slower and uses more resources but produces many more offspring. adlard and lester ( ) found simple rebrooding in an externalattaching isopod. found complex rebrooding in a gill chamber-dwelling isopod, williams and williams ( a) in an externally attaching isopod, and aneesh et al. ( ) in a buccal cavity-dwelling isopod. isopods producing larger broods may require the energy resources afforded by vegetative supra-female instars. of course, many other, undiscovered, reproduction scenarios probably exist. external isopods do not live as long as their hosts, as evidenced by empty attachment scars. additionally, some isopods become covered with encrusting organisms. these are likely old isopods which have ceased moulting and are about to die. bakenhaster et al. ( ) found that glossobius hemiramphi live for a year in south florida (usa). adlard and lester ( ) found anilocra pomacentri lived for a maximum of . months. bakenhaster et al. ( ) found monthly . - . % (ave. . %) prevalence throughout a year, in ballyhoo with the highest prevalences in the summer, with small, young-of-the-year hosts. with larger hosts, the prevalence exponentially declined. adlard and lester ( ) found anilocra pomacentri changed the behaviour of the great barrier reef chromis making them not migrate with uninfected cohorts. we (williams and bunkley-williams unpublished data) observed a similar phenomenon in brown chromis infected with anilocra chromis. the infected adults stay under coral heads with the juvenile brown chromis, while the uninfected adults go out above the reef slope to feed on plankton. meadows and meadows ( ) similarly found foureye butterflyfish infected with anilocra chaetodontis stayed in low-energy areas where their offspring were more available to young potential hosts. the diel migratory reef fish, french grunt, usually migrates from reef to seagrass habitat at dusk. those infected with anilocra haemuli were less likely to migrate than their uninfected schoolmates (welicky and sikkel ) . adlard and lester ( ) interpreted this as a depression of the host reproductive response to move to spawning areas on the reef slope. we interpret the observed behaviour of brown chromis to be because the infected adults cannot feed and survive in open waters (williams and bunkley-williams unpublished data). meadows and meadows ( ) suspected this was also a host behavioural change caused by the isopod. welicky and sikkel ( ) were not certain if infected fish had less energy to migrate or if uninfected cohorts harassed them. however, all four behaviour modifications, caused by the isopods, accomplished the same availability of manca juveniles to infect juvenile and young fishes. we will call this effect nursery hiding. these are newly recognised life cycle innovations caused by parasite modification of its host behaviour. among the nine species of anilocra described by williams and williams ( ) and two from japan (williams and williams a) , no males have been reported. after juveniles were found in apparent copula with females, we assumed that juveniles were functioning as males. to test this assumption, individual juveniles were raised on their host, periodically preserved, sectioned, and stained. the infective, -leg juvenile was a functional male as were all intermediates up to and including the -leg juvenile. however, as soon as a juvenile began to obtain an adult shape (juvenile-male transitional), it began to lose its male characters. only juveniles were reproductive males. this resolved the 'mystery of missing males'. we refer to this reproductive juvenile as a micro-male. juveniles with full male characters have been found in several species, but these have never been suspected to be sexually active. many species are known to use paratenic hosts (thatcher ; bakenhaster et al. ) , which may predispose them to becoming micro-males. six-legged manca juveniles are released from the marsupium (williams and williams c) , swim to the surface in the daytime, and descend near the bottom at night. they find small hosts, which do not go to cleaner fish or shrimp (blennies, gobies, cardinalfishes), attach, feed, and moult once, or several times, into -legged juveniles. in off-reef areas in venezuela, these juveniles were found on adult glasseye (bunkley- williams et al. ) . they may sense the pheromones of receptive females, leave this micro-male host, swim to the female, crawl under her, and mate belly-to-belly. we have observed them in copula and found a semen string when they were separated. they may hide under the female either before or after copulation, return to their micro-male host, or find another small host. they may eventually locate a juvenile of their definitive host and develop into a female as their host develops. no adult male stage exists. juveniles develop directly into juvenile-female transitionals, immature females, and females. we sometimes found juveniles under females or very close to females on the final host, but usually we found them on cardinalfishes, gobies, and other small fishes (williams and bunkley-williams unpublished data) . adlard and lester ( ) found anilocra pomacentri juveniles on the final host, blennies, and cardinalfishes. they apparently spend most of their time attached and feeding on resting hosts and only visit the female to mate. this life cycle is unique, not just among isopods, but in parasites in general. we are not aware of anything similar. it is a modification of juveniles on resting hosts probably driven by cleaner pressure. it seems to be widespread and highly successful. while we have not seen males associated with many other anilocra, some anilocra do have associated males. possibly, this life cycle and other host specificity differences will place the micro-male anilocra in a different genus. we are exploring this possibility. other cymothoids share this life cycle, e.g. livoneca ovalis (williams and bunkley-williams unpublished data), anilocra apogonae (see fogelman and grutter ) , and a. pomacentri (see adlard and lester ) . the advantages of this live cycle have allowed anilocra spp. to be the only large, external cymothoid isopod of larger caribbean coral reef fishes (fig. . ) . nerocila spp. occur around but are unable to penetrate any distance into the caribbean (bunkley- ; and renocila spp. only infect a few small species (fig. . ; williams and williams ) . the apparent advantages of this life cycle include the following: . it allows the female to grow larger on the host because no resources are used by a male partner (fig. . ). . two females may be supported by a host, instead of a male-female pair (fig. . ). . larger females produce more offspring and two females twice as many. . it protects vulnerable juveniles from cleaners. . it produces greater genetic diversity as many micro-males are available at different times to fertilise each female, instead of one permanent male partner. . the abundance and close proximity of numerous micro-males solve the problem of finding a mate. . micro-males form a quick reserve available for developing new females. . micro-males become sexually mature quicker than true males. . the energy necessary for a female to hormonally control a male partner is unnecessary. . larger-growing females quickly become too large for cleaner organisms to threaten. . micro-males can parasitise smaller hosts that do not seek out cleaner organisms. . micro-males can easily change host specimens and/or species and are thus more flexible and resilient. . micro-males could potentially use up the resources of one host and just move to another with little effort or danger. . the lack of host specificity in micro-males allows them to exploit a broader and more available food supply and to be more flexible and resilient. . the time a planktonic reproductive form has to locate a final host is almost infinitely extended by being able to exploit a broad range of more available smaller fishes. . copulation while on the outside of a fish host is easier and safer with a tiny micro-male than with a larger and more bulky 'true' male. . this life cycle also resolves the classic question of the expense of sexual reproduction, since all adults produce eggs. . it maintains, or even multiplies, the advantage of diversity in sexual reproduction while not sacrificing the productivity advantage of asexual reproduction. new life cycle: prey-predator transfer life cycle* many intriguing questions regarding the cymothoid life cycle still exist. these include the following: how can little, slow, juvenile fish isopods (cymothoids) possibly chase and infect fast-swimming pelagic fishes? why do juvenile isopods infect and develop into non-swimming forms in the mouths of fishes too small for them to develop into adults? juvenile isopods may not chase large, fast hosts, but rather rest and wait for small, slow ones. are little hosts dead-ends or 'bait'? apparent prey-to-predator transfer has been observed in king mackerel , shortfin smooth lanternshark , and red lionfish (aguilar-perera et al. ). connors et al. ( connors et al. ( , experimentally showed sea lice would abandon their host, when it was attacked by a predator (~ % of the time), and reattach to the predator. juvenile isopods infect the mouths of common, small, easily infected, 'bait' fishes. they feed and pass through moults from the -leg juvenile to a post-juvenile, non-swimming stage on this transfer host. at any point during this development, if the host is eaten by a predator, they flee their (prey) host and attach in the mouth of the predator host (final host). the first isopod to arrive becomes the female and the second a male, as was once thought to happen in all other cymothoids (mladineo and valic ; mladineo ; aneesh et al. ) . isopods in trap, net, and trawl fisheries are well documented to frequently abandon their host and sometimes enter a different fish host. three cases of natural prey-to-predator isopod transfers have been described aguilar-perera et al. ) , and adult and juvenile isopods have successfully, experimentally, been transferred between the same and different host species (e.g. . the attack of a predator or its feeding action may dislodge an isopod from a transfer host or the isopod may abandon the host. swallowing a single prey fish may be too fast for a transfer, but with a mouthful of prey fishes, adequate time may exist for a transfer. all transfers need not be successful, just enough. cymothoa spp. are quite common (~ %) in the two most abundant bait fishes in the caribbean and in five small species of cardinalfishes in okinawa. can all these juveniles be 'wasted' in dead-end hosts? they cannot develop into adults in these small fishes, and they can no longer swim. we have collected these juveniles from the plankton and found they attach to any available fish in aquarium experiments. is this a desperate survival mechanism or something more? we have described burst release (williams and williams c) , which is female isopods reacting to predator attack by dumping, and thus saving all her juveniles in her marsupium. these juveniles immediately attach to anything including humans. if juveniles react this way to attack, and adults are known to switch hosts, why would juveniles in a prey host not transfer to a predator? in field experiments, we found juvenile isopods first attached all over host fishes and then crawled to their adult positions. transferring juveniles could attach anywhere in the mouth or throat of predators and then crawl to their adult position. in contrast, adult transfers attach wherever they can and do not migrate aguilar-perera et al. ). infecting small, slow-swimming, schooling, abundant fishes seems rather easy as evidenced by~ % success rate. infecting fast-swimming, pelagic fishes seems almost impossible. we have observed many diverse cases where the isopods appeared to slow down their hosts and make them more susceptible to predation. in this case, it is a benefit for the completion of the parasitic life cycle and another form of a parasite modifying the behaviour of its host. the proposed life cycle occurs in cymothoa spp. and livoneca ovalis and may also occur in others. it may also occur as a supplemental life cycle to isopods that also have normal life cycles and could explain some of their unusual hosts. isopods of most predacious fishes infect juvenile hosts and mature with them. only a few older host specimens are infected. these could have been parasitised by preypredator transfer. this life cycle is unique, not just among isopods, but in parasites in general. we are not aware of anything similar, although pascual et al. ( ) reported an accidental prey-predator transfer in decapod isopods. the transfers may have begun as accidents and then gradually evolved into an important pathway. this allowed isopods to infect a variety of hosts that they could not have possibly otherwise reached. fish gnats are small ( - mm) ectoparasites on marine and estuarine fishes (smit and davies ; tanaka ) . as protelean parasites, they are only parasitic as juveniles and, due to their typically brief associations with hosts, may best be termed 'micropredators' (e.g. lafferty and kuris ) . fish gnats are perhaps best known as the main food of cleaner fishes on coral reefs (grutter ) . approximately species of fish gnats (most in gnathia leach, ) in genera and a single family are known around the world from the marine shallows to the deep sea. they occur at all latitudes but are more diverse and abundant in the tropics. the study of fish gnats has had a 'split personality' until recently, with benthic ecologists studying the adults and parasitologists the juveniles. only recently has the morphology of juveniles been included in the taxonomy (hadfield et al. ; farquharson et al. ) . however, the life cycles of six species are known (smit et al. ; tanaka ; hadfield et al. ; hispano et al. ) . even the most basic life cycle of fish gnats has only very recently been discovered (fig. . ; smit and davies ) . zuphea are the first juvenile stage of fish gnats that leave the female, find a host, gorge and swell up with blood, and become the second stage called 'praniza'. the praniza eventually drops off the host and finds a secluded place on the bottom to develop into the next zuphea (z ). z swims up, finds a host, and repeats the cycle (p ). zuphea feeding times vary from a few hours to a few days with z s taking the longest. they can be as short as an hour on coral reef fishes. zuphea may attack, feed off (snack*), and kill larval or juvenile fishes as smaller predators*. eventually, p moults into an adult (fig. . ) . in some species, in genera elaphognathia monod, , gnathia, and paragnathia omer-cooper & omer-cooper, , this occurs in one moult. in one species in genus caecognathia dollfus, , the first moult results in a pre-adult, which later moults into an adult. we call these feeding and metamorphing units serial parasites, which is somewhat similar to the life cycle found in ticks (arachnids). praniza may only stay on bony fishes for a few hours but on sharks and rays for weeks. complete life cycles vary from short to lengthy, directly in relation to seawater temperature: polar (~ - years), temperate (~ years males,~ year females), and tropical (~ months) (smit et al. ) . life cycle fluctuations appear to be seasonal in most species but could be dependent on host availability in some. adults are benthic, nonfeeding, and semelparous. global warming could make them more successful and more damaging (hispano et al. ) . some z have mouthparts too small to swallow blood cells and must feed on lymph. at z , they can feed on blood (hispano et al. ) . ota et al. ( ) appear to have solved the mystery of why p (praniza iii) were only found on elasmobranchs. they collected hundreds of praniza i and ii of gnathia trimaculata coetzee, smit, grutter, & davies, on bony fishes (teleosts) and hundreds of praniza iii only on sharks. this host switch seems to be the life strategy of all fish gnats so far found on sharks and rays. praniza iii takes the most time to feed. attaching to an elasmobranch protects this stage from cleaners. several recent studies on australian and caribbean recently settled, larval, coral reef fishes found gnathiid micropredation [¼ our smaller predator] damaged and/or killed them (artim et al. , and references therein) . this can have great importance in fish recruitment (artim et al. ) . sikkel et al. ( ) suggested gnathiids are micropredators [¼ our minipredators*] but act like parasites by not leaving the host during each larval life cycle instar (protelean). barnard, on its host fish, clinus superciliosus (linnaeus, ). image from smit et al. ( ) fish gnats are also implicated in the spread of a parasitic protozoa such as haemogregarina bigemina laveran & mesnil, , between hosts (smit and davies ) . juvenile fish gnats on fishes often cannot be identified morphologically as the taxonomy is based on adult males, although this is beginning to change in recent studies (jones et al. ) . fish gnats on coral reefs appear most active at night (sikkel et al. ) , although this may not be the case in other environments. at least in the eastern caribbean, diel activity appears to be related to both life history stage and sex (sikkel et al. ) . fish gnats appear to be generalists. they are known to parasitise families in orders of fishes; however, some fishes are infected more often and more heavily (tanaka ; coile et al. ). this could be attributable to some combination of preference, host susceptibility, and/or host behaviour. fish gnats may even appear on pelagic fishes . for example, amberjack (carangidae), which fed near the bottom, have been infected with fish gnats, and fish species which did not feed near the bottom were free of gnathiids (williams and bunkley-williams unpublished data). coile et al. ( ) found fish gnats, which fed on more susceptible hosts, produced larger, presumably more successful offspring. fish gnats can attach all over the body of fishes. smit et al. ( ) suggested where they first attach is where they stay. some studies suggested site preferences, but whether this is by selective settlement or migration is unknown. fish gnats are repelled from the skin of toxic gobies by their poison glands but do attach to their fins. it seems likely that they also avoid the toxic skin of trunkfishes (ostraciids, fishkill toxin) and also puffers (tetraodontids, fugu toxin). adult fish gnats do not feed. males attract females, usually young females with pheromones, and there are some reports of males defending or acquiring harems of females (smit and davies ) . in reality, of the few relationships known, in only two species have males been reported with many females. males do appear to fight with the big, impressive, giant mandibles, at least not under lab conditions. brood sizes vary from a few to almost and may vary by female size and environmental conditions (coile et al. and citations) . we suggested that another reason mudskippers leave the water during low tides was because fish gnats are concentrated in the remaining, small, tide pools (williams et al. ). the small atlantic cleaner gobies, with small mouths, swallow fish gnats whole. in contrast, the bigger, indo-pacific cleaner wrasses, with larger mouths, appear to bite them into pieces . some of these cleaners selectively feed on larger gnathiids (grutter ). with implications for our understanding of cleaning symbioses, cleaner fishes often do not eat the fish gnats that are not gorged with blood. is this because the blood-swollen ones are more easily found? or is this due to preference by cleaners for fish blood rather than gnats without blood? the category 'epicardeans' was formerly considered a suborder but is now an infraorder placed in suborder cymothoida. it contains described species of crab isopods and cryptic isopods. crustacean isopods are almost unique in using crustaceans as their intermediate and final hosts, with the exception of a few corallanids and a cirolanid (bruce pers. comm.) . williams and boyko ( ) call them partial castrators because reproduction is often not completely blocked. this is nutritional sterilisation, not hormonal sterilisation. boyko and williams ( ) reviewed the methods to find, collect, and preserve crustacean isopods. cryptic isopods are poorly studied, but interesting, with most of their species hyperparasitic on other isopods in their own order, or in parasitic barnacles (fig. . ) . they also parasitise a variety of free-living crustaceans. there are species in genera and nine families. they are of little commercial interest, except as potential hyperparasitic controls for other crustacean parasites. adult female cryptic isopods usually have neither pereopods nor oostegites. their epicaridian larvae must develop within the female since there is no marsupium. her body ruptures to free the larvae. they find and attach to copepods, and their life cycle is the same as in decapod isopods, except the males stay in the cryptoniscus larval form. cryptic isopoda are ecto-, meso-, or endoparasitic. liriopsids are hyperparasites of parasitic barnacles and parasites of other symbiotic crustaceans. lovrich et al. ( ) found liriopsis pygmaea (rathke, ) ( fig. . ) infected . % of the externa on the parasitic barnacle, briarosaccus callosus boschma, , parasitising false king crabs in argentina. these were mostly ( of ) cryptonicus larva. this suggests to us that most of the infective larvae, even finding a correct host, fail to infect the host. larvae were highly aggregated with . % inside empty externae, suggesting these sites attracted cryptonici. only a few early subadult females, late subadult, and one late subadult were found along with adult females. parasitic barnacles recovered from hyperparasitic sterilisation once the cryptic isopods died. cabiropsids, with species, are parasitic on free-living isopods and hyperparasitic on other crustacean isopods. the family may also include a few parasites of cumaceans. hemioniscids (barnacle isopods*), with eight species, are parasites of barnacles. dajids (backpack isopods*), with species, are external parasites of krill (euphausiids and mysids) and midwater shrimp; however, ohtsuka et al. ( ) found an endoparasitic species alternate host sharing with a copepod (see copepod section above). the parasites look like backpacks on their hosts and are often mistaken as fleshy growths or tumours. a few occur in the gills. cyproniscids, with species, are hyperparasitic on parasitic barnacles (some formerly liriopsids), occasionally directly parasitic on decapod hosts, and parasites (hypersymbiotic) on symbiotic crustaceans. podasconids (amphipod isopods*), with four species are parasites of amphipods. asconiscids only have a single species parasitic on a mysid. crinoniscids, with three species, also only have one species on a cirripede barnacle. they are parasitic on sessile and pedunculate thoracican barnacles. entophilids, with two species, are endoparasitic in callianassid shrimp and munidid squat lobsters. other cryptic isopods, besides the one we found (williams and williams ) , hyperparasitically infect the brood pouches of isopods. stone and heard ( ) found a new cryptic isopod in the serial fish isopod* excorallana delaneyi stone & heard, . many species of cryptic isopods remain undescribed. there are more than species of decapod isopods (bopyroidea). members of families bopyridae and ionidae cause a noticeable swelling of the gill chamber or carapace . the bulges they cause in the carapace of decapods make them among the most impressive and distinctive crustacean parasites. the deformities named kanthyloma crusta klompmaker, artal, van bakel, fraaije, & jagt, (ichnotaxa-trace fossil evidence), date these parasites in the fossil record back to the lower jurassic ( . - . million years ago; klompmaker et al. ) , and they have been found in~ species of fossil decapods (klompmaker and boxshall ) . they slow the growth and nutritionally sterilize some commercially important crabs and can cause the collapse of a population but have also been used as bioindicators (williams and boyko ) . eggs develop into free-swimming larvae within a ventral brood pouch (marsupium) formed of lamellar outgrowths of the female pereopodal coxae (oostegites). larvae of a single brood mature synchronously and are released simultaneously as microniscan larva (epicaridium, microniscus or microniscid). the microniscan attaches externally to a pelagc calanoid copepod, pierces its cuticle to feed on its blood, and undergoes six moults and becomes a cryptoniscan (cryptoniscus and cryptoniscid). when it drops off its copepod host, it seeks a crab or shrimp definitive host in the earliest post-larval stage. on the host, it develops into a juvenile (bopyridium) and then into a female. the first cryptoniscan arriving at a host becomes a female and the second, a male (epigametic sex determination). the female attracts a male with pheromones and hormonally controls it to remain a male similar to cymothoids. the female grows large, while the male remains a dwarf attached to the female. they are ectoparasites. females look nothing like isopods, except for some vague segmentation. males look more like isopods. they attach in the branchial chamber of their hosts. some adults do not moult to grow. partial or complete sterilisation of hosts is due to energy loss from parasitic feeding, not hormonal control as in parasitic barnacles. it is temporary and reversible once the parasite is gone. williams and boyko ( ) summarise the papers following the introduction of a nonindigenous decapod isopod, a drastic population decline of a mud shrimp, and possible collapse of a north-west pacific ecosystem. williams and boyko ( ) found double parasitism of two species in the gills and abdomen of a specimen of hermit crab in indonesia. only eight species are known and are ectoparasitic on the gills or under the abdomen of ghost shrimp. similar to bopyrids, they cause a noticeable swelling of the gill cavities. in most respects, they are like the decapod ectoparasitic isopods (above). there are species of crab mesoparasitic isopods. they are mesoparasites in the haemocoel of brachyuran and anomuran crabs. they make a small, chiselled hole through the host's exoskeleton to communicate with the environment. they release larvae, through an exit pore near the base of the fourth pereopods of the host, which follow the typical bopyrid life cycle. apparently, this also involves a copepod intermediate host, and they presumably settle as cryptoniscid larvae in the branchial chamber and then penetrate their hosts. they first become endoparasitic and later mesoparasitic. however, their life cycle is poorly and incompletely known (williams and boyko ) . females produce a posterior stalk that extends to the external environment of the host through the branchial region or eyestalks. as in bopyrids, females look nothing like isopods, except for some vague segmentation. males look more typical. some adults do not moult to grow. partial or complete sterilisation of hosts is due to energy loss from parasitic feeding, not hormonal control as in parasitic barnacles. they have been suggested as biological control agents for introduced crabs (williams and boyko ). kuris et al. ( ) suggested portunion maenadis (giard, ) could be used as a biological control of the green crab. squid are intermediate hosts for marine anchor worms and have been reported to attract many forms of crustacean parasites accidentally. pascual et al. ( ) surmised a prey-predator transfer of larval isopods when patagonian squid ate the intermediate host copepods. their cryptoniscus larvae successfully penetrated and colonised the squid. they were entirely embedded in the oral bulb at the beginning of the oesophagus of two female squid. they suspected this was only accidental parasitism; however, this could represent a strategy for host switching, new resting host, or even eventual speciation. a shift of phyla in hosts is always of interest. tanaids are a large, free-living group with only one species, exspina typica lang, , often found in the intestine and body cavity of deep-sea holothurians, assumed to be a parasite (e.g., kudinova-pasternak ) . alvaro et al. ( ) confirmed it was a parasite. many species associate with invertebrates and hexapleomera robusta moore, , even with sea turtles and the caribbean manatee from which we have collected it. most are similar to fish cymothoid isopods with a free-swimming manca leaving from the marsupium. two manca life cycle stages of one species were found in the gut of a deep-sea polychaete in the florida straits (suárez-morales et al. ). these stages are probably endoparasitic. the adults show few modifications to a parasitic life. however, the full reduction of maxillule setation only occurs in e. typica, and the bifurcated and sharply tipped dactyli in the pereopods is probably a parasitic adaptation for anchoring the crustacean in the soft tissue of the host (alvaro et al. ). the unmodified life cycles would suggest a parasitic mode of life is in the early developmental stages. many tanaidacean species make sand tunnels. this behaviour could have predisposed them to making tunnels in the body wall of holothurians. several families of shrimp are well known as associates of other invertebrates, notably sponges and corals, also bivalve molluscs and echinoderms. these species are generally categorised as commensal and not considered parasites as such. a review of these taxa is beyond the scope of the present work. shrimps* shrimp that inhabit sponges are usually referred to as commensals largely because very little is known about their habits. only a few parasitic species have been determined from two superfamilies; however, many more species are parasitic and probably not just in sponges. small, eusocial synalpheus spence bate, , carry one to a few dozen large eggs that hatch directly into benthic juveniles (duffy and macdonald ) . large, malepaired synalpheus sp. females release several hundred small, planktonically dispersing nauplii from a clutch (duffy and macdonald ) . eggs hatch into nauplii, which are feed on yolk reserves (lecithotrophic larvae), and metamorphose into zoeae. zoeae feed on algae (planktotrophic larvae) and metamorphose into myses, which look like tiny adults, and feed on algae and zooplankton. the final instar is post-larvae. typton carneus holthuis, , form heterosexual pairs and exclude conspecifics and other shrimp (negative precursor). duffy et al. ( ) found synalpheus regalis duffy, , excluded heterospecific shrimp. he also demonstrated this species had a colony hierarchy with only one reproductive female and hundreds of helpers. this was the first case of eusociality noted in a marine animal. six more, probably parasitic, species in the same genus have been found to practice eusociality. we suggest these are the first known eusocial parasites*. williams and boyko ( : fig. a ) illustrated a crab isopod, probably bopyrella harmopleon bowman, , on a sponge shrimp, synalpheus fritzmuelleri coutière, , from panama. this may be a hyperparasite, but we cannot be certain. we do not know if the shrimp was collected from a sponge and this shrimp species is not an obligate parasite. anker ( ) published a photograph of a decapod isopod on synalpheus brevicarpus (herrick, ), which is definitely a hyperparasite. several other species of sponge shrimp have hyperparasitic decapod isopods. duffy et al. ( ) showed that shrimp of the synalpheus gambarelloides group, and s. regalis, in particular, were sponge parasites. Ďuriš et al. ( ) studied typton carneus in caribbean fire sponges in belize and found it was parasitic. they also examined two other species of typton costa, , and three species in three other genera of palaemonids (pontoniinae) from the western and eastern atlantic and indo-pacific and one alpheid from the indo-pacific and found them to be parasites. their results suggested parasitism by sponge shrimp was widespread. zitzler and cai ( ) reported the first obligate sponge shrimp*, caridina spongicola zitzler & cai, , in freshwater. this spectacularly coloured, now popular aquarium, shrimp infects an undescribed spongillinid sponge in an ancient lake in indonesia. we believe it is also the first obligate parasitic sponge shrimp reported from freshwater. zitzler and cai ( ) only found diatoms in six shrimp stomachs and called them commensal. however, they have a life cycle like eusocial sponge shrimp [a few ( - ) black eggs directly develop and are released as immature adults with no planktonic phase]. have numbers (up to /host) like eusocialists. we doubt diatoms alone would support any shrimp, much less this many. all marine obligate sponge shrimp, studied thus far, are parasitic. true crabs do not make very good parasites. we would call them semi-parasites or kleptoparasites, at best. we believe this is because their basic adult structure is so very unsuited for parasitic adaption and modification. coral gall and pea crab parasites have a long fossil record but remain low in diversity. their life cycles also show no adaption for parasitism. they mostly steal their host's food, may slow host growth, but do little, if any, physical damage to the host. many reside at the uneasy border between parasitism and commensalism. we have seen indicators of the potential for the development of 'better' parasitism, for example, a superinfection of crab zoea living in the gills of a gray angelfish at mona island, puerto rico, and adult burrowing crabs living in the gill chambers and feeding on the gill filaments of two gray snappers in colombia . however, these examples were rare, in incapacitated hosts, and proved impossible to duplicate. jelly crabs benefit by their life cycles associating with gelatinous plankton through protection, transportation, food, and development faster in warmer waters and saving energy (towanda and thuesen ) . their relationships with their hosts are more complicated. ohtsuka et al. ( ) summarised the symbionts of gelatinous plankton. they found crab larvae were only associates, not parasites. however, at least four jelly crabs feed on their hosts, and they spend their entire life cycles on one host. this seems to us to be protelean parasitism. they also steal the food of their hosts, which seems to us kleptoparasitism. we know too little about most jelly crabs to determine their kind of symbiosis. towanda and thuesen ( ) closely studied one species, graceful rock crab, but did not diagnose their type of symbiosis. not much is known. adults are benthic and planktonic zoea search for and attach to jellyfish. the megalopae and juveniles develop on the host. eventually, the cypris drops off the host and develops into adults. sometimes hundreds of megalopae occur on the host. fewer juveniles are seem, which might suggest some cannabilism occurs (towanda and thuesen ) . jelly crabs steal food and eat tissues of their hosts; however, they eat jelly parasitoids, which greatly benefit their hosts. is parasitism rated as a proportion of good vs evil? we do not think so. this is mutualistic parasitism*. klompmaker et al. ( ) objected to the existing common name 'gall crab' because so few actually form real galls. whether these crab are parasites or commensals, if they damage corals, and even what they eat remains unknown (vehof et al. ). there are species in genera and a single family, which form galls in shallow reef corals and in some deep-water corals (castro ) . we have seen these crabs on coral reefs all over the world. they have separate sexes, different internal fertilization, and mate belly-to-belly. mating takes place just after the female has moulted and is still soft. females only mate once and store the sperm to fertilise all their batches of eggs. the eggs are released onto the female's abdomen, below the tail flap, secured with a sticky material, and protected there during embryonic development. females with eggs are called 'berried' (as are all egg-carrying decapods) because the eggs resemble round berries. when development is complete, the female releases the newly hatched larvae into the gall; they pass out into the water and become part of the plankton. zoea have a tall dorsal spine and may have additional spines for predator deterrence. the zoea of most species must find food (planktonic), but some crabs provide enough yolk in the eggs that the larval stages can live off the yolk (lecithotrophic). larval development is scarcely known for coral enveloped crabs but is thought to consist of at least five, and possibly seven, planktonic larval stages (van der meij ). different species may have various numbers of zoeal stages, separated by moults, before they change into a megalopa stage. this last larval stage resembles an adult crab, except for having the abdomen (tail) sticking out behind. megalopae settle in hole, cracks, or creases in living corals. after one more moult, the crab is a juvenile. the coral grows around the crab, and somehow the crab forms a gall to its particular specifications. females become sexually mature after the th instar in the gall but continue to enlarge until the th instar. they somehow manage to enlarge the gall. they produce multiple broods of eggs fertilised by the first mating. many feed on mucus secreted by the corals, inadvertently a little coral tissue, and detritus. some filter feed. males are smaller than females. females cannot leave their gall, but males can. sometimes pairs live together in one gall, in adjacent galls, or even in interconnected galls. they cause no real harm to corals. these crabs form their gall to their own size and design. related crab species form similar galls; therefore, the galls have phylogenic importance (wei et al. ) . some galls are too closed to be accessible to males. these females copulate before the gall closes, store sperm, and produce up to eight broods over the next months (vehof et al. ) . they found evidence of recent mating of females in more open galls. castro ( ) discussed all the publications concerning the food habits of gall crabs. they were largely based on supposition, and castro concluded, like vehof et al. ( ) , that we still do not know what they eat. he also found the question of their types of symbiosis unresolved. badaro et al. ( ) observed mucus feeding in the laboratory and suggested all enveloped crabs fed this way and that they are not parasites. we disagree with badaro et al. ( ) because corals are their obligate hosts and they feed off coral-produced products, if not tissues, which the corals need. that they cause little harm to corals may be true but is not relevant to their type of symbiosis. the most famous species in this group is the oyster pea crab. they are cosmopolitan, but more common in the tropics and subtropics, and speciose with species in genera and families. they are tiny soft-bodied crabs that live parasitically largely in the mantle of bivalve molluscs and in a few large gastropods, sometimes inside sand dollars and sea urchins, in the rectum of sea cucumbers, tubes of parchment worms, burrows of mud shrimp, and gills of sea squirts (castro ) . some have been reported commensally in, on, or in tubes or burrows of various invertebrates. many of these relationships are not well studied, and some may be parasitic. they retard the growth of some commercial molluscs by % causing serious problems and millions of dollars in losses in aquaculture (trottier and jeffs ) . many males venture out of their hosts to visit females in other hosts, mostly at night. this is because hosts are more active and sensitive during the day and can squash males. trottier and jeffs ( ) observed males being crushed. additionally, they are subject to predation less in the dark, than in the light of day. however, we documented the first record of predation on a male, and probably at night, since it was by a cardinalfish, on a coral reef in okinawa (williams and williams c) . pea crabs find females by their pheromones. male crabs sometimes must rub, or tickle, bivalve mollusc hosts for hours to make them open up (trottier and jeffs ) . once in the host, they copulate with the female, who never leaves the host. thus, females can become relatively larger (still only pea-size) to produce eggs, while the polygamous males are smaller and flatter to sneak in and out of hosts. in hosts with more roomy accommodations, males may reside with females. females carry egg masses attached to the pleopods, where they develop until they hatch into zoeae. usually, there are five zoeal stages, but some only have one. the zoeal and one megalopal stages are usually separated by only a few days. hernández et al. ( ) found extended parental care and the suppression of the free-living megalopa in a species associated with western atlantic ascidians. they undergo a complex metamorphosis during the post planktonic development. the male passes through two forms after the invasive stage, the pre-hard stage with a soft bare, carapace, and no swimming setae on the pereopods and the hard-stage with a hairy, hard carapace and natatory setae on some legs. the female goes through two very similar stages, only differing in the number of abdominal appendages, and five more feminine stages. population biology of few species has been studied, those in tropical and subtropical regions reproduced year round and in temperate regions seasonally during the summer. reproduction and the presence of juveniles were not related to water temperature or salinity. the greatest abundance of juveniles (pleopods poorly developed) occurred just after the peaks of abundance in gravid females. very few recruits (megalopal i) were found (peiró and mantelatto ) ; therefore, they must pass through this stage quickly. the female-juvenile correlation also suggests they do not disperse very far. de bruyn et al. ( ) examined dissodactylus primitivus bouvier, , ectoparasitic on two spatangoid echinoid (heart urchins) hosts, meoma ventricosa (lamark, ) and plagiobrissus grandis (gmelin, ), which have the entire life cycle on m. ventricosa and only adults on p. grandis, but with more fecundity. crabs detected hosts by olfactory cues. crabs from p. grandis were more attracted by this host (where the entire life cycle can be fulfilled, possible imprinting). crabs from m. ventricosa are equally attracted to either host. host switching may explain asymmetrical infection rates and specialisation on p. grandis may be in progress. jossart et al. ( ) characterised pea crabs ectoparasitic on sea urchins at discovery bay, jamaica, in which both sexes changed hosts, searched for sexual partners, and had a polygamous mating system. most mate by polygynandry between large females and wandering small males, although some by monogamy, or temporary monogamy between adults of similar sizes, and a few by swarming of males (castro ) . ambrosio and baeza ( ) found the pea crab, tunicotheres moseri (rathbun, ), did not attempt to infect previously, conspecific infected ascidian hosts, styela plicata (lesueur, ), to avoid conflict, even though this host was scarce and defence of the host was minimal. this is another variety of negative precursor. we wonder if defence of the host was once fiercer, incited this avoidance, and then faded with non-use. castro ( ) listed many damages attributed to pea crabs. the most common was slowed growth, and the most harmful, sterilization and sex reversal. he concluded that most associations were parasitic and only some more loosely associate forms in tube-or burrow-dwelling hosts might be commensal. the california bay pea crab has the distinction of being one of only two marine crustaceans on the iucn red list (wikipedia), and it is the only possible parasite on this list. the life strategies of most parasitic crustaceans are not very modified from their freeliving ancestors. with a few notable exceptions, their life cycles suggest they just do not make very good parasites. most would seem to have recently evolved into parasitism, yet fossil evidence shows otherwise. the most grossly modified females still metamorphose from simple life cycles. even the most successful group, the copepods, is still hindered in exploiting different classes of hosts by the simplicity of their life cycles. the rigidity of their life cycles seems a major limitation of crustacean parasites. part of this apparent situation may be a result of our lack of knowledge and understanding. here we described four new and innovative life cycles, complex rebrooding, mesoparasite, micro-male, and prey-predator transfer; four instances of a new life cycle host behaviour modification, nursery hiding*; a common, but undescribed, life cycle form, mid-moult stage*; two instances of parasite intraspecies facilitation, positive precursor*; parasite intraspecies antagonism, negative precursor*; an ambush life cycle strategy, opossum attack*; doubling of the normal reproductive set on a host, double parasitism*; and separated male-female pairs, duplex arrangement*. we also named and redescribed a known life cycle, simple rebrooding*. possibly, many more life cycles and modifications remain to be discovered. however, these still represent rather minor modifications. crustacean parasites lag far behind the other major parasite groups in both complexity and modification of their life cycles. mid-moult* individuals are an important new means to identify juvenile life cycle instars. they are also very useful in determining exactly what changes occur in a moult and can also be used to distinguish between supra-female instars. our term displaced parasites* refers to life cycle forms which matured in the wrong locations on their host due to their normal sites being occupied. this is also linked to superinfections*, which are mass infections of many parasites on or in a host (often resulting in the death of the host and the parasites). these occurrences strain the fabric of normal parasite relations, and life cycles, often revealing unimagined changes and trends madinabeitia and nagasawa ; ismail et al. ) . we used the term proportional stunting* to describe the slowing of growth in fishes caused by fish isopods (cymothoids). this cannot be evaluated by host condition factors, which have confused many into believing these parasites do little harm. actually, they are quite detrimental and cause major economic loses to aquaculture and commercial fisheries. the hypothesis of the first crustacean cymothoid being external attaching and then forms moving into the gills or mouth has not been supported by molecular phylogeny. the gill chamber appears to be a much more inviting and less hostile habitat for initial colonisation, as we have seen in two cases of unusual crustacean infections . our displacement cases seem to also suggest this scenario. schmid hempel ( ) did not find well-adapted parasites became harmless but rather more efficient at countering the defences of their host. many recent authors have suggested parasite evolution favours virulence. however, we report the first hyperparasite ever known to evolve into a mutualist and explain the pathway. poulin ( a) thought that parasites with few adaptions could revert to a free-living existence but cited few examples. he found no parasite reversals* to mutualism. predation has only recently been shown to occur within parasitic crustacean life cycles and cause damage and death of hosts. parasitism and predation are difficult enough to distinguish when isolated, much more so within a life cycle. as we learn more details of life cycles, predation may become important phases. we attempt to define the types involved (table . and annotated glossary (sect. . ) below). cleaners feeding on fish gnats (gnathiids) may be accessory vampires. they may more easily find and/or select larger, swollen fish gnats filled with blood. eckes et al. ( ) suggested cleaners benefited more from consuming fish mucus than fish gnats. we certainly believe they benefit more from eating blood-filled gnathiids, than ones without fish blood. we found copepod pre-adult life cycle stages were common on western pacific fishes but very rare on caribbean ones. this indicates that smaller cleaner gobies may be more efficient than larger cleaner wrasses and a factor in parasites completing life cycles. contrary to the literature, we find the wormlike copepod on sea turtles, manatees, and whales are not parasites. we described how pennella exocoeti may have speciated. flying fishes are food for many offshore large predators, which host pennella species. flying fishes were probably a downward incorporated intermediate host for pennella spp. at one point. eventually, a form became isolated and speciated into pennella exocoeti on flying fishes. fish isopods (cymothoids) seem on the verge of evolving a real intermediate host. fish gnats (gnathiids) may also be exploring intermediate hosts through micropredation. a new ergasilid copepod seems to be becoming an endoparasite. tongueworms are completely parasitic with no free-living stages, endoparasitic in an intermediate and a final host, and so modified to parasitism we cannot even equate their life cycle stages to free-living ones. all other parasitic crustaceans are incompletely parasitic in one way or another. therefore, these life cycles suggest tongue worms are not crustaceans. they further suggest tongue worms are not even related to crustaceans, and this needs further investigation. fish lice and tongue worms have long been suspected to be related on the basis of their sperm morphology. recent molecular work also finds them similar. their life cycles are the same in having no free-living larval stages but are otherwise worlds apart. fish lice are not even good ectoparasites, flitting around from host-to-host as juveniles and adults with females free-living off hosts. tongue worms are good endoparasites every step of their way. the life cycles of fish lice and tongue worms suggest they have no phylogenetic relationship. the few fossil 'tongue worms' only recently discovered are larval parasites of marine invertebrates. equating these with tongue worms of present terrestrial vertebrates with no larval stages is impossible. these fossil tongue worms may be related to extant tongue worms, but they are not their ancestors. a parallel, and completely separate, evolution is more likely. octopus copepods (harpacticoida) and tunnelling tanaidaceans (tanaidacea) have similar life histories tunnelling through the tissues of octopuses and sea cucumbers, respectively. they also represent the rare parasitic forms in their largely free-living orders. both also have commensal species on sea turtles and manatees. however, their life cycles are quite different (lópez-gonzález et al. ; alvaro et al. ) , and they reside in different classes of crustaceans. their modes of living and feeding must represent parallel evolution. anchor worms (lernaeids) and marine anchor worms (pennellidae) are an astonishing example of parallel evolution. so much so that they were originally classified together in lernaea linnaeus, . they are also the only copepods to make major host group switches: amphibia and reptilia by the anchor worm and mammalia by the marine anchor worm. even their life cycles are similar with intermediate hosts, except that it is not an obligate intermediate in the anchor worm. furthermore, the anchor worm is freshwater, and marine anchor worms are marine, and they are classified in different orders. lafferty and kuris ( ) recognised four life cycle strategies (a bit too simple) and ten trophic strategies. poulin ( b) arranged all parasites into six major life strategies (see table . ). all categories, except vector transmission, apply to crustaceans. poulin and randhawa ( ) further defined and defended the categories but made little more use of them. half were named for transmission methods and half for effects on the hosts, which seems confusing as they are not, necessarily, mutually exclusive. in order to standardise his terms, we rephrase parasitoid to 'adult injection transmitted', parasitic castrator to 'larval penetration/injection transmitted', and table . ). we also add a seventh strategy. most microbial parasites have no animate transmission agent. they contaminate potential hosts in incidentally shed host products or by long-lived free-living stages. some symbionts on sea turtles and manatees spend their entire life histories on their hosts, never leaving, never transmitted. poulin and randhawa ( ) call their categories 'dead ends', but we do not believe micropredator is necessarily a parasitological dead end. if we use the seven new transmission-standardised names to categorise crustacean parasites: . adult injection transmitted-larval parasitic copepods (sect. . . ) have a life cycle similar to parasitoids but do not kill the host, and jelly parasitoids (sect. . . ) are almost parasitoids. . larval penetration/injection transmitted-some parasitic barnacles (sect. . . ), some echinoderm copebarnacles (sect. . . ), and some crab barnacles sterilise their hosts. sterilisation is not one strategy but two. hormonal sterilisation is a permanent, parasite chemical control of a host. nutritional sterilisation is a temporary parasite use of the host resources to the extent that host reproduction cannot occur. . directly transmitted parasite-represents almost all of the crustacean parasites. poulin and randhawa ( ) found these forms were the simplest and least parasitically evolved of the parasites, which agrees with our analysis. . trophically transmitted parasite-is only found in tongue worms (sect. . , which are probably not crustaceans). marine anchor worms (pennellids) have this strategy, except the intermediate host is not eaten by the trophically higher predator. our prey-predator life cycle follows this strategy, except the parasite juvenile only moults to an adult in a paratenic (not intermediate) host, before it is eaten. . vector-transmitted parasite-does not occur in crustacean parasites. . micropredator transmitted parasite-occurs in fish lice (sect. . ), sea lice (sect. . . , caligids), jelly parasitoids (sect. . . ) and some juvenile fish isopods (sect. . . ) that are micropredators. . inanimate transmitted parasite-occurs in non-swimming fish lice (sect. . ) and whale lice (sect. . . ). williams and bunkley- made the first, large-scale comparison of caribbean and western pacific parasites of coral reef fishes using the same collection and examination techniques. in terms of crustacean parasites, they found less aegid associates and more tongue worms in the pacific. cymothoids, fish gnats, copepods, and barnacle associates were approximately equal. in contrast, non-crustacean parasites were less diverse and abundant in the pacific than the caribbean. fish lice, tongue worms, larval parasitic copepods, isopods, and whale lice lack larval dispersal stages. fish lice and isopods have free-swimming juveniles to make up for this limitation, somewhat. larval parasitic copepods, fish lice, and sea lice have free-swimming adults. many recent authors seem to equate, or even confuse, the complexity of parasitism with its severity. sometimes these do go hand-in-hand, but they are different. complexity makes parasites more resilient, adaptable, and in the case of marine anchor worms more able to switch major host groups. severity is how voraciously and efficiently parasites use host resources to successfully reproduce the most. some of the most severely parasitic crustaceans actually have rather simple life cycles (e.g. parasitic barnacles and sarcotaces). the elaborate modification of adults is also sometimes equated to severity. again, these may co-exist but are different. many crustacean parasites, in general, seem to infect young hosts and 'grow up' with them. this has been shown in many parasites where the younger hosts, even planktonic juveniles, are much more often parasitized than the larger, older ones. in addition, host tissues growing around their parasites indicate long-term association. younger hosts are easier to find and infect often occurring in inshore schools. our prey-predator transfer may be one of the only ways older host can be infected. to evolve towards greater parasitism, the life cycle stages of crustaceans must be less and more. they must be less like the free-living crustacean stages (e.g. fish lice and fish isopods) and/or must add more parasitic stages in real intermediate hosts (e.g. tongue worms). they can either convert their free-living stages to parasitism or metamorphose new parasitic stages. those few that have developed parasitic larval or juvenile parasitic stages are progressing. however, the free-living adults many retain must become parasitic. adults must also become more modified and adapted to a parasitic existence. crab barnacles have done a pretty good job of this, and a few copepod adults are well modified, but in general, crustaceans have done a terrible job of adapting to and exploiting parasitism. currently, we know only a minute fraction of the crustacean life cycles. additional studies may turn what we think we know on its head, upside down, or throw it out the window. we are fooling ourselves that the little we know is typical. we expect most known group life cycles are not only unknown but probably false. not only do we know few life cycles, but we also know few of the species in most groups, and these may have even different life cycles. also, major groups are still springing into existence. life cycle study is a field where magic is still awaiting discovery. crustacean and nematode parasites will soon dominate metazoan parasitology. we call the present dominating parasites (flatworms, tapeworms, thorny-head worms, etc.) as "climax parasites". they are ancient, stable, and perfect to take maximum advantage of the current conditions. unfortunately, they are also practically unchangeable in having no free-living forms to become parasitic, no adults in invertebrates, major parasitic modifications, and low species abundance. we call crustaceans and nematodes (ecdysozoans) as "transitional parasites". they would eventually evolve into climax parasites but now possess just the opposite of the climax characters stated above. the climax parasites have traded flexibility and the ability to innovate for stability and the maximum parasite experience. they are rigid and vulnerable with exposed multiple hosts and complex life cycles, unable to make major host group shifts, life cycle reductions or additions, and too involved in, and dependant on, stability. major global changes will leave them behind, at worse in extinction or, at best, in remnant triviality. crustaceans and nematodes will inherit the new world of parasites. an astonishing number and variety of recent authors misapply and misuse common life cycle terms. this inaccuracy creates misunderstanding and undermines scientific precision. we here precisely define these terms. abandon host-when parasites evacuate a host that has been captured, injured, incapacitated, or poisoned. accidental attachment-(a) a host-specific parasite rarely attaching to an accidental host or (b) a site-specific parasite rarely attaching in a different position. accidental (incidental) host-(a) a host on which the parasite cannot complete its life cycle; (b) also used for very rarely infected hosts on which the life cycle can be completed. aegathoa-a juvenile genus used for cymothoidae juveniles that could not be identified to genus and as such is a form genus. this should no longer be used, but inexplicably, recent uses exist. aesthetascs-chemosensory organs on the antennae and other structures of larval crustaceans used to locate appropriate hosts, virgin females, etc. aggregation-(a) usually refers to the distribution of parasites within hosts. often, these are not uniformly distributed among host but aggregated in a few hosts; (b) can also refer to a group of hosts or to a cloud of parasite infective stages. alternate host sharing-a life history strategy where two different species of parasites infect the same host, but not at the same time. ambushing-a manner of searching for hosts used by larvae and free-swimming adults, generally, resting still in the water column arched upwards at a body angle and then suddenly attacking an unaware host. used in the daytime by some forms. opossum attack is a form of ambushing. see cruising and hover and wait. androdioecy-having dwarf males and larger hermaphrodites. the small male is usually attached on or near a larger hermaphrodite. antennae-the second pair of antennae, usually longer than the first. antennulae-the first pair of antennae, which are usually shorter than the second. biological control-an agent killing or sterilising unwanted organisms. biphasic moult-a moult occurring in two posterior (first) and anterior (second) parts. this occurs in all peracarida. see moult, mid-moult stage. body-is in three sections: the head (cephalon), thorax (pereon), and abdomen (pleon). see metasome and protosome. bopyridium (juvenile)-the third life cycle stage of epicaridian crustacean isopods. brood mortality-loss of some individuals in developmental stages in the marsu-called a capsule. in some cases, both contain a parasite. some life cycle forms are encysted and/or encapsulated. see gall. definitive host (final host, primary host)-the usual, or normal, host of the parasite and the one on which it can complete its life cycle. demarsupiation-the releasing of the swimming or infective or reproducing stage from the marsupium (brood pouch). direct life cycle-involves a single host. direct develop larvae (crawl-away larvae)-larval stages that have very low dispersal potential and usually appear like the adult form of the animal. eusociality-a colony system where only the queen is reproductive and is served and protected by relatives. life cycles are direct lacking a planktonic stage. sponge shrimp (carids) are the first known eusocial parasites. exoskeleton-(a) the outer covering of crustaceans; (b) the old shell left after moulting or the exuviae. externa (plural: externae)-the outside, egg-, larvae-holding, sac of a female of parasitic barnacles (rhizocephalans). exuviae (formerly only plural, but exuvia is coming into use)-see exoskeleton. facultative intermediate host-infecting the host is not necessary to complete the life cycle. facultative parasite-may parasitise a host, but the host is not necessary to complete its life cycle. feeding stage-see vegetative stage. feminisation-parasitic barnacles (rhizocephalans) change the pleon configuration of male crabs they infcct into the female configuration to better protect their externa. cryptic isopods (cryptoniscoids) residing in the marsupium of other isopods may control their host to retain this brood pouch. final host-see definitive host. founder pair-two infective stages attaching to a host together, excluding other cohorts, and successfully developing into a male and female pair (e.g. cymothoids). fugu toxin-tetrodotoxin (ttx) in puffers, which repels gnathiids from the skin of puffers, and we believe some other crustacean parasites. gall-a plant term. it is sometimes misused for large, parasitic cysts. usually galls contain more than one parasite, often both sexes and life cycle stages. see cyst. good parasite-see true parasite and strict parasite. growth stage-a distinctly different morphological form not resulting from a moult. some pre-adults may qualify. many crustaceans have different growth stages as adults age or grow in size. see supra-female. hover and wait-a form of ambush host seeking employed by fish lice (branchiurans) during the day. see ambushing. hormonal sterilisation-a permanent, parasitic chemical control of a host. see nutritional castration and partial sterilisation. host hopping-is adults, and/or larval life cycle stages, leaving one host and finding the same, or another, within a life cycle stage. host switching-can be (a) a long-term evolutionary change usually to quite a different kind of host; (b) part of the prey-predator transfer life cycle sequence; (c) casual parasite survival (e.g. connors et al. connors et al. , ; or (d) even within a life cycle (ota et al. ) . hyperparasite (epiparasite)-a parasite parasitising another parasite. hypersymbiotic (episymbiotic)-a symbiont, usually a parasite, associating with/ infecting another symbiont, usually a commensal. immature-a young individual and another name for post-larva or juvenile. indirect life cycle-see complex life cycle. infective stage-the larval stage, which attaches to or injects infective material into a host. in many forms a cypris. infection-(a) parasitologically, a parasite or microbe invading a host (formerly referred only to endoparasites; see infestation); (b) medically, invasion of endoparasites; and (c) microbiologically, invasion of an organism that multiplies within the host (e.g. bacteria, fungi, protozoans, viruses). infestation-(a) parasitologically, ectoparasite on host can still be refer to parasites in the environment; (b) invasion of any parasite; (c) medically, sometimes used for ectoparasites; (d) medically, sometimes refers to the initial stage of parasite invasion followed by infection; and (d) common usage, aggregations of pests (e.g. mosquitoes, rats). inquiline-an animal living in another species of animal's nest, burrow, den, or resting place. instar-a larval stage between moults. for example, there are usually five naupliar instars. intermediate host-a host that a larval stage infects, feeds on, and undergoes at least one moult upon. see micro-male host, resting host, and transfer host. interna (plural: internae)-the internal and anastomosing part of a female parasitic barnacle (rhizocephalans). iteroparous-females have multiple broods, e.g. cymothoids. juvenile (immature, immature adult, post-larva)-a miniature adult. in cymothoids, the juvenile escaping the female marsupium has six pairs of legs (six-legged juvenile). see larva and manca. juvenile-like larvae-look like adults with four pairs of active thoracopods, but no suction disks. they are larvae of fish lice (branchiurans). kentrogon-an infective stage, metamorphosed from a crypis, of parasitic barnacles (rhizocephalans). the kentrogon injects a vermigon. kleptoparasites-steal the food or food stores of its host. larva (plural: larvae)-an immature greatly differing from the adult form, which must go through a metamorphosis to be an adult. some authors use the terms juvenile and larvae interchangeably; however, these are different and distinct stages, which should not be confused. lecithotrophic larva-do not feed but use their yolk for nourishment. leg-see pereopod. lice (singular: louse)-technically phthirapteran insects; fish lice are branchiurans; and sea lice are either micro stinging jellyfish (since ), bird schistosomes (since ), or caligid copepods (since~ ); and whale lice are cyamid amphipods. tongue-eating louse is an incorrect name for the famous tonguereplacing isopod. life cycle-development from conception until the organism produces its own offspring. its study often emphasises stages of development. see life history. life cycle abbreviation-the brooding or elimination of normally free-living larval stages. life cycle truncation-the elimination of parasitic larval life stages. life history-reproductive strategies and traits plus other key events in the life of an organism. see life cycle. lifestyle-the mode of life. habit (behaviour) and habitus (form). mobility, feeding, nutrition, habitat, activity period, etc. major host group switch-parasitising a new class of host. manca juvenile (plural: mancae) (aegathoa, manca, micro-male, pullus ii)-the post-larval juvenile of many pericardians (not amphipods), which leaves the brood pouch. see aegathoa. marsupium (brood pouch)-formed of lamellar outgrowths of the female pereopodal coxae (oostegites). this pouch is under the ventral body surface of the female. see complex rebrooding and simple rebrooding. marsupium stages-various egg, embryo, and larva stages. this embryology is seldom considered in life cycle studies. megalopa-a post-larval crab. extreme transformations occur in this stage. mesoparasite-(a) inside their host but retains a pore or hole connecting to the outside; (b) half-in-and-half-out as anchor worms (lernaea); and, quite confusingly, (c) also used for parasites that enter host orifices (ear, nares, etc.). the latter might be less confusingly called orifice parasites. metanauplus (pleural: metanauplii)-see nauplius. metanauplus-like larvae-are similar to crustacean nauplii, but not metanauplii because of post-mandibular appendages and differentiated first thoracopods. they are larvae of fish lice (branchiurans). metasome (tail)-the posterior part of the protosome. micro-male-a -, . -, . -, . -, -leg juvenile functional male of anilocra spp., livoneca ovalis, and possibly other cymothoids. micro-male host-a resting host for some juvenile fish isopods (cymothoids) on which moults from the -leg to -leg micro-male stage can occur. this is not an intermediate host because no larval stages are involved. the micro-male leaves this host to fertilise a female on the final host. it may return to this host specimen or another micro-male host. microniscan larva (epicaridium, microniscus or microniscid)-the first larval stage of epicaridian crustacean isopods. micropredator-like a mosquito; a small predator feeding on a big host and not spending much time with its macroprey. predator less than times size of prey. see minipredator, smaller predator, and table . . mid-moult stages-a form of half delayed moulting found in some parasitic forms that must stay firmly attached to a host. the first half moults as usual, but the second half moult is delayed until the first half hardens. minipredator-a small predator that may, or may not, kill its prey and spends little time with it (feeds and leaves) but is greater than / of its host's size, but less than / (e.g. gnathiids, cirolanids, some corallanids, some leeches). see micropredator, smaller predator, and table . . molt (apolysis, ecdysis)-the splitting of the outer covering (cuticle carapace, etc.) in order for a larger and/or different form to emerge in the life cycle. outside the usa, the preferred british spelling is moult. multiple parasitism-when one host is infected with two, or more, different species of parasites representing, three or more reproductive sets. see double parasitism. mutualistic parasitism-when the symbiont is both harmful and helpful. similar to sickle cell anaemia and malaria. mutualistic symbolism-when both host and associate benefit from the other. natural history-the interactions of an organism with its environment that influence behaviour, forms, function, and abundance. see life history, life cycle, and lifestyle. nauplius stage (plural: nauplii)-is characterised by the use of the appendages of the head (antennae) for swimming. this first series of larvae has been shared by almost all crustaceans for the last half billion years. negative precursor-when one species of parasite or commensal infects a host first and causes a second species of parasite to be less successful in infecting the same host. see positive precursor. non-swimming larvae-the odd larvae of chonopletis, a fish louse (branchiurans). nursery hiding-infected adult hosts staying with juveniles or young of the same species instead of migrating elsewhere with non-infected adults. nutritional sterilisation-is a temporary parasite use of the host resources to the extent that host reproduction cannot occur. see hormonal castration and partial castration. nymphs-are small - -legged, larval forms of tongue worms (pentastomids). obligate parasite-is a species that must infect a particular host to complete its life cycle. onychopodid larva-replaces the on-host copepodid larva in gonophysema gullmarensis bresciani & lützen, , a copepod parasite of a tunicate. oostegites (marsupium coverlets)-see marsupium. opossum attack-when a manca juvenile swimming dorsal side up, stops swimming, falls to the bottom, lands dorsal side down, and does not move. when a small fish comes near, the manca springs to life and attaches. see ambushing. orifice parasite-a term we prefer in place of mesoparasite for parasites living in natural orifices or openings in hosts. orthonauplius-a nauplius but has a shorter head, antennula, and two additional pairs of limbs. ova-eggs. overdispersed (aggregated, clumped)-in parasitology is a distribution with a higher variance than expected (e.g. when most hosts have a few parasites, but a few have many). pantochelis larva-the first larva of jelly parasitoids (hyperiideans) with four cheliform legs (pereopods) and an unsegmented and limbless metasome and urosome. it metamorphoses into a protopleon larva. prey-predator transfer host (prey host)-a resting host for some juvenile fish isopods (cymothoids) on which moults from the six-legged manca juvenile to a post-juvenile stages can occur. primary host-see definitive host. proportional stunting-parasites slowing the growth of their host in a way that cannot be discerned in length-weight condition factors. protelean organisms-have larvae that are parasites, usually endoparasites, and free-living adults. protopleon larva-the second larval series of jelly parasitoids (hyperiids) with a segmented metasome and imperfect pleopods. it metamorphosed from a pantochelis larva and gives rise to a juvenile. pseudoparasite-our term for something that almost everyone accepts as, and calls, a parasite, but is not (e.g. most leeches). pullus ii (plural pulli)-see manca larva. pupa-the unique, cigar-shaped, endoparasitic nauplii of larval parasitic copepods (monstrilloids). reproductive stage-see vegetative stage. recently encysted (copepodids - )-pandarid copepodid stage (lewis ) . see late encysted. resting host-a host on which infective larvae or juveniles rest and feed. larvae do not go through a metamorphosis on this host. see intermediate host, micro-male host, and transfer hosts. sea lice-caligoid copepods that infect marine aquaculture fishes, especially salmon. many recent authors have called them fish lice, which is incorrect. see lice. secondary host-a host less often infected than the preferred host or definitive host but on which the parasite may complete its life cycle. semelparous-females that have only one brood. serial parasitoid-similar to a parasitoid in brooding its young in a host, but may use and kill more than one host as a nursery, and also may feed on, and kill, multiple hosts, as an adult. serial parasite-an obligate parasite that largely depends on host, or hosts, for nutrition. it is not simply a minipredator, because it associates with a host longer than to feed and leave and has one or more life cycle stages that exist multiple times potentially on and off the same or different hosts. see temporary parasite. simple life cycle-see direct life cycle. simple rebrooding-new marsupial reproduction after a single moult. the internal organs are only flattened by a brood pouch with a moderate number of off spring. the female can feed as soon as she moults. see complex rebrooding. site specific-when a parasite almost always attaches in the same position on or in a host. see accidental attachment. smaller predator-not a great deal smaller than its prey, attaches to, feeds on, and kills the host. it is an, hitherto, unrecognised life cycle phase (minipredator phase) in parasitic crustacean life. see micropredator, minipredator, and table . . spillover-a term for the parasites, principally salmon sea lice, produced in fish farms, infecting native fishes. 'spillover' suggests the pen is so full of free-swimming, infective parasites (copepodids) that a few are forced out. see filtering. sterilisation-the act of making an animal unable to reproduce. see partial sterilisation, hormonal sterilisation, and nutritional sterilisation. strict parasite-see true parasite. superinfection-a mass infection with about as many parasites that can fit on or in a host. this usually results in the death of the host and the parasites. supra-females (post-adult stages, supra-adult)-stages after the first adult female stage with size and/or morphological differences with the first adult and each other. these can be growth stages or instars and often occur in crustaceans. supra-males-see supra-females. tantulus larva-the only larval stage of minute crustacean parasites (tantulocarids) with a dorsal head shield, six-segmented thorax, and biramous urosome. temporary parasite-(a) a facultative parasite; (b) a parasite that survives for a time after ingestion by a host species other than its customary host; (c) a minipredator, associated with a prey item just long enough for it to be called a host but largely free-living and rarely found associate with a host; or (d) a parasite with any freeliving stage. thoracopod-see pereopod. transfer host (prey host)-a resting host for some juvenile fish isopods (cymothoids) on which moults from the -leg manca juvenile to a post-juvenile stages can occur. this is not an intermediate host because no larval stages are involved. see paratenic host, micro-male host, and prey-predator life cycle. transitional parasite-a crustacean or roundworm (nematoda) in the clade ecdysozoa. they are relatively new parasites with many free-living relatives and little evolved towards a climax parasite state. see climax parasite. transport host-see paratenic host. trichogon-injected into a female by a male crypis, of parasitic barnacles (rhizocephalans), and becomes a dwarf male. trophic transmission-see parasite-induced trophic transmission. true parasite (good parasite, strict parasite)-a small organism, which feeds from and harms a larger organism. true parasites have a definitive host, which they never leave. true predator (carnivore)-an organism that hunts, kills and eats other organisms (prey) . see ambushing, opossum attack, and vector mutualists-when a disease or parasite transmitted to a host also benefits the vector. when crustacean parasites are vectors, the infective stage of the crustacean benefits by the invasive transferred organism confusing the immune system of the host and increasing infective crustacean survival. see viral crustacean mutalists. vegetative stage-is a term we borrowed from botany to describe a feeding and nonreproductive female stage of fish isopods (cymothoids) between stages with a marsupium. see complex rebrooding. vermigon-a migratory internal stage injected by the kentrogon stage of a parasitic barnacle (rhizocephalans). viral crustacean mutualists-a virus benefiting its crustacean host and doing little or no harm to the host. virgin female-a term used in the literature for an adult female lacking a marsupium. a vegetative stage is a better term since these stages can occur both before and after stages with a marsupium. whale lice-see lice. y-cypris (plural: y-cyprii)-see y-nauplius. y-nauplius (plural: y-nauplii)-the first larval stage of y-parasites (facetotectans) and y-cypris the second. ypsigon-a slug-like, unsegmented, and limbless form, which may be the infective stage for y-parasites (facetotectans). zoea stage (plural: zoeas or zoeae)-the first larval stage of a decapod characterised by the use of the thoracic appendages for swimming and a large dorsal spine. zuphea-the first juvenile of fish gnats (gnathiids) with obvious segmentation, which leaves the female, finds a host, feeds, and becomes the second stage, a praniza (p ). common names are listed alphabetically in bold with and family names in square brackets: and the larvae feed off the host and eventually kill it and escape as adults. larval parasitic copepods (monstrilloids) have a similar life cycle, but do not kill their host. paratenic host (transfer host, transport host)-like an intermediate host parthenogenesis-asexual reproduction by females without the need of males. partial sterilisation-a term used by williams and boyko ( ), as 'nutritional castration', where some reproduction still occurs. pereon-the thorax of the thorax (cephalothorax, pereon) of crustaceans. periodic parasite-acting like a facultative parasite, on a host for short periods of time, but free-living most of the time. does not obtain most of its nutrition from a host or hosts. not a serial parasite, temporary parasite, or true parasite. permanent parasite-is an obligate parasite, which spends more than one generation without leaving a host. see temporary parasite. planktotrophic larva-feeds on plankton ) the abdomen (pleon) of crustaceans. positive precursor-when the presence of one parasite facilitates infection by another. the interspecific form is fairly common; however, intraspecific forms are rare. this is when one species of a parasite or commensal infects a host first and causes a second species of parasite to be more successful in infecting the same host post-larva (plural: post-larvae) (immature, juvenile, pre-adult)-resembles the adult and characterised by the use of abdominal appendages (pleopods) for propulsion. praniza (formerly pranzia)-see zuphea predisposition-(a) some hosts of crustacean parasites appear to be predisposed to the presence of parasites (williams et al. ); (b) also may refer to a pre-existing condition or behaviour preferred host-see secondary host and definitive host pre-larvae (pre-zoae)-the first stage after an egg hatches. usually held in a marsupium in isopods pre-manca-the isopod brood-pouch stage after eyed embryo before manca and is less developed and has less setae. prevalence collection effect-when infected hosts are impaired by their parasites and are more readily to be collected (e.g. dip net, seine, trawl), or less likely to be collected (e.g. hook and line, baited traps), than uninfected ones. prey-predator transfer-fish isopods (cymothoids) from prey fishes can transfer to the predators that eat them brown mussel, perna perna (linnaeus, ) epinephelus chlorostigma (valenciennes, ) california bay pea crab, parapinnixa affinis holmes caribbean fire sponges, tedania spp trichechus manatus manatus linnaeus scomberomorus regalis (bloch, ) common fish louse, argulus foliaceus (linnaeus, ) epinephelus fulvus (linnaeus, ) cressey's sea louse, caligus rogercresseyi caranx hippos (linnaeus, ) acanthurus chirurgus (bloch, ) coryphaena hippurus (linnaeus, ) epaulette shark, hemiscyllium ocellatum (bonnaterre, ) false king crab, paralomis granulosa (hombron & jacquinot, ) chaetodon capistratus linnaeus haemulon flavolineatum (desmarest, ) heteropriacanthus cruentatus (lacepede, ) [priacanthidae]; goldfish, carassius auratus (linnaeus, ) graceful rock crab, metacarcinus gracilis (dana, ) (cancer sometimes still used) serranus tigrinus (bloch, ) japanese louse (also goldfish louse), argulus japonicus thiele jelly isopod, anuropus spp pomacanthus arcuatus (linnaeus, ) lutjanus griseus (linaeus, ) eschrichtius robustus (lilljeborg, ) green crab, carcinus maenas (linnaeus, ) king mackerel, scomberomorus cavalla (cuvier, ) micropterus salmoides (lecepede, ) marine anchor worm, pennella spp non-swimming fish lice, chonopeltis spp balaenophilus manatorum oyster pea crab, zaops ostreus (say, ) loligo gahi (orbigny, ) cichla ocellaris (bloch & schneider, ) [cichlidae]; pink salmon, salmo gorbuscha (walbaum, ) tegastes acroporanus humes epinephelus guttatus (linnaeus, ) pterois volitans (linnaeus, ) holacanthus tricolor (bloch, ) epinephelus adscensionis (osbeck, ) salmon sea louse, incorrectly called 'salmon louse', lepeophtheirus salmonis (krøyer, ) sea firefly, cypridina hilgendorfii (müller, ) 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(copepoda: monstrilloida) and other parasites in the brown mussel perna perna from brazil discovery of a new genus of tanaidacean (crustacea: tanaidacea: mirandotanaidae) found associated with a deep-sea terebellid polychaete late postnaupliar development of monstrilla sp. 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(copepoda: harpacticoida) from the antarctic fish rhigophila dearborni dewitt key: cord- -u zjzqbr authors: demos, terrence c.; webala, paul w.; goodman, steven m.; kerbis peterhans, julian c.; bartonjo, michael; patterson, bruce d. title: molecular phylogenetics of the african horseshoe bats (chiroptera: rhinolophidae): expanded geographic and taxonomic sampling of the afrotropics date: - - journal: bmc evol biol doi: . /s - - - sha: doc_id: cord_uid: u zjzqbr background: the old world insectivorous bat genus rhinolophus is highly speciose. over the last years, the number of its recognized species has grown from to , but knowledge of their interrelationships has not kept pace. species limits and phylogenetic relationships of this morphologically conservative group remain problematic due both to poor sampling across the afrotropics and to repeated instances of mitochondrial-nuclear discordance. recent intensive surveys in east africa and neighboring regions, coupled with parallel studies by others in west africa and in southern africa, offer a new basis for understanding its evolutionary history. results: we investigated phylogenetic relationships and intraspecific genetic variation in the afro-palearctic clade of rhinolophidae using broad sampling. we sequenced mitochondrial cytochrome-b ( bp) and four independent and informative nuclear introns ( bp) for individuals and incorporated sequence data from additional individuals on genbank that together represent of the currently recognized afrotropical rhinolophus species. we addressed the widespread occurrence of mito-nuclear discordance in rhinolophus by inferring concatenated and species tree phylogenies using only the nuclear data. well resolved mitochondrial, concatenated nuclear, and species trees revealed phylogenetic relationships and population structure of the afrotropical species and species groups. conclusions: multiple well-supported and deeply divergent lineages were resolved in each of the six african rhinolophus species groups analyzed, suggesting as many as undescribed cryptic species; these include several instances of sympatry among close relatives. coalescent lineage delimitation offered support for new undescribed lineages in four of the six african groups in this study. on the other hand, two to five currently recognized species may be invalid based on combined mitochondrial and/or nuclear phylogenetic analyses. validation of these cryptic lineages as species and formal relegation of current names to synonymy will require integrative taxonomic assessments involving morphology, ecology, acoustics, distribution, and behavior. the resulting phylogenetic framework offers a powerful basis for addressing questions regarding their ecology and evolution. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. we remain in an era of biological discovery [ ] , even for supposedly well-known vertebrate groups such as mammals. in the last years alone, the total number of mammal species has grown by fully %, while the accumulation of new bat species ( . %), especially in tropical regions, has grown even faster [ , ] . the discovery of new bat species in the afrotropics (africa south of the sahara, including madagascar and continental shelf islands) has paralleled these global trends, buoyed by continuing geographic and taxonomic surveys of bats across the region, a growing number of systematic investigations using molecular phylogenetic and integrative taxonomic approaches, and the use of more powerful and objective means of assessing species boundaries. the species limits of morphologically conservative or cryptic lineages of bats have been greatly clarified by an integrative approach using multi-locus genetic delimitation methods as a starting point for identifying candidate species and then testing them using additional, corroborative data from behavioral, morphological, distributional, and/or ecological information ( [ ] , cf. [ ] ). new species have also come to light via collecting in previously unsampled regions and through genetic analysis of ancient dna using new methods [ ] [ ] [ ] [ ] . the genus rhinolophus offers an instructive example. the sole living genus of the paleotropical (and southern palearctic) family rhinolophidae, rhinolophus is the second-most speciose genus of bat (after myotis). over the last years, the number of its recognized species has grown from ( of them afrotropical; [ ] ) to (with afrotropical; [ , ] , an enormous % increase. in this study, recent intensive surveys in east africa and neighboring regions of africa, coupled with parallel studies by others in west africa and in southern africa, permit a new region-wide multi-locus phylogenetic study of the genus.. rhinolophidae has been arranged taxonomically on the basis of molecular and morphological data into subgenera by csorba et al. [ ] . of these subgenera, the subgenus rhinolophus is restricted to africa and the palearctic; it includes species groups whose names represent the nomenclatural framework for this study: ( ) r. landeri group (landeri, alcyone, guineensis, lobatus); ( ) r. euryale group (euryale, blasii, mehelyi); ( ) r. capensis group (capensis, denti, gorongosae, rhodesiae, simulator, swinnyi); ( ) r. adami group (adami, maendeleo); ( ) r. ferrumequinum group (ferrumequinum, bocharicus, clivosus, damarensis, deckenii, hillorum, horaceki, nippon, sakejiensis, silvestris); ( ) r. maclaudi group (maclaudi, hilli, kahuzi, ruwenzorii, willardi, ziama); ( ) r. fumigatus group (cohenae, fumigatus, darlingi, eloquens, hildebrandtii, mabuensis, mossambicus, smithersi). of the currently recognized afrotropical rhinolophus species [ , ] , our study includes at least named taxa (fig. ) . multiple well-supported and deeply diverged clades are also revealed by our phylogenetic analyses. coalescent species delimitation methods incorporate phylogenetic uncertainty in gene trees and jointly infer species limits and species phylogenies. they have been shown to be conservative in that high delimitation posterior probabilities are consistent indicators of species status ( [ ] and references therein). briefly, methods such as the software bpp [ , ] infer statistical support for genetic isolation on an evolutionary timescale. however, species delimitation based exclusively on molecular data is controversial. it has been shown that multispecies multilocus coalescent delimitation methods can confound species-level and population-level processes and delimit population structure rather than species when the speciation process is protracted ( [ ] but see also, [ , ] ). however these debates on the status of delimited lineages are resolved, the multispecies coalescent remains a powerful method for inferring the evolutionary independence of lineages that can be subsequently tested with independent data (e.g., morphology, and bioacoustics data in bats) to assess species status in an integrative taxonomy [ ] . in this study we carry out lineage delimitation as a foundation for subsequent taxonomic revisions (see [ ] ). we do not claim that lineages distinguished by our analyses substantiate the boundaries of species; for this reason, we do not formally name these delimited lineages pending integrative taxonomic revision. the goals of this study are to identify evolutionary lineages among the afrotropical rhinolophus and to assess their phylogenetic relationships. lineages for which we were unable to assign confident names (here considered putative species) are considered hypotheses for later testing via integrative taxonomy. more than half of the sequence data used in this study are newly generated, extending the multi-locus analysis of dool et al. [ ] with substantial new material obtained in bat surveys of western, central, eastern, and southern africa. our expanded geographic, taxonomic, and population level sampling enables a more robust assessment of phylogenetic relationships and population structure among afrotropical rhinolophus. the intron data set used here has strong advantages over using mitochondrial loci alone and offers independent representation of the nuclear genome as each of the four introns are found on different chromosomes [ ] . incorporating independent genomic regions into phylogenetic analysis of this monogeneric family [ , ] and assessment of species relationships and limits is crucial because several instances of mitochondrial introgression have been documented within rhinolophidae [ , [ ] [ ] [ ] . other instances of possible mitochondrial introgression were investigated via comparisons between our intron phylogenies and those generated with mitochondrial sequences. finally, using our well resolved nuclear gene tree and species tree, we assess support for broad biogeographic patterns in a comparative context to studies of other afrotropical bats [ , , , , ] . all new genetic data from tissue samples used in this study (n = ) were obtained from specimens previously catalogued and part of the permanent collections of the following natural history museums: field museum of natural history, chicago, usa; biodiversity research and teaching collections, texas a&m university, college station, usa; royal ontario museum, toronto, canada; national museums of kenya, nairobi, kenya; and durban natural science museum, durban, south africa. no animals were collected in this study; all tissues were parts of permanent research collections. tissue samples available from kenya and tanzania was especially dense (fig. ) . initial assignments to species were based on the bats of east africa key in [ ] . an additional cytochrome-b (cyt-b) sequences and nuclear intron sequences for each of the introns acox , cops a, rogdi, and stat a of rhinolophus were downloaded from genbank from a total of individuals. a species in the recently resurrected genus macronycteris [ ] , m. vittatus (hipposideridae) was used as an outgroup. in total, individuals with - genes were analyzed for our study (see additional file for voucher numbers, locality data, and genbank accession numbers). to avoid adding to taxonomic confusion in rhinolophus, we purposefully took a conservative approach to the nomenclatural consequences of our analyses. where an apparent group's taxonomic identity is unknown or fig. type localities for recognized species of rhinolophus (black circles), as well as subspecies and synonyms (white circles); label names represent the specific epithets of currently recognized species. biomes of africa and neighboring regions indicated by color shading, dark yellow: tropical and subtropical moist broadleaf forests; orange: flooded grasslands and savannas; gray: tropical and subtropical grasslands, savannas, and shrublands; olive brown: deserts and xeric shrublands; gray-green: tropical and subtropical moist broadleaf forests; peach: mangroves; ochre: mediterranean forests, woodlands, and shrub; dark tan: tropical and subtropical dry broadleaf forests [ ] ambiguous, we refer to it as a numbered clade. this approach was applied to specimens provisionally assigned to combined rhinolophus fumigatus and r. eloquens clades that are labeled fumigatus/eloquens. these names are used as explicit labels for our analysis but cannot vouch for their validity with respect to other taxa that might have nomenclatural priority. morphological assessment of the clades supported by our analyses will be necessary to determine which existing names can be applied to them. we generally used the methodology previously described by demos et al. ( ) and patterson et al. ( ) in the generation and analyses of genetic data for this study. whole genomic dna was extracted using the wizard sv genomic dna purification system (promega corporation, wi, usa). specimens were sequenced for mitochondrial cytochrome-b (cyt-b), using the primer pair lgl f and lgl r [ , ] , and four unlinked autosomal nuclear introns: acox intron (acox ), cops a intron (cops a), and rogdi intron (acox , cops a, rogdi; [ ] ); and stat a intron (stat a; [ ] ; table , supplemental material). internal primers were designed for the cyt-b gene to amplify degraded dna from a museum skin of putative rhinolophus landeri from cameroon, the nearest topotype available from a voucher specimen (additional file ). pcr amplifications were carried out using the same thermocycler protocols as in [ ] . amplified products were purified using exosap-it (thermo scientific, ma, usa). sequencing was performed on an abi prior distributions on τ represent two relative divergence depths (deep and shallow) and on θ represent two relative mutation-rate-scaled effective population sizes (large and small) thermocycler (applied biosystems, ca, usa) at the pritzker laboratory for molecular systematics and evolution, field museum of natural history (fmnh). sequences were assembled and edited using gen-eious pro v. . . (biomatters ltd.). sequences were aligned using muscle [ ] with default settings in geneious. protein coding data from cyt-b were translated to amino acids to set codon positions and confirm the absence of premature stop codons, deletions, and insertions. several gaps were incorporated in the alignments of the nuclear introns, but their positions were unambiguous. gene trees, species trees, and summary statistics jmodeltest [ ] on cipres science gateway v. . [ ] was used to determine the sequence substitution models that best fit the data using the bayesian information criterion (bic) for cyt-b and the four nuclear introns. uncorrected sequence divergences (p-distances) between and within species/clades were calculated for cyt-b using mega x . . [ ] . maximum likelihood estimates of cyt-b gene trees and a concatenated alignment of the four partitioned introns were made using the program iq-tree version . . [ ] on the cipres portal. we conducted analyses using the ultrafast bootstrap algorithm to search for the bestscoring ml tree algorithm [ ] with bootstrap and topology replicates. bayesian gene-tree analyses were carried out in mrbayes v. . . [ ] on the cipres portal to infer individual gene trees for cyt-b, the four individual nuclear introns, and the concatenated partitioned alignment of four nuclear introns. two replicates were run to assist proper mixing. nucleotide substitution models were unlinked across partitions and then allowed to evolve at individual rates for each locus in the concatenated alignment. four markov chains with default heating values were run for × generations and sampled every th generation. stationarity of mrbayes results was assessed using tracer v. . [ ] . majority-rule consensus trees were inferred for each bayesian analysis. african taxa assigned to species or clades and named based on support for such clades in the bayesian and ml analyses of the cyt-b and nuclear intron datasets. thus, results from gene-tree analyses were used to define populations to be used as 'candidate species' (as in [ ] ) in a coalescent-based species-tree approach implemented in starbeast [ ] , an extension of beast v. . . [ , ] . species-tree analysis was conducted using the four nuclear intron alignments. substitution, clock, and tree models were unlinked across all loci. a lognormal relaxed-clock model was applied to each locus with a yule tree prior and a linear with constant root population size model. analyses were replicated four times with random starting seeds and chain lengths of × generations, with parameters sampled every , steps. for the starbeast analyses, evidence for convergence and stationarity of the posterior distribution of model parameters was assessed based on ess values > and examination of trace files in tracer v. . . burn-in was set at %, and separate runs were assembled using logcombiner v. . . and treean-notator v. . . [ ] . we conducted joint independent lineage delimitation and species-tree estimation using the program bpp v. . [ , ] . this analysis was carried out to guide future investigations of the lineages inferred here, using an integrative species taxonomic approach to include fixed differences in phenotypic characters, acoustics, ectoparasitic associations, and geographic distributions. bpp analyses were carried on those populations obtained from the concatenated gene-tree analyses and were identical to specimens assigned to lineages in the species-tree analyses. each population was designated as a putative independent lineage to be evaluated under the multispecies coalescent model [ and references therein]. separate analyses were carried out for lineages within each of four different rhinolophus species groups: capensis group, six lineages; ferrumequinum group, six lineages; fumigatus/eloquens group, eight lineages; and landeri group, four lineages. the validity of our assignment of specimens to populations was tested using the guide-tree-free algorithm (a ) in bpp. two replicates were run for each of four different combinations of priors on divergence depth and effective population sizes (τ and θ, respectively; table ), as the probability of delimitation by bpp is sensitive to these two parameters [ , ] . all bpp analyses were run for × generations, with a burn-in of generations and samples drawn every th generation. in total, eight bpp runs were carried out for each of the aforementioned species groups using nuclear intron loci (n = ). lineages were considered to be statistically well supported when the delimitation posterior probabilities generated were ≥ . under all four prior combinations. all newly generated sequences were deposited in genbank with accession numbers mn -mn ; (see also additional file ). sequence alignments used in this study have been made available on the figshare data repository (doi: https://doi. org/ . /m .figshare. ). the alignment of cyt-b sequences used in the ml and bi gene-tree analyses had a total number of base pairs (bp) ranging from to , and averaged % coverage of the complete cyt-b gene ( bp). to aid in visualizing the phylogenies inferred from this matrix, we reduced a matrix of individuals to a set of mostly unique sequences, resulting in a final alignment of individuals. the number of base pairs for the sequence alignments used in individual ml and bi gene trees and bayesian species tree analyses were: acox (n = ml and bi, species tree), - bp; cops a (n = ml and bi, species tree), - bp; rogdi (n = ml and bi, species tree), - bp; stat a (n = ml and bi, species tree), - bp; and intron concatenated alignment (n = ), - bp. the best supported substitution models for each locus estimated by jmodelt-est were: sequence cyt-b = gtr + i + g; acox = k + g; cops a = hky + i; rogdi and stat a = hky + g. uncorrected cyt-b p-distances for african rhinolophus in the sequence cyt-b alignment (removing eurasian sequences except for r. hipposideros and r. xinanzhongguoensis) ranged from . to . between species/clades, while within species/clade distances ranged from . to . (additional file ). maximum likelihood (ml) and bayesian inference (bi) inferred trees with similar topologies; the ml gene tree is shown for the sequence cyt-b alignment of rhinolophus species/clades ( fig. ; see also additional file for the phylogeny with all terminals labeled). in the cyt-b gene tree, a majority of sub-saharan taxa were strongly supported as monophyletic (i.e., maximum likelihood bootstrap support [bs] ≥ %, bayesian posterior probability [pp] ≥ . ), with several exceptions detailed here. for sub-saharan african rhinolophus, there were four major well-supported monophyletic endemic haplogroups: a) the fumigatus species group that includes eight r. fumigatus/eloquens clades, two r. hildebrandtii clades, and r. darlingi; b) the maclaudi species group that includes r. ruwenzorii and r. willardi, whose phylogenetic position is unresolved; c) the capensis species group that includes two r. simulator clades, r. denti, r. capensis, r. swinnyi, and two clades provisionally labeled as cf. denti/simulator and cf. simulator distributed widely south of the sahara; and d) the landeri species group consisting of two r. landeri clades, r. lobatus, and r. alcyone. the phylogenetic position [ ] of r. damarensis, recently elevated because it rendered r. darlingi paraphyletic [ ] , is uncertain. rhinolophus damarensis as currently known is associated with arid southern african habitats. however, a newly available specimen collected in western democratic republic of congo (drc; guineo-congolian rainforest in [ ] ) is unexpectedly nested within the r. damarensis cyt-b clade. of the two species groups whose members include both african and palearctic species, the ferrumequinum species group is strongly supported as sister to fumigatus + maclaudi + damarensis while the monophyly and position of the euryale species group is poorly supported. species from eastern eurasia + australasia cluster outside of african clades in the cyt-b tree with two notable exceptions. first, r. nippon [formerly r. ferrumequinum; ]) from eastern eurasia is strongly supported as sister to four endemic afrotropical r. clivosus clades specific epithets in parentheses following clade names indicate sequences from specimens used in recent species descriptions that were not supported as monophyletic and are subordinate to other clades and would render them paraphyletic. branch colors indicate individual clade membership; species groups are from [ ] and two r. ferrumequinum + r. clivosus clades whose distributions include north africa, europe, and the middle east. second, eastern eurasian r. xinanzhongguoensis [ ] has mixed support as sister to taxa in the fumigatus, maclaudi, and ferrumequinum groups (bs = %, pp = . ). finally, within the euryale group, r. blasii includes a clade distributed in eastern and southern africa that is sister to a north african + middle eastern clade. a majority of the deeper nodes are strongly supported ( of ). several currently recognized species scarcely differ genetically (~ % or less cyt-b uncorrected p-distances); and render other species paraphyletic (see additional file for a detailed cyt-b tree that depicts all labeled terminal branches). in the maclaudi species group, r. kahuzi is genetically identical to three sequences of r. ruwenzorii. in the fumigatus species group, r. smithersi, r. cohenae, and r. mabuensis all differ by < % in cyt-b from r. hildebrandtii clade and, if they are valid, would render that species paraphyletic (cf. [ ] ). in the capensis species group, five newly sequenced r. gorongosae specimens and three r. rhodesiae specimens differed from r. simulator clade by only . and %, respectively, in cyt-b. moreover, they are not reciprocally monophyletic and likewise would render r. simulator paraphyletic [cf. , where r. gorongosae is . % cyt-b distant from r. simulator; also see fig. , supplemental material]. we resequenced a specimen assigned to r. landeri by taylor et al. [ ; dm , genbank mg ], along with another newly obtained specimen from the same locality in liberia, and found that they nest deeply within r. blasii clade (fig. and additional file ) . finally, a monophyletic clade of three specimens from three separate countries in the central african guineo-congolian rainforest region (cf. denti) was unexpectedly inferred as nested within the capensis group, otherwise distributed in eastern and southern africa. all other members of the capensis group are considered to be savanna/woodland species [ ] with the exception of the subspecies r. simulator alticolus (see discussion). the ml gene tree inferred from concatenation of the nuclear genes acox , cops a, rogdi, and stat a ( individuals; matrix > % complete) is shown in fig. (individual intron gene trees from ml and bayesian analyses are depicted in additional file ). this tree was very similar to the bi tree with most nodes recovered as well supported. topological differences with the cyt-b gene tree exist, including the indeterminate placement of r. hildebrandtii within the fumigatus/eloquens group and strong support for r. landeri from mali as sister to r. cf. landeri + r. lobatus + r. alcyone. incomplete lineage sorting and/or gene flow between recently diverged sisters may account for the lack of monophyly for a) r. fumigatus/eloquens clades + + , r. fumigatus/eloquens clades + , r. fumigatus/eloquens clades + , r. hildebrandtii clades + , and r. ferrumequinum clades + . the remaining clades supported as monophyletic in the cyt-b gene tree are moderately or strongly supported as monophyletic in the concatenated nuclear gene tree with the exception of r. alcyone which is not supported as monophyletic. rhinolophus gorongosae is not monophyletic and is nested among minimally diverged specimens identified as r. simulator by [ ; sequences, ; sequence], and in this study as r. simulator . this simulator clade is distributed in tanzania, malawi, zambia, mozambique, and south africa. rhinolophus rhodesiae is likewise nested within r. simulator clade that includes sequences from drc, botswana, zambia, malawi, mozambique, zimbabwe, and south africa. there is no indication of mitochondrial introgression involving either r. gorongosae or r. rhodesiae. rhinolophus cf. denti/simulator is a deeply diverged, monophyletic clade from southeast africa with uncertain relationships to r. denti (southern africa) and sympatric r. simulator + . the membership of r. deckenii in the ferrumequinum group is challenged by its well supported sister relationship to the fumigatus group, rather than to r. clivosus + r. ferrumequinum (fig. ) . the position of r. ruwenzorii and r. willardi (maclaudi group) is uncertain, although r. ruwenzorii + r. willardi + r. deckenii are strongly supported as sister to members of the fumigatus group. the four starbeast runs in the multilocus coalescent species tree analyses converged within × generations. we discarded the first % of each run, resulting in , trees in the posterior distributions. ess values for all posterior parameters were greater than in the combined species tree analysis of , trees. the species tree (fig. ) is largely in agreement with the concatenated nuclear tree (fig. ) in the following respects: a) strongly supports r. hipposideros as sister to all other rhinolophus species in the tree; (b) strongly supports the sister relationship of the r. landeri group to the remaining african groups in the tree; c) strongly supports r. landeri as sister to r. alcyone + r. lobatus + r. cf. landeri; and d) strongly supports all the species group assignments made by csorba et al. , with the exception of r. deckenii (which had been uncertainly placed in ferrumequinum group) and r. ruwenzorii and r. willardi (maclaudi group), but here recovered in the fumigatus group. in contrast to the concatenated analysis, r. alcyone is poorly supported as sister to r. lobatus + r. cf. landeri clade. r. gorongosae and r. rhodesiae are respectively members of well supported monophyletic clades that also include specimens assigned to simulator and simulator in the cyt-b tree ( fig. and additional file ), although r. simulator clades and in the species tree analysis (fig. ) have different memberships than the two clades in the mitochondrial gene tree (fig. ) with the same labels. the clades labeled simulator and simulator in the species tree analysis (fig. ) have largely overlapping distributions although simulator also includes specimens from drc and gabon that were provisionally assigned to r. cf. denti in the cyt-b gene tree (fig. ) . the enigmatic placement of these guineo-congolian rainforest [ ] specimens within r. simulator , otherwise distributed in savanna/ woodland, warrants further investigation. results from the replicated bpp analyses show that prior choice had minimal effect on delimitation probabilities for most of the tested species/clades (table ) . however, for the four clades whose mean pp in the four summed partition schemes (see table for prior scheme definitions) fell below a threshold of . , ps and had the most influence. the clades that were not delimited all had pp ≤ . but ≥ . and thus had marginal support. most of the unsupported clades had short branches and weak node support in the species tree analysis (fig. ) . distinguishing robustly defined lineages by congruence across all prior schemes, evolutionarily independent lineages are delimited including all six lineages analyzed in the capensis group (these include two possibly new species; three of five lineages in the ferrumequinum group, including possibly new species; two of four lineages in the landeri group, including strong support for recently recognized r. lobatus (distinct from r. landeri [ ] and the newly sequenced r. cf. denti/ simulator; and finally of lineages in the fumigatus group, including three possibly new species as well as support for the recent recognition of r. damarensis as a valid species [ ] . however, there was no pp support for alternative delimitations of clades; that is, all alternate delimitations that statistically tested the merger of two or more putative species had pp ≤ . ). the eight strongly delimited clades that could not be confidently named are candidates to be evaluated as potentially valid species using independent datasets. this is the broadest phylogenetic study of afrotropical species in the genus rhinolophus published to date. multiple phylogenetic studies have confirmed the monophyly of rhinolophus and the rhinolophidae [ , , ] . csorba et al. [ ] presented a phylogenetic hypothesis for the monophyly of african rhinolophus, with r. blasii and r. clivosus extending from the afrotropics to the western palearctic, and r. euryale, r. ferrumequinum, and r. hipposideros having distributions in both north africa and the western palearctic. several studies have placed the most recent common ancestor of rhinolophus at~ mya [ , ] ; cf. ([ ] , at mya). it has long been considered that rhinolophidae originated in the african or asian tropics, although csorba et al. [ ] presented data supporting a european origin of the family when tropical conditions prevailed in southern europe during the miocene. however, dool et al. [ ] argued instead for a middle eastern origin for the basal lineage r. hipposideros. owing to poor resolution of basal nodes in their multi-locus phylogeny [ ] , they refrained from speculating on the ancestral origin of rhinolophidae but, did find support for accelerated diversification within afrotropical rhinolophus over the last mya. subsequently, the new species r. xinanzhongguoensis, described from southwestern china [ ] , was strongly supported as having affinities to the ferrumequinum, fumigatus, and maclaudi groups. this eastern palearctic/indomalayan species is phylogenetically nested deeply within the african rhinolophus radiation (fig. ) ; it has mixed support (bs = %, pp = . ) as sister to the african fumigatus and maclaudi species groups plus the afro-palearctic ferrumequinum group that includes another eastern palearctic species, r. nippon [ ] . both of these eurasian species are closer to the endemic african groups fumigatus and maclaudi than to the endemic afrotropical capensis and landeri groups. thus, the membership of r. xinanzhongguoensis and r. nippon in a predominantly african clade seems to indicate a complex historical biogeographical relationship between the afrotropics and eastern eurasia, terminal tips in the tree that are statistically well-supported (pp ≥ . ) from bpp are indicated by "*" preceding the clade name, and terminal tips that had pp < . are indicated by "?" preceding the clade name. species groups are from [ ] possibly supporting additional dispersal events between the continents. however, it should be noted that data is still lacking from independent nuclear loci for r. xinanzhongguoensis and r. nippon. the phylogenetic relationships of afrotropical rhinolophus species inferred here are in broad agreement with the study of dool et al. [ ] , based on six introns. to extend their findings, we sampled four of their introns for vouchered specimens representing eight monophyletic cyt-b clades not present in their study. we also sequenced members of nine clades represented in their study with samples from new afrotropical localities. our expanded data set is the largest yet for afrotropical rhinolophus, and infers support for up to independent evolutionary lineages as candidate species for future assessment with corroborative data. results from coalescent delimitation and species tree analysis suggest three named species may be synonyms (r. kahuzi and either r. gorongosae or r. rhodesiae). although we did not have access to tissue samples from r. smithersi, r. cohenae, and r. mabuensis, and so lack intron data, cyt-b sequences from genbank indicate that these recently described taxa are minimally divergent from r. hildebrandtii (< % in cyt-b), and their recognition would render it paraphyletic. taylor et al. [ ] recently argued for species status for two african rhinolophus names long regarded as synonyms: r. lobatus (peters, ) from r. landeri and r. rhodesiae (roberts, ) from r. swinnyi. they described the new species r. gorongosae on the basis of integrative data that included a suite of morphological variables, but their molecular phylogenetic analyses relied solely on cytochrome-b. their putative r. gorongosae (dm , dm ) had anomalously long branches (p-distance . and . % from r. simulator and putative r. rhodesiae, respectively; see their fig. ) and nested within r. simulator, which motivated us to re-examine these deeply diverged specimens. in addition, the sister relationship they determined of r. landeri and r. gorongosae ( % p-distance), instead of with alcyone and lobatus [other members of the landeri group; ], led us to compare this sequence to genbank accessions using blastn. the blastn query showed % identity of their r. landeri (genbank accession mg , dm , liberia) with bos taurus. when tissue from this voucher specimen and another (dm ), also from liberia, was extracted and new sequence data generated, those individuals were found to nest deeply within the r. blasii clade (additional file ). we extracted and sequenced five samples identified as r. gorongosae from the durban museum, including dm from [ ] , and found them to be - . % cyt-b diverged from r. simulator and r. rhodesiae. this strongly suggests that the genetic arguments in [ ] for the newly described r. gorongosae and for elevation of r. swinnyi rhodesiae to species rank were based on sequencing error (see additional file for comparison of genbank sequences versus newly sequenced material). several studies have demonstrated instances of mitochondrial introgression (i.e. mitochondrial capture) among populations of r. ferrumequinum and r. clivosus [ ] ; r. sinicus, r. rouxii, r. pearsonii pearsonii, and r. p. chinensis, restricted to eastern eurasia (mao et al. [ , ] , and the r. macrotis species complex in china [ ] . potential mitochondrial introgression is apparent in our study in the cyt-b gene tree for ferrumequinum clades and and clivosus clade as discussed in [ ] . taylor et al. [ ] suggested historical genetic introgression might account for the discrepancy between the morphological disparity of r. simulator and r. rhodesiae and their lack of genetic differentiation ( . %). however, they did not test this hypothesis with genetic data. in this study, mitochondrial, concatenated nuclear loci, and the species tree (also inferred with nuclear data only) all strongly infer the very close relationship of r. rhodesiae to r. simulator clade . although the cyt-b tree recovers both gorongosae and rhodesiae as paraphyletic, the concatenated nuclear phylogeny recovers them in two separate monophyletic clades (simulator and ; fig. ). to understand these conflicting signals, and to determine which of these clades actually represents true r. simulator, a geographically expanded integrative taxonomic assessment will be necessary. the broad phylogenetic and geographic sampling in our study uncovered a number of range extensions for described species and also suggests possible niche divergences of putative undescribed species based on their genetic and geographic relationships. in the capensis group, a clade from cameroon (rom ), gabon (fmnh ), and western drc (fmnh ) is strongly supported in the cyt-b gene tree (fig. ) as sister to r. denti, otherwise known from savanna and woodlands of southern (r. denti) and western (r. denti knorri) africa. the specimens from drc and gabon were included in the concatenated nuclear intron tree and found to nest well within r. simulator, which is sister to r. denti. rhinolophus alcyone alticolus sanborn, [ ] was allocated to r. simulator by koopman [ ] , who was followed by subsequent authors, but csorba et al. [ ] suggested that r. simulator alticolus might prove to be a separate species. the type specimen (which is now lost) was from mt. cameroon, and western cameroon is the only lowland rainforest distribution for the savanna woodland simulator [ ] . the specimen we sequenced from mt. cameroon was from a~ -year-old skin and nuclear genes were not successfully amplified. nonetheless, the strong support for the drc and gabon specimens (from the same clade as the mt. cameroon specimen in the cyt-b tree) with r. simulator suggests that our cf. denti clade may be introgressed r. simulator whose range now extends well into the western african rainforest habitat [ ] . independent nuclear data from additional specimens are needed to confirm the status of this clade. also within the capensis group, all analyses in our study strongly support the existence of an undescribed species provisionally designated cf. denti/ simulator. at present, populations are known from tanzania, mozambique, and malawi where they are sympatric with its close relative r. simulator and presumably differ from it ecologically. in the euryale group, two newly sequenced individuals from liberia are strongly supported as sisters of populations identified as r. blasii populations from southeastern africa. this extends the range of sub-saharan populations clade > km west of their current distribution. however, r. blasii has a highly disjunct distribution, and individuals from southern europe (its type locality is italy) were not included in our analysis; if conspecific with moroccan populations of r. blasii, the liberian records document a km range extension. both specimens had been identified as r. landeri; if mitochondrial introgression was responsible, it is unclear where contact may have occurred. in the maclaudi group, the range of r. ruwenzorii is now extended to the mountains west of lake kivu in kahuzi-biega np, as strong support from mitochondrial and nuclear data indicate that r. kahuzi [ ] is a synonym of r. ruwenzorii. as in csorba et al. [ ] and dool et al. [ ] , the most basal lineage within the african radiation is the landeri group (alcyone, landeri, and lobatus), whose partial distribution in rainforest habitats has been hypothesized to be indicative of the habitat affinities of early colonizers [ ] . however, this hypothesis has not been tested with ancestral-area reconstruction analyses. as in [ ] , resolution of deeper nodes in our analysis was inconsistent, weakening any attempts at ancestral reconstructions. also, uneven geographic sampling in our nuclear dataset indicates that additional populations should be incorporated before carrying out quantitative analysis. as in the bat genus scotophilus [ ] , the populationlevel phylogenetic analyses presented here document repeated patterns of clade replacements between eastern and southern africa. in the fumigatus group, paired clades support replacement between eastern and southern africa (fig. ; fumigatus/eloquens + + vs. fumigatus/eloquens and fumigatus/eloquens + vs. fumigatus/eloquens ). this relationship is also supported by one clade-pair (clivosus vs. clivosus ) in the ferrumequinum group, and one pair (cf. landeri vs. lobatus) in the landeri group. in the capensis group, phylogeographic patterns appear more complex (probably owing to better sampling) and multiple lineages exhibit sympatry. at least one is a putative but undescribed species in sympatry with its sisters in the capensis group (cf. denti/simulator; figs. and ). sampling is still limited in the rain forests of central and west africa, but an enigmatic relationship is apparent in the cyt-b tree (fig. ) , where three newly sequenced specimens from the western guineo-congolian rain forest (cf. denti in fig. ) are sister to arid-land r. denti. the concatenated nuclear tree recovers two members of this clade from drc and gabon as nested within r. simulator. in both datasets an unexpectedly close relationship is inferred for a savanna/woodland habitat species with poorly surveyed populations living in humid rainforest. additional insights to afrotropical rhinolophus are now possible with this greater phylogenetic understanding. the genus is interesting from a public health standpoint owing to various associated viral pathogens [ , [ ] [ ] [ ] . its constant-frequency echolocation calls have been widely studied for their value in communication [ , ] , adaptation and speciation [ ] [ ] [ ] , and resource subdivision [ , ] . their noseleaves, cranial morphology, dentition, and bacula are all richly diversified morphological systems [ , ] hardly studied from developmental or evolutionary perspectives. continued efforts to characterize these newly documented lineages across all of these phenotypic dimensions will offer greater understanding of their evolutionary development and diversification. additional file : maximum likelihood gene tree inferred for cyt-b using iq-tree that includes two sequences of r. gorongosae deposited in genbank [ ; indicated by red font] and five specimens newly sequenced for cyt-b in this study (indicated by blue font). dm is included twice in the tree (both the genbank sequence and a newly generated sequence from this study). nodal support is indicated above branches. our analysis was strengthened with samples collected by the late w. t. (bill) stanley, and this paper is dedicated in his memory. we acknowledge with special thanks the assistance of peter taylor (university of venda) and leigh richards (durban natural science museum) in loaning samples of taxa included in [ ] , and jessica light and duane schlitter (texas a&m university), jacqui miller and burton lim (royal ontario museum), leigh richards (durban natural science museum), and simon musila (national museums of kenya) for tissue loans. carl dick, ruth makena, david wechuli, richard yego, and aziza zuhura all helped collect museum vouchers. new genetic data were generated in the fmnh pritzker laboratory, managed by kevin feldheim. the efforts of curators and collection managers in all the institutions cited in additional file are acknowledged for maintaining the museum voucher specimens that enable follow-up studies, sometimes on different datasets, keeping our science verifiable and our errors correctable. authors' contributions bp, td conceived the project; td, bp analyzed the data; td, bp, pw, jkp, smg, mb contributed samples and provided interpretation of their context; td, bp, pw, jkp, smg, mb participated in discussion and interpretation of the results; td, bp wrote the paper with input from all authors. all authors have read and approved the manuscript. collections in east and southern africa were funded by a variety of agencies in cooperation with the field museum, especially the jrs biodiversity foundation. field museum's council on africa, marshall field iii fund, and barbara e. brown fund for mammal research. we thank the generous support of bud and onnolee trapp and walt and ellen newsom. publication costs were paid by the integrative research center. thanks to the john d. and catherine t. macarthur foundation, fulbright program of us department of state, wildlife conservation society, and the centers for disease control and prevention who sponsored and assisted in obtaining vouchers from drc, malawi, and uganda for accession to fmnh. we thank wwf gabon and partenariat mozambique-réunion dans la recherche en santé: pour une approche intégrée d'étude des maladies infectieuses à risque épidémique (mozar; fond européen de développement régional, programme opérationnel de coopération territoriale) for funding support. the dna sequence data generated for this article are available on genbank with the following accession numbers: mn -mn . the dna sequence alignments used in the analyses for this article have been deposited on figshare under accession doi: https://doi.org/ . /m . figshare. ). ethics approval and consent to participate no animals were used in this study. not applicable. a new age of discovery mammal species of the world: a taxonomic and geographic reference how many species of mammals are there? coalescent-based species delimitation in an integrative taxonomy a genomic evaluation of taxonomic trends through time in coast horned lizards (genus phrynosoma) nuclear introns outperform 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with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no competing interests.author details key: cord- -hxxizipk authors: roberts, katherine e.; hadfield, jarrod d.; sharma, manmohan d.; longdon, ben title: changes in temperature alter the potential outcomes of virus host shifts date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: hxxizipk host shifts–where a pathogen jumps between different host species–are an important source of emerging infectious disease. with on-going climate change there is an increasing need to understand the effect changes in temperature may have on emerging infectious disease. we investigated whether species’ susceptibilities change with temperature and ask if susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with an rna virus and measured how viral load changes with temperature. we found the host phylogeny explained a large proportion of the variation in viral load at each temperature, with strong phylogenetic correlations between viral loads across temperature. the variance in viral load increased with temperature, while the mean viral load did not. this suggests that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. we found no significant relationship between a species’ susceptibility across temperatures, and proxies for thermal optima (critical thermal maximum and minimum or basal metabolic rate). these results suggest that whilst the rank order of species susceptibilities may remain the same with changes in temperature, some species may become more susceptible to a novel pathogen, and others less so. emerging infectious diseases are often the result of a host shift, where a pathogen jumps from one host species into another. understanding the factors underlying host shifts is a major goal for infectious disease research. this effort has been further complicated by the fact that host-parasite interactions are now taking place in a period of unprecedented global climatic warming. here, we ask how host shifts are affected by temperature by carrying out experimental infections using an rna virus across a wide range of related species, at three different temperatures. we find that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. this has important consequences for our understanding of host shift events in a changing climate as it suggests that temperature changes may affect the likelihood of a host shift into certain species. temperature is arguably the most important abiotic factor that affects all organisms, having both indirect and direct effects on physiology and life history traits [ ] [ ] [ ] . there is much to be learned about the impact of climate change on infectious diseases [ , , ] . changes in temperature can impact both host and parasite biology, leading to complex and difficult to predict outcomes [ , ] . host shifts, where a parasite from one host species invades and establishes in a novel host species, are an important source of emerging infectious disease [ ] . a successful host shift relies on a number of stages occurring [ ] . firstly, exposure of the host to the new pathogen species must occur in such a way that transmission is successful. secondly, the pathogen must be able to replicate sufficiently to infect the novel host. finally, there must be sufficient onwards transmission for the pathogen to become established in the new host species [ , , ] . some of the most deadly outbreaks of infectious diseases in humans including ebola virus, hiv and sars coronavirus have been linked to a host switch event [ ] [ ] [ ] [ ] and many others have direct animal vectors or reservoirs (e.g. dengue and chikungunya viruses) [ , ] . the potential for novel host shifts may increase with changing temperatures due to, fluctuations in host and/or parasite fitness, or changes in species distributions and abundances [ , ] . distribution changes may lead to new species assemblages, causing novel contacts between parasites and potential hosts [ ] [ ] [ ] . susceptibility to infection is known to vary with temperature, due to within individual physiological changes in factors such as the host immune response, metabolic rate or behavioural adaptations [ ] [ ] [ ] [ ] . thermally stressed hosts may face a trade-off between the resource investment needed to launch an immune response versus that needed for thermoregulation, or behavioural adaptations to withstand sub-optimal temperatures [ ] [ ] [ ] [ ] . temperature shifts could also cause asymmetrical or divergent effects on host and parasite traits [ ] . for example, changes in temperature may allow differential production and survival of parasite transmission stages, and changes in replication rates, generation times, infectivity and virulence [ ] [ ] [ ] . temperature is also known to impact vector-borne disease transmission through multiple effects on both vector life cycles and transmission behaviours [ , [ ] [ ] [ ] [ ] . host shifts have been shown to be more likely to occur between closely related species [ ] [ ] [ ] , but independently of this distance effect, clades of closely related hosts show similar levels of susceptibility [ , ] . thermal tolerances − like virus susceptibility − are known to vary across species, with groups of closely related species having similar thermal limits, with a large proportion of the variation in these traits being explained by the phylogeny [ ] [ ] [ ] [ ] . previous studies on host shifts have assayed the susceptibility of species at a single temperature [ , , , ] . however, if the host phylogeny also explains much of the variation in thermal tolerance, then phylogenetic patterns in virus susceptibility could be due to differences between species' natural thermal optima and the chosen assay temperatures. therefore, for experiments carried out at a single temperature, phylogenetic signal in thermal tolerance may translate into phylogenetic signal in thermal stress. any apparent phylogenetic signal in susceptibility could potentially be due to the effects of thermal stress, and may not hold true if each species was to be assayed at its optimal temperature. if this was indeed the case this would have implications for species distribution models that aim to use estimates of environmental conditions to predict host and pathogen ranges [ , , ] . here, we have asked how species' susceptibilities change at different temperatures and whether susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with drosophila c virus (dcv; dicistroviridae) at three different temperatures and measured how viral load changes with temperature. viral load is used here as a measure of dcv's ability to persist and replicate in a host, which has previously been shown to be tightly correlated to host mortality [ ] . we are therefore examining one of the steps ("ability to infect a novel host") needed for a host shift to successfully occur [ , , ] . we also examine how proxies for thermal optima and cellular function (thermal tolerances and basal metabolic rate) relate to virus susceptibility across temperatures, as increasing temperatures may have broad effects on both host and parasite [ ] [ ] [ ] . dcv is a positive sense rna virus in the family discistroviridae that was originally isolated from drosophila melanogaster and in the wild has been found in d. melanogaster and d. simulans [ ] [ ] [ ] . dcv infected flies show reduced metabolic rate and activity levels, develop an intestinal obstruction, reduced hemolymph ph and decreased survival [ ] [ ] [ ] [ ] . this work examines how temperature can influence the probability of host shifts, and looks at some of the potential underlying causes. we used drosophila c virus (dcv) clone b a, which is derived from an isolate collected from d. melanogaster in charolles, france [ ] . the virus was prepared as described previously [ ] ; briefly dcv was grown in schneider's drosophila line cells and the tissue culture infective dose (tcid ) per ml was calculated using the reed-muench end-point method [ ] . flies were obtained from laboratory stocks of different species. all stocks were maintained in multi generation populations, in drosophila stock bottles (dutscher scientific) on ml of their respective food medium at ˚c and % relative humidity with a hour lightdark cycle (table a in s text). each day, two vials of - day old male flies were randomly assigned to one of three potential temperature regimes; low, medium or high ( ˚c, ˚c and ˚c respectively) at % relative humidity. flies were tipped onto fresh vials of food after days, and after days of acclimatisation at the experimental temperature were infected with dcv. flies were anesthetized on co and inoculated using a . mm diameter stainless steel needle that was bent to a right angle~ . mm from the end (fine science tools, ca, usa) [ , , ] . the bent tip of the needle was dipped into the dcv solution (tcid = . × ) and pricked into the pleural suture on the thorax of the flies. we selected this route of infection as oral inoculation has been shown to lead to stochastic infection outcomes in d. melanogaster [ ] . however, once the virus passes through the gut barrier, both oral and pinpricked infections follow a similar course, with both resulting in the same tissues becoming infected with dcv [ ] . one vial of inoculated flies was immediately snap frozen in liquid nitrogen to provide a time point zero sample as a reference to control for relative viral dose. the second vial of flies were placed onto a new vial of fresh cornmeal food and returned to their experimental temperature. after days (+/- hour) flies were snap frozen in liquid nitrogen. this time point was chosen based on pilot data as infected flies showed little mortality at days post infection, and viral load plateaus from day at ˚c. temperatures were rotated across incubators in each block to control for incubator effects. all frozen flies were homogenised in a bead homogeniser for seconds (bead ruptor ; omni international, georgia, usa) in trizol reagent (invitrogen) and stored at - ˚c for later rna extractions. these collections and inoculations were carried out over three replicate blocks, with each block being completed over consecutive days. the order that the fly species were infected was randomized each day. we aimed for each block to contain a day and day replicate for each species, at each temperature treatment ( species × temperatures × experimental blocks). in total we quantified viral load in , flies over biological replicates (a biological replicate = change in viral load from day to day post-infection), with a mean of . flies per replicate (range across species = - ) . of the species, had biological replicates and three species had biological replicates. the change in rna viral load was measured using quantitative reverse transcription pcr (qrt-pcr). total rna was extracted from the trizol homogenised flies, reverse-transcribed with promega goscript reverse transcriptase (promega) and random hexamer primers. viral rna load was expressed relative to the endogenous control housekeeping gene rpl (rp ). rpl primers were designed to match the homologous sequence in each species and crossed an intron-exon boundary so will only amplify mrna [ ] . the primers in d. melanogaster were rpl qrt-pcr f ( '-tgctaagctgtcgcacaaatgg - ') and rpl qrt-pcr r ( '-tgcgcttgttcgatccgtaac - '). dcv primers were f ( '-gacactgccttt gattag- ') and r ( 'ccctctgggaactaaatg- ') as previously described [ ] . two qrt-pcr reactions (technical replicates) were carried out per sample with both the viral and endogenous control primers, with replicates distributed across plates in a randomised block design. qrt-pcr was performed on an applied biosystems steponeplus system using sensifast hi-rox sybr kit (bioline) with the following pcr cycle: ˚c for min followed by cycles of: ˚c for sec followed by ˚c for sec. each qrt-pcr plate contained four standard samples. a linear model was used to correct the cycle threshold (ct) values for differences between qrt-pcr plates. any samples where the two technical replicates had cycle threshold (ct) values more than cycles apart after the plate correction were repeated. to estimate the change in viral load, we first calculated Δct as the difference between the cycle thresholds of the dcv qrt-pcr and the rpl endogenous control. for each species the viral load of day flies relative to day flies was calculated as -ΔΔct ; where ΔΔct = Δct day -Δct day . the Δct day and Δct day are a pair of Δct values from a day biological replicate and a day biological replicate. calculating the change in viral load without the use of the endogenous control gene (rpl ) gave equivalent results (spearman's correlation between viral load calculated with and without endogenous control: ρ = . , p< . ) we carried out two assays to measure the thermal tolerances of species; a cold resistance measure to determine critical thermal minimum (ct min ) under gradual cooling, and a heat resistance measure through gradual heating to determine critical thermal maximum (ct max ). - day old males were collected and placed onto fresh un-yeasted cornmeal food vials. flies were kept for days at ˚c and % relative humidity and tipped onto fresh food every days. in both assays individual flies were placed in ml glass vials (st , ampulla, uk) and exposed to temperature change through submersion in a liquid filled glass tank (see fig a in s text). for ct max the tank was filled with water and for ct min a mixture of water and ethylene glycol ( : by volume) was used to prevent freezing and maintain a constant cooling gradient. five biological replicates were carried out for each species for both ct max and ct min . temperature was controlled using a heated/cooled circulator (txf , grant instruments, cambridgeshire, uk) submerged in the tank and set to change temperatures at a rate of . ˚c/min, always starting from ˚c (the rearing temperature for stock populations). flies were monitored continually throughout the assay and the temperature of knock down was ascertained by a disturbance method, whereby a fly was scored as completely paralysed if on gentle tapping of the vial wall the fly did not move any of its body parts. to examine how cellular function changes with temperature, we estimated the resting metabolic rate of each species at ˚c, ˚c and ˚c to examine if changes in general cellular processes were related to changes in viral load. following the same methods as the viral inoculation assay, groups of , - day old male flies from species were acclimatised at the three experimental temperatures for days (d. pseudoobscura was excluded as not enough individuals could be obtained from stocks for sufficient replication). every days flies were tipped onto fresh vials of cornmeal food. this was repeated in three blocks in order to get three repeat measures of metabolic rate for each of the species, at each of the three experimental temperatures. flies were collected in a randomly assigned order across the three blocks. closed system respirometry was used to measure the rate of co production (vco ) as a proxy for metabolic rate [ ] . flies were held in ml - airtight plastic chambers constructed from bev-a-line v tubing (cole-parmer instrument company, uk). all measures were carried out during the day inside a temperature controlled incubator, with constant light, that was set to each of the experimental temperatures that the flies had been acclimatised to. the set up followed that of okada et al. ( ) [ ] . compressed air of a known concentration of oxygen and nitrogen ( % o : % n ) was scrubbed of any co and water (with ascarite ii & magnesium perchlorate respectively) and pumped through a sable systems rm eight-channel multiplexer (las vegas, nv, usa) at ml/min - (± %) into the metabolic chambers housing the groups of flies. the first chamber was left empty as a reference cell, to acquire a baseline reading for all subsequent chambers at the start and end of each set of runs, therefore seven groups of flies were assayed in each run. air was flushed into each chamber for minutes, before reading the previous chamber. readings were taken every second for minutes by feeding the exiting air through a licor li- infrared gas analyser (lincoln, ne, usa). carbon dioxide production was measured using a sable systems ui analog-digital interface for acquisition, connected to a computer running sable systems expedata software (v . . ) [ ] . the metabolic rate was calculated from the entire -minute recording period by taking the co reading of the ex-current gas from the chamber containing the flies and subtracting the co measure of the incurrent gas entering the chamber. these values were also corrected for drift away from the baseline reading of the empty chamber. volume of co was calculated as vco = fr (fe co -fi co ) / ( -fi co ). where fr is the flow rate into the system ( ml/ min - ), fe co is the concentration of co exiting and fi co is the concentration co entering the respirometer. species were randomly assigned across the respiration chambers and the order in which flies were assayed (chamber order) was corrected for statistically (see below). to check for any potential effect of body size differences between species on viral load, wing length was measured as a proxy for body size [ ] . a mean of (range - ) males of each species were collected and immediately stored in ethanol during the collections for the viral load assay. subsequently, wings were removed and photographed under a dissecting microscope. using imagej software (version . ) the length of the iv longitudinal vein from the tip of the proximal segment to where the distal segment joins vein v was recorded, and the mean taken for each species. the host phylogeny was inferred as described in longdon et al ( ) [ ] , using the s, adh, amyrel, coi, coii, rpl and sod genes. briefly, any publicly available sequences were downloaded from genbank, and any not available we attempted to sanger sequence [ ] . in total we had rpl sequences for all species, s from species, adh from species, amyrel from species, coi from species, coii from species and sod from species (see www.doi.org/ . /m .figshare. full details). the sequences of each gene were aligned in geneious (version . . , [ ] ) using the global alignment setting, with free end gaps and a cost matrix of % similarity. the phylogeny was constructed using the beast program (version . . , [ ] ). genes were partitioned into three groups each with their own molecular clock models. the three partitions were: mitochondrial (coi, coii); ribosomal ( s); and nuclear (adh, sod, amyrel, rpl ). a random starting tree was used, with a relaxed uncorrelated lognormal molecular clock. each of the partitions used a hky substitution model with a gamma distribution of rate variation with categories and estimated base frequencies. additionally, the mitochondrial and nuclear data sets were partitioned into codon positions + and , with unlinked substitution rates and base frequencies across codon positions. the treeshape prior was set to a birth-death process. the beast analysis was run twice to ensure convergence for million mcmc generations sampled every steps. the mcmc process was examined using the program tracer (version . , [ ] ) to ensure convergence and adequate sampling, and the constructed tree was then visualised using figtree (version . . , [ ] ). all data were analysed using phylogenetic mixed models to look at the effects of host relatedness on viral load across temperature. we fitted all models using a bayesian approach in the r package mcmcglmm [ , ] . we ran trivariate models with viral load at each of the three temperatures as the response variable similar to that outlined in longdon et al. ( ) [ ] . the models took the form: where y is the change in viral load of the i th biological replicate of host species h, for temperature t (high, medium or low). β are the fixed effects, with β being the intercepts for each temperature, β being the effect of basal metabolic rate, β the effect of wing size, and β and β the effects of the critical thermal maximum (ct max ) and minimum (ct min ) respectively. u p are the random phylogenetic species effects and e the model residuals. we also ran models that included a non-phylogenetic random species effect (u np:ht ) to allow us to estimate the proportion of variation explained by the host phylogeny [ , , ] . we do not use this term in the main model as we struggled to separate the phylogenetic and non-phylogenetic terms. our main model therefore assumes a brownian motion model of evolution [ ] . the random effects and the residuals are assumed to be multivariate normal with a zero mean and a covariance structure v p � a for the phylogenetic affects and v e � i for the residuals (� here is the kronecker product). a is the phylogenetic relatedness matrix, i is an identity matrix and the v are × (co)variance matrices describing the (co)variances between viral titre at different temperatures. the phylogenetic covariance matrix, v p, describes the inter-specific variances in each trait and the inter-specific covariances between them. the residual covariance matrix, v e, describes the within-species variance that can be both due to real within-species effects and measurement or experimental errors. the off-diagonal elements of v e (the covariances) can not be estimated because no vial has been subject to multiple temperatures and so were set to zero. we excluded d. pseudoobscura from the full model as data for bmr was not collected, but included it in models that did not include any fixed effects, which gave equivalent results. diffuse independent normal priors were placed on the fixed effects (means of zero and variances of ). parameter expanded priors were placed on the covariance matrices resulting in scaled multivariate f distributions, which have the property that the marginal distributions for the variances are scaled (by ) f , . the exceptions were the residual variances for which an inverse-gamma prior was used with shape and scale equal to . . the mcmc chain was run for million iterations with a burn-in of million iterations and a thinning interval of , . we confirmed the results were not sensitive to the choice of prior by also fitting models with inverse-wishart and flat priors for the variance covariance matrices (described in [ ] ), which gave qualitatively similar results ( . /m .figshare. ). all confidence intervals (ci's) reported are % highest posterior density intervals. using similar model structures we also ran a univariate model with bmr and a bivariate model with ct min and ct max as the response variables to calculate how much of the variation in these traits was explained by the host phylogeny. both of these models were also run with wing length as a proxy for body size as this is known to influence thermal measures [ ] . we observed significant levels of measurement error in the metabolic rate data; this was partially caused by respiratory chamber order during the assay. we corrected for this in two different ways. first, we fitted a linear model to the data to control for the effect of respiratory chamber number and then used this corrected data in all further models. we also used a measurement error model that controls for both respiratory chamber number effects and random error. both of these models gave similar results although the measurement error model showed broad cis suggesting the bmr data should be interpreted with caution. all datasets and r scripts with the model parameterisation are provided as supporting information (s text). to investigate the effect of temperature on virus host shifts we quantified viral load in , flies over biological replicates, from species of drosophilidae at three temperatures ( fig ) . dcv replicated in all host species, but viral load differed between species and temperatures (fig ) . species with similar viral loads cluster together on the phylogeny (fig ) . measurements were highly repeatable (table ) , with a large proportion of the variance being explained by the inter-specific phylogenetic component (v p ), with little within species or measurement . we also calculated the proportion of between species variance that can be explained by the phylogeny as v p /(v p + v s ) [ ] , which is equivalent to pagel's lambda or phylogenetic heritability [ , ] . we found the host phylogeny explains a large proportion of the inter-specific variation in viral load across all three temperatures, although these estimates have broad confidence intervals due to the model struggling to separate the phylogenetic and non-phylogenetic components (low = . , % ci: . to examine if species responded in the same or different way to changes in temperature we examined the relationships between susceptibilities across the different temperatures. we found strong positive phylogenetic correlations between viral loads across the three temperatures (table ). our models showed that the variance in viral load increased with temperature, however the mean viral load showed no such upward trend (table ). this suggests that the changes in variance are not simply occurring due to an increase in the means, that is then driving an increase in variance. the high correlations suggest the rank order of susceptibility of the species is not changing with increasing temperature. however, the change in variance suggests that although the intercepts are the temperature-specific intercepts when the other covariates (e.g. wing size) are set to their temperature specific means. they can be interpreted as the expected viral loads at the root of the phylogeny at each temperature. v p is the variance in between-species effects, which are structured by the phylogeny, and v r is the variance in within species effects attributable to between individual differences and measurement error. reaction norms are not crossing they are diverging from each other as temperature increases i.e. the most susceptible species are becoming more susceptible with increasing temperature, and the least susceptible less so [ ] . for example, d. obscura and d. affinis are the most susceptible species at all three temperatures. the responses of individual species show that some species have increasing viral load as temperature increases (fig , e.g. z. taronus, d. lummei) , while others decease (e.g. d. littoralis, d. novamexicana). the changes we observe could be explained by the increase in temperature effectively increasing the rate at which successful infection is progressing (i.e. altering where in the course of infection we have sampled). however, this seems unlikely as at days post infection at the medium temperature ( ˚c), viral load peaks and then plateaus [ ] . therefore, in those species where viral load increases at higher temperatures the peak viral load itself must be increasing, rather than us effectively sampling the same growth curve but at a later time point. likewise, in those species where viral load decreased at higher temperatures, viral load would need to first increase and then decrease, which we do not observe in a time course at ˚c [ ] . to check whether this also holds at higher temperatures we carried out a time course of infection in a subset of six of the original experimental species at ˚c, where we would expect the fastest transition between the rapid viral growth and the plateau phase of infection to occur (fig b in s text) . this allowed us to confirm that the decreasing viral loads observed in some species at higher temperatures are not due to general trend for viral loads to decline over longer periods of (metabolic) time. we quantified the lower and upper thermal tolerances (ct min and ct max ) across all species with replicates per species. neither ct max nor ct min were found to be significant predictors of viral load (ct min - . , % ci: - . , . , pmcmc = . and ct max . , % ci: - . , . , pmcmc = . ). when treated as a response in models we found the host phylogeny explained a large proportion of the variation in thermal maximum (ct max : . , % ci: . , ) and thermal minima (ct min : . , % ci: . , . , see s text fig c) . we also measured the basal metabolic rate of flies from species, across the three experimental temperatures, to examine how cellular function changes with temperature. bmr was not found to be a significant predictor of viral load when included as a fixed effect in our model (slope = . , % ci = - . , . , pmcmc = . ). bmr increased with temperature across all species (mean bmr and se: low . ± . , medium . ± . , high . ± . co ml/min - , see s text fig d) . when bmr was analysed as the response in models, the phylogeny explained a small amount of the between species variation (low . , % ci: × − , . , medium . , % ci: × − , . , high . , % ci: × − - . , s text fig e) indicating high within species variation or large measurement error. consequently the mean bmrs for each species, at each temperature, were used in the analysis of viral load will be poorly estimated and so the effects of bmr will be underestimated with too narrow credible intervals. to rectify this we ran a series of measurement error models, the most conservative of which gave a slope of - . but with very wide credible intervals (- . , . ) . full details of these models are given in the supporting information (s text). we found that susceptibilities of different species responded in different ways to changes in temperature. the susceptibilities of different species showed differing responses as temperatures increased (fig ) . there was a strong phylogenetic correlation in viral load across the three experimental temperatures (table ) . however, the variance in viral load increased with temperature, whereas the mean viral load did not show the same trend. this suggests that the rank order of susceptibility of the species remains relatively constant across temperatures, but as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. changes in global temperatures are widely predicted to alter host-parasite interactions and therefore the likelihood of host shifts occurring [ , , , , ] . the outcome of these interactions may be difficult to predict if temperature causes a different effect in the host and pathogen species [ , , [ ] [ ] [ ] . our results show that changes in temperature may change the likelihood of pathogens successfully infecting certain species, although they suggest that it may not alter which species are the most susceptible to a novel pathogen. the increase in phylogenetic variance with temperature is effectively a form of genotypeby-environment interaction [ , [ ] [ ] [ ] . however, it varies from the classically considered ecological crossing of reaction norms, as we do not see a change in the rank order of species susceptibly across the range of experimental temperatures. instead, we find the species means diverge with increasing temperatures and so the between species differences increase [ , ] . it is also important to note that temperature may not simply be causing a change in effect size when considering the biological processes occurring during host-parasite interactions [ , ] . for example, virus replication may plateau at higher temperatures due to resource limitation. the observed level of susceptibility may be the combined outcome of both host and parasite traits, which may interact nonlinearly with temperature. we also note that by using a limited range of temperatures for practical reasons we may have not captured all unimodal relationships between viral load and temperature. as temperature is an important abiotic factor in many cellular and physiological processes, we went on to examine the underlying basis of why viral load might change with temperature. previous studies that found phylogenetic signal in host susceptibility were carried out at a single experimental temperature [ , ] . therefore, the patterns observed could potentially be explained by some host clades being assayed at sub-optimal thermal conditions. we used ct max and ct min as proxies for thermal optima which, due to its multifaceted nature, is problematic to measure directly [ ] [ ] [ ] . we also measured basal metabolic rate across three temperatures to see if the changes in viral load could be explained by general increases in enzymatic processes. we found that these measures were not significant predictors of the change in viral load with temperature. this may be driven by the fact that all temperature related traits are likely to be more complex than what any single measure can explore. traits such as host susceptibility are a function of both the host and parasite thermal optima, as well as the shape of any temperature-trait relationship [ , ] . the host immune response and cellular components utilised by the virus are likely to function most efficiently at the thermal optima of a species, and several studies have demonstrated the outcomes of host-pathogen interactions can depend on temperature [ , , , ] . however, the mechanisms underlying the changes in susceptibility with temperature seen in this study are uncertain and a matter for speculation. our results show that in the most susceptible species, viral load increases with temperature; this may be due to the virus being able to successfully infect and then freely proliferate, utilizing the host cells whist avoiding host immune defences. in less susceptible species viral load does not increase with temperature, and in some cases it actually appears to decreases. here, temperature may be driving an increase in biological processes such as enhanced host immunity, or simply increasing the rate of degradation or clearance of virus particles that have failed to establish an infection of host cells. we have investigated how an environmental variable can alter infection success following a novel viral challenge. however, temperature is just one of the potential environmental factors that will influence the different stages of a host shift event [ ] . using a controlled method of viral inoculation allows us to standardize inoculation dose so we can ask, given equal exposure, how does temperature affect the ability of a pathogen to persist and replicate in a given host? however, in nature hosts will be faced with variable levels of pathogen exposure, infected through various modes of transmission and often by multiple strains or genotypes [ ] . such variables may have consequences for the establishment and subsequent infection success of any potential host shift event. it is known that oral infection by dcv is stochastic and immune barriers such as the gut are important [ , , ] , therefore establishing the relevance of infection in the wild in this system would require further study using different potential routes of infection. the geographical distribution of a host will also influence factors such as diet and resource availability [ , [ ] [ ] [ ] [ ] , and so further work on the role of nutrient and resource availability would therefore be needed to further explore the impact of these on potential host shifts. in conclusion, we have found changes in temperature can both increase or decrease the likelihood of a host shift. our results show the rank order of species' susceptibilities remain the same across temperatures, suggesting that studies of host shifts at a single temperature can be informative in predicting which species are the most vulnerable to a novel pathogen. changing global temperatures may influence pathogen host shifts; for example changes in distributions of both host and pathogen species may generate novel transmission opportunities. our findings suggest that increases in global temperature could increase the likelihood of host shifts into the most susceptible species, and reduce it in others. climate change may therefore lead to changing distributions of both host and pathogens, with pathogens potentially expanding or contracting their host range. understanding how environmental factors might affect broader taxonomic groups of hosts and pathogens requires further study if we are to better understand host shifts in relation to climate change in nature. climate warming and disease risks for terrestrial and marine biota global warming and temperature-mediated increases in cercarial emergence in trematode parasites climate change and evolutionary adaptation climate change and infectious diseases: from evidence to a predictive framework environmental-mechanistic modelling of the impact of global change on human zoonotic disease emergence: a case study of lassa fever. freckleton r, editor climate oscillations and the structure of natural communities emerging pathogens: the epidemiology and evolution of species jumps the evolution and genetics of virus host shifts host phylogeny determines viral persistence and replication in novel hosts population biology of emerging and re-emerging pathogens virus rna structure specialization facilitates host adaptation rapid spread of emerging zika virus in the pacific area ebola in west africa: the outbreak able to change many things impact of climate change on global malaria distribution chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes the many projected futures of dengue rapid range shifts of species associated with high levels of climate warming host and parasite thermal ecology jointly determine the effect of climate warming on epidemic dynamics how will global climate change affect parasite-host assemblages? global temperature constraints on aedes aegypti and ae. albopictus persistence and competence for dengue virus transmission. parasit vectors evolution in action: climate change, biodiversity dynamics and emerging infectious disease thermal biology in insect-parasite interactions host thermal biology: the key to understanding host-pathogen interactions and microbial pest control? variation in the immune state of gammarus pulex (crustacea, amphipoda) according to temperature: are extreme temperatures a stress? environmental temperature variation influences fitness trade-offs and tolerance in a fish-tapeworm association the influence of ambient temperature on the course of myxomatosis in rabbits some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket gryllus texensis host-parasite and genotype-by-environment interactions: temperature modifies potential for selection by a sterilizing pathogen environmental stressors alter relationships between physiology and behaviour empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics parasites and global warming: net effects of temperature on an intertidal host-parasite system some (worms) like it hot: fish parasites grow faster in warmer water, and alter host thermal preferences global change, parasite transmission and disease control: lessons from ecology understanding uncertainty in temperature effects on vector-borne disease: a bayesian approach detecting the impact of temperature on transmission of zika, dengue and chikungunya using mechanistic models short title: temperature predicts zika, dengue, and chikungunya transmission impact of human mobility on the emergence of dengue epidemics in pakistan rethinking vector immunology: the role of environmental temperature in shaping resistance phylogenetic signal in plant pathogen-host range phylogenetic determinants of potential host shifts in fungal pathogens host phylogeny constrains cross-species emergence and establishment of rabies virus in bats the causes and consequences of changes in virulence following pathogen host shifts phylogenetic studies of coadaptation:preferred temperatures versus optimal performance temperature of lizards phylogenetic constraints in key functional traits behind species' climate niches: patterns of desiccation and cold resistance across drosophila species upper thermal limits of drosophila are linked to species distributions and strongly constrained phylogenetically upper thermal limits in terrestrial ectotherms: how constrained are they? fox c, editor infection success in novel hosts: an experimental and phylogenetic study of drosophila -parasitic nematodes spatial, seasonal and climatic predictive models of rift valley fever disease across africa global trends in emerging infectious diseases studies on drosophila c and a viruses in australian populations of drosophila melanogaster the discovery, distribution, and evolution of viruses associated with drosophila melanogaster twenty-five new viruses associated with the drosophilidae (diptera) physiological and metabolic consequences of viral infection in drosophila melanogaster drosophila c virus systemic infection leads to intestinal obstruction the novel genome organization of the insect picorna-like virus drosophila c virus suggests this virus belongs to a previously undescribed virus family the toll-dorsal pathway is required for resistance to viral oral infection in drosophila existence in drosophila of groups of picornavirus with different biological and serological properties host shifts result in parallel genetic changes when viruses evolve in closely related species a simple method of estimating fifty per cent endpoints measuring metabolic rates: a manual for scientists longevity, calling effort, and metabolic rate in two populations of cricket genetic architecture of metabolic rate: environment specific epistasis between mitochondrial and nuclear genes in an insect sexual size dimorphism in a drosophila clade, the d. obscura group geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data bayesian phylogenetics with beauti and the beast . mcmc methods for multi-respoinse generalized linear mixed models: the mcmcglmm r package r: a language and environment for statistical computing. vienna, austria: r foundation for statistical computing the phylogenetic mixed model maximum-likelihood estimation of evolutionary trees from continuous characters phylogenetic analysis and comparative data: a test and review of evidence inferring the historical patterns of biological evolution the role of genotype-by-environment interactions in sexual selection how will global climate change affect parasite-host assemblages? identifying climate drivers of infectious disease dynamics: recent advances and challenges ahead complex effects of temperature on mosquito immune function infection risk decreases with increasing mismatch in host and pathogen environmental tolerances the thermal mismatch hypothesis explains host susceptibility to an emerging infectious disease genotype-environment interaction and the evolution of phenotypic plasticity quantitative genetics and the evolution of reaction norms genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in drosophila melanogaster coexistence of similar genotypes of daphnia magna in intermittent populations: response to thermal stress temperature checks the red queen? resistance and virulence in a fluctuating environment phenotypic variance, plasticity and heritability estimates of critical thermal limits depend on methodological context making sense of heat tolerance estimates in ectotherms: lessons from drosophila validity of thermal ramping assays used to assess thermal tolerance in arthropods the evolution of transmission mode costs and benefits of sublethal drosophila c virus infection entry is a rate-limiting step for viral infection in a drosophila melanogaster model of pathogenesis impact of environmental variation on host performance differs with pathogen identity: implications for host-pathogen interactions in a changing climate host nutrition alters the variance in parasite transmission potential measuring parasite fitness under genetic and thermal variation immunity in a variable world many thanks to darren obbard and frank jiggins for useful discussion and vanessa kellerman and johannes overgaard for discussion about thermal assay methods. thanks to dave hosken for use of bmr chambers and to the drosophila species stock centre for supplying flies. thanks to two anonymous reviewers for constructive comments. key: cord- -ximzvqbm authors: forsdyke, donald r. title: chargaff’s gc rule date: - - journal: evolutionary bioinformatics doi: . / - - - - _ sha: doc_id: cord_uid: ximzvqbm evolutionary selective pressures sometimes act to preserve nucleic acid features at the expense of encoded proteins. that this might occur in the case of nucleic acid secondary structure was noted in chapter . that this might also apply to the species-dependent component of the base composition, (g+c)%, was shown by sueoka in [ ]. the amino acid composition of the proteins of bacteria is influenced, not only by the demands of the environment on the proteins, but also by the (g+c)% of the genome encoding those proteins. chargaffs "gc rule" is that the ratio of (g+c) to the total bases (a+g+c+t) tends to be constant in a particular species, but varies between species. sueoka further pointed out that for individual "strains" of tetrahymena (ciliated protozoans) the (g +c)% (re ferred to as "gc" ) tends to be uniform throughout the genome: " if one compares the distribution of dna molecules of tetrahymena strains of different mean gc contents, it is clear that the difference in mean values is due to a rather uniform difference of gc content in individual molecules. in other words, assuming that strains of tetrahym ena have a common phylogenetic origin, when the gc content of dna of a particular strain changes, all the molecules undergo increases or decreases of gc pairs in similar amounts. this result is consistent with the idea that the base composition is rather uniform not only among dna molecules of an organism, but also with respect to different parts of a given molecule." again, this observation has since been shown to apply to a wide variety of species, although many organisms have their genomes finely sectored into regions ("homostability regions" or " isochores") of low or high (g+c)% (see later). sueoka also noted a link between (g+c)% and reproductive isolation for strains of tetrahym ena: "dna base composition is a reflection of phylogenetic relationship. furthermore, it is evident that those strains which mate with one another (i.e. strains within the same 'variety ') have similar base compositions. thus strains of variety i ..., which are freely intercrossed, have similar mean gc content." it seems that, in identifying (g +c)% as the component of the base composition that varies between species, chargaff had uncovered what can now be recognized as the " ho ly grail " of speciation postulated by the victorian physiologist george romanes [ ] . romanes had drawn attention to the possibility of what we would now call non-genic variations (germ-line mutations that usually do not affect gene products). as manifest in the phenomenon of hybrid sterility, these would tend to isolate an individual reproductively from most members of the species to which its close ancestors had belonged, but not from individuals that had undergone the same non-genic variation . romanes held that, in the general case, this isolation was an essential precondi-tion for the preservation of the anatomical and physiological characteristics (genic characteri stics) that were distinctive of a new species. in the early dec ade s of the twentieth century william bateson als o postulated non-genic inherited vari ati ons that tend to remain relatively constant (vary only within narrow limits) with in a species, but would vary between species (i.e. a species member would not d iffer from its fellow species member s, but would differ from members of allied species). t he non-genic variation s, in whatever was responsible for carry ing hereditary information from generatio n to generation (not known at that time), would have the potential to lead to spec ies differentiation, so that variant individuals (con stituting a potential " not-self' incipient species) would end up not being able to reproduce with members of the main speci es ("sew' species). reproduction bein g unsu ccessful, the main species can be viewed as const ituting a "reproduct ive env ironment" that moulds the genome phenotype ("reprotype") by negatively se lect ing (by den ying reproducti ve success to) variant organisms that attempt (by mating and producing healthy, fertile , offspring) to recross the eme rging interspecies boundary. thu s, the main specie s positively selects itself by negati vely selecting variants. should these variants find compatible mates, then they might accumulate as a new species that, in turn , would positiv ely select itself by negatively selecting further variants. this is " spec ies se lection," a form of group se lection that many biologists have found hard to imagine. indeed, richard dawkins, hav ing sco rned the " argume nt from personal incredulity," was obliged to resort to it when confro nted with the possibility of species se lection: " it is hard to th ink of reasons why species survivabi lity should be decoupled from the s um o f the surv ivabilities of the individual members of the spec ies" [ ] . when the latter sentence is parsed its logic see ms imp eccabl e. hold tight , and we will see if we can work it out. "the spec ies" is the establi sh ed main species, members of which imp eril themselves onl y marginally, if at all , by mating with (denying reproductive success to) members of a small potentially incipient species . thu s, in reproductive interactions between a main and an incipient spec ies, survivability of the main spec ies is coupled negatively to the sum of the survivabilit ies of ind iv idua l members of the incipient specie s (i.e. it surv ive s when they do not survive), much more than it is coupled positively to the sum of the survivabilities of its own individual members (i .e. it s urv ives when they survive). in this sense, main spec ies survivability is coupled to the sum of the survivabilities of individual members of the incipient species, and decoupl ed from the sum of the survivabilities of its own individual members. of course, by individual survivabilities is meant, not just mer e survival, but survival permitting unimpeded production offertile offspring. survival of members of an incipient species occurs, not only when cla ssical darwinian phenotypic interactions are favourable (e.g. escape from a tiger), but also when reprotypic interactions are favourable (e .g. no attempted reproduction with members of the main species). tigers are a phenotypic threat. members of the main species are a reprotypic threat [ ] . individual members of a main species that are involved (when there is attempted crossing) in the denial of reproductive success to individual members of an incipient species, are like individual stones in the walls of a species fortress against which the reproductive arrows of an incipient species become blunted and fall to the ground . alternatively, the main species can be viewed as a gulliver who barely notices the individual lilliputian incipients brushed off or trampled in his evolutionary path. just as individual cells acting in collective phenotypic harmony constitute a gulliver, so individual members of a species acting in collective reprotypic harmony constitute a species. that harmony is threatened, not by its own members, but by deviants that, by definition, are no longer members of the main species (since a species is defined as consisting of individuals between which there is no reproductive isolation). these deviants constitute a potential inc ipient species that might one day pose a phenotypic threat to the main species (i.e. they will become part of the environment of the latter). it is true that a member of a main species that becomes irretrievably pairbonded with a member of an incipient species (e .g. pigeons) will leave fewer offspring, so that both members will suffer the same fate (have decreased survivability in terms of number offertile offspring). but, in the general case, one such infertile reproductive encounter with a member of an incipient species will be followed by many fertile reproductive encounters with fellow members of the main species. members of the main species are most likely to encounter other members of the main specieshence, there will be fertile offspring. members of an incipient species, being a minority, are also most likely to encounter members of the main species -hence, there will be infertile (sterile) offspring. much more rarely , a member of an incipient species will encounter a fellow incipient species member with which it can successfully reproduce -an essential precondition for species divergence. once branching (reproductive isolation) is initiated (fig. - ) , the natural selection of darwin should help the branches sprout (extend in length). natural selection would favour linear species differentiation by allowing the survival of organisms with advantageous genic variations, and disallowing the survival of organisms with disadvantageous genic variations. these genic variations would affect an organism's form and function (the classical phenotype). darwin thought that natural selection might itself suffice to bring about branching. indeed, it appears to do so in certain circumstances, as when segments of a species have become geographically isolated from each other. however, here the branching agency is whatever caused the geographical isolation , not natural selection. speciation requires isolation in some shape or form. the probl em of the ori gin of species is that of determining what form isolation takes in the general case . in his faith in the power of natural selection , darwin wa s like the early chemists who were s atisfied w ith atoms as the ultimate basi s of matter. but for some chemists phenomena such as swinging compass needles (magnetism) , falling apples (gravity), and (lat er) radioactivity, were manifestations of som ething more fundamental in chemistry than atom s. likewise, for some biologists the phenomenon of hybrid ste rility seemed to manifest something more fundamental in biology than natural selection [ ] . romanes referred to his holy grail (speciating factor) as an abstract " intrinsic peculiarity" of the reproductive system. bateson described his as an abstract " res id ue" with which genes were independ ently assoc iated. goldschmidt's was an ab stract chromosomal " patte rn" caused by "s ys temic mutations" that would not necessaril y affect genic function s (see chapter ). these are just what we might expect of (g +c)%. indeed, in bacteria, which when so inclined inte rmitte ntly tran sfer dna in a sex ual fashion [ ] , differences in (g +c)% appear early in the spec iation process [ ], in keeping with sueoka's above obs ervations in ciliates. as show n in chapter , where different levels of genetic information were considered , a metaphor for the role (g +c)% might play in keeping individuals reproductively isolated from each oth er, is their acc ent [ ] . a common language brings people together, and in this way is conducive to sexual reproduction . but languages can vary , first into diale cts and then into independent sub-lang uages . lin gu istic differen ces keep people apa rt, and this difference in the reproductive environment can militate against sexual reproduction . at the molecular level , we see similar force s acting at the level of meiosis -the dance of the chromosomes. in the gonad sim ilar paternal and maternal chromosomes (homologues) align . the early microscopists referr ed to this as "c onj ugation." if there is sufficient seq uence identity (i.e. the dna " accents" match), then the band plays on . the chromosomes continue their minuet, progressing through various check-points [ ] , and gametes are formed. if there is insufficient identity (i .e. the dna " accents" do not match) then the music stops. meiosis fails , gametes are not formed , and the child is ster ile -a " mule." thus, the parents of the child (their " hy brid") are reproductively isolatedfrom each other (i.e. unable to generate a line of descendents due to hybrid sterility), but not necessarily from other members of their species. at least one of the parents has the potential to be a founding member of a new spec ies, provided it can find a mate with the same dna " accent." differences in (g+c)% have the potential to initiate the speciation process creating first " incipient species" with partial reproductive isolation, and then " species" that, by definition , are fully reproductively isolated. to see how this might work, we consider the chemistry of chromosome alignment at meiosis [ ]. in muller suggested that the pairing of genes as parts of chromosomes undergoing meiotic synapsis in the gonad might provide clues to gene structure and replication [ ] : "it is evident that the very same forces which cause the genes to grow [duplicate] should also cause like genes to attract each other [pair] .... if the two phenomena are thus dependent on a common principle in the make-up of the gene, progress made in the study of one of them should help in the solution of the other." in he set his students an essay "how does the watson-crick model account for synapsis?" [ii] . the model had the two dna strands " inwardlooking" (i.e. the bases on one strand were paired with the bases on the other strand). crick took up the challenge in with his " unpairing postulate" by which the two strands of a dna duplex would unpair to expose free bases in single-stranded regions [ ] . this would allow a search for sequence similarity (homology) between two chromosomes (i.e. between two independent duplexes). others later proposed that the single-stranded regions would be extruded as stem-loops. the " outward-looking" bases in the loops would be available to initiate the pairing process [ ] [ ] [ ] . thus, for meiotic alignment, maternal and paternal chromosomal homologues should mutually explore each other and test for "self' dna complementarity, by the " kissing" mechan ism noted in chapter [ ] [ ] [ ] . under this model ( fig. - ), the sequences do not commit themselves, by incurring strand-breakage, until a degree of complementary has been recognized. the mechanism is essentially the same as that by which trna anticodon loops recognize codons in mrnas, except that the stem-loop structures first have to be extruded from dna molecules that would normally be in classical duplex form . in all dna molecules examined, base-order supports the formation of such secondary structures (see chapter ). if sufficient complementarity is found between the sequences of paternal and maternal chromosome homologues (i.e. the genomes are "reprotypically" compatible), then crossing over and recombination can occur (i.e. the " kissing" can be "consummated") . the main adaptive values of this would be the proper assortment of chromosomes among gametes, and the correction of errors in chromosome sequences (see below and chapter ). "kissing" turns out to be a powerful metaphor, since it implies an exploratory interaction that may have reproductive consequences . as negative supercoiling progressively increases, the strands of each duplex synchronously open to allow formation of equivalent stem-loop secondary structures so that "kissing" interactions between loops can progress to pairing. at the right, paternal and maternal duplexes differ slightly in (g+c)% (x, and x + ). the maternal duplex of higher (g+c)% opens less readily as negative supercoiling increases, so strand opening is not synchronous, "kissing" interactions fail, and there is no progress to pairing. in this model, chromosome pairing occurs before the strand breakage that accompanies recombination (not shown). even if strand breakage were to occur first (as required by some models), unless inhibited by single-stranded dna-binding proteins the free single strands so exposed would rapidly adopt stem-loop conformations . so the homology search could still involve kissing interactions between the tips of loops the model predicts that, for preventing recombination (i.e. creating reproductive isolation), a non-complementarity between the sequences of potentially pairing strands, in itself, might be less important than a noncomplementarity associated with sequence differences that change the pattern of stem-loops. this implies differences in the quantities of members of the watson-crick base pairs in single strands (i.e. a parity difference).this is because parity between these bases would be needed for optimum stem formation . parity differences should correlate with differences in stem formation, and hence, different stem-loop patterns, as will now be con sidered. what role does the (g+c)% "accent" play in meiotic pairing? from calculated dna secondary structures, it has been inferred that small fluctuations in (g +c)% have great potential to affect the extrusion of stem-loops from duplex dna molecules and , hence, to affect the pattern of loops which would then appear ( fig. - ) . a very small difference in (g+c)% (reprotypic difference) would mark as "not-self' a dna molecule that was attempting to pair meiotically with another dna ("self'). this would impair the kissing interaction with the dna [ , ] , and so would disrupt meiosis and allow divergence between the two parental lines , thus initiating a potential speciation event. the total stem-loop potential in a sequence window can be analysed quantitatively in terms of the relative contributions of base composition and base order, of which base composition plays a major role (see chapter ). of the various factors likely to contribute to the base composition-dependent component of the folding energy of an extruded single stranded dna sequence, the four simplest are the quantities of the four bases. two slightly more complex factors are the individual bases, from each potential watson-crick base pair, that are present in lowest amounts. for example, if the quantities of a, g , c and t in a nucleotide sequence window are , , and , respectively , then what may be referred to as "a t min " would be , and the corresponding "gc min " would be . these numbers would reflect the upper limit on the number of base pairs that could form stems, since the quantity of the watson-crick pairing partner that was least would placc a limit on the possible number of base pairs. this value might be expected to correlate positively with folding stability. conversely, the excess of bases without a potential pairing partner (in the above example a-t = and g-c = ) might provide an indication of the maximum number of bases available to form loops . since loops tend to destabilize stem-loop structures, these "chargaff difference" values might be expected to correlate negatively with folding stability. although the bases are held in linear order, a vibrating single-stranded dna molecule has the potential to adopt many structural conformations, with watson-crick interactions occurring between widely separated bases. accordingly, pairing can also be viewed as if the result of random interactions between free bases in solution. this suggests that the two products of the quantities of pairing bases could be important ( x , and x , in the above example). the products would be maximal when pairing bases were in equal proportions in accordance with chargaffs second parity rule . in an attempt to derive formulae permitting prediction of folding energy values directly from the proportions of the four bases , jih-h . chen [ ] examined the relative importance of e ight of the above ten factors in determining the base composition-dependent component of the folding energy (fors-m; see chapter ). these factors were a, g , c, t , at mi ." cg mi ,,, a x t, and g x c (where a, c , g, and t refer to the quantities of cach particular base in a sequence window). the products of the quantities of the watson-crick pairing bases (a x t, and g x c) were found to be of major importance, with the coefficients of g x c (the strongly interacting s bases), greatly exceeding those of ax t (the weakly interacting w bases). less important were at min and cg min , and the quantities of the four bases. all ten parameters were exam ined in an independent study, which confirmed the major role of the product of the quantities of the s bases in a segment ( of particular importance is that it is not just the absolute quantities of the s bases, but the product of the multiplication of these absolute quantities. this should amplify very small fluctuations in (g+c)%, and so should have a major impact on the folding energy of a segment and, hence, in the pattern of stem-loops extruded from the duplex dna in a chromosome engaging in a "kissing" homology search for a homologous chromosome segment. if stem-loops are of critical importance for the initiation of pairing between segments of nucleic acids at meiosis, then differences in (g +c)% could strongly influence the establishment of meiotic barriers, so leading to speciation . but barriers may be transient. having served its purpose, an initial barrier may be superseded later in the course of evolution by a more substantial barrier (see figure - ). in this circumstance evidence for the early transient barrier may be difficult to find. however, in the case of different, but related, virus species (allied species) that have the potential to co infect a common host cell , there is circumstantial evidence that the original (g+c)% barrier has been retained. modern retroviruses, such as those causing aids (hiv - ) and human t cell leukemia (htlv-i), probably evolved by divergence from a common ancestral retrovirus. branching phylogenetic trees linking the sequences of modern retroviruses to such a primitive retroviral " eve" are readily constructed, using either differences between entire sequences, or just (g +c)% differences [ ] . the fewer the differences, the closer are two species on such trees. unlike most other virus groups, retroviruses are diploid. as indicated in chapter , diploidy entails a considerable redundancy of information, a luxury that most viruses cannot afford. they need compact genomes that can be rapidly replicated, packaged and dispersed to new hosts. however, different virus groups have evolved different evolutionary strategies. the strategy of retroviruses is literally to mutate themselves to the threshold of oblivion ("mutational meltdown"), so constituting a constantly moving target that the immune system of the host cannot readily adapt to . to generate mutants, retroviruses replicate their nucleic acids with self-encoded enzymes (polymerases) that do not have the error-correcting ("proof-reading") function that is found in the corresponding enzymes of their hosts. indeed, this is the basis of aids therapy with azt (azidothymidine), which is an analogue of one of the nucleotide building blocks that are joined together (polymerized) to form linear nucleic acid molecules ("polymers;" see chapter ) . azt is recognized as foreign by host polymerases, which eject it. but retroviral polymerases cannot discriminate, and levels of mutation (in this case termination of the nucleic acid sequence) attain values above the obi iv-ion threshold ("hypermutation") from wh ich it is impossibl e to recover ("error catastrophy"). below the thre shold, there is a most effective mech ani sm to counter mutational damage. the retroviral counter-mutation strategy requires that two complete sing lestrand retroviral rna genomes be packaged in each viru s particl e (i .e. diploidy). each of these genomes will be seve rely mut ated but, since mutations occur randomly, there is a chance that each genome w ill have mutations at different sites. thus, in the next host cell there is the possibility of recombination (cutting and splicing) betw een the two genomes to gen erate a new genome with many less, or zero, mutations [ ] . the copackaging of the two genomes requires a proc ess analogous to meiotic pairing. on each genome a "d imer initiation" nucleotide sequenc e folds into a stem-loop struc ture. " kissing" interact ions between the loop s preced e the form ation of a short length of duplex rna , so that the two genomes form a dimer. this allows packaging and , in the next host , recomb ination can occur. wh at if two diploid viru ses both infected the same host ce ll, thu s releasin g four geno mes into an environment conducive to recombination? in many cas es th is would be a most favorable circumstance, sinc e there would now be four damaged genomes from which to regenerate, by repeated acts of recombination, an ideal ge nome . thus, it would seem maladaptive for a viru s with this particularly strate gy to evo lve mechanisms to prevent entry of anoth er virus ("sup erinfect ion") into a cell that it was occupying, at least in the early stage s of infection [ ] . this presupposes that a co-infec ting viru s will be of the same spec ies as the virus whi ch first gain ed entry . however, h v- and htlv -l are retroviruses of alli ed , but distin ct, species. th ey have a common host (humans) and common host cell (known as the cd t-iymphocyte). when in the cou rse of evo lution these two virus spec ies first began to diverge from a common ancestral retroviral species, a barrier to recombination had to develop as a cond ition of successful div ergence. yet, these two virus types needed to retain a common host cell in which they had to perform sim ilar tasks. thi s meant that they had to retain similar gen es. many simi lar gene-encoded function s are indeed found . similar genes implies similar sequences, and sim ilar sequences implies the possibility of recombination betw een the two genomes. thu s, coexisten ce in the same host cell could result in the viruses destroying each oth er, as distinct species members, by mutually recombining (shuffl ing their genomes tog ether). without a recombination barrier each virus was part of the selective environment of the other. this should have provided a pre ssure for genomic changes that, while not interfering with conventional phenotypic functions , would protect against recombination with the other type. if (g+c)% differences could create such a recombination barrier (while maintaining, through choice of appropriate codons, the abilities to encode similar amino acid sequences), then such differences would be selected for. when we examine the (g +c)% values of each of these species there is a remarkable difference. j- iv-i is one of the lowest (g+c)% species known (i.e. it is at-rich). j- tlv-i is one of the high est (g+c)% species known (i.e. it is gc-rich). this might be regarded as just a remarkable coincidence save for the fact that, in some other situations where two viruses from different but allied species occupy a common host cell , there are also wide differences in (g +c)% [ , ] . as set out above, th ese (g+c)% differences alone should suffice to prevent recombination. the plant which gives us tobacco, nicotiniana tabacum, is a tetraploid which emerged some six million years ago when the two diploid genomes of nicotin iana sylvestris and nicotiniana tomentosiform is appeared to fuse . nicotiniana tabacum is designated an allotetraploid (rather than an autotetraploid) since the two genomes were from different so urce species (greek : allos = other; autos = same). the two species are estimated to have diverged from a common ancestral species million years ago . as allied species they should have retained some sequence similarities; so within a common nucleus in the tetraploid there should have been ample opportunity for recombination between the two genomes. yet , the genomes have retain ed their separate identities. this can be shown by backcrossing to the parental types. half the chromosomes of the tetraploid pair at meiosis with chromosomes of one parent type. thus, recombination of the other chromosomes of the tetraploid with chromosomes of that parent type is in some way prohibited. in goldschm idt noted [ ]: "c lausen ... has come to the conclusion that n. tabacum is an allotetraploid hybrid, one of the genomes being derived from the species sylvestris, the other from tomentosa. by continuous backcrossing to sylvestris the chromosomes deriv ed from sylvestris can be tested because they form tetrads with the sylvestris- the surv ival of a duplicate copy of a gene depends on a var iety of factors , including (i) natural selection favouring organisms where a function encoded by the gene is either increased or changed (i.e . there is either concerted or divergent gene evolution), (ii) a recombination-depend ent proc ess known as gene conversion , and (iii) a recombination-dependent process that can lead to copy-loss (see fig. - ) . these intragenomic recombination s can occur when there is a successful search for similarity between dna strands . thi s is likely to be greatly influ enc ed by the (g+c)% environment of th e or iginal gene and the (g +c)% env iro nment wh ere the duplicate copy locates. once a (g +c)%-dependent speciation proc ess has begun, factors oth er than (g +c)% are likely to replace the original difference in (g +c)% as an intergenomic barrier to reproduction (i .e . a barri er to recombination between diverged paternal and maternal genomes within their hybrid, if such a " mule" can be generated; fig . - ) . in this circumstance, (g +c)% becomes free to adopt oth er roles, such as the prevention of recombination within a genome (intragenom ic recombination). this can invo lve the differentiation of regi ons of relatively uniform (g+c)%, that japane se physicists aki yo shi wada and ak ira suy ama referred to as having a " homosta bizing propensity" and g iorg io bernardi and his coworkers named " isoc hores" (greek : iso = sa me; choras = group) [ , ] . th ese hav e the potential to recombinatio nally isolate different part s of a genome. t hus, the attempted duplication of an ance stral g lobin gene to gen erate the a-globin and [ -globin gen es of mod ern primates might have fa iled sinc e sequ ence sim ilarity would favour recombination between the tw o gen es and incipi ent differences (early sequence divergence) co uld have been e liminated (" gene conversion;" fig. - ). how ever, the dup lication app ears to hav e involved relocation to a d ifferent isochore with a different (g +c)%, so the two genes became recombinationally isolated to the ext ent that initi ally the sequ ences flanking the genes d iffered in (g +c)% . later the new gene would have increased its recombinational isolation by mutating to acquire the (g+c)% of its host isochore. as a con sequence of the differences in (g+c)% the correspond ing mrnas today utilize different codons for correspond ing amino ac ids, even though both mrnas are tran slated in the same cell using the same ribosomes and same trna populations. so it is most unlikely that the primary pressure to differentiate codons aro se at the translational level. ig. - . model for possible outcomes of a gene duplication. the duplication from (a) can result in identical multicopy genes (b) that confer an ability to produce more of the gene product. if this is advantageous, then the multicopy state will tend to be favored by natural selection. if unmutated (white box in (b)) or only slightly mutated (light grey striped box in (c)) , there are not sufficient differences between the duplicates to prevent a successful homology search (d). this allows the mutation (c) to be reversed to (b) by the process known as gene conversion (see fig. - ). this maintains identical copies, so allowing concerted evolution of the multicopy genes to continue. however, the recombination necessary for gene conversion can also result in removal of a circular intermediate (e, f), and restoration of the single copy state (g). the risk of copy-loss due to recombination (d-g) can be decreased by further mutation (dark grey striped box in (h)). this will decrease the probability of a successful homology search. being protected against recombination (i.e. preserved), the duplicate is then free to differentiate further by mutation (black box in (i)). if the product of the new gene confers an advantage, then the duplicate will be further preserved by natural selection (divergent gene evolution). in the general case, mutation facilitating recombinational isolation (h) precedes mutation facilitating functional differentiation (ij under positive darwinian selection < atgctgcggctatcgcagcat s + m ' ---i t-a-g--g-a-g-g-g-g g i t a g-c--g+g-g-i=-a > ' '< ' atgctg~cag ca t (b) > ' ( ) e-e-e-~e-e-e-.t '----+--a-f"l-ffi-ih -fi---f;- '::-(.,; r=f-"f-'~re-fi-., ' in the alternative shown here, the status quo is restored to the top duplex (an a is mutated to t), but in the bottom duplex the t-t non-watson-crick basepair is replaced with an a-t watson-crick base-pair (i.e. a t is mutated to an a). thus, there has been conversion of the sequence of the original m allele to that of the p allele. there has been a loss of heterozygosity (as in (a)) and a gain of homozygosity (as in (d)). in this example, gene conversion involves copies of homologous genes (alleles) on different chromosomes. however, gene conversion can also involve homologous genes (non-allelic "paralogues") on the same chromosome (see fig. - ). note that, in chapter , sequence . (p above) is shown to form a stem-loop with the central bases being located in the loop (sequence . ). since the single base-pair difference between p and m versions is in this loop, then the m version has the potential to form a similar stem-loop. because the loops differ slightly, during the initial homology search loop-loop "kissing" interactions might fail and prohibit subsequent steps. however, cross-over points can migrate (e.g. (b) to (c)), so that if crossing over is prohibited in one region there is some possibility of a migration from a neighboring region that would reveal mismatches. thus, multiple incompatibilities (base differences) are most likely to inhibit the pairing of homologous chromosomes and the repairing of multiple mismatches each isochore would have arisen as a random fluctuation in the base composition of a genomic region such that a copy of a duplicated gene that had transposed to that region was able to survive without recombination with the original gene for a sufficient number of generations to allow differentiation between the copy and its original to occur. thi s would have provided not only greater recombinational isolation, but also an opportunity for functional differentiation. if the latter differentiation were advantageous, organisms with the copy would be favoured by natural selection. the regional base compositional fluctuation would then have "hitch-hiked" through the generations by virtue of its linkage to the successful duplicate (i.e. the copy would have been positively selected). by preserving the duplicate copy from re-combination with the original copy, the isochore would, in turn , have itself been preserved by virtue of its linkage to the duplicate copy. when functional differentiation of a duplicate is necessary for it to be selected (divergent evolution), there is the danger that, before natural selection can operate, recombination-mediated gene conversion will rev erse any incipient differentiation, or intragenic recombination between the copies (paralogues) will result in copy-loss. in the case of duplicate eukaryotic genes that have diverged in sequence, koichi matsuo and his colleagues noted that divergence was greatest at third codon positions, usually involving a change in (g+c)% [ - ]. thus, there was a codon bias in favour of the positions of least importance for the functional differentiation that would be necessary for the operation of natural selection. where amino acids had not changed, different gene copies used different synonymous codons. it wa s proposed that the (g+c)% change was an important "line of defence" against homologous recombination between the duplicates. thus, recornbinational isolation of the duplicate (largely involving third codon position differences in (g+c)%) would protect (preserve) the duplicate so allowing time for functional differentiation (largely involving first and second codon position differences), and hence, for natural selection to operate. in the general case, isolation would precede functional differentiation, not the converse. (g+c)% differentiation, largely involving third codon positions, would precede functional differentiation, largely involving first and second codon positions under positive darwinian selection . from all this it would be predicted that, if a gene from one isochore were transposed to an isochore of different (g+c)%, and its ability to recombine with its allele were advantageous, then the gene would preferentially accept mutations converting its (g+c)% to that of the new host isochore (i .e. organisms with those mutations would be genetically fitter and thus likely to leave more fertile offspring than organisms without the mutations). indeed, there is evidence supporting this. the sex chromosomes (x and y) tend not to recombine at meiosis except in a small region (the "pseudoautosomal" region; see chapter ). transfer of a gene from a non-recombining part of a sex chromosome to the pseudoautosomal region forces the gene rapidly to change its (g+c)% value [ ] . for various reasons (e.g. large demand for the gene product), certain genes are present in multiple identical copies. but, in the absence of some restraint, copies that are initially identical will inevitably diverge in sequence [ ]. so how can multicopy genes (e .g. rrna genes) preserve their similarity to each other? to prevent divergence through the generations (i.e . to allow "concerted evolution"), they should mutually correct each other to eliminate deviant copies. this is likely to occur by a recombination-dependent process -"gene conversion" (figs. - , - ; see chapter ). thus, multicopy genes should all be, either in the same isochore, or in isochores of very close (g+c)%, so that recombination can occur. before dna sequencing methods became available, " isochores" were described as dna segments that could be identified on the basis of their distinct densities in samples of duplex dna obtained from organisms whose cells had nuclei (eukaryotes). the method involved physically disrupting dna by hydrodynamic sheering to break it down to lengths of about kilobases. the fragments were then separated as bands of distinct densities by centrifugation in a salt density gradient. the densities could be related to the average (g+c)% values of the segments, since the greater these values, the greater the densities. this way of assessing the (g+c)% of a duplex dna segment distinguished one large segment from another, and largeness became a defining property of isochores. isochores, as so defined, were not identified in bacteria, which do not have distinct nuclear membranes (prokaryotes; see chapter ). since prokaryotes (e.g. bacteria) and eukaryotes (e.g. primates) are considered to have evolved from a common ancestor, does this mean that the ancestor had isochores that were subsequently lost by prokaryotes during or after their divergence from the eukaryote lineage (isochores-early)? or did the ancestor not have isochores, which were therefore freshly acquired by the eukaryotic lineage after its divergence from the prokaryotic lineage (isochores-late)? if prokaryotes could be shown to have isochores, then this would favour the isochores-early hypothesis. indeed, prior to modern sequencing technologies, physical methods demonstrated small segments of distinct (g+c)% in the genomes of prokaryotes and their viruses. the kb duplex genome of phage lambda (see chapter ) was extensively sheered to break it down to subgenome-sized fragments. these resolved into six distinct segments, each of relatively uniform (g+c)%, by the density method [ ] , and into thirty four "gene sized" segments by another, more sensitive, method (thermal denaturation spectrophotometry) [ ] . with the advent of sequencing technologies, in mervyn bibb and his colleagues were able to plot the average (g+c)% values of every third base for small windows in the sequences of various bacteria (fig. - ) [ ] . three plots were generated, the first beginning with the first base of the sequence (i.e. bases in frame i, , , etc.), the second beginning with the second base of the sequence (i.e. bases in frame , , , etc.), and the third beginning with the third base of the sequence (i.e. bases in frame , , , etc.) . in certain small regions (g+c)% values were relatively constant within each frame. these regions ofconstant (g+c)% corresponded to genes. note that the relative constancy of (g+c)% is most for the third codon position (mainly independent of the encoded amino acids), and least for the second codon position (most dependent on the encoded amino acids). the fluctuation in values at the second codon position is more apparent when a window size equivalent to codons is used (b) than when a window size equivalent to codons is used (a). this figure was redrawn from ref. [ ] thus, individual genes have a relatively uniform (g +c)% and each codon position makes a distinctive contribution to that uniformity . this is not confined to bacteria. wada and suyama noted that, whether prokaryotic or eukaryotic, "every base in a codon seems to work cooperatively towards realizing the gene's characteristic value of (g+c) content." this was a "homostabilizing propensity" allowing a gene to maintain a distinct (g+c)%, relatively uniform along its length , which would differentiate it from other genes in the same genome [ ) . thus, each gene constitutes a homostabilizing region in dna . stated another way , if large size is excluded as a defining property, many bacteria have isochores. when isochores are defined as dna segments of relatively uniform (g+c)% that are coinherited with specific sequences of bases, then bacteria have isochores. to contrast with the classical isochores of bernardi, these are termed "rnicroisochores," and their length is that of a gene, or small group of genes (see chapter ). thus, classical eukaryotic isochores ("macroisochores") can be viewed as constellations of microisochores of a particular (g +c)%. the proposed antirecombination role of (g+c)% would required that , unless they represent multicopy genes, microisochores sharing a common macroisochore (i.e. they have a common (g+c)%) have other sequence differences that are sufficient to prevent recombination between themselves [ ). within an organism, genes with similar (g+c)% values may sometimes locate to similar tissues, so that there is a tissue-specific codon usage tendency [ i). since both prokaryotic (e.g. bacterial) and eukaryotic (e.g. primate) lineages have some form of isochore, this appears most consistent with the isochores-early hypothesis. while not endorsing a particular role for (g+c)%, this underlines the fundamental importance of (g +c)% differences in biology . let metaphors multiply! a given segment of dna is coinherited with a "coat" of a particular (g+c)% "color." a given segment of dna "speaks" with a particular (g+c)% "accent," (and hence has a distinct potential vibrational frequency; see fig. - ) . a fundamental duality of information levels is again manifest. as will be further considered in chapter , it is likely that differences in (g+c)% serve to isolate recombinationally both genes within a genome, and genomes within a group of species (a taxonomic group). the power to recombine is fundamental to all life forms because, for a variety of reasons, it is advantageous (see chapter ). however, the same power threatens to homogenize (blend) genes within a genome, and to homogenize (blend) the genomes of members of allied species within a taxonomic group (i.e. genus). this would countermand evolution both within a species and between spe-cies. thus, f unctional differentiation. be it between genes in a genome. or between genomes in a taxonomic gro up (spec iation), must. in the general case, be preceded (or closely accompanied) by the establishment of recombinational barriers. species have long been defined in terms of recombinational barriers (see chapter ). in some cont exts, genes are defined sim ilarly. a species can be defined as a unit of recombination (or rather, of antirecombination with respect to other species). so can a gene . most definitions of the "gene" contain a loose or explic it refe renc e to function. thus, biologists talk of a gen e encod ing information for tallness in peas. biochemists ta lk of the gene encoding information for growth hormone (a prot ein), and relate this to a segment of dna (se e legend to fig . - ) . however, before it can function , information must be preserved. classical darwinian theory proposes that function , through natural selection, is itself the preserving agent. thus, function and preservation go hand-in-hand, but fun ction is more fundamental than preservation . in biol ogi st ge org e williams in the usa , an originator of the "se lfis h gen e" con cept, seem ed to argue the converse wh en arriving at a new definition. the function of any multipart entity, which needs more than one part for this function , is usually dependent on its parts not being se parated. preservation can be more fundamental than function . williams propo sed that a gen e should be defined entire ly by its property of remaining intact as it passes from generation to generation. he identified recombination as a major thr eat to that intactness. thus, for williams, "gene" meant any dna segment that has the potential to persist for enough generations to serve as a unit for natural selection; this requires that it not be easily disruptable by recombination . th e gene is a un it of reco mbination (or rather, of antirecombination with respect to other gen es) [ ] . " socrates' ge nes may be with us yet, but not his genotype, becau se meiosis and recombination destroy genotypes as surely as death. it is only the meiotically dissociated fragments of the genotype that are transmitted in sex ua l reproduction , and these fragments are further fragm ented by meio sis in the next generation . if there is an ultimate indivisible fragm ent it is, by definition, ' the gene ' that is treated in the abstract discussions of population genetics. various kind s of suppress ion of recomb ination may cause a major chromosomal segment or even a whole chromo some to be transmitted entire for many generations in certain lines of descent. in such cases the segment, or chromosome, behaves in a way that approximates the population genetics of a s ingle gene . . . . i use the term gene to mean ' that which segregates and recombines with appreciable frequency ' .... a gene is one of a multitude of meiotically dissociable units that make up the genotypic message." despite this, williams did not invoke any special chromosomal characteristic that might act to facilitate preservation . pointing to "the now discredited theories of the nineteenth century," and lamenting an opposition that " arises . . . not from what reason dictates, but from the limits of what the imagination can accept," his text adaptation and natural selection made what seemed a compelling case for "natural selection as the primary or exclusive creative force ." no other agency was required. this tendency, which can be infectious, to bolster the scientific with the ad hominem in otherwise rational discourse, will be considered in the epilogue.jn contrast, we have here considered intergenomic and intragenomic differences in (g+c)% as an agency, essentially independent of natural selection, which preserves the integrity of species and genes, respectively . within a species individual genes differ in their (g +c)%. relative positions of genes on the (g +c)% scale are usually preserved through speciation events. if, in an ancestral species, gene a was of higher (g+c)% than gene e, this relationship has been sustained in the modern species that resulted from divergences within that ancestral species. accordingly, when the (g+c)% values of the genes of one of the modern species are plotted against the corresponding (g+c)% values of similar (orthologous) genes in the other modern species, the points usually fit a close linear relationship (c.f. fig. - ) . species with intragenomic isochore differentiation can themselves further differentiate into new species. in this case, a further layer of intergenomic (g+c)% differentiation would be imposed upon the previous intragenomic differentiation . again, when a sufficient degree of reproductive isolation had been achieved this initial barrier between species would usually be replaced by other barriers, thus leaving (g+c)% free to continue differentiating in response to intragenomic demands. however, (g+c)% is never entirely free. it can itself be constrained by demands on gene function (i .e. natural selection) that primarily affect first and second codon positions. furthermore, as we shall see next , in extreme environments, natural selection can make direct demands on (g +c)%, which might then conflict with its role as a recombinational isolator. there are few environments on this planet where living organisms are not found . hot springs, oceanic thermal vents, and radioactive discharges of nuclear reactors, all contain living organisms ("extremophiles"). fortunately, since heat and radiation are convenient ways of achieving sterilization in hospitals, none of these organisms has been found (or genetically engineered to become) pathogenic (so far) . thermophiles are so-called because they thrive at high temperatures. proteins purified from thermoph iles may show high stability at normal temperatures, a feature that has attracted commercial interest (i.e. they have a long "shelf life"). hence, the full genomic sequences of many prokaryotic thermophiles (bacteria and archaea) are now available . some thennoph iles normally live at the temperature of boiling wat er. nucleic acid s in solution at this temperature soon degrade. so how do nucleic acid s survive in thermophil es? the secondary structure of nucleic acids with a high (g+c)% is more stable than that of nucl eic acids with a low (g+c)%. this is con sistent with watson-crick g-c bonds being strong, and a-tor a-u bonds being weak (see table - ). do thermophiles have high (g +c)% dna ? in the case of gen es corresponding to rnas whose structure is vital for rna function , namely rrnas and trnas, the answer is affirmative. free of cod ing con straints (i .e. they are not mrnas), yet required to form part of the precise structure of ribo somes where prot e in synthesis occurs, gen es corresponding to rrna s appear to have had the flexibility to accept mutation s that increase g +c (i.e. organisms that d id not accept such mut ations perished by natural selection, presumably acting again st organ isms w ith less effic ient prot ein synthesis at high temperatures). the g +c content of rrnas is directly proportional to the normal growth temperature, so that rrna s of thermophilic prokaryotes are highly enriched in g and c [ ] [ ] [ ] . yet, althou gh optimum growth temperature correlates positively with the g+c content of rrna (and hence of rrn a genes), optimum growth temperature does not correlate positively with the overall g+c content of genomic dna , and hence with that of the numerous mrna populations transcribed from the genes in that dna (fi g. - a). instead, optimum growth temperature correlates positi vely with a+g content (fi g. - b ; see chapter ) [ ] . the finding of no consistent trend tow ards a high genomic (g +c)% in thermophilic organi sm s has been interpreted as supporting the " neutralist" argument that vari at ions in genomic (g +c)% are the consequences of mutational biases and are , in themselves, of no adapti ve value, at least with respect to maintaining duplex stability [ , ] . however, the finding is also consistent w ith the argument that genomic (g +c)% is too important merely to follow the dictates of temperature, since its prim ary role is related to other more fundamental adaptations. the stability of duplex dna at h igh temperatures can be ach ieved in ways other than by an increase in g +c content. these include association with small basic peptides (polyam ines) and relaxation of tor sional strain (supercoiling) [ , ] . thus, there is ev ery reason to believe that , whatever their (g+c)% content, thermophiles are able , both to maintain their dna s in class ica l duplex stru ctures with watson-crick hydrogen-bonding between oppo-site stra nds, and to adopt any necessary extruded secondary structures involving intrastrand hydrogen-bonding (i .e. stem-loops). this will be further considered in chapter . reflect the fact that relatively few thermophiles have been sequenced at this time. note that, whereas in (a) only % of the variation between points can be explained by growth temperature (~= . ), in (b) % can be explained on this basis (r" = . ; see appendix ) darwin held that biological evolution reflected the accumulation of frequent very small variations, rather than few intermittent large variations. that nature did not work by means of large jumps was encapsulated in the latin phrase "natura non facit saltum ." however, huxley, while supporting most of darwin's teachings, considered it more likely that evolution had proceeded in jumps ("natura facit saltum"). according to the arguments of this chapter, both are correct. within some members of a species small variations in the genome phenotype (i .e. in (g+c)%) accumulate, so that these members become progressively more reproductively isolated from most other members of the species, initially without major changes in the conventional phenotype. as it accrues, reproductive isolation increasingly favors rapid change in the conventional phenotype, often under the influence of natural selection. so, when their appearance is viewed on a geological time scale, new species can seem to "jump" into existence. the rate increase reflects better preservation of frequent phenotypic micromutations rather than of infrequent phenotypic macromutations (i.e . of "hopeful monsters," to use goldschmidt 's unfortunately term). in other words, while there is continuity of variation at the genotype level , as far as speciation is concerned variants (mutant forms) seem to emerge discontinuously at the phenotype level. being infrequent, and hence unlikely to find a member of the opposite sex with the same change, organisms with macrornutations are not the stuff of evolution. single strands extruded from duplex dna have the potential to form stemloop structures that, through exploratory loop-loop "kissing" interactions, may be involved in the homology search preceding recombination. the total stem-loop potential in a sequence window can be analyzed quantitatively in terms of the relative contributions of base composition and base order, of which base composition, and particularly the product of the two s bases (g x c), plays a major role . thus, very small differences in (g +c)% should impair meiotic pairing, resulting in hybrid sterility and the reproductive isolation that can initiate speciation (i.e. because their hybrid is sterile, the parents are, in an evolutionary sense, "reproductively isolated" from each other). in chemical terms, chargaff's species-dependent component of base composition, (g+c)%, may be the "holy grail" responsible for reproductive isolation (non-genic) as postulated by romanes, bateson and goldschmidt. once a speciation process has initiated, other factors (often genic) may replace (g+c)% as a barrier to reproduction (preventing intergenomic recombination between species). this leaves (g+c)% free to assume other roles, such as defined as long segments of relatively uniform (g+c)% that are coinherited with specific sequences of bases. these may facilitate gene duplication. indeed, each gene has a " ho mostabilizing propensity" to maintain itself as a "microisochore" of relatively uniform (g+c)%. protection against inadvertent recombination afforded by differences in (g+c)% facilitates the duplication both of genes, and of genomes (speciation). george williams' definition of a gene as a unit of recombination rather than of function is now seen to have a chemical basis key: cord- -z c zgp authors: garden, jenni g.; mcalpine, clive a.; possingham, hugh p.; jones, darryl n. title: habitat structure is more important than vegetation composition for local‐level management of native terrestrial reptile and small mammal species living in urban remnants: a case study from brisbane, australia date: - - journal: austral ecol doi: . /j. - . . .x sha: doc_id: cord_uid: z c zgp abstract as urban areas continue to expand and replace natural and agricultural landscapes, the ability to manage and conserve native wildlife within urban environments is becoming increasingly important. to do so we first need to understand species' responses to local‐level habitat attributes in order to inform the decision‐making process and on‐ground conservation actions. patterns in the occurrence of native terrestrial reptile and small mammal species in sites located in remnant urban habitat fragments of brisbane city were assessed against local‐level environmental characteristics of each site. cluster analysis, multidimensional scaling ordination, and principal axis correlation were used to investigate relationships between species' occurrences and environmental characteristics. native reptiles were most strongly associated with the presence of termite mounds, a high amount of fallen woody material, and a moderate amount of weed cover. native small mammals were most strongly associated with the presence of grass trees (xanthorrhoea spp.), and both reptiles and small mammals were negatively influenced by increased soil compaction. significant floristic characteristics were considered to be important as structural, rather than compositional, habitat elements. therefore, habitat structure, rather than vegetation composition, appears to be most important for determining native, terrestrial reptile and small mammal species assemblages in urban forest fragments. we discuss the management implications in relation to human disturbances and local‐level management of urban remnants. over the past years, habitat loss and fragmentation have had the greatest influence on terrestrial ecosystems and biodiversity worldwide (sala et al. ; millennium ecosystem assessment ) . agricultural land use has previously been regarded as the primary cause of habitat loss and fragmentation, yet as areas of suitable arable land are exhausted, agricultural expansion is declining (millennium ecosystem assessment ) . comparatively, urban development is expanding rapidly, encroaching on agricultural and natural, non-arable, landscapes (levia & page ; lugo ) . it therefore seems likely that urban development will surpass agricultural land use as the primary anthropogenic driver of land use change and habitat loss and fragmentation. areas deemed suitable for urban development often coincide with those areas that also support high native species richness and endemism (lugo ) . the impacts of urbanization on fauna populations are multicausal and multiscaled, altering the in situ structure and composition of habitat fragments, as well as their spatial patterning within the landscape context. these habitat modifications potentially have important consequences for concomitant fauna assemblages, with significant differences being apparent between urban and preurban assemblages (jones & wieneke ; van der ree ; tait et al. ) . within the urban matrix, introduced species and a handful of native generalist species tend to dominate, while habitat and dietary specialists and migratory species tend to decline in numbers or become locally extinct (how & dell , white & burgin ; tait et al. ). this problem is particularly pertinent to australia, where more than % of the human population currently resides in urban areas (united nations ) . for native fauna assemblages, native terrestrial reptile and small mammal species are considered to be the fauna groups most sensitive to urbanization and its associated disturbances (how & dell , van der ree ; white & burgin ; tait et al. ) . although many native mammals and reptiles are negatively influenced by urban development, some native mammal and reptile species, such as the common brush-tail possum (trichosurus vulpecula), blue-tongue lizard (tiliqua scincoides), and fence skink (cryptoblepharus virgatus) may adapt, and even thrive, within the built environment (koenig et al. ; matthews et al. ; garden et al. ) . in contrast, urban-sensitive species (e.g. dunnarts, sminthopsis spp., antechinus, antechinus spp., geckos, and large reptiles) face significant dispersal, predator avoidance and, resource adaptation challenges within the human-dominated urban matrix. consequently, these urban-sensitive species are restricted to the often isolated, remnant native vegetation patches that occur within the urban matrix or in the fringing peri-urban landscapes (how & dell tait et al. ; garden et al. ) . effective conservation in urban remnants requires scientific knowledge underpinning management decisions and on-ground actions. much of the current available scientific knowledge is based on non-urban ecological research (garden et al. ) . in non-urban environments there is a strong global consensus that, regardless of the disturbance pressure, many native reptile and small mammal species depend more on structurally complex habitats, than compositionally diverse floristics (e.g. bennett ; southgate et al. ; flemming & loveridge ; lenders & daamen ; spencer et al. ; kanowski et al. ) . similar findings have also been reported for native species within australian urban landscapes (e.g. dufty ; jellinek et al. ; white & burgin ) . although, fischer et al. ( ) and jellinek et al. ( ) found that vegetation composition, in addition to structure, was important for reptile species in grazed and urban affected habitats. in contrast, wilson et al. ( ) reported that within coastal heathlands of victoria, several native rodent and dasyurid species displayed no floristic or vegetation structural preferences. such discrepancies in research findings, coupled with the distinct lack of urban-based ecological research (garden et al. ) , limit our ability to make generalizations about the habitat requirements of urban reptiles and small mammals, and hence management recommendations. this limitation is further compounded by differences in the type, rate and intensity of disturbances within urban landscapes compared with non-urban landscapes. a further complication is the large variation in habitat requirements among species (e.g. jellinek et al. ; monamy & fox ), but also within species according to age. for example, although fischer et al. ( ) found that four-fingered skinks (carlia tetradactyla) responded to habitat structure and composition in grazing-affected landscapes, they also found that the relative importance of different attributes varied between juveniles and adults. consequently, australian urban conservation managers face significant uncertainty regarding the most appropriate management strategies for achieving long-term conservation outcomes for a diversity of native fauna species. if native fauna diversity is to be conserved in the face of rapid urban expansion, it is vital that we understand the habitat requirements and sensitivities of species living within urban remnants. this requires understanding how both species' composition and species' richness are influenced by local-level habitat factors. however, local-level habitat management is often focused on maintaining overall species' diversity within the landscape by managing habitat patches so that current concomitant assemblages of native fauna are conserved before they become locally extinct, thereby avoiding the need for expensive reintroduction programs. conserving native fauna in urban landscapes first requires understanding what local or in situ factors are important for maintaining diverse assemblages of native fauna. this knowledge may then by used to inform urban conservation managers and planners about priority habitat management decisions and activities so that native fauna occupying urban habitat fragments are adequately conserved. this was the focus of our study. this paper investigated correlations between the composition of native terrestrial reptile and small mammal fauna within urban habitat fragments and local-level (< ha) habitat factors such as habitat structure, vegetation composition, fire and human disturbances. these habitat factors were considered ecologically important for the target species and also can be manipulated and managed by conservation managers. investigations were based in urban habitat fragments located within the brisbane city council (bcc) local government area, where local government is responsible for setting within-patch management priorities and actions.we applied cluster analysis, multidimensional scaling and principal axis correlation to identify significant habitat attributes and examine their importance for reptile and small mammal assemblages. mammal and reptile assemblages were analysed separately. based on our findings, we discuss the implications for conservation management within brisbane city. the study focused on queensland's capital city, brisbane ( ° ′s, °e; area km , population approximately million) (fig. ). brisbane has a subtropical climate and is australia's third largest and most rapidly growing capital city (commonwealth of australia ). as european settlement in the early s approximately two-thirds of the original woody vegetation has been cleared for agricultural, industrial and/or urban development purposes (brisbane city council ) . more than % of this clearing was of lowland forests (< m from sea level) resulting in the current, highly fragmented and isolated lowland remnant vegetation patches that vary in their internal condition and disturbance and management histories (catterall & kingston ; brisbane city council ) . of the remaining % remnant vegetation cover, about % is protected for conservation purposes, yet much of this area is concentrated in contiguous forest on the city's outskirts, particularly in the d'aguilar ranges to the west (brisbane city council ). contemporary urban growth has concentrated along major transport networks to the north, south and south-west, with coastal wetlands and moreton bay to the east and mountain ranges to the west constraining urban expansion in these directions. accord-ingly, the highest concentration of urban development pressure currently occurs in the lowland outer suburbs to brisbane's south and south-east (brisbane city council ) . despite recent rapid urban expansion and subsequent habitat destruction and modification, brisbane still supports high floristic and faunal diversity and endemism, boasting the highest diversity of native vertebrate species of any of australia's capital cities (queensland museum ) . however, in the face of rapid urbanization, the continued persistence of this species' diversity is uncertain. our study focused on lowland remnant habitat fragments situated within - km of brisbane's central business district, in the southern (karawatha) and southeastern (burbank) suburbs (fig. ) . we selected sites located within regional ecosystem (re) type . - . , which is dominated by scribbly gum (eucalyptus racemosa) woodland located on sedimentary rocks and sandy soils (young & dillewaard ) . although classified as 'not of concern' by the queensland environmental protection agency (queensland environmental protection agency ), this re is extensively cleared and fragmented within brisbane city (young & dillewaard ) . bcc spatial data and satellite imagery were used to select sites located on private and council owned land. potential site locations were ground-truthed to assess their suitability. site locations were considered unsuitable if they were: (i) difficult to access; (ii) had been largely cleared or disturbed; (iii) patches were too small to fit a survey site; or, (iv) had a high likelihood of human interference to fauna survey equipment. survey sites measured m ¥ m and consisted of three parallel transects m apart, along which fauna traps were placed and habitat assessments were conducted. where possible, sites were positioned perpendicular to the landscape slope and a minimum of m from patch edges. this distance was selected to concentrate on interior rather than edge and matrix habitats. sites were also located at least m from designated walking tracks and recreational areas. wildlife surveys were conducted over three consecutive nights, during fine weather conditions in the spring and summer of . initial surveys ( were conducted at all sites, yet repeat surveys ( ) occurred at only sites, as nine sites were abandoned owing to recent fire or substantial human interference. native reptile and small mammal species were identified at each site using a combination of live-trapping, direct observation and trace survey methods, to maximize the probability of detecting the range of target species. each site was surveyed using: eight cage traps, elliott traps (two sizes: ¥ ¥ mm; ¥ ¥ mm), five dry pitfall traps ( l buckets) and three hair funnels. opportunistic species identifications from scats, visual observations and vocalizations were also recorded. cage traps, elliott traps and hair funnels were baited with the standard australian native small mammal bait mixture (menkhorst & knight ) . pitfall traps were unbaited. the dense vegetation and fallen woody debris at several sites prevented the use of drift-fences, which have previously been used with pitfall traps to improve capture success (e.g. mengak & guynn ; crosswhite et al. ; menkhorst & knight ) . cage and elliott traps were set and baited each afternoon, checked for captures each morning and closed during the day. pitfall traps remained open for the entire trap cycle ( days and nights) and were checked for captures each morning and afternoon. hair funnels were set at the beginning of a trap cycle and left undisturbed until collection at the end of the trap cycle. scat collection, direct observations and vocalization records occurred opportunistically throughout each site visit. at the point of capture, animals were identified to species-level using a relevant field guide (menkhorst & knight ; wilson ) , photographed, weighed, sexed (if possible) and immediately released. scat and hair samples were identified ex situ by one of two independent experts (initial surveys analysed by michiala bowen; repeat survey samples analysed by barbara triggs ) . habitat assessments were conducted at all sites between the initial and repeat wildlife survey periods. twenty-three habitat variables (table ) were measured at each site, following a protocol similar to that detailed by eyre et al. ( ) .the number of large trees (d.b.h. Ն cm) within the site perimeter was visually counted from the centre of each site, and the total basal area was recorded using the bitterlich variable radius method (sensu mueller-dombois & ellenberg ). a visual appraisal of the total number of termite mounds, approximate weed cover and, the presence or absence of fire (e.g. tree scars, charcoal, ash) and human disturbances (e.g. litter, garden waste, sawn logs) were recorded over the duration of each site assessment. the remaining habitat variables were measured along each of the three transects and the measurements either totalled or averaged for the whole site. the total number of acacia spp., allocasuarina spp., banksia spp., pteridium esculentum, callistemon spp., xanthorrhoea spp., and melaleuca spp. within m of the transect lines were recorded. similarly, all fallen woody material within m of each transect line was tallied and categorized into five diameter classes (< cm, - cm, - cm, - cm, > cm), eight decay classes (recently felled, sound, bark peeling off, to % decay, - % decay, - % decay, > % decay, debris), and three complexity classes (simple, complex, stump). these records were later used to calculate the relative volume (m ) of fallen woody material for each site.the total number of pieces of fallen wood that contained hollows was also recorded. the presence of canopy, mid-storey, and understorey cover was recorded every m along each transect using a gimballed sighting tube. understorey density was also measured every m using an adaptation of methods described by macarthur and macarthur ( ) and haering and fox ( ) . this involved a m screen, divided into cm grid cells, viewed at waist height from a -m distance. three understorey measurements were recorded: (i) the total number of grid cells obscured by vegetation; (ii) the highest grid row obscured by vegetation ( cm- cm); and (iii) the highest grid row with at least one grid cell Ն % obscured by vegetation. these six stratum records were later averaged to provide measures of average per cent cover for each stratum and average understorey density at each site. the per cent of ground cover, number of ground cover types (e.g. coarse leaf litter, casuarina needles, rocks, herbs), ground cover depth, and soil compaction were recorded every m within a ¥ m quadrat along each transect. per cent and types of ground cover were assessed visually. a -cm ruler was used to record ground cover depth. soil compaction was measured using a penetrometer (e.g. fox et al. ) and counting the number of hits taken (up to hits) to drive a weighted probe mm into the soil. for both ground cover depth and soil compaction, six measurements were made every m. the records of per cent ground cover, ground cover depth and soil compaction were later averaged for each site. analyses were based on the species detected and habitat variables recorded at each site. total species occurrence data were derived by combining the species detected from each wildlife survey method and during both survey periods.the overall species' occurrence data were divided into two separate data sets: (i) native reptile species; and (ii) native mammal species. exotic species were not included in the data analysis. continuous habitat variables were initially compared using the spearman rank correlation test in r version . . (the the r development core team ). a correlation coefficient > . was chosen to identify highly correlated habitat variables.where variables were highly correlated, one variable was removed from the data set. the remaining variables were used for subsequent statistical analyses. we applied a multivariate statistical approach using patn version . . for windows (belbin ) to examine the relationships between species' occurrence and site habitat characteristics. similarities between sites in terms of species' composition were first investigated using the bray & curtis index (bray & curtis ) . clusters were derived from the data set using the flexible unweighted pair group using arithmetic averaging method (upgma) with a beta (b) value of - . . the upgma clustering method is considered superior as it considers more than one species at any fusion (wardell-johnson & williams ; podani & schmera ) . at a beta value of - . , the upgma clustering method is space-dilating (cf. spacecontracting), thereby creating even-sized groups and preventing the formation of a single large group (belbin ; wardell-johnson & williams ) . we examined the acceptability of the resulting site groups and patterns using semi-strong hybrid multidimensional scaling ordination. the ordination was considered in conjunction with the minimum spanning tree (mst) and dendogram.the mst represents pairwise associations between group objects. its use is comple-mentary to ordination and may be used practically to confirm or deny close relationships or identify trends in the data (belbin ) . the dendogram schematically displays the hierarchical clustering of groups.the number of site groups was adjusted until maximum congruence was obtained between the ordination, mst and dendogram, thereby indicating that the most robust site groupings had been attained. relationships between the intrinsic species occurrence and the ordination groups were examined using principal component correlation (pcc) with permutations. pcc uses multiple linear regression to fit selected variables to the ordination space, showing the direction of best fit and the correlation with that direction (r ) (belbin ) . monte-carlo attributes in ordination (mcao) procedure with iterations was then used to test the reliability of these correlations with the ordination space by calculating the proportion of r values that exceed the true r in a given number of iterations (belbin ). an mcao value less than % was considered significant. variables identified as having significant correlations with the species groups were then overlaid on the ordination space. this analysis procedure was repeated to investigate relationships between the extrinsic habitat variables and the ordination groups. species and habitat variables that were found to be highly correlated with the ordination were examined further using the kruskal-wallis statistic function in patn to determine how well each variable discriminated between the ordination groups. the larger the kruskal-wallis statistic for a variable, the more significant that variable's contribution was to the differentiation of ordination groups. significant variables according to the kruskal-wallis statistic (p < . ) were compared with mcao values in order to test the strength of the relationships. we recognize, however, that the reliability of the kruskal-wallis statistic is proportional to the number of objects in the smallest group (belbin ) . as such, the kruskal-wallis statistic results are included, based on expert advice, as a comparative value to investigate the strength of mcao results, but these values should be treated with caution. a total of native species (eight mammals, nineteen reptiles) were identified from trap nights (table ). an additional native mammal (melomys sp.) was identified at two sites from scat and hair samples, but positive identification to the species level was not possible. instead, the genus was included in the analysis of the mammal data set. in total, these represented native family groups. the most common mammal species was the common brushtail possum identified in of the sites ( %). however, it was considered a predominantly arboreal species (how & kerle ) and was not included in further analysis.the common dunnart (sminthopsis murina) and the northern brown bandicoot (isoodon macrourus) were the most commonly detected terrestrial mammal species (n = sites each). detections of the common planigale (planigale maculata), common dunnart, and the yellow-footed antechinus (antechinus flavipes) were of particular interest as these dasyurids are relatively uncommon in brisbane's lowland habitats and are classified as significant species within the local government area (brisbane city council ).the fence skink (c. virgatus) and garden skink (lampropholis delicata) were the two most common reptile species (n = and n = sites, respectively). larger-bodied and more cryptic reptile species such as snakes, lace monitors (varanus varius), and dragons (e.g. bearded dragon, pogona barbata, and eastern water dragon, physignathus lesuerii) were identified at significantly fewer sites than small-bodied skink species (table ) . correlation analysis revealed the three measures of understorey density were strongly correlated (> . ). we eliminated two variables and retained total understorey density. although not highly correlated with other variables, average ground cover was also removed from the data set as it showed very little variation between sites with sites having an average cover > %. each species' common name and the total number of sites at which they were detected are also shown. for reptiles, four groups of sites (fig. a ) yielded the lowest ordination stress level (stress = . ) and maximum congruence between the ordination, mst and dendogram. the two most commonly captured reptile species, c. virgatus and l. delicata, dominated group sites (n = ), being identified at % and % of sites, respectively (fig. a) .this indicates a high degree of overlap in the distribution of these species within group . however, these skink species showed little or no overlap in the and sites that, respectively, comprised groups and . group sites were characterized by the ubiquitous presence of l. delicata (detected in all sites) and the absence of c. virgatus (fig. a) . the presence of c. virgatus rather than l. delicata dominated group ( % and %, respectively) (fig. a) . species' richness also varied between groups, with group containing the highest overall native species richness ( species), more than % greater species richness than group (fig. a) . group represented the fewest sites (n = ) and was characterized by a total absence of native reptile species. mcao reported eight native reptile species that were highly correlated with the ordination of reptile groups: carlia foliorum, carlia vivax, cryptoblepharus virgatus, dendrelaphis punctulata, diporiphora australis, l. delicata, carlia pectoralis, and v. varius (fig. b) . of these species, the presence of c. virgatus, l. delicata, and c. vivax explained the largest amount of variation in the ordination (r = . , . and . , respectively) and were highly significant at p < . (table ) . c. foliorum, and d. australis were less significant (p < . ). although c. pectoralis, d. punctulata, and v. varius were highly correlated with the ordination, these species were not found to be statistically significant (table ). the results further suggest that calyptotis scutirostrum and lampropholis amicula discriminate well between the groups (p < . and p < . , respectively), yet mcao indicated these species had a low correlation with the ordination groups (table ) . four habitat variables: soil compaction, total number of termite mounds, wood volume and weed cover ( - %) were significantly correlated with reptile groups (fig. c) . of these variables, only soil compaction and weed cover ( - %) discriminated well between the groups according to the kruskal-wallis statistic (table ) . three statistical groups of native mammal species (fig. d) produced the best ordination fit (stress = . ) and maximum congruence between the ordination, mst and dendogram. group was comprised of the sites at which no native mammals species were detected, indicating that native mammal species were undetected at the majority of the sites. comparatively, groups and were dominated by the dasyuridae and peramelidae families, respectively. native rodent species (family: muridae) were represented in group and group (fig. b ). all native mammal species were correlated with the ordination (fig. e) and, with the exception of northern brown bandicoots, explained between % and % of the group variation (table ) . northern brown bandicoots explained more than % of the group variation (r = . ), being found at all sites comprising group (fig. b) . the statistical significance of all but one species (long-nosed bandicoot, perameles nasuta) was supported by the kruskal-wallis statistic (table ) . both northern brown bandicoots and common dunnarts had the highest discrimination between groups (p < . ). the number of grass trees (xanthorrhoea spp.) and soil compaction were the only habitat variables that were significantly correlated with the mammal ordination (fig. f) . however, the associated kruskal-wallis values indicated that neither of these variables discriminated well between the groups (table ) . this study considered a range of local-level environmental factors and their correlation with native terrestrial reptile and small mammal species' compositions within fragmented urban bushland habitats. our results initially appear to imply that both vegetation composition (weed cover and grass trees) and habitat structure (termite mounds, wood volume and soil compaction) are important for native reptile and small mammal species. however, when previous research and species' behaviours and life history traits are examined, it seems likely that species in this study were responding to the structural role fulfilled by weed cover and grass trees rather than the compositional or floristic role. therefore, we conclude that at the local-level, habitat structural complexity is more important than vegetation composition for the occurrence of terrestrial, native reptile and small mammal species in brisbane's lowland remnant habitat fragments. this overall result is consistent with several other studies of various fauna species (including birds, reptiles and mammals) living within natural and disturbed (non-urban) habitats (e.g. grover & slater ; catterall et al. ; scott et al. ; webb & shine ; tait et al. ; vesk & macnally ). many of these and similar studies indicate distinct speciesspecific responses to particular habitat attributes, yet it was not possible in our study to identify speciesspecific relationships because of the low detection rate of many species in several sites. the low detection of several species may potentially be explained by localized species' declines owing to urbanization and its associated disturbances and/or, owing to the cryptic nature of habitat use, dietary preferences and seasonal population fluctuations resulting in a high degree of false-absences. additional surveys conducted over a longer time period are likely to have improved detection rates, added certainty to our results and, provided more information on the habitat preferences of individual species. however, such long-term surveys were not possible in this study owing to time and resource constraints. consequently, our analysis focused on the influence of habitat characteristics on native reptile and small mammal species' compositions rather than individual species' occurrences. reptile captures were dominated by two main species, l. delicata and c. virgatus. both species are also found throughout the urban matrix, although c. virgatus apparently more so than l. delicata (j. garden, pers. obs. garden, pers. obs. - . the recorded dominance of these species during fauna surveys played a significant role in determining the subsequent reptile groups in the patn analysis, indicating that these skink species may influence the composition of skink assemblages within urban patches. similar reptile inter-species interactions were reported by fischer et al. ( ) who found that c. tetradactyla more frequently inhabited sites occupied by at least two other small reptile species. other small reptile species and l. delicata and/or c. virgatus is a hypothesis that warrants further investigation in order to clearly delineate existing relationships and examine the influence of inter-species interactions relative to habitat suitability. the significance of termite mounds and fallen woody material is likely explained by the habitat and resource requirements of reptiles. reptiles are ectothermic and ) , mcao (%) and chi-squared (derived from kw statistic) are shown. significant mcao variables and values are indicated in bold font. chi-squared significance is indicated by: * . > p > . ; ** . > p > . ; ***p < . ; n/s, not significant. the ordination group with which each species and habitat variable was most highly correlated is also shown. so are dependent on habitat attributes that enable them to regulate their body temperature to achieve optimal performance, which is essential for foraging, breeding and predator-avoidance behaviours (huey ; bauwens et al. ; vitt et al. ; burrow et al. ; singh et al. ; lenders & daamen ) . termite mounds and fallen woody material both provide suitable basking locations, habitats for prey species and, numerous nesting and refuge niches. it is not surprising therefore that these structural attributes were most strongly associated with sites dominated by the presence of sun-loving reptile species such as c. vivax, ctenotus taeniolatus and, d. australis (group ). the association of reptiles with termite mounds is consistent with previous studies, both in australia and elsewhere. for example, a lizard species (metroles cuneirostris) from the namib desert of coastal namibia, was found to commonly utilize newly formed termite mounds as foraging locations (murray & schramm ) .the use and importance of termite mounds as refuge locations has also been reported for different reptile species, such as inactive monitors (varanus bengalensis) in sri lanka (wikramanayake & dryden ) and frillneck lizards (chlamydosaurus kingii) in australia's northern territory (griffiths & christian ) . griffiths and christian ( ) particularly demonstrated the importance of termite mounds as refuges by showing that c. kingii individuals who utilized termite mounds to avoid high intensity fires had a % survival rate, compared with the increased mortality and injury of individuals that sought refuge in tree canopies. as supported by previous research, the significance of fallen woody material is likely to provide similar resources as termite mounds for a suite of smallbodied, reptile species (e.g. smith et al. ; fischer et al. ; jellinek et al. ) . other ground-level structural attributes, such as ground cover/leaf litter (e.g. burrow et al. ; singh et al. ) and bushrocks (e.g. schlesinger & shine ; webb & shine ; white & burgin ) have also been reported to provide suitable basking, shelter and foraging opportunities for various native terrestrial reptile species (e.g. snakes, skinks, agamids and geckos). these structures are naturally occurring yet, in degraded urban remnants, human-introduced materials such as discarded metal and wood, and even old car bodies may work equally well (j. garden, pers. obs. ) . the occurrence of native reptiles was positively associated with a moderate amount of weed cover ( - %). weediness was particularly associated with group which was characterized by comparatively 'secretive' reptile species such as l. delicata, l. amicula and burton's snake-lizard (lialis burtonis). although all species require habitats that facilitate thermoregulation, group species appear to tolerate of a moderate amount of certain habitat disturbances (as indicated by weediness), so long as adequate vegetation cover is available either to facilitate thermoregulation or to provide rapid refuge from predators.this is consistent with bragg et al. ( ) who found that l. delicata was more likely to inhabit forested areas, which had more leaf litter, ground and shrub cover than the adjoining open habitat of regenerating minedisturbed areas. furthermore, in our study l. delicata, unlike c. virgatus, were more likely identified from pitfall captures than direct observations, a trend that is indicative of the more cryptic nature of l. delicata. similar capture trends were noted by singh et al. ( ) for l. delicata and c. virgatus surveyed in contiguous forest near brisbane. l. burtonis and pseudechis porphyriacus were also identified from direct observations as pitfall traps were not large enough to trap these larger-bodied species. however, upon detection, these species were observed to actively seek refuge within relatively dense thickets of lower-stratum vegetation. the positive association therefore between the occurrence of certain reptile species and a moderate amount of weed cover is most likely owing to the shelter provided by low, weedy vegetation rather than the weed species' composition per se. these findings support those of fischer et al. ( ) who noted that juvenile and some adult c. tetradactyla were found in moderate to highly weed-infested habitats, concluding that these species are able to tolerate a certain degree of habitat disturbance and potentially benefit from the associated structural changes. other researchers have similarly commented on the importance of lowerstratum vegetation cover for supporting important reptile prey species and also for the safe shelter provided from predators while foraging and dispersing (e.g. burrow et al. ; fischer et al. ) . although it appears that some reptile species respond positively to a certain degree of weed cover, hadden and westbrooke ( ) and jellinek et al. ( ) reported that overall reptile species' richness was negatively associated with increased weediness. our results provide some agreement with these previous findings. moderate weed cover was most associated with group which, compared with groups and , also had the lowest overall native species richness. further, the highest amount of weed cover ( - %), although not significantly correlated with the ordination, appeared to be associated with group (no reptiles detected). jellinek et al. ( ) discuss the possible influence of weediness and time since isolation, commenting on smaller and older remnants being more likely to be dominated by weeds than larger and younger remnants. this may potentially be critical for effectively managing reptile species' compositions investigated in the current study. as weed invasions are indicative of disturbed habitats, it seems likely that, as highlighted by jellinek et al. ( ) and suggested by the current study's findings, certain reptile species are sensitive to habitat disturbances and so will respond negatively to even a low amount of weed cover. conversely, some reptile species are able to tolerate a certain degree of habitat disturbance and may even benefit from the cover provided. it is therefore difficult to make generalizations of the importance of weeds for all reptiles. increased soil compaction was characteristic of habitats in which few or no native reptile species were detected, with the hardest soils occurring at sites at which no native reptile species were detected (group ). there are two possible explanations for this finding. the first directly implicates soil compaction and considers its impact on species' behaviours, whereas the second considers indirect implications of soil compaction, its associated disturbances on vegetation structure, and the resulting influence on reptile species. soil compaction was least associated with group , indicating that reptiles in this group occur more frequently in habitats with soft soils. this is consistent with the two fossorial skink species identified, c. scutirostrum and anomalopus verreauxii, which were found only at sites within this group. owing to their burrowing behaviour, these species do no inhabit or persist in areas with hardened soils where burrowing is difficult. soil compaction also has a significant influence on vegetation growth and regenerative ability (amrein et al. ; bassett et al. ) . increased soil compaction is often a result and a consequence of decreased vegetation cover (e.g. groves & keller ; hadden & westbrooke ) . this cyclic condition is intensified by external disturbances which directly compact the soil and destroy ground cover vegetation. hence, habitats with compacted soils are also likely to be indicative of highly disturbed habitats. inappropriate fire regimes (including arson fires) and aesthetic clearing which decrease vegetation cover and do not promote vegetation regeneration are likely to increase soil compaction and, in turn, make it more difficult for plant species to regenerate (amrein et al. ) . as a result, the habitat structure and soil condition degrades, negatively impacting on various reptile species dependent on structurally complex habitats. similarly, off-track trampling within urban bushland habitats compacts soil and destroys or degrades ground cover vegetation, making these habitats less suitable for reptiles. mammal species were found to be influenced primarily by habitat structure rather than vegetation composition. this is consistent with previous studies (e.g. bennett ; haering & fox ; monjeau et al. ; vernes ; monamy & fox ) which concluded that vegetation structure, rather than vegetation composition, was more important for small mammal occurrence, although species-specific responses to various aspects of structure were evident. for instance, small-bodied mammals, such as dasyurids, are likely to be more capable of moving rapidly through dense undergrowth, whereas largebodied mammals such as kangaroos are impeded by dense midstorey cover, but are less likely to be affected by dense understorey and ground cover. comparatively, large areas of dense undergrowth may present a significant locomotor (and escape) obstacle for medium-bodied mammals, such as bandicoots, and may also inhibit bipedal vigilance behaviours (garden ) . vernes ( ) found that northern bettongs (bettongia tropicana) avoided areas of dense ground cover, particularly dense cover within . m above ground, which approximates the height of an adult bettong. keiper and johnson ( ) similarly reported that short-nosed bandicoots (isoodon obesulus peninsulae) in north queensland forests avoided habitats with a tall, dense grass understorey. such a response is also likely to be true for bandicoots in urban landscapes and so these animals may actively select habitats that are structurally complex, yet are not vegetatively dense. therefore, as suggested by previous bandicoot studies (e.g. dufty ; scott et al. ; chambers & dickman ) , optimal habitats for peramelidae are structurally complex and encapsulate a mosaic of open foraging areas and denser shelter sites that are not so dense as to impede locomotor ability. grass trees also appear to be an important factor for the occurrence of native mammal species. this finding supports previous studies that have identified grass trees as important structural elements for several australian small terrestrial mammal species. vernes and pope ( ) , for instance, found that prior to fires, almost half the number of b. tropicana nests were located in dense vegetation cover such as the skirts of grass trees. spencer et al. ( ) similarly found that native bush rats (rattus fuscipes) responded negatively to the decrease in grass tree cover following fires. likewise, lunney ( ) commented that 'cover-seeking' r. fuscipes prefer habitats with dense ground and understorey cover -a structural requirement that would be partially filled by the presence of grass trees in the understorey layer. grass trees have also been documented as providing important nesting habitats for small dasyurid species such as the common dunnart (fox ) and the yellow-footed antechinus (marchesan & carthew ) . our results support these previous findings, in that the presence of grass trees was most strongly associated with group , which was dominated by dasyurids and r. fuscipes. soil compaction, as for reptiles, was also associated with mammal species occurrences. like reptiles, mammal species also appear to avoid habitats with very hard soils and the reasons may be similar. first, certain mammal species such as some dasyurids may nest in terrestrial burrows (woolley ; j. garden, pers. obs. ) and harder soils would inhibit their ability to burrow. however, unlike the reptile results, certain mammal species appear to respond positively to compacted soils. mammal group was most strongly associated with moderately compacted soils rather than very hard soils. this group was dominated by the peramelidae (bandicoot species). bandicoots are omnivorous and most commonly feed on subterranean invertebrate prey and plant structures such as roots and hypogeal fungi (vernes ; keiper & johnson ) , a behaviour that is obvious from the conical diggings left from foraging (triggs ; j. garden, pers. obs. j. garden, pers. obs. - . their ability to obtain these food resources is possible owing to specialized strong claws on their forefeet which make them powerful diggers. this morphological trait may provide them with an advantage over other species in enabling them to penetrate into harder soils and therefore utilize habitats with increased soil compaction. however, if soils become too hard, vegetation cover decreases (amrein et al. ) , resulting in decreased soil moisture and microbial activity, and hence less food availability and increased energy output (for foraging). structurally complex habitats are likely to occur where there is low to moderately compacted soils influencing vegetation floristics and cover. in addition, although bandicoots appear to prefer structurally open habitats for foraging, they have also been reported to require structurally dense habitats for diurnal shelter (e.g. dufty ; southgate et al. ; chambers & dickman ; vernes ) . therefore, as soils harden and vegetation cover decreases, important food resources decrease as does critical vegetation cover. however, in the urban landscape, dufty ( ) reported that the easternbarred bandicoot (perameles gunii) used a range of natural and artificial structures as shelter sites, implying a certain degree of disturbance tolerance, including 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- . . queensland government global biodiversity scenarios for the year choosing a rock: perspectives of a bush-rock collector and a saxicolous lizard ecology and population biology of long-nosed bandicoots (perameles nasuta) at north head effect of control burn on lizards and their structural environment in a eucalypt open-forest the effect of habitat fragmentation and livestock grazing on animal communities in remnants of gimlet eucalyptus sulabris woodland in the western australia wheatbelt population and habitat characteristics of the golden bandicoot (isoodon auratus) on marchinbar island adult free zones in small mammal populations: response of australian native rodents to reduced cover changes in species assemblages within the adelaide metropolitan area, australia r: the r project for statistical computing (v . . ) [freeware] language and environment copyright tracks, scats and other traces the impact of urbanisation on the mammals of melbourne -do atlas records tell the whole story of just some of the chapters? fine-scale habitat preferences and habitat partitioning by three mycophagous mammals in tropical wet sclerophyll forest, north-eastern australia stability of nest range, home range and movement of the northern bettong (bettongia tropica) following moderate-intensity fire in a tropical woodland, north-eastern queensland the clock is ticking: revegetation and habitat for birds and arboreal mammals in rural landscapes of southern australia the impact of individual tree harvesting on thermal environments of lizards in amazonian rain forest a floristic survey of the tingle mosaic, south-western australia: applications in land use planning and management paving the way for habitat restoration: can artificial rocks restore degraded habitats of endangered reptiles? current status and future prospects of reptiles and frogs in sydney's urban-impacted bushland reserves thermal ecology of habitat and microhabitat use by sympatric varanus bengalensis and v. salvator in sri lanka a field guide to reptiles of queensland ecology of small mammals in coastal heathland at anglesea, victoria. aust nest location by spool-and-line tracking of dasyurid marsupials in new guinea southeast queensland this research was a collaborative project between the university of queensland and brisbane city council. financial support was provided by brisbane city council and a uq postgraduate research scholarship. fauna surveys were conducted under uq aec permit, gsp/ / /bcc/uqgs, and queensland government epa scientific purposes permit, wisp . we thank private and council land owners for kindly allowing access to their properties. thanks also to the volunteers who helped establish survey sites and, to the project's two research assistants, michelle walton and alison howes. we gratefully acknowledge valuable statistical advice and comments provided by the uq patn-users group, particularly dr grant wardell-johnson, dr ben lawson and hussein bashir. we thank the editor for useful comments provided on the manuscript. key: cord- -ztdz a s authors: bologna, mauro title: biological agents and bioterrorism date: - - journal: detection of chemical, biological, radiological and nuclear agents for the prevention of terrorism doi: . / - - - - _ sha: doc_id: cord_uid: ztdz a s for this very stimulating course, i want to share with you some of my studies and even some of my scientific and phylosophical considerations on biological agents living in the environment and their relations with humans, in the very wide concepts of ecological relationships, parasitism, immunolgical defenses and infectious disease mechanisms. all these concepts must be studied and considered in the event of criminal use of biological agents (bioterrorism) aimed at harming human populations in time and in geographical space. terrorism is the use of violence to condition societies or governments in their political choices. bioterrorism is the use (or menace of use) of biological agents to enact terrorism events and induce generalized fear concerning negative consequences in target populations. (a) use of poison darts/arrows (primitive populations): mostly for hunting, but also for battles against enemies. from here derived many of the advances of toxicology, the science of toxic substances (the word "toxicology" derives from the greek words "toxon" = arch and "farmakon" = poison; toxicon farmakon = poison for arch hunting): (b) from such practice derives the knowledge we have of stricnin, curare, ouabain, aconite, other plant/animal poisons primarily devised for hunting and fi ghting. (c) impingement of darts in decomposing cadavers or putrefaction soil (or manure >> tetanus) before throwing at enemies (new guinea, tribal combats; sciites b.c.) (d) last but not least, the use of fear that humans have of beasts. here comes the example of hannibal (from carthago), leading the ships of prusia i, king of bithynia (west turkey) in a battle of year b.c. against eumene ii (attalides, pergamon); he won that naval battle because he managed to throw canisters full of reptiles at the enemy ships, causing terror and uncoordinated reactions leading to his victory. he therefore used fear as a weapon: snakes were not even harmful (not poisonous), but big was the surprise and reactions were out of control! (e) in recent history, we can see that first world war (also named the war of chemistry) contributed to the development and use of nervine gases , chemical weapons banned everywhere but still existing in some countries. second world war (also named the war of physics) led to the development and use of the atomic bomb. thepeace treaties: geneva protocol ruled against chemical weapons ( ) and was followed later by additions concerning bacteriologic war. not all the states however subscribed it. most states anyway have banned, in time, chemical and bacteriological weapons by . today, how scared should we be of biological and chemical terrorism? well, since these are lethal and cheap weapons, they are of considerable concern, because they may be seen as the atomic bomb of poors and represent remarkable threats to peace in local confl icts and in terroristic attacks worldwide. we should all know more on the subject and do extensive prevention. albert einstein once said "i do not know by what weapons the third world war will be fought, but for sure the fourth will be fought with stones". well, we do not want any more world wars, for sure, period. life began on earth with unicellular beings: primitive bacteria and algae, ∼ . billion years ago (archean age). biological evolution of species produced today's forms of life, as we observe them. millions of species co-exist and share the stage (biosphere). humans (we) pretend to have absolute priority, but … share the stage in such a crowded environment on earth means to learn, respect, understand and prevent. on this planet we have millions of different species of live beings, with variable proportions in the biosphere and in different ecosystems: they are all co-existing, interacting and competing for food and survival. this encompasses the very universal phenomena of competition and of parasitism. one of the most recent and precise evaluations of the number of species existing on planet earth ( ), but still very approximate and provisional, (well illustrated and summarized in national geographic, ) fi nds evidence of more than , species of mammals, , species of birds, , species of reptiles, , species of amphibians, , species of fi sh, , species of crustaceans, , species of mollusks, , species of aracnids, fi ve million species of insects and many, many millions (unestimable indeed) species of bacteria, viruses and other microrganisms. are we many on earth? is there enough work for the immune system of each living multicellular organism to distinguish "self" from possibly harmful "not self"? species of microrganisms ascertained as pathogenic for humans are indeed a very small fraction of the existing species: we come to know them better because we study the diseases connected with them, but we ignore a lot about the great number of other, presumably innocuous species. in general, different species interacting may set a parasitic relationship , in which the larger animal ( the host ) may receive harm (food loss or disease) and the smaller one ( the parasite ) may get advantages (more food, protection). both must preserve their identity and prevent contamination by foreign genetic material (immunologic surveillance, bilaterally). some parasites can even live within the hosts (endoparasitism), like some bacteria and all viruses. three types of interactions may occur between a microrganism and a human host: (a) symbiotic relationship, in which the microrganism and the host both benefi t; (b) commensal relationship, in which the microrganism gains but the host suffers no harm; and (c) a true parasytic relationship, in which the microrganism gains and the host is harmed. symbiosis offers frequently mutual advantages and remains very stable in time. pathogens are a minimal part of existing microrganisms. we humans host some advantageous bacterial populations (intestine, surface germs on the skin, commensal germs on the mucosae): we indeed are also made of the germs living in/on our body. indeed, only one cell out of ten in our body is a human cell: the rest are bacteria (the so-called micro-biome). among these, we count billions of bacteria in the intestines, useful for many functions (vitamin production, competition with pathogens, contribution to metabolism, etc.). the balance between host and parasites depends on two basic forces, an aggressive force by the parasite depending on survival/proliferation/invasion capacity of the parasite itself and a defensive force by the host depending on the immune mechanisms (phagocytosis, cellular and humoral immune reactions). in this balancing of opposite forces the parasitic relationship is played by the contendents. if we have prevalence of parasite, we may have disease (and eventually death) of the host, but if we have prevalence of host defence we may have control (and eventually elimination) of parasites. in the light of recent concern and interest about the potential for biological terrorism (biofarware) there are several diseases and bacterial toxins that must be considered in particular, like anthrax [ , ] , smallpox [ , ] , plague [ ] , botulinum toxin [ ] , and tularemia [ ] . a very detailed discussion of such diseases and other infectious diseases with similar risks in terms of bioterrorism goes beyond the scopes of this concise chapter, but some features of these and other infectious diseases representing important threats in the biofarware fi eld will be mentioned. in this respect, we may distinguish in time diseases which are: smallpox is a highly contagious disease (incubation - days) caused by the smallpox virus, an orthopoxvirus. it causes death in up to % of infected subjects. indigenous infection has been eradicated (last case, ethyopia, -who). the main concern for outbreaks of smallpox is today from bioterrorism. smallpox is characterized by severe constitutional symptoms (fever, headache, extreme malaise) and a characteristic pustular rash. treatment is supportive; prevention involves vaccination, which, because of its risks (eczema, encephalitis, etc.), is done selectively. pathogenesis of smallpox demonstrates that the virus is transmitted from person to person by direct contact or inhalation of droplet nuclei. clothing and bed linens can also transmit infection. most contagions are in the fi rst - days after the skin rash appears. once crusts form, infectivity declines. the virus invades the oropharyngeal and respiratory mucosa, multiplies in regional lymphnodes, causing viremia and localization in small blood vessels of the skin (rash) and rarely in cns (encephalitis). offi cially, smallpox is dead on earth. there are no longer cases detected in the world population since , but can we destroy the samples of smallpox virus existing in some virology laboratories around the world? certainly not [ ] , because we could no longer prepare vaccine doses without live virus samples to start from. and without vaccine, a small amount of wild virus could ignite a wide epidemic killing a large proportion of the human population, since the vaccination is no longer mandatory in any country and a large percentage of young populations have no longer been vaccinated after the early s. poliomyelitis is an acute infection caused by a poliovirus. manifestations include a nonspecifi c minor illness (abortive poliomyelitis), sometimes aseptic meningitis without paralysis (nonparalytic poliomyelitis) and, less often, fl accid weakness of various muscle groups (paralytic poliomyelitis). diagnosis is clinical, although laboratory diagnosis is possible. treatment is supportive. vaccination is available, still mandatory in many countries, although soon legislations may change. childhood vaccination produces immunity in % of recipients. declared cases worldwide have diminished remarkably, but some areas with particularly poor sanitary services or with confl icts preventing health services to operate are recording increased numbers of cases recently (syria, ; china ). polioviruses have three serotypes. the virus enters the mouth via the fecal-oral route, then enters the lymphoid tissues of the gi tract. if not contained, infection may enter the cns with signifi cant damage in spinal cord and brain, specifi cally to nerves controlling motor and autonomic function (breathing). spreading is through the enteric route. vaccine is live, attenuated virus, able to immunize many contacts respect to the vaccinated subjects (community vaccination strategies; problems in nomad populations). anthrax is caused by bacillus anthracis , toxin producing, encapsulated, aerobic or facultative anaerobic organisms. anthrax, an often fatal disease of animals, is transmitted to humans by contact with infected animals or their products (woolsorter's disease). in humans, infection tipically occurs through the skin. inhalation infection is less common; oropharingeal, meningeal and gi infections are rare. for inhalation and gi infections, nonspecifi c local symptoms are typically followed in several days by severe systemic illness, shock and often death. empyric treatment is with cyprofl oxacin or doxycycline. a vaccine is available (antitoxin). pathogenesis of anthrax takes place since bacillus anthracis readily forms spores when germs encounter dry environment -a condition unfavorable for growth . spores resist destruction and can remain viable in soil, wool, and animal hair for decades. spores germinate and multiply in favourable conditions (wet skin, tissue, blood) and can give human disease by contact (papules, black eschars, contagious also via fomites) ingestion (raw meat > fever, nausea, vomiting, diarrhea), and inhalation (fl u-like illness, respiratory distress, cyanosis, shock, coma). of note is the anthrax bioterrorist attack through mailings (using spores in powder form) that took place in the usa in (us postal service, washington dc), event that highly sensitized the public to the global theme of bioterroristic attacks. plague is caused by yersinia pestis (formerly named pasteurella pestis ). short bacillus with hairpin shape, infects wild rodents and can infect humans via tick bites. symptoms are either severe pneumonia or massive lymphadenopathy with high fever, often progressing to septicemia. diagnosis is epidemiologic and clinical, confi rmed by culture and serologic testing. treatment is with streptomycin or doxycycline. unfortunately, a vaccine is not available for plague. tularemia is a febrile disease caused by francisella tularensis ; it may resemble typhoid fever: symptoms are a primary local ulcerative lesion, regional lymphadenopathy, profound systemic symptoms, and, occasionally, atypical pneumonia. diagnosis is primarily epidemiologic and clinical and supported by serologic tests. treatment is with streptomycin, gentamycin and other antibiotics. tetanus is an acute poisoning from a neurotoxin produced by clostridium tetani. symptoms are intermittent tonic spasms of voluntary muscles. spasm of the masseters accounts for the name "lockjaw" (trismus). incubation requires - days. diagnosis is clinical. treatment with immune globulin and intensive support. only unbound toxin can be neutralized. a vaccine is available, with a good extent of preventive protection. botulism is a neuromuscular poisoning due to clostridium botulinum toxin. botulism may occur without infection if toxin is ingested. symptoms are symmetric cranial nerve palsies accompanied by a symmetric descending weakness and fl accid paralysis without sensory defi cits. diagnosis is clinical and by laboratory idenifi cation of toxin. treatment is with antitoxin and support therapies. tbc is a chronic, progressive infection by mycobacterium tuberculosis , often with a long period of latency following initial infection. it occurs most commonly in the lungs, with productive cough, chest pain and dyspnea. diagnosis is most often by sputum culture and smear. tbc can involve any tissue (organ disease). treatment is with multiple antimicrobial drugs. forms of multiresistant tb bacteria are becoming more and more frequent. coronavirus infections in humans most frequently cause common cold symptoms; however in , a relatively new coronavirus caused an outbreak of severe acute respiratory syndrome (sars), which was much more severe than other coronavirus infections. sars is an infl uenza-like disease leading to progressive respiratory insuffi ciency with signifi cant mortality rate. first detected in china (guandong, ), the sars epidemic spread to more than countries. in mid-july , there were > , cases with > deaths ( % mortality). then the outbreak subsided and no new cases have been identifi ed from to . in a new similar epidemic (sustained by the virus ncov, novel coronavirus) started in middle east (arabia), with an estimated mortality above %. later the ncov epidemic has been named mers (middle east respiratory syndrome) and is being studied as a new zoonosis trasmitted to humans from dromedary camels. studies are currently in progress, with great attention by the international sanitary authorities [ ] . who in indeed alarmed many countries against the new sars-like coronavirus responsible of mers, that infected at the moment of this writing (december ; www.who.int/en ) more than persons (arabia, great britain, france, germany, tunisia, italy, abu dhabi, united arab emirates, etc.) with reduced infective capacity as compared to sars, but still highly lethal and communicable via close contacts (family members). the latest available numbers call for ascertained diagnoses in humans, with deaths (mortality, . %). updates can be found at the following web sites: www.who.int/en ; www.cdc.gov and (recommendations for clinicians) emergency.cdc.gov , emphasizing the need to consider the novel (ncov) coronavirus when treating patients with a severe respiratory illness who have recently traveled to the arabian peninsula (or close contacts of the travelers). marburg and ebola are fi loviruses that cause hemorrhage, multiple organ failure and high mortality rates. diagnosis is with enzyme-linked immunosorbent assay, pcr or electron microscopy. treatment is supportive. strict isolation and quarantine measures are necessary to contain outbreaks. incubation - days. marburg virus has been identifi ed in bats and in primates. human to human transmission occurs via skin and mucous membranes contact (humans/primates). filoviruses can affect intestines (nausea, vomiting, diarrhea), respiratory tract (cough, pharingitis), liver (jaudice), cns (delirium, stupor, coma), and cause hemorrhagic phenomena (petechiae, frank bleeding) with high mortality rates (up to % with ebola virus). survivors recover very slowly and may develop long lasting complications (hepatitis, uveitis, orchitis) with only supportive care available: no specifi c antivirals nor vaccines are available fo fi lovirus infections. bunyaviridae contain the genus hantavirus (four serogroups, nine viruses) causing hemorrhagic fevers with renal and pulmonary consequences, starting with fl u-like symptoms and evolving with severe renal and pulmonary consequences. lethal in - % of cases. lassa fever is an often fatal arenavirus infection occurring mostly in africa. it may involve multiple organs, except cns. treated with ribavirin. no vaccinations are available so far for hantavirus infections. outbreaks of such infections have been recorded in nigeria, liberia, central africa, with some rare imported cases in the usa and in the united kingdom. the animal reservoir of such viruses is in wild african rats ( mastomys natalensis ), frequently found in african houses. direct human to human transmission is documented via urine, feces, saliva or blood. mortality (up to %) can be reduced by prompt ribavirin treatment. universal hygiene precautions, airborne isolation and surveillance of contacts are essential. last but not least … we must mention now infl uenza! flu viruses are in nature among the most rapidly changing (mutating) organisms through their ability to infect a variety of hosts: birds (migrating waterfowl -ducks-, stantial poultry -chickens-), mammals (pigs, felines) and humans. in south east asia (mostly in china, but also in viet-nam, laos, thailand, etc.) it is very common to have mixed farms of pigs, poultry and ducks, attended by humans. every year, new strains appear in se-asia, favoured by the recyprocal passage between migrating birds (mostly fowl), pigs and chickens, with exposure of many humans in farms, markets, rooster fi ghting sports, and food preparation places. a common say in china tells that "anything with four legs (except chairs) and anything that fl ies (except airplanes), can be eaten". with this phylosophy, there is generally a lot to be desired in food safety and in general hygienic prevention in such geographical areas. after the avian fl u h n of - , highly lethal but unable to give human to human contagion, new combinations of fl u strains are expected and feared, with high lethality and high human to human transmissibility. on this widely interesting theme for the world diffusion of new virus strains with pandemic potential, i wrote in together with the colleague virologist aldo lepidi a book entitled "pandemics -virology, pathology and prevention of infl uenza" (bollati boringhieri publisher, turin, italy , ) [ ] . in summary, we can see that a continuous surveillance is being devoted worldwide to the appearance of new strains of infl uenza viruses, in order to isolate as soon as possible potentially pandemic new strains and to prepare biological stocks suitable for massive vaccine preparations in due time to prevent the global spreading of potentially lethal new variants of the infl uenza viruses. examples in time recall the cases of the highly lethal pandemics known as "spanish fl u" in - (in excess of million deaths worldwide), "asian fl u" in (in excess of , deaths worldwide) and "hong kong fl u" in (in excess of , deaths worldwide). the basic question is: when the new pandemic will strike ? sometimes soon, as international experts say. the so called "avian fl u" came close to that, but sometimes in the future new mutations may emerge with the potential of being much worse. in conclusion of this wide although rapid overview of the most frequent or alarming causes of microrganism-related human diseses with potential interest for bioterrorism, i hope to have provided suffi cient matter for discussion and for further future diffusion of medical and microbiological culture that may be useful for prevention and the betterment of human social relationships and for peace promotion. anthrax as a biological weapon: medical and public health management anthrax as a biological weapon, : updated recommendations for management smallpox as a biological weapon: medical and public health management addressing the unthinkable: preparing to face smallpox plague as a biological weapon: medical and public health management. working group on civilian biodefense botulinum toxin as a biological weapon: medical and public health management tularemia as a biological weapon: medical and public health management the merck manual of diagnosis and therapy, th edn deadly mers coronavirus not yet a global concern pandemie¸ virologia, patologia e prevenzione dell'infl uenza (pandemics -virology, pathology and prevention of infl uenza) key: cord- -k i wgs authors: woolhouse, mark e.j.; gowtage-sequeria, sonya title: host range and emerging and reemerging pathogens date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: k i wgs an updated literature survey identified , recognized species of human pathogen, % of which are zoonotic. of the total, are regarded as emerging or reemerging. zoonotic pathogens are twice as likely to be in this category as are nonzoonotic pathogens. emerging and reemerging pathogens are not strongly associated with particular types of nonhuman hosts, but they are most likely to have the broadest host ranges. emerging and reemerging zoonoses are associated with a wide range of drivers, but changes in land use and agriculture and demographic and societal changes are most commonly cited. however, although zoonotic pathogens do represent the most likely source of emerging and reemerging infectious disease, only a small minority have proved capable of causing major epidemics in the human population. a recent, comprehensive literature survey of human pathogens listed > , different species ( ), more than half known to be zoonotic, i.e., able to infect other host species ( , ) . the survey data showed that those pathogens regarded as emerging and reemerging were more likely to be zoonotic than those that are not ( , ) , confirming an association between these characteristics which had long been suspected ( , ) , but which could not be formally demonstrated without denominator data as well as numerator data. here, we revisit these calculations, using updated information on the biology and epidemiology of recognized human pathogens. we pay close attention to possible differences between the major pathogen groups-viruses, bacteria, fungi, protozoa, and helminths. we also examine in detail the relationship between host range and pathogen emergence or reemergence, considering both the type and diversity of nonhuman hosts. we catalog the kinds of proximate factors or drivers that have been linked with pathogen emergence and reemergence and ask whether these differ between the major pathogen groups or between zoonotic and nonzoonotic pathogens. we focus mainly on pathogen diversity (as numbers of species) rather than on the effects of disease that they impose, noting that many diseases, e.g., infant diarrhea, can be caused by more than one species of pathogen. however, we comment on the transmissibility of pathogens once they have been introduced into the human population because transmissibility is an important determinant of the potential public health problem. we obtained counts of pathogen species from an updated version of the previously published database ( ) . as before, we defined a human pathogen as "a species infectious to and capable of causing disease in humans under natural transmission conditions." we included pathogens that have only been reported as causing a single case of human disease and those that only cause disease in immunocompromised persons. we also included instances of accidental laboratory infection but excluded infections resulting from deliberate exposure in the laboratory. we added recently recognized pathogens listed online by the centers for disease control and prevention, the world health organization (who), promed, and elsewhere ( ) ( ) ( ) ( ) . we obtained taxonomic classifications online from the international committee on taxonomy of viruses, the national centre for biotechnology information, the cab international bioscience database of fungal names, and from standard texts ( ) ( ) ( ) ( ) ( ) ( ) . pathogen species were categorized as emerging or reemerging based on previously published reviews of the literature ( , ), again updated from online sources ( ) ( ) ( ) . a species was regarded as emerging or reemerging if any recognized variant fell into this category (e.g., escherichia coli o , h n influenza a). we considered the following pathogen groups: viruses (including prions), bacteria (including rickettsia), fungi (including microsporidia), protozoa, and helminths. we did not consider ectoparasites (ticks and lice). each group was further divided into subgroups (families) to test whether biases existed in numbers of emerging and reemerging species at this level. the viruses were also divided according to genome type (e.g., negative singlestranded rna viruses). we examined aspects of host range, both for all pathogens combined and separately for each of the viruses, bacteria, fungi, protozoa, and helminths. first, we distinguished pathogen species according to whether they were known to be zoonotic, using the who definition "diseases or infections which are naturally transmitted between vertebrate animals and humans" ( ) . note that this definition includes pathogens for which humans are the main host and other vertebrates are only occasional hosts, as well as the opposite, but excludes purely human pathogens that recently evolved from nonhuman pathogens, e.g., hiv. we then compared the fraction of emerging or reemerging species that were or were not zoonotic across the major pathogen groups and within each group by family. second, for all zoonotic species we identified the types of nonhuman vertebrate host they are known to infect, using the following broad categories: bats, carnivores, primates, rodents, ungulates, and other mammals and nonmammals (including birds, reptiles, amphibians, and fish). we excluded vertebrate intermediate hosts of parasites with complex life cycles. host types were ranked by the number of zoonotic pathogen species associated with them, and rankings were compared by using spearman rank correlation coefficient. third, we obtained a crude index of the breadth of host range by counting the number of the host types that each pathogen species is known to infect: (i.e., not zoonotic), , , and or more. we compared the fraction of emerging and reemerging species across these classes. for the emerging and reemerging pathogen species, we identified the main factors believed to drive their increased incidence, geographic range, or both, by conducting a systematic review of the emerging diseases literature. we allocated these drivers to or more broad categories (table) . note that although we chose categories that we considered to be useful and informative for our immediate purposes, and which were similar to those listed elsewhere ( ) , this is inevitably a subjective procedure and alternative categorizations may be equally valid. we then ranked the drivers (by number of emerging and reemerging pathogen species associated with each) and compared the ranking of drivers for the major pathogen groups and for zoonotic versus nonzoonotic pathogens. for the zoonotic species, we distinguished those known to be transmissible between humans, allowing that this might be through an indirect route (e.g., a vector or an intermediate host), from those for which humans can only acquire infection (directly or indirectly) from a nonhuman source. for the transmissible zoonotic species, we further distinguished those that are sufficiently transmissible to cause major epidemics in human populations from those that cause only relatively minor outbreaks. this classification was intended to distinguish between pathogens with r > in humans from those with r < , where r is the basic reproduction number, i.e., the average number of secondary infections produced by a single primary infection introduced into a large population of previously unexposed hosts. direct estimates of r are unavailable for most zoonotic pathogens. throughout the study, we quantified associations as the relative risk (rr) and tested for statistical significance using a standard χ test (with correction for small expected values). although these statistical analyses are susceptible to bias introduced by related species (e.g., several species of hantavirus exist, most of which are zoonotic and many of which are regarded as emerging or reemerging), the analysis at the family level is an indication of the extent of any such bias. the survey of human pathogens produced a count of , human pathogen species, with ( %) species regarded as emerging or reemerging (online appendix, available at www.cdc.gov/ncidod/eid/vol no / - _app.htm). of all pathogen species, are viruses or prions, including ( %) regarded as emerging or reemerging. for bacteria, the counts were and ( %), respectively; for fungi, and ( %), respectively; for protozoa, and ( %), respectively; and for helminths, and ( %), respectively. these numbers differ slightly from those previously published ( , ) as a result of adjustments to taxonomies and the discovery of previously unknown pathogen species. clear differences were found between the pathogen groups (χ = . , p<< . ), with viruses greatly overrepresented among emerging and reemerging pathogens and helminths underrepresented. more than virus families contain human pathogens, with just , the bunyaviridae, flaviviridae, togaviridae, and reoviridae, accounting for more than half of the species affecting humans and, likewise, more than half of the emerging and reemerging species. overall, no significant difference was found between the largest families (pooling the remainder) in the fraction of species regarded as emerging or reemerging (χ = . , p = . ). nor were any significant differences found according to genome type, e.g., between rna and dna viruses (χ = . , p = . ) or between positive and negative single-stranded rna viruses (χ = . , p = . ). more than bacteria families contain human pathogens; the enterobacteria and the mycobacteria account for the most species and for the most emerging and reemerging species. overall, no significant difference was found between the largest families (pooling the remainder) in the fraction of species regarded as emerging or reemerging (χ = . , p = . ). numbers of species of emerging and reemerging fungi, protozoa, and helminths were too small for meaningful comparisons between families, but no indication was found that emerging and reemerging species are concentrated in any particular taxa. of the , human pathogen species, ( %) are known to be zoonotic. in comparison, of the emerging or reemerging pathogens, ( %) are known to be zoonotic. this corresponds to an rr of . and confirms the expectation that zoonotic pathogens are disproportionately likely to be associated with emerging and reemerging infectious diseases. this pattern varies somewhat across the different pathogen groups: for bacteria and fungi the association is strongest with rrs of . and . , respectively; for viruses and protozoa, no obvious association was found, with rrs of . and . , respectively; and for helminths (which are almost all zoonotic but very rarely emerging or reemerging), rr is . . however, the numbers involved are small (particularly for protozoa and helminths), and these differences were not statistically significant (χ = . , p = . ). all the defined host types are potential sources of zoonotic infections, but differences occurred in their importance (ranked by number of pathogen species supported) across viruses, bacteria, fungi, protozoa, and helminths and no type consistently dominates ( figure a) , although ungulates are the most important overall, supporting over species of human pathogen. emerging and reemerging pathogens show similar trends ( figure b) , with ungulates again the most important overall, supporting over species. in general, ranking of host types in terms of numbers of species correlates well both overall (r s = . , n = , p< . ) and individually for each pathogen group. the general impression is that the emerging and reemerging zoonotic pathogens are not unusual in the types of nonhuman hosts they infect. however, when the fraction of emerging and reemerging species is compared with the breadth of host range (as the number of host types other than humans), a pattern becomes apparent (figure ) . overall, the fraction tends to increase with host range: > % of pathogens with the broadest host ranges ( or more types of nonhuman host) are emerging or reemerging (exact p = . ). however, this trend does not hold for the protozoa and helminths (although the numbers for these groups are small). we identified main categories of drivers of emergence and reemergence and ranked these by the total number of pathogen species associated with them ( ranking of drivers across different categories of pathogen showed poor concordance (e.g., spearman rank correlation for bacteria vs. viruses, r s = . , n = , p = . ). the most striking discrepancies were as follows: ) the marked association of emerging or reemerging fungi with hospitalization, poor population health, or both; ) the greater importance of pathogen evolution and contaminated food and water and the lesser importance of international travel and changes in land use and agriculture for bacteria in comparison with viruses; ) the greater importance of changing land use and agriculture for zoonoses than for nonzoonoses. overall, most zoonotic pathogens are either not transmissible (directly or indirectly) between humans at all (i.e., humans are a dead-end host) or are only minimally transmissible. examples include rabies virus, rift valley fever virus, and borrelia burgdorferi (the agent of lyme disease). a small minority (≈ %) of pathogen species that are technically zoonotic are, in fact, spread almost exclusively from person to person (e.g., mycobacterium tuberculosis or measles virus) or can do so once successfully introduced from a nonhuman source (e.g., some strains of influenza a, yersinia pestis, or severe acute respiratory syndrome (sars) coronavirus). however, a substantial minority of zoonotic pathogens (about %, i.e., species) are capable of some person-to-person transmission but do not persist without repeated reintroductions from a nonhuman reservoir (e.g., e. coli o , trypanosoma brucei rhodesiense, or ebola virus). this pattern is fairly consistent across the major pathogen groups. humans are affected by an impressive diversity of pathogens; , pathogenic species of viruses, bacteria, fungi, protozoa, and helminths are currently recognized. of this total, ( %) pathogen species are considered emerging or reemerging. this number must be viewed with some caution, given that these terms are still used somewhat subjectively. more rigorous definitions of emerging and reemerging have been proposed ( , , ) , but these are difficult to apply universally because they require long-term data on distributions and incidences which are available for only a small subset of infectious diseases (e.g., malaria [ ] and tuberculosis [ ] ). moreover, the counts of emerging and reemerging pathogen species reported here are subject to ascertainment bias. despite these caveats, our results suggest that pathogens associated with emerging and reemerging diseases share some common features. first, emerging and reemerging pathogens are disproportionately viruses, although they are not disproportionately different kinds of viruses. numerically, rna viruses dominate, comprising % of all emerging and reemerging pathogens. rna viruses are also prominent among the subset of emerging pathogens that have apparently entered the human population only in the past few decades, such as hiv or the sars coronavirus ( , ) . a possible explanation for this observation is that much higher nucleotide substitution rates for rna viruses permit more rapid adaptation, greatly increasing the chances of successfully invading a new host population ( , ) . second, emerging and reemerging pathogens are not strongly associated with particular nonhuman host types, although emerging and reemerging pathogens more often are those with broad host ranges that often encompass several mammalian orders and even nonmammals. this pattern is consistent across the major pathogen groups. the determinants of host range in general remain poorly understood, but among viruses for which the cell receptor is known, an association exists between host range and whether the receptor is phylogenetically conserved (as measured by the homology of the human and mouse amino acid sequences) ( ) . emerging and reemerging pathogens have been likened to weeds ( ) , and that the associations reported above are likely reflecting underlying "weediness," that is, a degree of biologic flexibility that makes certain pathogens adept at taking advantage of new epidemiologic opportunities. this characteristic seems to be reflected in the broad range of drivers of the emergence or reemergence of pathogens, ranging from changes in land use and agriculture, through hospitalization to international travel. although some drivers are numerically more important than others, the overall impression is that pathogens are exploiting almost any figure . relationship between breadth of host range (as number of nonhuman host types, as listed in figure ) and the fraction of pathogen species regarded as emerging or reemerging. a total of change in human ecology that provides new opportunities for transmission, either between humans or to humans from a nonhuman source. even if a pathogen is capable of infecting and causing disease in humans, most zoonotic pathogens are not highly transmissible within human populations and do not cause major epidemics. the possible magnitude of an infectious disease outbreak is related to the basic reproduction number, r ( figure ). for pathogens that are minimally transmissible within human populations (r close to ), outbreak size is determined largely by the number of introductions from the reservoir. for pathogens that are highly transmissible within human populations (r >> ), outbreak size is determined largely by the size of the susceptible population. for pathogens that are moderately transmissible within human populations (corresponding to r ≈ ), notable outbreaks are possible (especially if multiple introductions occur), but the scale of these outbreaks is very sensitive to small changes in r . in other words, small changes in the nature of the host-pathogen interaction can lead to large increases (or decreases) in the scale of the public health problem (figure ). such pathogens may be likely sources of emerging infectious disease problems in the future. however, we currently have no way of predicting whether a novel human pathogen will behave like rabies (frequently introduced into the human population, but not capable of causing major epidemics) or hiv (probably rarely introduced, but capable of causing a global pandemic). in conclusion, this study suggests that biologic and epidemiologic correlates of pathogen emergence or reemergence may be identified. however, the most striking feature of emerging and reemerging pathogens is their diversity (online appendix). for this reason, surveillance and monitoring of infectious disease trends may have to be broadly targeted to be most effective. given that threefourths of emerging and reemerging pathogens are zoonotic, in many cases this targeting might usefully be extended beyond at-risk human populations to include populations of potential animal reservoirs. emerging infectious diseases • www.cdc.gov/eid • vol. , no. , december figure . expected relationship between outbreak size (as fraction of the population affected) and key epidemiologic parameters: i is the number of primary cases of infection introduced into the human population from an external source such as a zoonotic reservoir (increasing in the direction indicated); r is the basic reproduction number, a measure of the transmissibility of the infection with the human population (see text). the curves are obtained from a modified version of the kermack-mckendrick equation and show that expected outbreak size is particularly sensitive to small changes in i or r when r is close to . examples of zoonotic pathogens with r > , r < and r close to are shown. rivf, rift valley fever virus. (reprinted with permission from [ ] ). risk factors for human disease emergence population biology of multi-host pathogens diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence factors in the emergence of infectious diseases microbial threats to health: emergence, detection, and response emerging infectious diseases world health organization. emerging diseases the promed-mail archives the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents international committee on the taxonomy of viruses. index virum topley & wilson's microbiology and microbial infection foundations of parasitology a summary of taxonomic recently approved by ictv zoonoses: second report of the joint who/fao expert committee. geneva: the organization population biology of emerging and reemerging pathogens -preface terrestrial animal health code- . general definitions the global distribution and population at risk of malaria: past, present and future the growing burden of tuberculosis: global trends and interactions with the hiv epidemic evolvability of emerging viruses emerging pathogens: the epidemiology and evolution of species jumps population biology of emerging and re-emerging pathogens emerging infectious pathogens of wildlife we thank louise taylor and sophie latham for their work on the original database and ben evans for his contribution to the updated database.dr woolhouse is professor of infectious disease epidemiology in the centre for infectious diseases at the university of edinburgh. his research interests include foot-and-mouth disease, e. coli o , scrapie, and sleeping sickness. he is an advisor to the uk government on issues relating to infectious disease epidemiology.dr gowtage-sequeira is a postdoctoral research assistant in the division of animal health and welfare at the university of edinburgh. her doctoral research, for the institute of zoology in london, was on the epidemiology of viral infections of canids in namibia. she is currently studying the ecology of wild dogs in eastern kenya. key: cord- - znmkvy authors: montecino-latorre, diego; goldstein, tracey; gilardi, kirsten; wolking, david; van wormer, elizabeth; kazwala, rudovick; ssebide, benard; nziza, julius; sijali, zikankuba; cranfield, michael; mazet, jonna a. k. title: reproduction of east-african bats may guide risk mitigation for coronavirus spillover date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: znmkvy background: bats provide important ecosystem services; however, current evidence supports that they host several zoonotic viruses, including species of the coronaviridae family. if bats in close interaction with humans host and shed coronaviruses with zoonotic potential, such as the severe acute respiratory syndrome virus, spillover may occur. therefore, strategies aiming to mitigate potential spillover and disease emergence, while supporting the conservation of bats and their important ecological roles are needed. past research suggests that coronavirus shedding in bats varies seasonally following their reproductive cycle; however, shedding dynamics have been assessed in only a few species, which does not allow for generalization of findings across bat taxa and geographic regions. methods: to assess the generalizability of coronavirus shedding seasonality, we sampled hundreds of bats belonging to several species with different life history traits across east africa at different times of the year. we assessed, via bayesian modeling, the hypothesis that chiropterans, across species and spatial domains, experience seasonal trends in coronavirus shedding as a function of the reproductive cycle. results: we found that, beyond spatial, taxonomic, and life history differences, coronavirus shedding is more expected when pups are becoming independent from the dam and that juvenile bats are prone to shed these viruses. conclusions: these findings could guide policy aimed at the prevention of spillover in limited-resource settings, where longitudinal surveillance is not feasible, by identifying high-risk periods for coronavirus shedding. in these periods, contact with bats should be avoided (for example, by impeding or forbidding people access to caves). our proposed strategy provides an alternative to culling – an ethically questionable practice that may result in higher pathogen levels – and supports the conservation of bats and the delivery of their key ecosystem services. the order chiroptera is the second largest order of mammals with more than identified species [ ] . the members of this order, bats, provide important ecosystem services (reviewed in [ , ] ). for example, insectivorous bats can reduce arthropod herbivory [ ] [ ] [ ] , increase agricultural yields [ ] , reduce the need for insecticides [ ] , and prevent large financial losses in agriculture [ ] [ ] [ ] . plant-visiting chiropterans provide pollination and seeddispersing services (reviewed in [ ] ), certain nectivorous bats are pollinators of economically important plants [ ] , and frugivorous bats can be important for reforestation [ ] . finally, cave-roosting bats produce guano, the main energy source in many cave ecosystems [ , ] , and the mining of this product is an income source in poor communities [ ] . however, current evidence supports that bats are a natural host of several disease-causing viruses across the globe, including zoonotic viruses, such as rabies virus (rhabdoviridae, genus lyssavirus); hendra and nipah viruses (paramyxoviridae, genus henipavirus); and marburg and ebolaviruses (filoviridae, genus marburgvirus and ebolavirus, respectively; [ , ] ). bats are also hosts of several viruses of the coronaviridae family [ ] [ ] [ ] [ ] . molecular evidence suggests that the severe acute respiratory syndrome betacoronavirus (sars-cov beta-cov) and the middle-east respiratory syndrome betacov (mers-cov) originated from bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . both viruses emerged in humans during the past two decades, specifically in china ( ) and saudi arabia ( ). the sars-cov pandemic included cases in countries with a~ % case fatality [ ] , while mers-cov has affected people in countries with a case fatality of % [ ] . incidental cases of mers-cov are still detected mainly in saudi arabia [ ] , and it is thought that camels are important for human infection [ ] [ ] [ ] . today it is known that: i) a high genetic diversity of coronaviruses (covs) is present in more than bat species, including viruses related to sars and mers cov [ ] ; ii) covs are prone to move and adapt to new host species [ ] ; iii) plausibly, all mammalian-adapted covs may have originated in bats [ , , ] , including a recently emerged highly fatal alphacoronavirus in piglets [ ] and the e human cov [ , , ] ; and iv) covs found in bats can use human receptors for cell entry [ , , , ] . these lines of evidence suggest that future spillover of coronaviruses humans is feasible. because covs are found in bat species that have adapted to be in close contact with humans, such as the straw-colored fruit bat (eidolon helvum) and the brazilian free-tailed bat (tadarida brasiliensis [ , ] ), high contact "bat-human" interfaces currently exist around the world. if the bats in these interfaces shed covs with the ability to infect humans, then opportunities for spillover through direct exposure to feces [ ] or the contamination of food are created, as these viruses can remain infectious in the environment for days [ ] . therefore, strategies aiming to mitigate human exposure to covs, and thus, the risk of spillover and disease emergence are needed, while supporting the conservation of bats and their important ecological roles. longitudinal sampling with specific species has shown that the proportion of bats shedding covs varies seasonally [ ] [ ] [ ] [ ] and that fecal cov-rna loads can also be heterogeneous over time [ , ] . if exposure through contact with bat feces is a main pathway for zoonotic cov spillover to humans but shedding of these pathogens is not uniform over time, then mitigation strategies aiming to prevent bat cov-shed exposure could be targeted temporally, directed especially at highrisk seasons. such a strategy could guide policy in limitedresource settings where sampling bats for cov testing is not feasible and it could support an ethically acceptable management to mitigate spillover risk. however, the few species and locations tested to date do not allow for identification of a potential seasonal shedding pattern to responsibly suggest temporal spillover risk management across species and geography. therefore, assessment of the cov dynamics in a broader range of bat species that show different life history traits, as well as in diverse geographic and ecological circumstances, could be extremely useful. to this end, we evaluated the dynamics of cov shedding in different bat species sampled in several locations in east africa at different times of the year. this geographical region has been identified as a hotspot of pathogen emergence [ ] , where cov host switching events seem to be higher compared to other areas [ ] , but, to our knowledge, no study on cov dynamics in bats has been conducted. specifically, we hypothesized that bat species exhibit seasonal trends in cov shedding that are associated with the reproductive season. we assessed this hypothesis by fitting bayesian statistical multivariable models to evaluate whether cov shedding in bats is positively associated with the time period when pups are becoming independent from the dam. beyond the inclusion of several species sampled in different countries at different times, we explicitly identified the reproductive events for each species at the time of sampling and also included other traits, such as the aggregation of individuals at the roost, that may be involved in cov dynamics. samples (rectal swabs and fresh feces) were collected from bats captured in uganda, rwanda, and tanzania ( fig. ) , between september and april with permission from local authorities and under the institutional animal care and use committee at the university of california, davis (protocol number: ). bats were captured in unique locations between latitudes − . and − . (fig. ). these locations were selected because they represented potentially high-risk interfaces for contact between bats and humans, such as areas of land-use change, human-dwellings, ecotourism sites, markets, and places with potential for occupational exposure [ ] . locations in close proximity (euclidean distance < km) in which sampling was conducted within the same week were considered a single sampling event. the remaining sampling events that occurred in the same location or spatially close to others but conducted in different weeks were considered independent sampling events. as result, we collected samples from unique sampling events. all captures were conducted using mist nets set in the early morning or at dawn. individuals were released after sample collection. samples were handled as previously described [ , ] . each sample was immediately transferred to vials containing viral transport media and nuclisens® lysis buffer (biomérieux, inc., marcy-i'Étoile, france), which were maintained in liquid nitrogen until storage in a - c freezer in each country. rna was extracted from all samples, and cdna was prepared as previously described [ , ] . two broadly reactive consensus pcr assays targeting non-overlapping fragments of the orf ab were used to detect both known and novel covs [ , ] . amplified products of the expected size were cloned and sequenced as described in [ ] . sequences were analyzed and edited using geneious prime . . [ ] . a sample was considered positive when at least one pcr assay yielded a sequence that matched coronaviruses in genbank. coronavirus sequences were classified as belonging to a specific taxa following previously described methodologies [ ] . bats were categorized as adults and juveniles based on size, and morphological and behavioral characteristics were observed at sample collection. the sex of the bats was also recorded. identification of some bat species can be challenging in the field. for this reason, field-identified species were confirmed by dna barcoding using the cytochrome b or cytochrome oxidase subunit mitochondrial genes [ ] . obtained sequences were compared against sequences in the genbank and barcode of life databases [ , ] . when possible or necessary, sequences from both genes were used for species identification. a threshold of % nucleotide identity was used to confirm the species; sequences with - % nucleotide identity were assigned a "conferre" (cf.) species status, and sequences below % nucleotide identity were either classified to the genus level or as unidentified. sequences with > % nucleotide identity to more than one species for either gene, were classified to the genus level unless they clearly clustered with sequences from other animals in the same geographic area. if barcoding results for all of the first ten bats tested per species were in agreement with the field identification, we assumed that the field identification for the remaining bats of that species in each country was correct. otherwise, all of the remaining samples were barcoded to ensure correct speciation. we recorded when sampled females were pregnant based on abdominal palpation, had attached pups (indicating recent parturition), and were lactating, as well as when juveniles were captured. therefore, we were able to track pregnancy, lactation, and recent birth pulses. moreover, we accessed the data in the pantheria [ ] and amniote [ ] databases, and we thoroughly reviewed the literature on the biology of the bats species we sampled for latitudes similar to our sampling locations. with the gathering of these information sources we established the timing of the birth pulses, lactation periods, and the weaning of pups for each species. for the details justifying the dates inferred for these three life history events for each species and the corresponding bibliographical references see additional file . once the timing of these events was confirmed or inferred, we were able to establish seasons that occur at least once during the year across all observed bat species: i) when juveniles are being weaned and female-pup contact decreases ("recent weaning" [rw]) and ii) the rest of the year (hereafter "n-rw" for "not recent weaning"). we chose to evaluate risk of cov shedding for the first period because past longitudinal studies with microchiropterans in germany and china found higher cov-rna loads approximately month after parturition [ , ] . similarly, peaks were found months after the formation of a maternity colony of myotis macropus [ ] , which would match a post weaning period for that species. here, we defined the end of the rw period as month after the last pups were weaned. we assumed that month would provide a reasonable time window for the colony to "clear" the cov susceptibility status of this period and acquire the susceptibility corresponding to the season(s) when weaning does not occur (n-rw period), if differences actually exist. finally, once we determined these two seasons, we categorized each bat sample into one of them depending on the week of the year in which they were taken. because some species had more than one litter per year, there could be more than one rw period during the year. it is worth noting that we were able to define these periods for those species in our dataset that have synchronized reproduction, whose biology was properly described, and whose taxonomy is generally accepted. when we could not assign a reproductive period to specific bats, this season was imputed (see methods: statistical analysis). we characterized specific traits of each bat species studied based on previous scientific literature on pathogen dynamics in bats [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] : colony size (small, medium, or large if a typical colony contains one to dozens, hundreds to thousands, or thousands to millions of individuals, respectively); roost type ("closed" if the species has been reported to use caves, mines, roofs, or other confined spaces; or "open" if the bats have been typically reported roosting in the foliage of trees); the aggregation of bats in clusters while roosting (no, yes); and the number of litters per year of the species at equatorial latitudes. references are provided in additional file . we also considered data from pantheria and amniote [ , ] . we could not include other species traits, such as the mating strategy (harem or another) and the segregation of females in maternity colonies, because available studies were incomplete or contradictory. we did not include factors, such as multi-species occupancy of the roost, because we did not observe all of the roosts. further, we did not assess postpartum estrus, as within the study area, it is only known to occur in some molossid bats [ ] , of which we only sampled a small number. to statistically estimate the association between the rw and cov detection we used a bayesian inference to model the detection of covs as a bernoulli process of the form: where cov i , the detection of cov in rectal swabs ( if detected, otherwise) from the i th bat (sample), is assumed to follow a bernoulli process parameterized by p i , the probability of cov detection on the i th bat. this parameter was related to a set of candidate covariates as: with s j~n ormal ( , σ s ) and sp k~n ormal ( , σ sp ). here x and y are binary covariates representing the rw season and juvenile age category. we specifically included these two terms to separate the potential association of the season with cov detection from the seasonal presence of juveniles. because it was not feasible to allocate all species in the rw or n-rw seasons based on previous research, we assumed that these unknown reproductive seasons were "missing at random" and they were imputed as a function of the latitude at sampling, the day of the year of the sampling event, the number of litters per year of the corresponding species (one litter per year versus more than one litter per year), and the historical precipitation of the month at the sampling event location. the description of the imputation model is provided in additional file . the terms s j and sp k represent the sampling eventand the species-specific intercepts, respectively, because we assumed that bats sampled in the same event and bats belonging to the same species were not independent with respect to cov detection. no sampling event involved the same bat colony in successive rw and n-rw seasons, therefore, we assumed that cov detection was not temporally correlated within sampling locations. we constructed the model by adding other covariates one-at-a-time to this working model: the c ...l categorical variables; and they remained in the model if they were judged to confound the relationship between cov detection and the reproductive seasons or between age and cov detection (i.e., their inclusion caused meaningful changes in the posterior probability distributions [ppds] of the specific reproductive season or age coefficients). finally, c ...l categorical variables could be retained as well if they were marginally associated with cov detection (the corresponding coefficient ppd did not include zero). to assess the goodness-of-fit of the models, we evaluated the congruence between cov detection in the data and in the posterior predictive distributions yielded by the models by: i) ages and seasons, ii) age, iii) per season, iv) per age and season, and v) per sampling event. all models were constructed using "stan" v. . . [ ] which was run from "r" v. . . [ ] through the package "rstan" v. . . [ ] . weakly informative priors were assigned for all coefficients: normal( , . ) for the estimates of α , β's, γ, and ρ's. the σ s and σ sp had a prior half-cauchy ( , ) following previous suggestions [ , ] . the ppds were estimated by sampling in parallel from mcmc chains for iterations following iteration warm-up for a total of saved samples for each parameter ppd. convergence was assessed by the gelman-rubin statistic [ ] and graphically using trace plots. the code to fit the models is available at https://github.com/ dmontecino/east-african-bats-and-cov-shedding. we sampled individuals, all of them aged and successfully identified to belonging to species. nycteris thebaica, pipistrellus hesperidus, and rhinolophus clivosus were assigned the "conferre" status. hipposiderids were assigned only to the genus level because barcoding did not provide certainty on species identification in line with previously recognized taxonomic difficulties [ ] [ ] [ ] ; however, the biology of the candidate hipposiderid species is similar ( fig. ; [ , ] ), and we were able to used them for estimation purposes. we excluded scotophilus viridis (n = ) from the analysis because their reproductive traits are unknown, and this species also has taxonomic difficulties for species identification [ ] . therefore, these six individuals were removed, and bats were included in the study. we were able to infer the reproductive season for all bats except for lissonycteris angolensis, rhinolophus cf. clivosus, and pipistrellus cf. hesperidus (n = , adults and juveniles) that had limited available knowledge on biology and reproductive season. these reproductive seasons were imputed as explained above. this imputation process did not substantively affect the proportion of bats in each reproductive period or the crude cov detection per age (additional file : figure s . ). moreover, we had partial data for neoromicia nana and triaenops persicus lactation period and we assigned one that is likely longer than it would be in reality based on the other species. even under this overextended period, none of these bats (n. nana and t. persicus) could have being sampled during the rw season, so this knowledge gap did not risk a misclassification (fig. ) . in the end, and bats were allocated in the n-rw and rw periods, respectively. the distribution of bats across the reproductive periods per age and sampling event was heterogeneous as expected due to the opportunistically nature of sampling. indeed, and adult bats were in the n-rw and rw periods, respectively, while and juvenile bats were sampled in these seasons, respectively. in total, . % bats were positive for covs (n = ). within the subset of adult bats, . % were positive (n = ) while . % of the juvenile bats were positive (n = ). the detection of cov shedding was variable across seasons and bat species, as well as across sampling events ( , , . , and . % for the minimum, maximum, median, and mean detection, respectively). the covs found per species are shown in additional file : table s . a summary of the roosting and reproductive traits of the bat species sampled is provided in table . all bats except n. nana (n = ) and e. helvum (n = ) roosted in "closed" structures, such as caves, abandoned mines, and roofs. within the group of bats using "open" structures, e. helvum was the only species with cov positive individuals. therefore, we did not use this covariate to assess a potential association with cov shedding. the models showed adequate sampling. the markov chain monte carlo chains converged graphically, whilst all gelman-rubin statistics were < . . the selected model had a number of effective samples for each coefficient of at least . the data were properly fitted, as well (additional file : figure s . ), although some predictions lacked precision. the ppds of the fixed coefficients are shown in additional file : figure s . . the selected model to assess periodic differences in cov shedding included season and age, species-specific intercepts, and sampling event-specific intercepts. beyond the species-specific terms, we included a binary categorical covariate equal to for e. helvum and t. persicus and otherwise. we incorporated this term because cov detection in these species was remarkably higher than the other species. as expected, this fixed effect was correlated with the corresponding species-specific intercepts (the remaining correlations were all low); however, we decided to keep it to assess if the main findings hold even when accounting for the bat species with highest detection. the corresponding means, standard deviations, and % hpdi are shown in table . the coefficients' ppds from the selected model indicate an association between age and cov shedding, with juveniles presenting . - . times higher odds to shed compared to adult bats ( % hpdi). the coefficients' ppds also point to an association between the reproductive season and cov shedding as well, with an estimated odds . - . times higher to shed during the period when pups are being compared to other seasons ( % hpdi). the proportions of cov shedders estimated by reversing the % hpdi of the logits were: . - . , . - . , . - . , and . - . for juveniles during the "n-rw" and "rw" periods, and adults during the "n-rw" and "rw" periods, respectively ( % hpdi). these values refer to bats not belonging to the species e. helvum or t. persicus. finally, the predicted cov detections, based on the posterior predictive distributions, were . - . , . - . , . - . , and . - . , for these same groups ( % hpdi; fig. , left) . in practical terms, these last values imply that juveniles during the "rw" period are, on average, . times more likely to be detected shedding covs compared to juveniles in the "n-rw" period. adults during the "rw" period are, on average, . times more likely to be detected shedding covs compared to adults in the "n-rw" period. in both seasons, juveniles are, on average, more likely to shed covs, than adults. the selected model suggests a higher odds of cov detection in e. helvum and t. persicus compared to other species. the species-specific intercept terms, once the e. helvum -t. persicus effect is included, suggest no further differences in terms of cov (fig. , right) ; however, the estimates are not precise. the sampling event-specific random intercepts suggest that a few specific locations could show differential cov shedding but that most of them do not explain further variation (additional file ). if coronavirus shedding by bats follows temporal patterns that are generalizable across species and locations, then mitigation strategies targeting the prevention of human exposure and potential spillover could be directed toward high-risk periods, through mechanisms that can also support bat-human coexistence and the provision of bat ecosystem services. previous research has focused on viral identification in specific locations and in few species [ ] [ ] [ ] [ ] , resulting in a limited representation of viral dynamics in association with few ecological settings, biological traits, and reproductive strategies. additionally, few studies of coronavirus shedding patterns have employed statistical models and, in consequence, the potential complex web of factors and causal relationships that may determine this process has not been fully explored. here, we aimed to address these issues by statistically modeling coronavirus rna detection in several bat species, captured at different times and locations in east africa and involving different ecological contexts and life histories. using data from several hundreds of bats, we found that, beyond spatial, taxonomic, and life history differences; i) the odds of coronavirus shedding is higher during the period when pups are being weaned (up to a month after the lactation period is over), and ii) juvenile bats have higher odds to shed these viruses. moreover, the ratios of predicted detections per bat category (age and reproductive season) suggest that juvenile bats during the recent weaning period have relatively higher shedding compared to bats out of this period no matter their age. caution must be taken with these ratios because we used a logit link and our data had high proportion of cov shedding in specific groups. however, our results are consistent, and they are in agreement with previous research conducted in a restricted number of species and locations. similar seasonality of coronavirus shedding has been observed in germany, australia, thailand, china, and ghana (west africa). in the specific species involved in these previous studies, higher coronavirus shedding and viral loads were detected weeks after the birth pulse [ ] [ ] [ ] [ ] [ ] [ ] ] . further, and consistent with our results, detection of higher levels of coronavirus in juveniles has been reported in micro-as well megabats from africa, asia, europe, and north and south america [ , , , [ ] [ ] [ ] [ ] . it has been proposed that the increased detection of coronaviruses after the birth pulse is attributable to the waning of passively-received maternal antibodies in juveniles [ ] . this idea has been frequently cited; however, we are not aware of any longitudinal age-specific sd standard deviation and %, hpdi = % high posterior density interval coronavirus seroprevalence study in bats. such studies are important to understand the drivers of pathogen persistence and spillover risk, and in consequence, to ethically manage and prevent bat pathogen exposure. nevertheless, this kind of research is difficult to conduct due to logistical challenges, our questionable ability to obtain statisticallyrepresentative samples across age groups, cross-reactivity of serological assays, and the difficulties to differentiate serodynamics derived from closed-population processes from those caused by migratory movements. although extrapolations for antibody dynamics across viruses and species are not simple [ ] , bat serodynamics for hendra virus are congruent with the increased detection of coronaviruses after the birth pulse. pups passively receive maternal hendra virus antibodies which decline after the first month of age up to months of age [ ] [ ] [ ] [ ] . this decline would lead to a period in which young bats tend to be more susceptible to infection, become infected, and then shed virus. consistently, coronavirus shedding peaked weeks after the birth pulse in a german and a chinese species [ , ] and immunologically naïve bats shed higher coronavirus loads [ ] . over time, as young bats clear hendra virus infection, they become seropositive again [ ] [ ] [ ] [ ] . concordantly, capture-mark-recapture studies support the clearance of coronaviruses in infected bats [ , ] , which would become seropositive. however, young bats may not reach adult seroprevalence levels until they are older than a year, as occurs with hendra virus [ , , ] . therefore, the population of juvenile bats would remain comparatively more susceptible to viral infection and shedding beyond the period immediately after weaning. age-specific henipavirus seroprevalence in african e. helvum is in agreement with the serodynamics described for hendra virus [ ] . additionally, coronavirus transmission may be favored by high colony density created by the birth pulse, as previously proposed [ , ] , and then the seasonal influx of susceptible juveniles could accelerate viral spread across the entire colony, including adult bats. indeed, adult myotis macropus in an australian colony showed a peak of coronavirus detection after the birth pulse [ ] . the peak of coronavirus detection for two hipposideros species and nycteris cf. gambiensis sampled in ghana occurred during the months that encompassed the birth pulse and nursing after accounting for the age of the sampled individuals [ ] . higher coronavirus infection has also been reported in lactating females [ , ] , which overlap with the period of pup weaning and decay of maternally-derived immunity; however, the opposite has also been found [ , , ] . in practical terms, public health managers could anticipate high risk periods for coronavirus shedding to target interventions. assuming that higher spillover risk is a function of higher viral shedding [ ] and that all coronaviruses with zoonotic potential behave ecologically similarly to coronaviruses detected in this study, managers could target the prevention of human-bat direct (consumption) or indirect (bat droppings) contact specifically during the high-risk season: around and just after weaning, the timing of observable juveniles or individuals smaller than adults. for the species and interfaces defined herein, those management periods have now been determined (fig. ) . for others, direct observation of bats at high-risk transmission interfaces could be used to identify time periods when non-adult sized bats are present. however, observation of dependent pups is not always easy [ ] . of course, for specific species, birth pulses and lactation seasons could also be used to more precisely establish high risk periods similar to the methods we used here, including a combination of direct observation, reports from previous literature, and consultation with knowledgeable bat biologists. our proposed risk-driven strategy i) is evidence-based, as it builds upon coronavirus shedding patterns observed across several chiropteran species present around the world; ii) does not require the advanced laboratory capacity often lacking in resource-restricted settings where intense bat-human interfaces usually occur; iii) is a good alternative to the ideal but expensive and resourceintensive longitudinal surveys; and iv) it may prevent the exposure to viruses belonging to other taxa whose observed bat shedding dynamics resemble our findings for coronaviruses (e.g. paramyxoviruses [ ] ), the lower coronavirus detections in african emballonurids (c. afra and t. mauritianus) and the higher detections in e. helvum, african hipposiderids (hipposideros sp. and t. persicus), and r. cf. clivosus that we found are consistent with previous reports [ , , , ] and should be considered by managers when providing risk-based spillover prevention strategies. moreover, sars-like coronaviruses in africa have been found in hipposiderid, rhinolophid, and molossid bats [ , , ] , and mers-related coronaviruses have been found in vespertillionid bats [ , , ] . therefore, it seems reasonable to prioritize the identification of birth pulses and lactation seasons, and thus determine high-risk periods of coronavirus shedding, for these bat families. interestingly, e. helvum roost in tight clusters that can contain hundreds of individuals [ ] , similar to t. persicus. on the other hand, emballonurids, showing the lowest crude detection levels across families (represented by coleura afra and taphozous mauritanus here) tend not to cluster while roosting [ , ] . we did find an association between coronavirus shedding and whether the species typically aggregate in clusters while roosting when the variable "e. helvum -t. persicus" was not considered, but we chose a different model not including this term because we did not directly assess bat roosts and our categorization may oversimplify the continuum from mostly solitary roosting (e.g., neromocia nana) to common tight aggregations of bats (e.g., mops condylurus). using this categorization could be misleading, as some species differentially cluster while roosting depending on temperature, colony size, colony type (e.g., maternity colony versus not a maternity colony), and season [ ] . future studies should consider the roosting habits of bats, as this trait could further support riskbased management to prevent or reduce human exposure. the risk-driven strategy we propose provides a contactreduction alternative that is ethically favorable compared to often-employed measures, such as culling or other reactive measures, that ensue when the public becomes aware of a health threat without a suggested practical option to reduce their risk for exposure. in addition to ethical concerns and being logistically difficult and expensive, culling has failed to reduce disease in wild populations and can result in even higher pathogen levels. for example, "badger culling can make no meaningful contribution to cattle tuberculosis control in britain" [ ] . a culling program to reduce echinococcus multilocularis prevalence in red foxes (vulpes vulpes) resulted in an increase of infection [ ] . this strategy has also failed to control rabies in canids around the globe [ ] . similar results have been observed in bats. culling failed to reduce rabies seroprevalence in desmodus rotundus in perú and could have increased the levels of exposure to the virus [ , ] . in argentina, the extermination of bats changed the direction of spread of rabies in livestock but did not prevent its advancement [ ] . in uganda, miners exterminated a colony of rousettus aegyptiacus bats after an outbreak of marburg virus in that involved miners in close contact with these bats. five years later, a new outbreak occurred in miners from the same mine. the second time, marburg virus rna was detected in a higher proportion in the r. aegyptiacus that recolonized the mine ( . %, n = ; [ ] ) compared to rna detection before culling in this cave ( . %, n = ; [ ] ) and other caves in uganda ( . %, n = ; [ ] ) and gabon ( . %, n = ; [ ] ) where culling has never been reported. culling can also cause demographic changes, leading to a higher proportion of juvenile individuals. this change may occur because of a disproportionate cull of older individuals; the potential increase in survival of pups at lower population densities, followed by higher recruitment of juvenile females into reproductive age [ ] ; the hypothetical increase of young dispersers immigrating from neighboring colonies into culled, less dense, and better resourced colonies [ , ] ; or by causing compensatory reproduction [ ] . this last possibility may have not been studied in bats but seems unlikely due to their high conception rates and usual litter size of one. examples of younger populations after culling have been reported in the red deer (cervus elaphus), racoon (procyon lotor), american mink (mustela vison), and australian brushtail possums (trichosurus vulpecula), among others [ ] [ ] [ ] [ ] . as our results and past research consistently show higher viral shedding and detection in young individuals, activities leading to a younger bat population are not advisable for viral spillover management. similar results are expected when fruit bats are culled based on being categorized as "agricultural pests"; therefore, this kind of management may create higher risk of viral exposure to the human population. virological, ecological, and epidemiological research on bats over the last years has helped to identify chiropterans as hosts of zoonotic viruses and to document that human-driven environmental change, human behavior, and human-to-human transmission are the key drivers for the creation of bat-human interfaces, spillover, and epidemics of emergent viruses, respectively [ ] [ ] [ ] . in the context of the current biodiversity and bat conservation crisis [ , ] , we must not omit these facts when attempting to effectively, and responsibly frame and communicate disease risks associated with bats. realistic, data-based risk communication is of paramount importance to avoid framing bats as a threat to humans and to support bat conservation given their important ecological roles [ , ] . with this background, it seems a proper time for the scientific community studying "bat-associated" viruses to move the conversation from bat spillover risk assessments to the planning of pro-biodiversity and subsequently pro-ecosystem strategies aiming to mitigate spillover risk. science is valued not only for the diagnosis of problems but because it finds solutions to them. here, we have attempted to aid the progress of scientific and management dialogue by proposing, not only a management strategy to limit potential coronavirus spillover, but one that is context-and logistically-grounded and pro-conservation, promoting the delivery of the key ecosystem services provided by bats. data from hundreds of bats collected in east-africa show that coronavirus shedding is expected to be more frequent when pups are becoming independent from the dam, independently of the age of the bats, their species, their location, and their life histories; however, the odds of shedding do differ by species. these results can guide temporal-based mitigation strategies to prevent bat-associated coronavirus exposure using non-lethal methods in limited-resource settings, where longitudinal surveillance is not feasible, by identifying high-risk periods for coronavirus shedding when contact with bats should be avoided. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : summary of the inferred start date of the birth pulse, the end of the lactation period, and the start date of the mating period per microchiropteran species. additional file : bibliographic references for the traits of the bat species included in the study. additional file : imputation of the reproductive season to those bats whose biology is insufficiently known. model methods, results, and literature cited. table s . summary of the coefficients' posterior probability distributions of the selected model for the imputation of the reproductive seasons of bats missing this data. figure s carlo iteration. right: the distribution of the crude coronavirus detection per reproductive season across the , markov chain monte carlo sampling iterations after imputing the periods when un-inferred. the black boxplots show the distribution of the coronavirus detection per period, while the light and dark colored boxes above and below show the interquartile detection in non-adults and adult bats, respectively, per period. additional file : table s . summary of the alpha-and betacoronaviruses (alphacov and betacov, respectively) found in the microbats tested. additional file : results of the model to assess the association between cov shedding and the "recent weaning" season in eidolon helvum and microbats. figure s authors' contributions dml = designed the project; collected, analyzed, and interpreted data; and drafted, edited, and prepared the final manuscript and figures. tg = guided laboratory analysis and generated data, especially barcoding and sample testing, and supported manuscript writing. kg, dw, evw, rk = organized data collection and supported manuscript writing. bs, jn, zs, mc = data collection and supported manuscript writing. the predict consortium = provided support in the design and implementation of surveillance and interpretation of data. jkm = designed and supervised the project, including sampling design and data collection; supported data analysis and interpretation; and drafted, edited, and prepared the final manuscript. all authors read and approved the final manuscript. this study was supported by the generous support of the american people through the united states agency for international development (cooperative agreement numbers ghn-a-oo- - - and aid-oaa-a − - ). the contents of this paper are the responsibility of the authors and do not necessarily reflect the views of the us agency for international development or the us government. mammal diversity database. . www.mammaldiversity.org increasing awareness of ecosystem services provided by bats ecosystem services provided by bats bats limit arthropods and herbivory in a tropical forest bats limit insects in a neotropical agroforestry system top-down control of herbivory by birds and bats in the canopy of temperate broad-leaved oaks (quercus robur) bats and birds increase crop yield in tropical agroforestry 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we appreciate the support of the american people through the united states agency for international development (usaid) emerging pandemic threats predict project (cooperative agreement number ghn-a-oo- - - ). we thank the governments of rwanda, tanzania, and uganda for permission to conduct this study, and the country predict teams that led and conducted the field activities. we are mostly grateful of all the technicians involved in the processing, testing, and barcoding of the samples, especially ruth maganga (sokoine university of agriculture) and brett smith (one health institute, university of california -davis). finally, we appreciate the help of dr. evan eskew for supporting with stan coding and dr. wes johnson for guidance with bayesian inference. key: cord- -j cecioe authors: fager, edward w. title: determination and analysis of recurrent groups date: - - journal: ecology doi: . / sha: doc_id: cord_uid: j cecioe nan the primary information available from an ecological survey usually consists of a mass of sampling results involving many localities and many species. in order to investigate the interspecific processes which may be involved in controlling the abundance and distribution of the species found, it is useful to be able to group together those species which frequently occurred together in the samples, which were in this sense a nearly constant part of each other's biological environment. the grouping will be most valuable as a classificatory device if it consists of defined units obtained by a procedure which can be repeated. whatever the method of grouping employed, the sampling procedure is certain to introduce subjective elements; the size of sample, the number and selection of sampling sites and the sampling methods used will always be influenced by the judgment of the person doing the study and they will in turn influence the composition of the groups. if, however, the sampling conditions are explicitly given, the probable extent and character of these influences can be taken into account and a meaning given to the groupings in this context. some of the methods of grouping which have been suggested will be briefly reviewed (see also macfadyen, ) and a new method will be outlined which overcomes some of the difficulties review of some methods of grouping animals have often been grouped on the basis of vegetation or of various physical or chemical factors in the environment, but many difficulties are involved in vegetational classification or in deciding what is a critical limit in respect to a certain physical or chemical factor. furthermore, animal groupings do not always conform to vegetation or other factors ; a group may extend over several recognizable vegetation or soil divisions or there may be several distinct assemblages of animal species within an environmental complex which is uniform in many other respects. the characterization of the habitat in which an animal group is often found must include the associated plants and other factors, but it seems better to use the animals as the primary basis for their grouping. gisin ( gisin ( , gisin ( , has suggested a procedure using what he has called "differential" species. in the hands of an experienced worker this method can lead to useful groupings, but the choice of the species which will be designated as "differential" is so subjective that comparison between groupings by different workers, even if based on the same material, may be impossible. moreover, using this method groups may be suggested the constituent species of which so seldom occur together that it seems unlikely that they could have any relation to each other. for example, gisin ( ) in his rearrangement of agrell's ( ) swedish lapland collembola suggested four groups. members of three of these were found together moderately often. the fourth group contained four species, representatives of which were recorded in a total of thirty-one samples ; no sample contained all four, one sample contained three, five samples contained different pairs and twenty-five samples contained only one of the species. the subjective element can be reduced if the grouping is based on a defined index of affinity. perhaps the simplest expressions which might be used for this purpose are those proposed by jaccard ( ) and sprenson ( ) as measures of the similarity between floras. as given, however, neither differentiates between a coefficient based on ten samples and one based on one thousand samples, though the latter would surely be a more reliable estimate. the expressions also fail to take account of changes in the relative number of total occurrences of the two species; as long as the sum of the total occurrences remains constant, the proportions can change without affecting the value of the coefficient. a method based on correlation between pairs of species is precisely definable but, as cole ( ) has pointed out, product moment correlation cannot be used directly because sampling results for most species are not normally distributed in regard to numbers of individuals per sample; there are qsually a few outsize samples which will have a disproportionate effect on the calculations. transformation of the data or the use of rank correlation could overcome this difficulty, but any method which involves a measure of abundance may, in certain cases, not lead to the desired results : two species may always occur together and never separately and yet, unless there is a nearly constant relation between the relative numbers of individuals of the two species, a correlation coefficient will indicate no relationship even though they are a constant part of each other's biological environment. for this reason it seems best to base species groupings upon presence and absence alone and to consider abundance relations within such groupings after they have been determined. in his paper, cole ( ) discussed the coefficients of association which had been proposed up to that time and indicated the deficiencies of each. he then proposed a new coefficient using only presence and absence and based on a x con-ti~ig-ency table for which a chi-square test of sigmficance can be calculated. a basic assumption of his expression is that all samples taken should be included; i.e., the probabilities of occurrence of the two species being tested for association are assumed to be uniform throughout the area sampled. under this assumption, the chi-square indicates the significance of the departure of the observed number of joint occurrences from the number expected on the hypothesis of independent distribution of the two species over all the samples. although cole did not use his coefficient for grouping species, it has been so used several times since. the following example shows that it is not a satisfactory criterion for grouping if the groups are to be composed of species which form a nearly constant part of each other's biological environment: species and show no evidence of association when examined by cole's method and yet they nearly always occur together-over % of the occurrences of each are in company with the other -and should be considered together in any grouping based on this set of samples. on the other hand, species and are significantly associated by cole's criterion even though their affinity is rather low-less than % of the occurrences of each are in company with the other. in general, two species which occur in most of the samples taken will show little or no evidence of association and two species with a low percentage of joint occurrences can show significant evidence of association if they are rare enough or if a large enough number of samples in which they could not occur are included. bray ( s ) has recently noted these difficulties connected with the use of cole's index and has attempted to overcome them by considering only quadrats within plots in which both species occurred. macan ( s ) using the method without modification found a very large number of positive associations in his analysis of collections of corixidae, a finding which, as he indicated, was unsatisfactory because the objective analysis produced a less well-defined picture than that which could be obtained from a subjective technique based on a knowledge of habitat features. an expression of the sort suggested by cole has certain desirable properties; in particular, it is sensitive to changes in the relative frequency of occurrence of the two species and a "significance a t-test is preferred to chi square because it can be used as a one-tailed test [ . s ,....., p (.os) ] for positive affinity; negative affinity, being based on the failure to find a species, seems potentially subject to too many unavoidable errors. there are both mathematical and biological reasons for setting an upper limit on the value of nn which will be considered with a given na: the closer the ratio nnjna is to , the closer the normal approximation will be to the true value; keeping the ratio close to ensures that whenever evidence of affinity is obtained the number of joint occurrences will be a considerable proportion of the total number of occurrences of both of the species. this is a desirable property for an index to be used as a basis for grouping if the purpose is to put together organisms which are a nearly const·mt part of each other's biological environment. an upper limit of for the ratio nn/na is therefore sug-level" can be defined for use as an objective criterion of the presence of affinity. it can be adapted for use as an index of affinity if it is assumed that the probability of finding species a is najna + nb and of finding species b is nbjna + nb, where na is the total number of occurrences of species a and nb is the total number of occurrences of species b; i.e., the probalilities of occurrence of the two species are related to the sum of their occurrences (na + nb) rather than to the total number of samples taken. the use of this assumption removes the premium on rarity and also makes it possible to find evidence of affinity between two species which occur in most of the samples taken. it seems neither more nor less arbitrary than the use of the total number of samples for the latter is usually determined arbitrarily by the person doing the work, often mainly on grounds of practicality or subjective judgment that the number taken is sufficient. if the preceding assumption is used and the species are assigned to the letters so that na is less than or equal to ne, the number of joint occurrences (j), on the hypothesis of independent distribution, will have a hypergeometric distribution with expected j equal to nanejna + ne and variance equal to (nanb) j(na + ne) (na + ne- ). for values of na greater than and p and q not too different from j , a normal approximation can be used to test the significance of the deviation of the observed number of joint occurrences from the expected number. for this purpose, the following expression can be taken as a normal deviate with mean and unit variance (pearson ): gested. the use of such a limit has the disadvantage of preventing the finding of affinity between such a pair of species as a rare parasite and its relatively abundant host, but the existence of a relationship of this kind can, in any case, probably be satisfactorily established only by field observation and breeding studies. minimum values of j which are significant at the .os level have been calculated for values of na from s to using exact probabilities obtained by leslie's method ( ss) . similar values have been calculated for each tenth value of na from to using the normal approximation. both sets are given in table in the appendix. these values and intermediate ones which can be estimated by interpolation will make it possible to quickly sort pairs of species into those which certainly show evidence of affinity, those which cer-tainly do not and those which are doubtful and require calculation. when applied to the examples used in the discussion of cole's coefficient, the index proposed above indicated significant evidence of affinity between species and but none between species and . it is, therefore, a better index of the pro-portion of occurrences which are joint occurrences and should provide a more satisfactory basis for grouping. it may be repeated that cole did not develop his index as a basis for grouping and that the two indices measure different things: cole's index measures the departure of the two species from independence of distribution on the assumption that the probability of occurrence of each is constant over all the samples taken ; the index proposed in this paper provides an objective measure for the word "frequently" in the statement "these species frequently occurred together." the procedure to be outlined for the determination of recurrent groups may appear complicated but with a little practice it can be gone through at least as rapidly as the "fitting by eye" of a trellis diagram. it has the virtue of repeatability ; given the same primary information, two workers will always arrive at the same groups. this means that if several workers using similar sampling methods make studies in different localities and find different recurrent groups, there is some assurance that the differences are real and not the result of differences in judgment or emphasis. any dichotomous index of relationship between species can be used as a basis for grouping by this procedure; the meaning of the groups will be determined by the nature of the attributes measured by the index. in this paper, a recurrent group is defined as one which satisfies the following requirements : ( ) the evidence for affinity is significant at the . level for all pairs of species within the group. ( ) the group includes the greatest possible number of species. ( ) if several groups with the same number of members are possible, those are selected which will give the greatest number of groups without members in common. ( ) if two or more groups with the same number of species and with members in common are possible, the one which occurs as a unit in the greater number of samples is chosen. these requirements are to be taken in order; i.e., the group or groups which fulfill requirements and are determined and then, if there are several possible combinations, a choice is made on the basis of , followed, if necessary, by . such groups will represent the largest, most frequent, separate units within which all the species formed a nearly constant part of each other's biological environment. they would be a likely choice if one wished to make an intensive study of interspecific processes which are quantitatively important in the habitats sampled. the following procedure will give a recurrent group or groups as defined above. the affinity information for the species concerned may be set out in a trellis diagram as shown for the example (table i) . if there are many species, it has been found more convenient to record the information for each species on a separate card. . =non-affinity the species are put in order in terms of the number of other species with which they have affinity. in the example this order is a, b, c, . . . p, q. starting with the species with the largest number of affinities, species are counted in the direction of decreasing number of affinities until the number of species counted (x) exceeds the number of affinities ( y) of the last species counted. in the example this occurs at species h where x = and y = . two possible relationships between x and y must now be considered: if this is true, as it is in the example, the largest potential group will contain y + species ; species in the example. it cannot contain more because species h has affinities with only other species and can, therefore, only form a group of species satisfying requirement ( ). if species h is omitted, only species are left. because the species were put ecology, vol. , no. in order in terms of the number of other species with which they had affinity, the species beyond h will either have the same number of affinities as h, in which case they are also potential members of a group of species, or fewer affinities than h, in which case they cannot be included in a group of species satisfying requirement ( ). if species h had had affinities with other species, the largest potential group would have contained the first species, a to h. [x > y + ] if this is true, the largest potential group will contain the x - species preceding the last one counted. for example, if species h had had affinities with or fewer other species, the largest potential group would have been composed of the species, a to g, for h and any species beyond it could only form a group containing or fewer species. the counting procedure thus selects those species which are potentially members of a group satisfying requirements ( ) and ( ). these must be further examined to determine whether such a group can be formed. two preliminary tests of the possibility of its formation can be applied. the first test can be stated as follows : in order to form a group of z members from v potential members, there must be at least z of these with more than z- affinities with others of the potential group. for this test the affinities of each of the selected species with all others of the potential group are tabulated. this tabulation for the example is shown below : a b c d e f g h v = ;z = . there are only species which have more than affinities and, therefore, a group of species cannot be formed. passing the preceding test is a necessary but not sufficient condition for the formation of a group as can be seen by consideration of the following : if the interrelationships among the potential members had been such that there was affinity between all species except the four pairs, a-b, c-d, e-f, and g-h, the tabulated affinities would have all been and yet no group containing species and satisfying requirement ( ) could have been formed. on the other hand, if the species a to g had had affinity with all others except h, the tabulated affinities would have been seven 's and one and a recurrent group of species could have been formed. in general, the affinities lacked by the species needed to form the group must be accounted for by species which are not necessary for its formation. the second test determines whether this condi-tion is met. it can be expressed algebraically as follows : in order to form a group of z members from v potential members the following inequality must hold: (v- ) ( z-v) < + ~ z largest affinities -~ the rest of the affinities. applied to the two cases discussed in the preceding paragraph, it gives the following results: incorrect, a group of cannot be formed. ( - ) ( [ ] - ) < + ( )- ( ); correct, a group of can be formed. this is a necessary and sufficient condition for the formation of a recurrent group when v = z or z + ; it is necessary but not sufficient when v = z + or more for in this case the two sums on the right hand side of the expression can vary independently to some extent, the more so as there are more possible pairs of species within the category "rest." although passing these tests does not make the potential group a certainty in all cases, the two tests will prevent unnecessary work in many instances in which no group can be formed. returning now to the example, no group of species could be formed so the possibility of the next smaller group, species, is investigated. two additional species, j and k, must now be considered for, having affinities with other species, they could be members of a group of . the affinities within the group of species are shown in the first line of the tabulation below. both tests indicate that it may be possible to form a recurrent group of species : there are over species with more than affinities; ( - )( [ ] - ) is less than + ( + + + + + ) -( + + + ). the affinity interrelationships must now be examined in detail in order to make the final determination. perhaps the easiest way to do this is to eliminate those species which do not have more than z - affinities, g and k in the example ; repeat the tabulation as shown in the second line below, eliminate species which now do not have more than z - affinities and repeat until no more species can be eliminated. if at the end of this process v is less than z + , the second test can be applied as a both necessary and sufficient condition for the formation of a recurrent group. v=lo z= v= z= v= z= v= z= in the last line ( - ) ( [ ] - ) is less than + ( ) - and as v = z this is a necessary and sufficient condition for the formation of a recurrent group. therefore, the species a-e, j constitute the group. the presence of j in the group emphasizes the necessity of considering all species which, on the basis of the number of other species with which they have affinities, might be part of the group. the initial counting procedure takes care of this automatically for the largest potential group, but the appropriate species must be added if this group cannot be formed and the next smaller must be considered. as there were no alternative groups of satisfying requirements ( ) and ( ), requirements ( ) and ( ) do not have to be considered. the species not included in the group are now examined to see if any have affinities only with members of the recurrent group. this is true of l and q. such species are considered associates of the recurrent group. the procedure is repeated on the remaining species, omitting their affinities with members of the first recurrent group (table ii) . the largest potential group contains species; x = and y = at species n. when the affinities within this group of are tabulated, however, p and n have only and a group of species cannot be formed. all of the species must be included when the next smaller group, species, is considered. none can be eliminated on the basis of the first test and as ( - ) ( [ ] - ) is less than + ( + + ) -( + + + ) a group may be possible. when the interrelationships are examined in detail, the following groups are found to satisfy requirements ( ) and ( ) : fgn, fgp, fhp, fkp, gmn. requirement ( ) will be satisfied if gmn and either fhp or fkp are selected. in order to decide between the latter two, the original sampling results would have to be investigated on the basis of requirement ( ) . it will be assumed here that fhp occurred as a unit more often than did fkp. the relation- ships between the species might be set out as shown in figure . this method has been applied to several studies reported in the literature and to an unpublished survey of the invertebrate fauna of decaying wood done by the author. in all cases the groups determined were consistent with what was known of the species' requirements, preferences, etc. the invertebrates from decaying wood were grouped using code numbers for the species in order to eliminate the possibility of bias ; the groups found were in general agreement with impressions gained during sorting and counting. recurrent groups based on the index of affinity proposed in this paper and determined by the procedure outlined above are composed of species which very frequently form a part of each other's biological environment. such interspecific concepts as dominance, concordance and correlation should, therefore, be particularly meaningful when examined within these groups. the several methods of analysis suggested below use ranking procedures which, as kendall ( ) has shown, are very useful for detecting general trends in material which includes some extreme values, as sampling results from natural populations nearly always do. before the analytical methods can be applied, samples representative of each recurrent group must be selected. for groups containing only a few species, selection can be based on the requirement that all species in the group are present in the sample. for groups containing many species, this may too greatly reduce the number of samples and some compromise must be accepted. the requirement should be set at a high percentage present but the exact value to be used will depend on the sampling results which are available. it should be explicitly stated in any case. table iii presents some sampling results, related to actual observations but somewhat modified in order to show the different results which dominance numerical dominance is often expressed as a mean percentage of total individuals, obtained from the summed percentages of the samples considered separately. such an expression, using percentages calculated for samples with different total numbers of individuals, is hard to interpret and gives no information about the constancy of the relation. the difficulties of interpretation are increased if the species are dissimilar in size, activity, etc. it is, therefore, suggested that determinations of dominance be restricted to species within recurrent groups which are, or appear to be, similar in regard to size, activity, food requirements, etc. and that the determination be based on ranks within samples, summed over the set of samples. this is equivalent to n things (species) ranked m times (number of samples in the set). the concordance among the rankings can be tested by the statistic w (kendall , pp. - ) , which can range from to ; from no agreement to perfect agreement among the rankings. details of the calculation of w and of an f-test of its significance are given in the appendix. the use of ranks within samples gives every sample an equal voice in the decision and eliminates the bias which one or two samples with exceptionally large or small percentages may impose on the per cent method. rankings within samples for the species a, b and c and d and e are shown in the second part of table iii. for the value of w is significant at the . level showing that there is concordance among the samples; i.e., the dominance relations between the species tend to be constant over the set of samples. the sum of ranks is for each species the best estimate, in the sense of least squares, of the over-all rank of that species and if there is significant evidence of concordance the species may be ordered by these sums. the results may be stated as c >a, b; i.e., c is constantly dominant to a and b, and a is probably dominant to b but their sums of ranks are so nearly equal that the relative positions are less certain. the value of w[w = . ; f(n = . ; n = . ) = . (p > . )] is not significant for d and e indicating that dominance relation between these two species is not constant. relative abundance over all the samples is related to but not the same as numerical dominance. for example, assume that the numbers of individuals of two species found in samples were as follows: species x would be found to be constantly dominant to species y and yet their relative abundances would not be significantly different. the difference between the relative abundances could be tested by the usual t-test of the difference between the means, but if there were any outsize samples they would exert a disproportionate influence. as these are often present among samples taken from natural populations, it is probably better to use the distribution-free statistic u and a t-test based on it (hoel , pp. - ). this tests the hypothesis that the distributions of the two populations of values are the same against the alternative that one distribution is situated to the left (lower valued) of the other. it is not affected by outsize samples. details of the calculation will be found in the appendix. in the case of species a and b the observed value of u is not significantly different from the expected value. there is, therefore, no evidence that the distribution of abundances of a was different from that of b. as is apparent by inspection, c was significantly more abundant than either a or b. the observed value of u is significant at the .os level, indicating that e was significantly more abundant than d even though determination of dominance had shown that it was not constantly dominant to d. the concordance among the species of a recurrent group on what constitutes a good habitat can be tested by the same methods ( w) as were used in examining numerical dominance except that in this case ranking is done within species and the meaning of n (number of samples in the set) and n (number of species) are the reverse of those used in the previous test. if the observed number of individuals of a species is taken as an estimate of the goodness of a sample for that species, the samples can be ranked in regard to each species ; this eliminates difficulties due to differences between species in average abundance, efficiency of collection, etc. the sum of these ranks over all species of the group is for each sample the best estimate, in the sense of least squares, of the rank of that sample among the samples selected as representative of the group. if there is significant evidence of concordance, the samples can be put in order. rankings within species are shown in the third part of table iii. for the recurrent group considered as a unit; showing that there is agreement among the species on what constitutes a good or bad habitat. the samples can be put in order of decreasing goodness as follows: > , > , , , , , > . an examination of the other characteristics of these samples, especially the extremes, might reveal a good deal concerning the preferences and requirements of the group as a whole. rank correlations between species should also be calculated because the preceding generalization may miss significant relations: if, in a group consisting of four species two rankings are alike and the other two are exactly the opposite, the concordance will be zero although the six possible pairs of species will give four perfect negative correlations and two perfect positive correlations. because of the possibility of parent correlations, nonsense correlations, etc., the interpretation of correlation analysis must always be viewed with reservations. if, however, correlation coefficients are calculated only for those pairs of species for which there are other reasons for expecting interrelationships, they may serve as useful guides to the mature and constancy of the relations. kendall's ( , pp. - , ) the nearly significant positive correlation between the two species of poduromorph collembola, ac, might be due to chance, might indicate that the species have a favourable effect on each other, or might result from their liking the same conditions and not interfering. the significant positive correlation shown by the presumed prey-predator pair, cj, indicates that the predator did tend to congregate in those places where its prey was abundant. the absence of significant negative correlation between similar species means that in none of the pairs of species was an increase in the number of individuals of one always accompanied by a proportionate decrease in the number of individuals of the other. more evidence than the presence or absence of correlation is necessary before biological relationships can be claimed, but such an analysis will suggest those which might best repay further investigation. this paper presents a defined, repeatable method for grouping together species which are frequent components of each other's biological environment. such a method makes it possible to compare groups found in different habitats or at different times or localities. as the composition of these recurrent groups is influenced by the method of sampling used and by the "level of significance" required of the index of affinity, the groups are abstractions. it has, however, been found that if sampling is related to the size, activity, etc., of the organisms and the significance requirement is made stringent, the groups formed have ecological unity in the sense of significant intragroup agreement on what constitutes a good or bad habitat. it seems reasonable, therefore, to consider them as natural assemblages, somewhat artificially delimited but nonetheless real. because the recurrent groups are composed of species which very often occur together, they represent particularly appropriate units within which to examine interspecific relationships. the analytical procedures suggested for this examination have two purposes; the provision of a summary description of relationships and the suggestion of working hypotheses upon which to base further field and laboratory work. the use of methods employing ranking gives each species an equal voice in the analyses, removes many of the difficulties due to non-normality of distributions and makes it possible to determine the constancy of general trends. a new index of affinity between species, based on presence and absence, is proposed and a table is provided from which the significance of an observed number of joint occurrences can be estimated. using this index and a four-part definition of a recurrent group, a procedure is outlined which leads to the largest, most frequent, separate groups within which all species formed a nearly constant part of each other's biological environment. ranking methods are suggested for the examination of interspecific concepts such as numerical dominance, relative abundance, concordance and correlation within these groups. the essentials of the methods of testing for con-cordance ( w) and correlation ( -rb) and of the u test are given below for the convenience of those to whom the texts may not be readily available. the expressions given for the first two tests include the corrections required when tied ranks are present. in both cases, t or u represent the extent of ties present; e.g., if a ranking has members with one rank and others with another rank, the summation is ( - ) + ( - ) in the case of wand ( - ) + ( - ) in the case of "b· for the calculation of w all ties are considered together ; for the calculation of 'rb the extent of ties in one ranking is represented by t and that in the other by u. when tied ranks are absent, the summations are and the expressions are considerably simplified. concordance (kendall ) : if n things are ranked m times, the ranks for each of the n things are summecl and the sum of the squares of the deviations of these sums from their mean is represented by s, the concordance between the rankings ( w) is given by the following expression : w- s m n(n - )ml'jt(t - ). this does not have to be modified for tied ranks unless the number and extent of ties is large. to determine significance the usual table of the variance ratio (f) is entered with degrees of freedom n and n • correlation (kendall ) : the rankings are arranged so that one is in the natural order- , , , ... n. following this arrangement, scoring is based entirely on the other ranking. starting with the lefthand member, + is scored for every larger rank and - for every smaller rank to the right of it. after this has been done for each member of this ranking, -rb is calculated from the sum of the scores ( s) as follmvs : s 'rb = y'[n(n- )l'jt(t- ) ][n(n- )l'ju(u- )] . t is the sum of ranks of the y's when the x's andy's are ranked together. these expressions can be used in a one-tailed t-test of the hypothesis that the distributions of the two populations are the same against the alternative that the x population is situated to the left (lower valued) of the y population. for m and n equal to or more, the following expression may be taken as a normal deviate with mean and unit variance : this test is nearly as efficient as the usual t-test when applied to normally distributed populations ; as it is distribution-free, it is applicable to nonnormal distributions. example: if na = and nb= , a j of at least is necessary for significant evidence of affinity; if na= and nb= (nb/na= . ), minimum significant j can be zur okologie der collembolen: untersuchungen im schwedischen lappland a study of mutual occurrence of plant species the measurement of interspecific association vegetationsforschung auf soziationsanalytischer grundlage analyses et syntheses biocenotiques . l'ecologie introduction to mathematical statistics nouvelle recherches sur ia distribution florale rank correlation methods a simple method of calculating the exact probability in x contingency tables with small marginal totals a contribution to the study of the ecology of the corixidae (hemipt the invertebrate fauna of the choice of statistical tests illustrated on the interpretation of data classed in a x table a method of stabilizing groups of equivalent amplitude in plant sociology based on the similarity of species content and its application to analyses of the vegetation on danish commons key: cord- - m qko authors: durkin, louisa; jansson, tobias; sanchez, marisol; khomich, maryia; ryberg, martin; kristiansson, erik; nilsson, r. henrik title: when mycologists describe new species, not all relevant information is provided (clearly enough) date: - - journal: mycokeys doi: . /mycokeys. . sha: doc_id: cord_uid: m qko taxonomic mycology struggles with what seems to be a perpetual shortage of resources. logically, fungal taxonomists should therefore leverage every opportunity to highlight and visualize the importance of taxonomic work, the usefulness of taxonomic data far beyond taxonomy, and the integrative and collaborative nature of modern taxonomy at large. is mycology really doing that, though? in this study, we went through ten years’ worth ( – ) of species descriptions of extant fungal taxa – , studies describing at most ten new species – in five major mycological journals plus one plant journal. we estimated the frequency at which a range of key words, illustrations, and concepts related to ecology, geography, taxonomy, molecular data, and data availability were provided with the descriptions. we also considered a range of science-demographical aspects such as gender bias and the rejuvenation of taxonomy and taxonomists as well as public availability of the results. our results show that the target audience of fungal species descriptions appears to be other fungal taxonomists, because many aspects of the new species were presented only implicitly, if at all. although many of the parameters we estimated show a gradual, and in some cases marked, change for the better over time, they still paint a somewhat bleak picture of mycological taxonomy as a male-dominated field where the wants and needs of an extended target audience are often not understood or even considered. this study hopes to leave a mark on the way fungal species are described by putting the focus on ways in which fungal taxonomy can better anticipate the end users of species descriptions – be they mycologists, other researchers, the public at large, or even algorithms. in the end, fungal taxonomy, too, is likely to benefit from such measures. taxonomy is the science that discovers, identifies, classifies, and describes organisms. like in any scientific field, the knowledge gained in taxonomy has a value in itself, but it also caters to the needs of other research areas. almost all studies in biology, and many other sciences, are performed on a taxon (often a species), derivatives from samples of a specific taxon (e.g., a protein), or pertain to the diversity of taxa. this view of the fundamental nature of taxonomy is certain to be shared by scientists and decision makers alike, but surprisingly this is not enough to guarantee a steady long-term supply of resources to taxonomy (drew ) . in fact, the funding for taxonomy is at a record low. in what has become known as the "taxonomy crisis" and the "taxonomic impediment" (wheeler ; de carvalho ) , taxonomists are finding themselves at nearly the same risk of extinction as the very species they are supposed to study. various mechanisms have been put forward to visualize and highlight the importance of taxonomy and to give credit to taxonomic work, such as citing authors of species names -and the underlying publications -when using those species names in publications (wägele et al. ) . however, the extent to which these suggestions seem to have taken effect appears to be limited, as taxonomy remains locked in a state of crisis (magoga et al. ) . since the "taxonomy crisis" has been acting out gradually during at least the last years, it is reasonable to think that few biologists are unaware of it. taxonomists, in particular, are certain to be all too familiar with it, often reporting feeling marginalized in comparison to ecological or molecular initiatives in the context of, e.g., grant writing and scientific funding (giangrande ; coleman ) . in this context it is important to focus on the values of taxonomy and how they can be communicated to a wide audience. in this way of thinking, every new species description is a potential outlet for information that is useful not only in taxonomy but also in ecology, conservation biology, agriculture, and so on. the potential of each and every such outlet should be maximized in the interest of taxonomy. but are taxonomic papers written accordingly? several of the present authors have spent significant time going through published species descriptions for key data on, e.g., taxonomy, ecology, and geography for compilation into community-driven efforts such as unite (nilsson et al. ) , plutof (abarenkov et al. ) , funguild (nguyen et al. ) , and wikipedia (https://www. wikipedia.org/). we have found that scrutinizing species descriptions for key information can be surprisingly frustrating and time-consuming, largely owing to the heterogeneous or implicit ways in which information is sometimes provided (or omitted altogether) in species descriptions. our experience is that straightforward questions such as "what does the new species do for a living?" are often not addressed at all, or are treated only very indirectly by statements such as "on dead branch of quercus" (perhaps implying a saprotrophic ecology). another highly relevant question -"where in the fungal tree of life does the new species belong?" -is similarly often hard to make out from the paper, often requiring a genus name query in ncbi taxonomy (federhen ) or some similar database. questions on, e.g., the geographical distribution of the new species are likewise often hard to answer. this lack of information is most unfortunate -certainly, species descriptions should be more or less self-sustained, such that they should not expect significant mycological experience or google searches on the part of the reader. our initial observations would indicate that species descriptions are written either for narrow intradisciplinary communication or are disconnected from the wants and needs of many readers. this lowers their impact considerably and is hardly in the interest of taxonomy or mycology at large. similarly, the field of fungal taxonomy would do good to show that it is, in fact, a vibrant, modern, and collaborative discipline -a discipline that cares little for country borders, where both genders take an active part, and where knowledge is readily shared with, and passed on to, aspiring researchers as well as the public at large. but is that really happening? to assess whether fungal species descriptions are attuned to both the wants and needs of a target audience beyond taxonomists and the sign of the times, we explored years' worth of fungal species descriptions of extant mycological taxa in five major mycological journals (plus one botany journal for reference) for a range of factors pertaining to inter-and intra-scientific terms and concepts, science-demographical aspects, and illustrations and visualisations (tables , ; suppl. materials , ). we processed the underlying pdf files using a text mining approach where specific keywords were used to simulate a non-taxonomic reader. we also scored each paper manually for a number of features deemed relevant to the overall reader experience -such as the presence of a color photo (or illustration) of the organism being described, whether a map or a habitus photo was provided, and whether we could access the paper from a computer not connected to a university network (table ). our results show that there is much that can be improved in taxonomic descriptions to increase their availability, appeal, and usefulness to a wider scientific and public community and thus the impact of the work and of taxonomy itself. similarly, fungal taxonomists should adapt their output to an increasing number of automated readers, including data aggregators and search engines. fortunately, many of these improvements can be implemented in manuscripts in a matter of minutes and at zero cost. other aspects of our results should make mycologists rethink who should be invited to our studies, and how we would like taxonomic expertise to be passed on to younger researchers. the present study seeks to table . estimates obtained by the automated and manual parsing of the pdf files, broken down to three individual years (columns - ) as well as overall (column ). column indicates our interpretation of the mycological repercussions of the trend in the data. suppl. material breaks down these estimates onto the individual , mycological papers, and suppl. material shows the full syntax used to query the individual papers for each parameter group in column . the following estimates are given in per cent. altitude -altitude of sampling. biodiversity -whether the term "biodiversity" was mentioned. climate -whether the word "climate" was used. climate zone -whether reference to climatic zone was used. collection -whether reference to "herbarium" etc. was used. distribution -whether reference to geographical distribution was used. ecological association -whether any ecological association was indicated. ecological mode -whether the ecological mode was indicated. ecology, the term -whether variations of the word "ecology" was used. family (classification) -whether a family name was provided. gis/gps -whether gis/gps co-ordinates were provided. index fungorum -whether this resource was mentioned. locality, the term -whether variations of "locality" were mentioned. molecular availability (treebase/dryad) -whether reference to treebase or dryad was made. molecular search (blast) -whether reference to blast was made. molecular database (e.g., genbank) -whether reference to, e.g., genbank was made. molecular data used -whether dna data was used. mycobank -whether mycobank was mentioned. order (classification) -whether order was provided. phylum (classification) -whether phylum was provided. societal implications -whether societal implications were alluded to. supplementary data -whether supplementary data were bundled with the paper. threatened (endangered) -whether the taxon was highlighted as threatened or endangered. color photo/illustrationwhether a depiction of more or less the entire fungus was provided, as opposed to only micromorphological details. determination key provided -whether a determination key was provided. discussion section present -whether a dedicated discussion section was provided. electron microscopy used -whether electron microscopy was used. fungal culture shown -whether a photo of a fungal culture was shown. lead author male -whether the lead author was male. macro-photo indicates size -whether macroscopic images used scale bars/fiducial markers. manual micromorphology illustration -papers illustrating micromorphological features using manual illustrations. map used -whether a map was provided. paper available -whether the paper was found to be openly accessible. phylogenetic tree shown -whether a phylogenetic tree was provided (molecular or otherwise). photo showing biological context -whether a photo or illustration indicating the biological context of the species was provided. photo of micromorphology -whether microscopic details were illustrated by photos. spore print provided -whether a spore print was provided. the following estimates are provided as averages. academic age, last author -the academic age of the last author as assessed through google scholar profiles. academic age, lead author -the academic age of the lead author. co-authors -the number of co-authors. co-author continents -the number of continents in the list of co-authors. co-author countries -the number of countries in the list of co-authors. co-author departments -the number of departments in the list of co-authors. female co-authors -the number of female co-authors. pages -the number of pages. statistical figures -the number of statistical figures/data visualizations. leave a mark on the way fungal species are described, but we also hope to provide food for thought for editors, reviewers, and members of scientific boards. we went through each issue ( - ) of five major, well-reputed mycological journals known to publish new species regularly (table ). the journals come from three different continents and are known for their high standards, such that the species descriptions we examined are likely to represent international mycology at its finest. for reference we also included a botanical journal, where we included the fungal descriptions in the fungal description corpus and the plant descriptions in the plant description corpus. all articles whose title made it clear that one or more new species were being described were examined more closely. we retained articles describing at most new species. efforts such as fungal planet (e.g., crous et al. ) -where + species are described in a single paper by + co-authors -were deemed to be too heterogeneous to score meaningfully in a semi-automated context, as we were interested in singular research efforts by coherent groups of taxonomy-oriented co-authors. descriptions of fossil taxa were excluded. we retained all descriptions of non-fungal taxa (e.g., myxomycetes and oomycetes), but studies where existing species were simply recombined into other genera were excluded. all individual papers that met our criteria were downloaded as pdf files (suppl. material ). the resulting , pdf files were converted to text using pdftotext version . . (https://pypi.org/project/pdftotext/). the text files were mined using a python script (suppl. material ) which searched for the presence of key words and terms related to ecology, geography, taxonomy, molecular data, and data availability (table ). in this process, the article titles, author names and affiliations, abstracts, acknowledgements, and literature cited were excluded from the search to reduce the risk of false-positive matches. out of the , papers that were scored automatically, we went through % manually to verify that the automatic parsing produced reliable results. a number of features relevant to the description of species were not amenable to straightforward algorithmic interpretation -such as the presence of a full-color photo or illustration showing the whole organism being described rather than just micro-morphological details -and these aspects were scored manually by going through each pdf file in adobe illustrator cc (www.adobe.com). the number of co-authors, distinct co-author departments, countries of origin and continents of origin (using the seven-continent system) of the co-authors were counted manually to quantify the extent to which taxonomy is practised as a collaborative pursuit. we sought to establish the gender of all co-authors by brief queries in google, google scholar, and orcid (https://orcid.org/). only articles where we could determine the gender of all co-authors were used to infer the proportion of female coauthors and lead male authors. in an attempt at quantifying recruitment of aspiring researchers into taxonomy, we made the admittedly coarse assumptions that the last author was the supervisor, mentor, or taxonomic expert, and that the first author was a student or a nascent taxonomist. google scholar was used to determine the academic age of an author: year-of-the-oldest-publication minus year-of-the-most-recent-publication, in a way that dismissed obvious homonyms and ambiguous entries. unresolved cases were left out from the comparison. for convenience we group our results and discussion under the headings ecology and geography, systematics and taxonomy, metadata and data availability, visualisation, and demographical aspects. the overall automated and manual estimates are found in table , whereas the full set of results broken down to each individual paper is found in suppl. material . most biologists would probably agree that taxonomy should be pursued in light of as many data sources as possible, including molecular, morphological, and ecological information. the output of taxonomic work should similarly be rich and many-faceted. however, the fact that the word "ecology" (and its variations) was used in only . % of the examined studies somehow speaks against this assertion. on a more positive note, explicit reference to host, substrate, habitat, or partner was made in . % of the cases, and a reference to the nutritional mode of the new species was made in . % of the cases. the word "ecology" and any of the other ecology-related keywords (suppl. material ) we used were completely absent in . % of the studies (suppl. material ), suggesting that only a very small number of species descriptions are nucleated in what seems to be either complete disregard or lack of ecological data, or in total ignorance of the wants and needs of the scientific community. we acknowledge that when a new species is described, there may be no or limited occurrence data beyond the type locality. still, variations of the words "distribution" and "geography" were mentioned in a strong . % of the studies. although explicit reference to variations of "climate" was found in only . % of the studies, a full . % of the studies featured climate-related words such as "temperate" or "tropical". gis/gps co-ordinates were provided in a much more modest . % of the studies, and . % of the studies provided a map. ( . %) of the studies that did not provide gis/gps co-ordinates provided a map instead. a total of . % of the studies provided neither gis coordinates nor a map, and . % lacked any relevant variation of the word "locality". this does little to facilitate recollection of the species at the type locality. altitude/elevation was mentioned explicitly in . % of the studies. it strains credibility that more than % of all fungi described during - were collected at sea level, suggesting that the absence of altitude information should not be taken to mean sea level. . % of the studies featured at least one photo or illustration that gave at least some sort of feeling for the biological context of the new species, typically by showing the collection site, the collection spot, or the substrate of collection. we feel that there is room for improvement here, particularly if taxonomy indeed seeks to produce results of relevance and interest that extend beyond the field. it is surprisingly common to describe a new fungal species without mentioning where in the fungal tree of life it belongs: a phylum-level name was found in . % of the studies; order, . %; and family, . %. the intersection of these estimates was . %. in a few cases, some of this information may be truly unknown for the species being described (e.g., torres-cruz et al. ), but we argue that phylum, order, and perhaps family assignment is known for the vast majority of fungi being described. by knowing where the new species belongs, but not writing it out explicitly, the underlying authors rely on the reader to fill in the mycological gaps. this strikes us as unfortunate, because non-mycologists as well as automated data extraction tools such as data aggregators and search engine indexation software may lack this expertise. in fact, even many mycologists will probably have to look up where, e.g., the family sphinctrinaceae belongs. the situation is not alleviated by the fact that index fungorum, mycobank, and ncbi taxonomy regularly disagree on family-and order-level placement of genera (owing to, e.g., differential updates and taxonomic opinion). thus, even readers who actively go looking for this information may be left clueless or misinformed. although the fungal family level is in a state of flux in many parts of the fungal tree of life, we can't think of any good reason not to mention at least the phylum and order level affiliations of new species. it is thus not surprising that catalogue of life (https://www.catalogueoflife.org/) and similar efforts often fall short of providing the precise taxonomic placement of individual species in the fungal tree of life, even when this information was known to the mycologists who described the species in the first place. clearly, mycology does not stand to benefit from the presence of incomplete taxonomic information in online repositories. although taxonomy represents a core aspect of biodiversity, variations of the word "biodiversity" are not commonly used in papers describing new species of fungi -only . % of the studies used it. this comes across as a missed opportunity to place the new species in a richer context -and to have the underlying paper indexed properly in search engines and automated classifiers of scientific papers. highlighting the importance or relevance of the new species to society (e.g., agriculture, forestry, or biotechnology) -if motivated -would similarly lead to a wider readership and better article indexation. however, a moderate . % of the studies featured such keywords. a much lower number of studies - . % -made a reference to the threatened or endangered nature of the new species or its habitat, although this may be difficult to know at the time of description. where is the underlying specimen or culture deposited? we found it quite common ( . % of the cases) to provide this information in a way that does not employ any variations of the words "herbarium", "fungarium", "museum", or "culture collection"an example would be "deposited in h". the reader would then have to know -or find out -that h is a herbarium at the university of helsinki, finland. this poses no challenge to a seasoned taxonomist (through recourse to, e.g., index herbariorum at http:// sweetgum.nybg.org/science/ih/ or grscicoll at https://www.gbif.org/grscicoll), but we imagine that other readers would struggle with this, as would data mining efforts to extract information from scientific papers. improving clarity by writing, say, "deposited in herbarium h" -and why not write out the name of the herbarium in full? -should be easy enough. . % of the papers mention "index fungorum" (http://www.indexfungorum.org/names/names.asp) and . % "mycobank" (robert et al. ) explicitly, showing that writing out names in full is common in other contexts. identification keys help define what exactly differentiates the new species from others, and . % of the papers we examined featured an identification key. there can be many reasons why a key would be premature or impossible to construct for various fungal taxa, such that whether . % is a comforting estimate or not is hard to say. determination keys are, however, becoming rarer over time (table ) . the proportion of species descriptions making use of dna sequence data -as deduced from the use of variations of keywords such as "pcr", "dna", and "sequencing" -is on the rise, from . % in to . % in (fig. ) . across all years, at least one dna sequence-related keyword was found in . % of the studies, an estimate that blends well with the % reported for fungi by miralles et al. ( ) , who examined papers from , , and . the proportion of dna-based studies depositing their multiple sequence alignments or phylogenetic trees in treebase (vos et al. ) or dryad (https://datadryad.org/) is similarly on the rise: overall, . % of the studies mention treebase/dryad, up from . % in but down from the . % of . the problem of poor data availability in systematic mycology has been made apparent before (nilsson et al. ; drew et al. ), so we were happy to note a gradual -albeit slow -improvement over time. if taxonomy is to be a modern, reproducible, integrative, and standards-compliant field (see, e.g., dimitrova et al. and the miapa standard, leebens-mack et al. ) , then there is simply no excuse for not sharing the underlying phylogenetic -and other -data in standardized ways (grandcolas ; miralles et al. ) . increased data deposition is furthermore likely to improve the visibility of the underlying article and thus taxonomy at large. to simulate whether the general reader could access the underlying pdf publications by google searches, we queried google by pasting the name of the paper in quotation marks and then scrutinizing the first two pages of hits manually (february ). we did this from computers not connected to any university network. we ac-cepted hits to pdf files and full-text papers in the html format of both the final, published paper and to any preprints in, e.g, biorxiv (https://www.biorxiv.org/), and we accepted both legal as well as juridically more dubious sources of pdf files. if any sort of registration was needed to access the pdf file, we scored it as "not avail- . the x axis depicts year and the y axis depicts proportion of studies (from to ) fulfilling a specific criterion. dark green -proportion of studies mentioning the word "ecology" or its variations; brown -proportion of studies giving a complete account of the taxonomic affiliation of the new species (family, order, and phylum); purple -proportion of studies with a macroscopic colour photo/illustration of the new species; pink -proportion of studies with macroscopic photos, that also indicate the size of the depicted object through a scale bar or a fiducial marker; light green -proportion of studies with an identification key; yellow -proportion of openly available papers for each year as assessed in b demographical and publication trends showing the average number of coauthors (dark green), departments (brown), countries (purple), continents (pink), and number of data visualizations (light green) over time. the bars indicate the yearly standard error c the average academic age of the first (green) and last (brown) co-author over time. the bars indicate the yearly standard error d the proportion of female co-authors (green) over time plus the proportion of female lead authors (brown). female authors leading female author d able". we found that . % of the studies could be accessed from outside university networks. the observation that more than % of the taxonomic output of the mycological community cannot be readily accessed by the general public comes across as unfortunate. however, all of the journals we targeted allow the submission of preprints to online repositories. thus, submitting a vetted preprint at least post-publication (in order not to confuse effective publication dates of names) is a way around this inaccessibility (cf. kwon ) . it is, evidently, a solution that is not being explored to the extent it could have been by the mycological community. many cases of taxonomic mistakes, redundant species descriptions, and laboratory contaminations would have been avoided if the authors had subjected the newly generated dna sequences to a simple blast search in, e.g., genbank (nilsson et al. ) . sadly, only . % of the studies mention blast, although the trend is positive (table ) . a clear majority ( . %) of the examined studies make explicit reference to one of the insdc repositories (genbank, embl, and ddbj), again in a positive trend. bundling supplementary material with species descriptions is a good way to increase reproducibility and provide additional, helpful information with respect to the new species without consuming valuable page space in print journals. here we envisage additional photos or drawings of fungal specimens or the collection site, or perhaps extended maps, field notes, or laboratory details. however, a somewhat disappointing . % of the studies saw fit to include at least one supplementary item. the average study was . pages long, although we did not correct for the number of described species in each paper. the studies grew more voluminous over time (table ) , possibly as a consequence of the inclusion of more analyses based on molecular data. a high . % featured at least one color photo or illustration showing more or less the whole fungus being described (in the sense of a full fruiting body rather than only microscopic details or a spore print). somewhat disappointingly, only . % of the macroscopic photos/illustrations featured a scale bar or a fiducial marker, leaving assessment of size problematical for more than a third. . % of all studies contained at least one photo of a fungal culture, and a spore print was presented in . % of the studies. . % of the studies featured at least one visualization of a micromorphological detail or structure. in total, micro-morphological details were illustrated by line drawings in . % of the studies; photos ( . %); and electron microscopy ( . %). . % of the studies featured both a micromorphological photo and a line drawing; a total of . % of the studies commendably used all three techniques. a phylogenetic tree was displayed in . % of the studies. since . % of the studies used molecular data, this means that a few studies used molecular data without presenting a phylogenetic tree. a cursory look at a few of these indicated that techniques such as rflp had been used to generate a fingerprint of the new species. the average study featured . data visualizations (e.g., a graph or a chart other than a phylogenetic tree). the average number of co-authors was . , which was higher than we expected given that taxonomy is sometimes touted as a solitary discipline. the average number of departments, countries, and continents were . , . , and . -again higher than we had expected. plotting the number of co-authors and countries over time (fig. ) suggests that fungal taxonomy is slowly becoming an increasingly collaborative and international discipline. it is, however, a discipline dominated by males: out of the papers for which we were able to establish the gender of all co-authors, . % were male-only papers and . % were female-only papers. these papers comprised a total of , co-authors, of which ( . %) were female. males were lead authors in . % of the papers for which we were able to determine the gender of the lead author. our gender-related estimates certainly leave room for improvement of inclusivity and career opportunities in mycology (cf. salerno et al. ) , although they slowly improve over time (table ). our admittedly crude attempts at quantifying the extent to which recruitment of aspiring researchers and the passing on of knowledge is going on in fungal taxonomy showed that the average academic age of the last author ( . years) is higher than that of the first author ( . years), perhaps hinting that knowledge does seem to be passed on to younger generations (or at least a younger generation) albeit somewhat slowly. one hundred non-taxonomical mycological papers from the same journals produced similar figures - and years, respectively -hinting that this issue may not be specific to taxonomy. in fact, the high academic age of the authors may be part of explaining the gender bias, as gender equality tends to decrease upwards in the academic hierarchy (potvin et al. ) . although the key terms and concepts to look for in a mycological species description will be somewhat different from those of a botanical counterpart, we did find some notable differences between the description of fungi and plants. it should be kept in mind that our botanical reference corpus was limited to a single journal and papers, and the extent to which our results can be extrapolated to botany at large remains unknown. nevertheless, botany comes out on top of mycology when it comes to specifying the type locality through either a map or gis/gps co-ordinates: % of the botanical studies did this, as compared to only . % of the mycological. on the other hand, the use of molecular data is more widespread in mycological species descriptions ( . %) than in botanical ( . %). . % of the mycological studies that relied on molecular data made these available in treebase/dryad, compared to . % of the corresponding botanical papers. full-color macro-illustrations of the species being described were more common in mycology ( . %) than in botany ( . %). the number of co-authors on botanical species descriptions is lower ( . ) than in mycology ( . ), and so is the average number of female co-authors ( . vs. . ). mycology comes across as a somewhat more collaborative discipline in that the average number of co-authors from different depart-ments, countries, and continents are all higher in mycology, but botany struggles somewhat less with recruitment of aspiring taxonomists (suppl. material ). these figures should be interpreted with the very limited size of the reference corpus in mind, but they do seem to suggest that botany and mycology can learn from each other when it comes to describing new species in the st century. joint meetings such as the meeting of the mycological society of america, american bryological and lichenological society, american fern society, american society of plant taxonomists, and botanical society of america (http:// .botanyconference.org/) are commendable in this respect. the semi-automated nature of our approach is not without potential shortcomings, and we are likely to have both under-and over-estimated some of our parameter values. as an example of an overestimation, a study could mention "dna" or perhaps "pcr" without actually making use of sequence data in the description of the new species. this would have led us to the incorrect conclusion that dna sequence data was used in the description of the species. as an example of an underestimation, a study could conceivably provide information on the ecology or nutritional mode of the new species without using any of the ~ terms we looked for, leading us to the erroneous conclusion that nothing was said about the ecology of the new species. since we processed nearly , mycological papers, such outlier cases will not have contributed much to our estimates. our manual verification of % of the papers did not reveal significant cause for concern with respect to over-or under-estimations. a potentially larger bias lies in our choice of journals. we purposely selected five major international mycological journals with significant impact factors, stringent editorial and review processes, and very detailed author instructions. the journals are not solely focused on taxonomy but cover a wide spectrum of mycological subdisciplines, and the papers published therein can therefore be expected to be geared towards a more general mycological audience. however, fungal species are described also in other outlets. for instance, there are mycological journals with a formal web of science impact factor for . indeed, schoch et al. ( ) found that the fungal ribosomal nuclear internal transcribed spacer (its) sequences in genbank stemmed from over different scientific journals. yet other journals would not even be represented in genbank because they have yet to publish their first species using sequence data (focusing instead on morphology-based descriptions). we speculate that at least some of these other journals may have less stringent editorial and review processes (in fact, several journals that publish new species of fungi do not use formal peer-review at all). along the same line, many of these journals are not available digitally, and some are printed in black and white. thus, our estimates pertain to the state-of-the-art species descriptions of fungi rather than the full spectrum of fungal species descriptions. while not all of our estimates are flattering for fungal taxonomy (e.g., table ), it is likely that they still paint an overly optimistic picture of fungal species descriptions at large. the international code of nomenclature for algae, fungi, and plants (turland ) stipulates the minimal requirements for publication of new names (species). notions of, for example, ecology or geographical distributions, or inclusion of illustrations, are not part of those requirements (seifert and rossman ; hawksworth ) . but rather than asking what the minimal requirements are, mycologists should strive to showcase taxonomy as a vibrant, exciting field where species are described in the richest possible way, where all data and metadata elements are machine readable with persistent identifiers, and where ample auxiliary data are posted to online repositories and community initiatives such as zenodo (https://zenodo.org/), figshare (https://figshare. com/), wikimedia commons (https://commons.wikimedia.org/), and open tree of life (https://tree.opentreeoflife.org/) in standards-compliant formats and data structures (mons et al. ) . to some extent, our results question whether this is how fungal species are described at present. the fact that a full . % of the studies did not mention any variations of the word "ecology", or that . % of the studies lack the word "biodiversity", is certainly not encouraging, for instance. we argue that fungal species should be described in the richest possible way; in fact, a moderate . % of the studies featured a formal "discussion" section, which for the remaining studies seems like missed opportunities to anchor the new species in a richer mycological and biological context. with an average of . co-authors on the , papers, there would clearly be room to invite one or more additional co-authors to add, e.g., ecological aspects to the description. similarly, many opportunities to bundle helpful supplementary data are waiting to be filled ( . % of the studies did not bundle any supplementary material). we do realize that there are situations where the, e.g., ecology or geographical distribution (or even origin) of a new species is entirely unknown; thinly annotated herbarium specimens come to mind. we are not against species descriptions based on legacy specimens, but a random selection of studies in suppl. material did not present a single such case. our dataset presumably still contains examples of this and other special cases, but the vast majority of the studies covered represented relatively recently collected material. this makes the lack of data on ecology and geography harder to swallow. we were happy to note that the proportion of species descriptions using sequence data is on the rise (fig. ) . dna data are important for taxonomic identification in many studies. failure to provide characters for taxonomic identification using dna sequencing (typically in the form of sequences deposited in a public repository such as genbank) is therefore limiting the value of a description. put negatively, . % of the studies in suppl. material were done without dna data. we ask the fungal taxonomists of the world to always bundle at least an its sequence (and preferably also an nlsu sequence) with all new species (and genomes, for that matter), even if those sequences were not analyzed or used in the study in question. we recognize that not all mycologists have access to dna sequencing equipment, and we hope that more well-equipped mycological laboratories will be able to assist nearby, less well-equipped mycologists with the generation of such data. the cost of generating a sanger sequence is low today, and all of mycology would stand to benefit from such generous acts (cf. womack ) . adopting a species description to be meaningful also to a non-taxonomic reader may be challenging enough, but we argue that mycological taxonomy needs to go one step further. in a world where information is increasingly culled through automatic means, mycologists should no longer assume that all readers of species descriptions are human to begin with. this means that all data and metadata items should be machinereadable and available online, come with globally unique and persistent identifiers (including orcids for humans, accession numbers/dois for sequence data/datasets, dois for cited publications, and open tree of life identifiers for phylogenies). the notion of automated readers also brings about changes in the way manuscripts should be written in that it becomes particularly important to provide clear and precise information, almost to the point of tabularization. we argue that standardized terms should be used even when they cannot be parameterized; "ecology: unknown" is incomparably more helpful to human and automated readers alike than simply leaving out the word "ecology" altogether. along the same lines, and although brevity may suffer somewhat, "in herbarium gb (university of gothenburg)" is immeasurably more helpful than "in gb". no automated reader, and few non-taxonomists, will be able to contextualize the acronym "gb" in a meaningful way. to assume that the reader will be able to extrapolate the position of the species in the fungal tree of life, or the gps co-ordinates of the collection spot, and so on, should similarly be avoided. our demographical estimates suffered from various potential shortcomings and biases: online information can be hard to find (particularly when it comes to authors who have not registered on orcid), google scholar profiles are not necessarily complete or correct in terms of their publication lists, the last author does not have to be a supervisor or a mentor figure, and so on. getting around these shortcomings in a study of the present kind is next to impossible, and we feel that our demographical results should be seen merely as rough estimates of trends. but a surprisingly strong signal still came out of them: taxonomy is no longer -and perhaps never really was -an entirely solitary discipline, but instead comes across as a reasonably collaborative, international discipline where knowledge seems to be passed on to younger researchers, at least to some extent. this offers hope for the future -taxonomy may actually be on its way to shake some of the misconceptions surrounding it (coleman ). still, taxonomy needs to be wary of gender aspects as evinced by our estimate that a total of . % of the co-authors in our dataset were female, indicating that a lot of potential competence and perspectives are neglected by the field. there may also be other inequalities in the discipline, not estimated here. our data generally, but not exclusively, indicate what we feel is a gradual improvement in the richness of species descriptions and in the demographical aspects of fungal taxonomy over time ( table ). the improvements come across as slow but at least steady. there is little in our data to invite complacency, though. on the contrary, we feel that our data highlight the need for fungal taxonomy to make a sincere effort to be aware and to improve. it is not enough that mycological species descriptions perhaps come across as a bit more robust and reproducible than plant descriptions in our somewhat compromised comparison. we should set the bar really high -this-and-that is what we want species descriptions to look like from the scientific, demographic, and popular science points of view, so this-and-that is indeed what we will deliver. we feel that there are several measures that fungal taxonomists can and should take to improve the impact and outreach of their work. our suggestions -with the exception of generating and bundling a dna sequence with each species description -do not come with any direct costs and do not call for acquisition of any machines or utensils. that said, we don't want our suggestions to be used as excuses for cutting down other parts of species descriptions -we don't feel that the inclusion of a macroscopic color photo justifies shortening the corresponding text-based description of the macromorphology, for instance. our suggestions will add somewhat to the time it takes to describe a new species -or serve as the editor or reviewer of a manuscript in which a new species is described -but we argue that it is a price worth paying (de carvalho et al. ) . indeed, we feel, it is a price that mycology simply must pay to make it a reproducible field at the heart of systematics and biodiversity at large. plutof -a web-based workbench for ecological and taxonomical research, with an online implementation for fungal its sequences taxonomy in times of the taxonomic impediment -examples from the community of experts on amphipod crustaceans fungal planet description sheets systematics must embrace comparative biology and evolution, not speed and automation taxonomic impediment or impediment to taxonomy? a commentary on systematics and the cybertaxonomic-automation paradigm openbiodiv: linking type materials, institutions, locations and taxonomic names extracted from scholarly literature are we losing the science of taxonomy? as need grows, numbers and 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data and metadata the taxonomist -an endangered race. a practical proposal for its survival taxonomic triage and the poverty of phylogeny. philosophical transactions of the royal society of london. series b: biological sciences research data in core journals in biology, chemistry, mathematics, and physics rhn acknowledges financial support from the swedish research council of environment, agricultural sciences, and spatial planning (formas, - - ). dmitry schigel and nils hallenberg are acknowledged for very valuable feedback on an earlier draft of the manuscript. key: cord- -chrxyrls authors: owen, jennifer title: trophic variety and abundance of hoverflies (diptera, syrphidae) in an english suburban garden date: - - journal: ecography (cop.) doi: . /j. - . .tb .x sha: doc_id: cord_uid: chrxyrls hoverflies of species were caught in a malaise trap in an english suburban garden during the eight‐year period – , and three additional species were hand‐netted. hoverfly larvae fall into five trophic categories all of which were represented by adults in the trap sample. . % of the hoverflies trapped have larvae that feed on aphids, . % feed on decaying organic material, . % eat living plants, . % scavenge in hymcnoptera nests, and . % are associated with tree sap or rotting wood. the relative frequency of the different trophic groups varied annually and seasonally although the aphid‐feeders were nearly always the most abundant. species are believed to breed in the garden, and a further in the surrounding area; species are regarded as casual and as chance visitors. it is suggested that the high plant diversity and spatial heterogeneity of gardens result in them supporting more species than would be found in a natural area. s hoverflies of species were caught in a malaise trap in an english suburban garden during the eight-year period - , and three additional species were hand-netted. hoverfly larvae fall into five trophic categories all of which were represented by adults in ihe trap sample. . % of the hoverflies trapped have larvae that feed on aphids, . % feed on decaying organic material, . % eat living plants, . % scavenge in hymcnoptera nests, and . % are associated with tree sap or rotting wood. the relative frequency of the different trophic groups varied annually and seasonally although the aphid-feeders were nearly always the most abundant. species are believed to breed in the garden, and a further in the surrounding area; species are regarded as casual and as chance visitors. it is suggested that the high plant diversity and spatial heterogeneity of gardens result in them supporting more species than would be found in a natural area. /. owen, scraptoft lane, leicester, england. gardens, under the general heading of domestic habitats, have been dismissed as being 'in the direction of biological deserts' (elton ) on the assumption that the many introduced plants growing there have tew animals associated with them. intensive studies of a suburban garden in leicester, england, have shown that this is not so and that insect diversity is high (owen . a ,b, owen and owen . as industrial, and accompanying residential, development encroaches on the english countryside, gardens become increasingly important as refuges for wildlife, particularly insects. most gardens are small, some are very large, but collectively they occupy an area of more than () ()() ha, more than the area set aside as national parks and nature reserves. housing patterns and social customs in england are such that the garden habitat is in no way threatened, and indeed its total area is increasing all the time. gardens are characterized by contrived plant diver-sity and spatial heterogeneity. casual introduction of native plants, i.e. weeds, together with planned planting programmes result in continual addition of new plants, but this is countered by weeding and management decisions as to what to grow, gardens are therefore maintained in a state of permanent succession, and not only is the resulting plant diversity high, but the composition of the flora changes from year to year. the average gardener demands much from a small patch of land: a display of colourful flowers, a supply of vegetables and other produce, space to sit or for children to play, somewhere to stroll, a pleasing view from the house, a screen from neighbours or traffic, and often a display site for his or her skills and tidiness. this produces, in even the smallest garden, a patchwork of shaded and open places, so close-packed that the garden becomes a system of ecotones, each micro-habitat grading into another on all sides. consequently, even a small garden can accommodate a wide variety of insects, simply because the garden habitat encompasses a multitude of ecological niches. the study garden, which covers an area of m^ is typical of english suburbia. it was created in iy and, although subject to many minor changes in lay-out of paths, lawns and flowerbeds and in the sorts of annual and biennial flowers and vegetables grown, it has probably remained essentially unchanged for at least years. there are paved paths, neat lawns (one of which was left uncut for a season to allow grasses to flower, and has since been converted to a flowerbed), herbaceous borders with an array of colourful and fragrant flowers, vegetable patches, a compost heap, a rockgarden, fruit bushes and a tangle of brambles, an old apple tree, many shrubs of a variety of species, some tall, exotic conifers screening the garden from a busy road, and until recently a small pond. absolutely necessary, maintaining continuous soil cover, whether by weeds or cultivated plants, and rigorously excluding herbicides, insecticides and olhcr poisonous chemicals. small-scale plant diversity is further enhanced by mixed planting of vegetables and ornamental flowers, an ancient gardening practice which is believed to reduce outbreaks of pests. in the five-year period - , species of flowering plants belonging to families were recorded, native species and aliens. all plants that have been introduced to the british isles by man, whether intentionally or accidentally as weeds of cultivation, together with hybrids of garden origin are regarded as aliens. some plants on the garden list are so widespread and familiar that their status as aliens is open to dispute: acer pseudoplatanus was brought to england by the romans; buddleia davidii. widely introduced to gardens in the early () s had become naturalized by the s; and matricaria matricarioides and conyza canadensis, although probably introduced accidentally, are common weeds. on the other hand. daphne mezereuni and potentilla fruticosa, both widely cultivated, are rare native species, and such common garden crops as cabbages, cauliflowers and broccoli are derived from the native brassica oleracea. the exact composition of the garden flora changes from year to year as species appear and disappear, by accident or intent, but plant diversity and dense cover are maintained. nevertheless, the garden is in no sense wild or overgrown, and its general appearance is similar to that of neighbouring gardens. since , a malaise trap has been operated continuously on the same site from april to october, and the large catch includes many hoverflies, among them several species which have never been observed alive in the garden. hoverflies are good indicators of the diversity of a habitat because different speeies and life stages exploit their surroundings in many different ways, yet the family is distinct and easily recognized. the type of malaise trap used is described in townes ( ) . it is an open-sided tent-like structure of fine netting, with a central baffle parallel with the open sides. the netting "roof of the trap is highest at one end where a collecting jar filled with % alcohol is attached. insects flying in through the open sides and striking the central baffle tend to fly upwards, and ultimately fall into the collecting jar at the apex. the trap is particularly suitable for sampling flying insects, such as hoverflies, because it is in operation continuously, day and night, in all weather conditions, and uses no attraetant, capturing only those insects that have independently entered its air space. the trap samples an area of about . m^ to a height of . m, and therefore removes from the garden only those flying insects that enter . m-* of air space. this is unlikely to affect the size or composition of the garden fauna; even if insects enter the garden to fill the space created by removing some, both removal and replacement are effectively rand(»m. during the eight-year period - , the malaise trap caught hoverflies of species, slightly more than a third of the british species (kloet and hincks ) . three additional species have been hand-netted in the garden. a total of species has been recorded in the county of leicestershire (owen ) , st) one small garden has yielded two thirds of the species known to occur in the vicinity, including known only from the garden. adult hoverflies arc predominantly nectar-and pollenfeeders and although there are some differences between species, the requirements of all are broadly similar. the larvae, however, vary considerably in their food and feeding places, and hence in their positions in garden food webs. five categories (derived from colyer and hammond ) can be recognised: consumers of living roots, bulbs, stems or fungi; predators of aphids and other homoptera; dwellers in (and presumably feeders on) rotting wood or sap from tree wounds; dwellers in (and hence feeders on) soft, or liquid decaying organic material; and scavengers in the nests of ants, bees or wasps, members of most groups are probably opportunist feeders: aphid-feeders occasionally attack other insects and each other; larvae associated with tree sap may eat small insects attracted to the sap; and scavengers in hymenoptera nests occasionally eat live larvae and pupae. hoverflies as a family are unusual in exploiting the environment in such diverse ways. ail five groups are represented by adults caught in the malaise trap and the numbers of individuals and species, and the trophic levels of the groups are shown in tab. . the larvae of a number of garden species are undescribcd. but they are assumed to have habits similar to those of closely related species. three genera trapped in the garden, merodon, eumerus. and cheilosia, fall into group . merodon equestris and the two species of eumerus feed by burrowing in living bulbs, and almost certainly breed in the garden. of the five species of cheihsia, c. bergenstammi feeds on ragwort (smith ) . and the others are believed to feed on living plants. group i is represented by individuals, less than % of the total catch. most garden hoverflies. and the majority of the common speeies, have aphid-feeding larvae, as far as is known, the larvae of all syrphinae and pipizini feed on aphids or other herbivorous insects. fifty-three species and over % of all individuals taken fall into group . gardens provide ample opportunities for aphid-feeding species, and seven species of syrphinae have been reared from larvae collected in the garden: syrphus ribesii, episyrphus balteatus, sphaerophoria scripta, metanostoma mellinum, m. scalare, platycheirus albimanus and p. scutatus. most of the abundant garden species range widely over many habitats and the adults of a few are migratory. group includes speeies and only individuals, but neither rotting wood nor sap runs on trees are common garden micro-habitats, and the species collected are unlikely to breed in the garden. exactly what their larvae feed on is a matter for conjecture; indeed placing them in a separate group may be unjustified. they probably feed on rotting plant material, but larvae that imbibe tab. . feeding sites and trophic levels of larvae of hoverflies caught in a suburban garden, - . ( ) ferdinandea ( ) notes: separation into irophic levels is based on the system suggested by wiegcrt and owen ( ) . additional species collected in garden: secondary consumers ( syrphinae, pipizinij and decomposer (eristalini). tree sap might better be placed in group i. and any that are predators of insects attracted to the sites where they occur should perhaps be placed in group with the aphid-feeders. the species in group are taxonomically diverse, but all have larvae that live in decaying organic material, often of animal origin. all are decomposers, but since the nature of their food varies, they operate at several trophic levels. larvae of erisialis and helo{?hilus are usually found in water feeding on minute particles of organic material, but eristahs max also occurs in dung, in sewage and in wet carrion. the larvae of syriira pipiens occur in garden compost, but they have also been recorded from dung of cows, horses and humans (skidmore ) . most of the species in this group undoubtedly breed in the study garden or its immediate vicinity, cither in compost, on dung or carrion, or in ponds and tree holes, and the proximity of farmland accounts for the regular capture of rhingia campestris whose larvae feed on and in cow dung. group , represented by species and individuals, % of the total catch, ranks second in importance to the aphid-feeders in group . volucella hombykms and v. pellucens are the only representatives of group . their larvae scavenge in bee and wasp nests, not only eating dead hymenoptera and detritus, but also stimulating the host larvae to produce excrement on which they feed. occasionally however, especially towards the end of the season, they eat live larvae, pupae or weakened adults. group constitutes less than one per cent of the total sample. most of the species of hoverflies on the british list can be assigned to groups to , if larval habits are inferred from descriptions and from known habits of other species in the same genera. group , species with aphidfeeding larvae, arc over-represented in the garden, and groups and are under-represented; groups and form about the same proportion of the british and garden lists. the trophic composition of the malaise trap catch varied to some extent from year to year, as shown in tab, , although hoverflies with aphid-feeding larvae (group ) constituted the majority of the sample in every year, their relative frequency was considerably lower in , . in this was associated with an increase in relative frequency of species with larvae that feed on decaying organic material (group ); the autumn of was warm and sunny, and species such as erisialis arbustorum, which continue flying late in the season, were particularly abundant. in , species with herbivorous larvae (group i) formed a greater proportion of the total catch than in other years, and merodon equestris and eumeriis strigaius were rela-tah. . annual flticluations in relative frequency of hoverflies with larvae in different trophic groups (sec tab. ), year year . year year year ly - . corollae formed about % of the total catch in and in , and£./>a/rf(jm over % in (fig. ) . this is not reflected in the relative frequency of group hovertlics in these years (see tab. ), largely because the numbers of resident species of melanostoma and platycheirus in the catch were far lower than usual; melanostoma mellinum, for instance, whieh breeds in the garden and is usually common, was represented by only two individuals in , and m. scalare, which also breeds, was absent altogether. in , when the numbers of m. corollae and e. halieatus fell, those of m. mellinum, platycheirus albimanus and p. scutatus reached unprecedented levels. thus the trophic composition of the garden hoverfly fauna was apparently unaffected by immigration of m. corollae and e. halteatus to the garden in , and . seasonal changes in the relative abundance of species wilh different larval habits is expressed in fig. as the percentage of eaeh month's catch composed of aphidfeeders (group ). they were first to appear and increase in numbers in spring, and their frequency relative to those with other larval habits was highest in april and may, it fell in june when the catch increased in variety, but had usually risen again by august. by october, species whose larvae feed on decaying organic material, e.g. eristalis and helophilus, were common and the relative frequency of aphid-feeders was lower. the greatest departure from this pattern was in when eristalis arbustorum and other species with larvae that feed on decaying organic material became particularly abundant in august such that, by september, the relative frequency of aphid-feeders had fallen io . o. in , the relative frequency of aphid-feeders fell to r/o in june when merodvn equestris. syritta pipiens and eumerus spp. were common, and rose to only , % in july with the appearance of a great variety of other species, especially eristalis and helophilus. in june , abundance of the same species accounted for the low relative frequency of aphid-feeders, . %, but this rose dramatically to . % in july when eristalis and helophilus became relatively scarce. was the drought year when seasonality differed from the usual pattern. in the other years, the number of individuals and of species in the hovertly sample reached a peak in august, but in both peaked in july and immigration of a/, corollae and e. balteaiu.s to the garden took place earlier than in other years when this occurred; few hoverflies were caught in september and october, and the usual seven-month season was compressed in to the period april-august. hoverflies with aphid-feeding larvae were relatively scarce in july and , . and . %, respectively; in july the total catch was small and aphid-feeders unusually scarce, but in , e. arbustorum and s. pipiens were relatively common. the usual situation in any month was for hoverflies with aphid-feeding larvae to outnumber those with other larval habits in the malaise trap sample, although in the late summer those whose larvae feed on decaying organic material became relatively more common. aphid-feeders were not only more abundant than the other but also showed a more pronounced seasonal peak in numbers (fig. ) . the size and diversity of the hoverfly sample from the malaise trap leads to speculation as to what so many individuals and speeies were doing in the garden. adults eat not only nectar but also pollen and. in at least some species, a pollen meal is necessary for the maturation of eggs and therefore precedes egg-laying (pollard while adult hoverflies exploit flowers, honeydew and fallen fruit in a garden, their larvae eat a variety of different foods. gardens can obviously accommodate species with aphid-feeding larvae, and to a lesser extent those whose larvae inhabit decaying organic material, bulbs and hymenoptera nests; but those whose larvae feed on tree sap or rotting wood are less likely to find egg-laying sites. the spatial complexity of gardens is important, for adult females show habitat preferences when egg-laying; for instance, syrphus ribesii females are more often seen lapping honeydew and laying eggs on aphid-infested plants oi epilobium angustijolium in the sun than in the shade. some garden hovertlies are resident; the rest are visitors, sometimes casual, but more often seeking food or egg-laying sites. of the speeies, (such as baccha, ferdinandea, criorhina and xylola) are usually associated with woodland or woodland edge, eight (such as rhingia and merodon) with more open places, and are found in both types of habitat (compiled from pollard , steele and welch , and stubbs and chandler . since gardens include both open and shaded places, they might be expected to be more hospitable to species with no marked habitat preferences. species characteristic of open ground may be attracted to lawns and paths; furthermore much of the land surrounding suburban gardens is open and sunny. it is less easy to explain the presence of so many woodland species. there are many sheltered and shady corners in the garden and nearby, but it is unlikely that woodland species breed or spend any length of time in the garden. twenty-six of the woodland speeies are rare visitors, presumably attracted by flowers as a source of food; the other four have aphid-feeding larvae and might therefore be inspecting potential breeding sites. on the basis of captures and breeding records together with the statements about habitat given above, the hoverflies ean be divided into four categories: species are regular, common and are either known to breed or assumed to do so; a further species are regularly present and are assumed to breed within the surrounding gardens and suburban areas; species have been recorded in more than one year, but only infrequently, and are probably casual visitors, not resident in the area adjacent to the garden; species have been recorded in one year only, of them represented by a single individual in the malaise trap sample, and rank as chance visitors (tab. ). species in the last two categories may visit gardens for food when dispersing or migrating, but the most obvious migrants. m. coroltae and e. halteatus, are also regarded as resident. to summarize, of the species caught arc regarded as part of the resident fauna of the garden and surrounding areas, while species, including most of the woodland speeies, are casual or chance visitors. the special significance of gardens for hoverflies is as sources of food in the form of abundant flowers which, in woodland or marshy sites, may be seasonally scarce. gardens provide abundant food not only for residents but also for dispersing or migrating hoverflies and for those seeking breeding sites. they also provide refuge for a diversity of species including rarities whose habitats arc threatened by encroaching urbanization. several rare species were caught in , when the proportion of species taken once only was unusually high; adverse, dry weather conditions evidently caused many species to range widely seeking food which was available in the garden. there is no reason to suppose that the fauna of the study garden differs greatly from that of other gardens. the implication is that the hoverfly fauna of suburbia is abundant and diverse, and this can be attributed to the way in which gardens are maintained. man-made they may be, and in that sense arc unnatural, but they are far from impoverished. indeed, the contrived plant and habitat diversity of gardens probably makes them attractive to a greater variety of species than are found in most "natural' areas. the pattern of animal communities abundance and diversity of bumblebees and cuckoo hees in a surburban garden hoverflies (diptera: syrphidae) of leicestershire: an annotated checklist hedges vl habitat diversity and crop pests: a study cii brevicorxne brassicae and its syrphid predators the lar\'a and puparium of cheilosia bergen.stanimi becker (diptera: syrphidae) with a summary of the known biology of the genus in europe monks wood: a nature reserve record, -the nature conservancy a dipterisfs handbook trophic structure, available resources and population density in terrestrial vs. aquatic ecosystems acknowledgements -the leicestershire museums. biology section, provided me with working facilities and access to their collections, and k, g. v, smith of the british museum (natural history) helped with problems of identification of species, h, townes supplied the original malaise trap, and d. whiteley, oxford polytechnic, drew the figures, a travel grant from the royal society (london) enabled me to attend the second european ecological symposium on urban ecology held in berlin in . am grateful to d. owen for helpful suggestions at)out the work and valuable discussion of the results. tab. . status of hoverflies recorded in a suburban garden, - , and number of individuals caught in malaise trap, (nomenclature after kloet and hincks ,) known or assumed to breed in garden key: cord- - twm hmc authors: vourc’h, gwenaël; plantard, olivier; morand, serge title: how does biodiversity influence the ecology of infectious disease? date: - - journal: new frontiers of molecular epidemiology of infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: twm hmc over the past years, biodiversity has been reduced on an unprecedented scale, while new infectious diseases are emerging at an increasing rate. greater overall biodiversity could lead to a greater diversity of hosts and thus of pathogens. yet disease regulation – due to the buffering role of host diversity – is considered to be one of the services provided by biodiversity. in this chapter, we ask how biodiversity is linked to infectious disease. first, we investigate the influence of the biodiversity of pathogens. we highlight that the number of pathogen species is not well known but that new findings are facilitated by the rapid expansion of molecular techniques. we show that, although there is a trend to find higher pathogen richness toward the equator, identifying a global pattern between the richness of all pathogen species and their latitudinal distribution is challenging. we emphasize that pathogen intraspecific diversity is a crucial factor in disease emergence and allows pathogens to adapt to the selective pressures they face. in addition, the selective pressure acting on hosts due to parasite, and reinforced by parasite diversity within hosts seems to be a major evolutionary and ecological force shaping hosts biodiversity. second, we investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing (“dilution effect”) or increasing (“amplification effect”) the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing ("dilution effect") or increasing ("amplification effect") the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. over the past years, human activity has altered habitats and reduced biodiversity on an unprecedented scale that comes close to mass extinction (mea ) . at the same time, new infectious diseases seem to be emerging at an increasing rate (wilcox and gubler ) . during this period, there has been a dramatic spread of highly pathogenic diseases such as aids and multi-drug resistant bacterial infections, and in more recent years sars, west nile in north america, and highly pathogenic influenza viruses (jones et al. ) . habitat loss, largely a result of the conversion of forests and savannas into agricultural land, cities, and industrial sites, is the major cause of change in biodiversity. biodiversity represents the diversity of life at all levels of biological organization, from the genes within populations, the species that compose a community, to the communities that compose ecosystems. intuitively, one might assume that greater overall biodiversity would lead to a greater diversity of pathogens and hosts, and thereby increase the incidence of infectious diseases (dunn et al. ). yet disease regulation is said to be one of the services provided by biodiversity because a high level of species diversity creates a buffer that reduces the risk of transmission (mea ; walpole et al. ). scientific evidence supporting both of these views is beginning to emerge, but the core question remains: how is biodiversity linked to infectious disease? this is the question addressed in this chapter. pathogens are organisms that have a negative impact on the fitness of their host(s), often, if now always, by producing visible symptoms (e.g. a disease). such trophic interaction between two organisms, a host and a parasite, is just one of several interactions that take place within communities and ecosystems, others being those of prey-predator and plant-phytophagous for instance (begon et al. ) . to date, more attention has been paid to these other interactions, and their roles in ecosystem functioning (e.g. steffan and snyder ) , than to pathogen-host interactions, and food web studies only recently have begun to take parasites into consideration (arias-gonzález and morand ; lafferty et al. ) . studies incorporating pathogens are scarce (hudson et al. ) , probably due to the difficulties of surveying pathogens (using intrusive or even destructive sampling methods…). moreover, the systematics and even basic aspects of parasite biology often are unknown. however, although numerous species of pathogens still need to be described (dobson et al. ) , there is no doubt that pathogens represent a large part of biodiversity on earth. given that each free living species is host to numerous pathogens, and that pathogens of pathogens also exist (consider, for example, phages that are virus affecting bacteria), several authors believe that pathogens may be the most diverse living group on earth (windsor ) . the link between biodiversity and the ecology of infectious diseases is not simple. in this chapter, we investigate how biodiversity influences the ecology of infectious diseases at the intraspecific level (genetic variability of pathogens and hosts) and at the level of communities (species composition). although we mainly provide examples from human and animal diseases, we also use some illustrations from plants. we describe patterns of biodiversity and the consequences of changes in biodiversity on the ecology of infectious diseases. lastly, we rapidly examine the role of biodiversity in the treatment of infectious diseases. infectious disease ecology? we shall consider infectious diseases caused by bacteria, virus, fungi, protozoa and endo-parasites, and exclude from our analysis ecto-parasites that are considered here as disease vectors. in the light of the discussion above, the pathogen status of a given living organism clearly is not a straightforward question (consider, for example, the case of some rickettsia species that are considered to be not only blood vertebrate pathogens, but also tick symbionts, perlman et al. ). this status is related to the host, and varies with individual hosts and species as well as in space and in time (different hosts, for example, can have different resistance or susceptibility). when pathogens have complex life-cycles, some stages may have a different biology (such as biotrophic or necrotrophic plant pathogens, morris et al. ). furthermore, horizontal gene transfer is so extensive in bacteria that many microbio logists question the existence of species in bacteria, preferring to consider bacteria as populations that exchange genes. however, the existence of core genes responsible for the maintenance of species-specific phenotypic clusters is an argument supporting the identification of bacterial species (riley and lizotte-waniewski ) . for these reasons, combined with the limited knowledge available of the systematics of many pathogens (brooks and hoberg ) , it is difficult to accurately estimate the number of pathogen species. estimations of pathogen species richness vary from % to % of living beings (de meeûs et al. ; poulin and morand ) . in estuaries, the biomass of macro and micro-parasites has been estimated as exceeding that of top predators . although the existence of pathogens has been known for a long time, lists of species only were compiled recently for human and animals (ashford and crewe ; cleaveland et al. ; taylor et al. ) , with an update on human pathogens completed in (woolhouse and gaunt ) . approximately , human pathogens were reported, livestock pathogens (cattle, sheep, goats, pigs and horses), and domestic carnivore pathogens (dogs and cats). no clear figure was given for wildlife (but see the global mammal parasite database at http://www.mammalparasites.org/). on average, over two new species of human viruses also are discovered each year (woolhouse et al. ) . pathogens affecting humans have received more attention than those affecting other species. if one assumes that other animal species are affected in a proportional manner, huge numbers of pathogens remain to be discovered. altogether, , , , and , of fungi, viruses and bacteria respectively have been described, which represent only %, %, and - % of the total estimated number of species of fungal, viral and bacterial species. it is difficult to know the number of plant pathogens, but a significantly proportion of the fungal, viral and bacterial species are likely to be plant pathogens (ingram ) . until recently, many new pathogen discoveries relied on the investigation of atypical symptoms. today, new findings are facilitated by molecular techniques that render it possible to detect and characterise unculturable pathogens and to investigate the presence of genes and genomes independently of individuals (metagenomics). although multi-host pathogens are more numerous than single hosts, interactions between pathogens and hosts can evolve towards the specialisation of pathogens on a given host species (cleaveland et al. ; huyse et al. ; pedersen et al. ) . such a specialisation can lead to speciation, id est the birth of a new pathogen species. co-cladogenesis, a process of parallel diversification in hosts and pathogens, also can give birth to numerous pathogen species (page ) . until the development of molecular tools, these species were very difficult to distinguish (cryptic species). systematic investigations using molecular tools have made it possible, however, to reveal a high diversity of pathogens. for instance, in a systematic inventory of viruses in various vertebrate hosts conducted over a year period in the central african republic, different viruses were isolated, including new ones (saluzzo et al. ) . two species of plasmodium, p. falciparum, infecting humans, and p. reichenowi, infecting chimpanzees, were long considered to be within the clade that includes humans and the great apes. however, recent studies of apes in their natural habitat have revealed a much higher diversity of species infecting great apes; in addition, it has been found that p. falciparum also infect gorillas (liu et al. ; prugnolle et al. ) and are at the origin of human malaria. metagenomic studies in ecosystems such as human faeces (zhang et al. ) and marine sediments (breitbart et al. ) also have revealed that the majority of viral sequences found did not match in the databanks. finally, new investigations have been launched to monitor people, animals and animal die-offs in areas where people have a high exposure to wildlife. generic, broad screening tools will be used to detect pathogen species (wolfe et al. ). to our knowledge, a similar approach has not yet been implemented for pathogens of animals or plants. in addition to the inventories of pathogen biodiversity, scientists have investigated which part of the world holds the highest diversity of pathogen species. many studies on plants and animals have shown that species richness decreases the further one moves away from the equator. the reasons most likely are linked to the area, energy, time and habitat heterogeneity, and geometric constraints (gaston and blackburn ) . comparative studies exploring pathogen species richness in the tropics compared to temperate zones are scarce and have produced discrepant results. guernier et al ( ) studied the worldwide distribution of human pathogens (bacteria, virus, fungi, protozoa, and helminths) according to environmental, demographic and economical factors. they found that parasite species richness decreased with latitude and had a spatially nested organization; i.e. some species are widely distributed and occur in many communities while others have more restricted distributions and occur only in a subset of locations. such findings were confirmed by the analysis of dunn et al. ( ) , who showed that human pathogen diversity was strongly related to both mammal and bird species richness. diseases that occur in temperate zones also tend to occur in the tropics, while some tropical diseases are restricted only to the tropics. in primates, the number of protozoa species, which primarily are vector-borne transmitted, increase as one approaches the equator, however, the same trend was not found for viruses and helminths (nunn et al. ). lindenfors et al ( ), who examined the parasite richness of parasite species and terrestrial carnivore species, found that helminth parasite species richness increased the further away one moved from the equator. the reason for this finding is unknown and may be related to a bias in sampling because carnivores inhabiting areas of industrialized countries in the northern hemisphere may have been sampled more intensely. poulin ( ) and bordes et al ( ) did not find any correlation between helminth species richness at intra or interspecific levels and latitude. some studies have shown higher tick species richness at lower latitudes (cumming ) . however, this is not the case for flea species, which have been found to have low richness at lower latitudes (krasnov et al. ) . a final example is ichneumonid parasitoid hymenoptera. although a higher specific host diversity is found in the tropics, the number of species of this parasitoid group is similar in both tropical and temperate regions. it has been hypothesised that this is due to habitat fragmentation (leading to a lower density of hosts); lack of seasonality (and thus of a host population dynamics with peaks and high density of hosts), or the higher content in toxic compounds of tropical plants and thus in phytophagous insects (the "nasty hypothesis") (gauld et al. ) . a meta-analysis of parasite-associated host mortality (robar et al. ) revealed that host mortality risk declined as one moves away from the equator, indicating that, in addition to parasitic load, the effect of parasites on host mortality might be determined by some abiotic factors. thus, although there is a trend to find higher pathogen richness as we move toward the equator, it is thus challenging to identify a global pattern between the richness of all pathogen species and their latitudinal distribution. however, it should be noted that of the pathogens that have been discovered since , most have a global distribution (woolhouse and gaunt ) . pathogens generally are characterised as having higher mutation rates and generation times than those of their hosts (hamilton et al. ). genetic variability also results from recombination during sexual reproduction of eukaryotic pathogens, and any other genetic exchange mechanisms such as bacterial conjugation or viral recombination. in addition, many animal and plant pathogens use a vector to increase gene flow among populations and to reach a new individual host. this genetic diversity is a crucial factor in disease emergence (cleaveland et al. ) and allows pathogens to adapt to the main selective pressures they face: hosts' immune systems, the need to be transmitted, and treatments or vaccines used to counter infections. the capacity of some pathogens to genetically diversify facilitates their ability to evade host immune systems. one of the best examples is the human immunodeficiency virus (hiv), which is able to change its appearance faster than the time its takes for the immune system to reply (drosopoulos et al. ). another example is p. falciparum, which generates high levels of variability in genes involved in antigenic variability and virulence (var genes) by producing frequent recombination events between heterologous chromosomes (freitas-junior et al. ) . high genetic variation of pathogens also is involved in the interspecies infection process as it facilitates the infection of a broader range of host species, which is another characteristic of emerging pathogens (cleaveland et al. ; woolhouse and gowtage-sequeria ) . the evolutionary potential of pathogens allows them to respond quickly to the directional selective pressure provided by the massive use of drugs (palumbi ) . in areas where selective pressure is important, such as in hospitals, multiresistant bacteria are very frequent (levy and marshall ) . for bacteria, resistant genes probably originated from environmental organisms with which they shared their ecological niche (aminov and mackie ) . these genes can be transferred between different species of bacteria and even between species that colonize different hosts (nikolich et al. ) . although vaccination is a major advance of modern medicine, it thus far has contributed to the eradication of only one infectious disease in humans (small pox, www.who.int/mediacentre/factsheets/smallpox/en) and one in cattle, buffalo and wildebeest (rinderpest, normile ) . as many vaccines do not totally block transmission, vaccination modifies the selective pressure on pathogens. depending on how vaccines act on the pathogen, the epidemiology consequences can differ (gandon and day ) . for instance, theoretical work has shown that vaccines that reduce the growth rate or toxicity of pathogens also reduce selection pressure against virulent pathogens, leading to higher intrinsic virulence (gandon et al. ). in the poultry industry, an increase in virulence of avian tumour viruses has followed the use of vaccines that reduce virus growth rates (witter ) . although plants lack an adaptive immune system, through evolution they have developed various strategies to stop plant pathogen infections. an induced or acquired systemic resistance occurs following host recognition of a pathogen, which triggers the production of a hypersensitive reaction (jones and dangl ) . through this mechanism, the plant provides itself protection from secondary infection in distal tissues, even if the plant faces a pathogen for which it does not have the resistance gene (durrant and dong ). the immune system of vertebrates acquires its efficiency by being exposed to a diverse array of pathogens. the striking increase in hygiene standards that began in the early twentieth century has considerably lowered humans' exposure to pathogens, at least in developed countries. the immune response triggered by a pathogen can provide some cross protection against other pathogens (e.g. huang et al. ) . a low exposure to a diversity of pathogens has had immediate consequences in decreasing the risk of disease. but could this low exposure also induce evolutionary change in the ability of a host to respond to infection? due to a trade off between investment in disease resistance and other traits linked to fitness, low exposure could decrease the frequency of resistance down through the generations, setting the stage for a potentially devastating outbreak (altizer et al. ; graham et al. ) . domestic species that are bred to be protected from pathogens might be more susceptible to infectious diseases (lyles and dobson ) . furthermore, it has been suggested that on islands, where some pathogens may be absent, hosts may have lower immune response abilities (island syndrome) (lee and klasing ) . however, studies that have tested this hypothesis, both using experimental and theoretical approaches, have had contrasting results (beadell et al. ; hochberg and møller ; matson ) . infections by different species of pathogens or by different strains of the same species within the same individual host or vector are common (abbot et al. ; cox ) . in fact, parasite diversity in hosts seems to be a major evolutionary and ecological force for hosts (bordes and morand ). these concomitant infections can trigger cross-effective immune responses between pathogens that are antigenically similar, having thus an impact on the issue of the infection (lee et al. ). an infection also can enhance susceptibility to subsequent infection (cattadori et al. ). in particular, individuals with already are in poor physical condition may be more susceptible to multiple infections (beldomenico and begon ; telfer et al. ) . furthermore, concomitant infection may allow the exchange of genetic material between strains of a given pathogen species or even between species through horizontal gene transfer (see sect. . . above), allowing the emergence of new virulent strains. an extreme case is one in which a pathogen drives the extinction of a population or species. such scenarios are rare but do occur, generally due to a conjunction of pathogens and other causes. for instance, the decline of amphibian populations around the world is thought to be linked to a fungal pathogen batrachochytrium dendrobatidis causing chytridiomycosis (crawford et al. ). amphibians could have an increased susceptibility to the fungus due to changes in temperature variability (rohr and raffel ) . another example is the dramatic decline of the native red squirrel in the uk that has been attributed to a combination of direct competition with the grey squirrel and disease-mediated competition because the grey squirrel is a reservoir host of the squirrelpox virus that causes disease in the red squirrel (tompkins et al. ) . the local extinction of a host also may have tremendous consequences on an entire ecosystem (see for example the case of the wildebeest /rinderpest interactions in the serengeti, holdo et al. ). disease ecology? a change in species richness or abundance can modify the dynamics of local infectious diseases by either reducing or increasing the risk of transmission to the targeted species. the first outcome has been named, the "dilution effect", the second, the "amplification effect". the term "dilution effect" has conveyed different meanings since its first use in disease ecology literature (see box in the paper keesing et al. ) . the broad definition of the dilution effect refers to "the phenomenon -the net effect -when increased species diversity reduces disease risk" that is produced by a variety of mechanisms ("amplification effect" refers to the opposite phenomenon) (keesing et al. ) . this applies to vector-borne and directly transmitted diseases, although the concept of dilution has been developed most with regards to the tick-borne lyme disease (allan et al. ; logiudice et al. logiudice et al. , . the hypothesis underlying the amplification and dilution effect is that for many diseases, the competence of reservoirs, i.e. the ability to become infected and retransmit the pathogen, varies according to the host species (haydon et al. ) . the composition of the host community thus can influence the transmission dynamic of the disease. similarly, since vectors have different competence to transmit pathogens, the composition of the vector community likely influences transmission dynamics. different mechanisms are thought to be involved, but they are difficult to differentiate (begon ; keesing et al. ) . one is the modification of the encounter rate (when reduced, this corresponds to the "dilution effect" sensu stricto). in the presence of species that are poorly competent, the transmission event that should link an infectious individual to a susceptible individual instead links infectious individuals to non-competent individuals. for vector-borne diseases, the increased diversity of a poorly competent host species on which the vector feeds increases the proportion of vector bites that are wasted. for direct transmission, the addition of non competent hosts can decrease transmission if these hosts remove infectious particles (begon ) . a second mechanism at work is that a high diversity of host species regulates the abundance of the competent host population. this regulation can be mediated by interspecific competition for limiting resources or by predation upon competent hosts. this typically is the idea that underlies biological controls, where organisms prey upon reservoir hosts, vectors or intermediate hosts (straub and snyder ) . a third mechanism is based on the link between species richness and host mortality. this is the case when predators modify the mortality rate of a host and lower pathogen transmission by feeding on a heavily diseased individual (packer et al. ) . two other mechanisms are cited by keesing et al ( ) , but they are difficult to demonstrate: (i) the modification of recovery when species added to a community facilitate the recovery of infected individuals by, for instance, providing resources, and (ii) the modification of transmission once the contact has occurred, for instance, when adding a species modifies the pathogen load within the host. the dilution effect has been demonstrated mainly through theoretical work; there have been few case studies. one of the main examples is lyme disease in the usa that is caused by pathogenic bacteria transmitted by ticks. these ticks feed readily on many species of vertebrates and these species vary in their degrees of reservoir competence. the white-footed mouse (peromyscus leucopus) is thought to be the most competent host and dominates in fragmented forests. in native forests, which harbour a higher diversity of species than fragmented forests, ticks have a higher probability to dilute their bite by feeding on a less competent host (allan et al. ; logiudice et al. logiudice et al. , . however, such a dilution effect has not been demonstrated in europe, probably because of the complexity of the disease ecology which involves numerous reservoir host and bacteria species (halos et al. ) . another example is the west nile virus, where an increased diversity of non passerine birds, which are less competent reservoir hosts compared to passerines, was associated with decreased west nile virus infection in mosquitoes and humans (ezenwa et al. ; swaddle and calos ) . to date, there have been few examples of directly transmitted diseases, but studies on hantaviruses have shown that higher diversity of small mammals appears to regulate reservoir host populations through competition or predation. high small-mammal diversity also might inhibit intraspecific aggressive encounters between reservoir hosts that result in hantavirus transmission (suzán et al. ). in plants, crop diversity can reduce the total burden of disease in agricultural systems. this results from the combined effects of (i) the limitation of pathogen dispersal thanks to the physical barriers provided by the presence of non-host plants (burdon and chilvers ) , (ii) induced systemic resistance, and (iii) competition among pathogens. the efficiency of crop mixtures is linked to the size of the area on which this method is used: a high level of success has been observed in a field trial with susceptible and resistant varieties of rice conducted on a large scale ( , ha) in china (zhu et al. ) . illustrations of amplification effects are typically the consequences of species introduction that radically modifies encounter rates. the added species can introduce new pathogens that infect native hosts (spillover) (bruemmer et al. ) or amplify the circulation of local pathogens (spillback) (kelly et al. ). the introduction of additional species also can provide sources of vector meals and increase vector numbers or activity (saul ) . for instance, the introduction of siberian chipmunks (tamias sibiricus) in suburban forests could increase the risk of lyme disease because this host seems to be more competent than native hosts (vourc'h et al. ). the introduction of the mosquito aedes albopictus in many parts of the world has facilitated the transmission of the chikungunya virus (benedict et al. ; charrel et al. ) . theoretical works based on deterministic modelling have looked at the conditions in disease transmission dynamics that are needed for the amplification or the dilution effect to occur (begon ; dobson ) . when there is a relationship between the risk of a disease, the abundance of the reservoir host, and the abundance of an additional host, the addition of a species does not necessarily decrease the risk. in the case of lyme disease, for example, tick abundance mainly is determined by the abundance of deer, which are in fact a non competent reservoir. an increased abundance of deer may reduce infection prevalence when immature ticks are feeding on the deer. at the same time, however, the overall number of adult ticks increase proportionally with the number of deer (begon ) . further research in this field are relying on the modelling of the global community competence of hosts and vectors (roche ) . scientists and societies are increasingly interested in the dilution effect (mea ) due to the link between habitat disturbance, generalist host characteristics, and their competence in disease transmission. disturbance seems to favour generalist hosts (hosts that use different types of habitats) (devictor et al. ; marvier et al. ) , and these hosts often have a broad geographical distribution (mckinney and lockwood ; smart et al. ). crucially, these species also seem to have a higher competence reservoir or vector reservoir than species that are not favoured by disturbance (mills ; molyneux et al. ; vittor et al. ) . for example, many murid rodents that are recognized hosts of hemorrhagic fever viruses are opportunistic species that seem to be favoured in disturbed environments. the question is whether there is a causal link between a species' generalist and opportunist character and its disease competence. why are murid species associated with hemorrhagic fever more generalist than those which are not? could it be possible that specialist species also carry hemorrhagic fever viruses, only these viruses have not yet been identified? or is there something intrinsic in opportunistic species that makes them more likely to evolve and maintain hemorrhagic fever viruses (mills )? only a very small subset of plant and animal species have been domesticated (diamond ) . many species of that small subset, for example, cattle (in animals) and maize (in plants), have seen their genetic diversity considerably reduced for the purpose of intensive production (the bovine hapmap consortium, matsuoka et al. ) . in the wild, small populations of endangered species often have a very reduced genetic diversity (keller and waller ) . this then raises the following question: to what extent does a reduction of the genetic diversity in a host species impact the ability of the host population to response to infectious diseases (may ) ? genetic loci associated with the major histocompatibility complex (mhc) plays a key role in the acquired immune response of vertebrates (altizer et al. ) . mhc genes code for molecules that recognize foreign peptides (antigens) and display them on the cell surface. when the mhc-protein is displayed, it can be presented to immune cells, such as t lymphocytes or natural killer cells, which subsequently can trigger an adaptive immune response. because mhc genes are faced with an important diversity of antigens, they must themselves be diverse. the measure of the genetic diversity of mhc based on an analysis of polymorphism sequences of mhc among individuals in populations has been widely used in conservation biology as a proxy to estimate the fitness of populations confronted by pathogens (alcaide et al. ; bernatchez and landry ; sommer ) . however, the level of genetic variation at mhc loci results from different evolutionary forces (selection, gene flow, mutation) varying both in space and time in co-evolutionary systems involving both hosts and pathogens, making conservation genetics of non-model organism a challenging task (stockwell et al. ) . we already have many examples where low genetic diversity of species has favoured the diffusion of, and/or susceptibility to, pathogens. for example, the low genetic diversity of the tasmanian devil could be involved in its susceptibility to facial tumor disease (mccallum ) . the low genetic diversity found in commercially traded bee queens has been hypothesised as being one of the factors explaining colony collapse disorder (le conte and navajas ). the problem is even more critical in intensive crops in which disease resistance has relied on the use of a very small number of genes. this selection strategy has proven to be ineffective as pathogens manage to overcome the resistance. for example, the resistance of brassica napus (canola, oilseed rape and colza) to leptosphaeria maculans (causing the blackleg disease) due to a major resistance gene was overcome in an area covering approximately , ha in south australia in a period of years (sprague et al. ). even with advances in synthetic chemistry, which provides many biologically active molecules, pharmaceuticals derived from nature remain an important part of pharmaceutical practice today (newman et al. ). all organisms have developed compounds to protect themselves against infectious diseases and to interact with individuals of their own species or other species (e.g. rogerio et al. ). these molecules, coming from all organisms (bacteria, fungus, animals, plants) in terrestrial, marine and extreme ecosystems, represent an amazing diversity that has been tested in the field for millions of years by involving millions of individuals. however, only a very small subset of plants and marine organisms has been investigated for novel bioactive compounds. furthermore, it is estimated that less that % of bacterial species and only % of fungal species are known. those which have not yet been identified could be sources of novel molecules (cragg and newman ) . observations of natural medicine practices used by indigenous people have led to the discovery of many drugs. the most well known and widely used pharmaceuticals are quinine, used as a model to synthesize anti-malarial drugs (chloroquine and mefloquine), and artemisinin, indentified as a potent anti-malarial drug by chinese scientists (newman et al. ) . animals also are a source of inspiration for drugs against infectious diseases. for instance, compounds of the sponge cryptotethya crypta inspired the elaboration of antiviral medication such as azt used in the treatment of hiv/aids (cragg and newman ) . observing great apes medicate themselves through the plants they eat also could help to reveal new active compounds (krief et al. ). pathogens constitute a large part of biodiversity on earth and are present in all ecosystems and at all trophic levels, where they have a large impact on ecosystem functioning and on the population dynamics and evolution of their hosts. the recent acceleration of biodiversity loss due to human activities deeply impacts host-pathogen dynamics. pathogens and hosts form co-evolving systems exercising major selective pressures on each other. furthermore, the virulence or pathogenicity of a given species depends on its environment -which includes the hosts -that is highly variable in space and time. in such a context, human beings will never be able to completely control or eradicate every pathogen species; rather, we should accept that we must coexist with pathogens. to better understand and predict the evolution of pathogens and the impact of human activities on them, more in-depth studies are needed on how pathogens interact with host communities within different ecosystems. to understand human, animal, and plant epidemics, two-species systems involving only a single host and a single pathogen species are no longer appropriate. the approach considering multi-host/multi-pathogen systems in their environment is the framework that now needs to be used to deepen our understanding of disease dynamics woolhouse et al. ) (fig. . ) . however, these dynamics are very complex and difficult to study because precise knowledge regarding the diversity of pathogens and of interactions taking place on several scales is lacking (lloyd-smith et al. ). in addition to intensive fieldwork to collect adequate data and modeling to understand the main processes, the use of molecular tools in a multi-host/multi-pathogen framework will facilitate investigations into pathogen-host community interactions. in particular, new generation sequencing techniques render it easier to characterize the genetic diversity of pathogens and hosts. for instance, metagenomic investigations allow an approach that does not require a priori assumptions that is useful to describe a community of pathogens and their interactions. molecular techniques also may be used to clarify the taxonomic status of pathogens, revealing cryptic species or host races. with suitable molecular fig. . schematic representation of the link between biodiversity and the ecology of infectious diseases. diseases results from the complex interactions between the three compartments corresponding to pathogens, hosts and vectors (in the case of vector-borne diseases). the biodiversity of these three compartments can be considered at the community level (each circle corresponding to a species) or at the intraspecific level (each circle corresponding to a population or an individual within a population). the gray shading off of each unit considered (species, population or individual) illustrates its genetic or phenotypic variability in space and time, while variations in size illustrate frequency or density differences within the ecosystem. interactions within each compartment can be direct (competition) or indirect (apparent competition…), synergic or antagonistic as illustrated by the different arrows markers (producing a high level of polymorphism), the analysis of genetic variability within a spatially explicit framework renders it possible to identify the routes followed by a given pathogen. moreover, molecular techniques can be used to identify genes involved in important life history traits of a pathogen such as virulence and transmission. better knowledge of the mechanisms involved in host-pathogen interactions, and the extent of their variability, will significantly advance our understanding of outbreaks. although our knowledge of the number and variety of pathogens is not complete, it appears that their diversity, like that of their hosts and vectors, is greater in tropical areas than in temperate ones, and in undisturbed habitats than disturbed ones (chaisiri et al. ; friggens and beier ) . the reason we are so concerned by the loss of species biodiversity is because a reduction of biodiversity seems to favour opportunistic species that are highly competent as pathogen reservoirs and vectors. however, this observation was derived from only a few studies and theoretical works, mainly undertaken in the temperate zones. further investigations should be launched to verify the link between, and understand the process involved in, biodiversity loss and disease dynamics. this especially should be done in the tropics to understand whether high levels of biodiversity create a buffer reducing the risk of disease transmission, and to understand the consequences of biodiversity loss in high biodiversity regions. with global changes, there is a high risk that diseases currently circulating in the tropics will reach temperate zones where species diversity is reduced and the availability of alternate hosts is limited. what could be the consequences of such a shift ? what may happen in a world where increased movements of hosts and pathogens, high population densities, and rapidly changing environments increase contact rates, spread, and selective pressures on pathogens and hosts while at the same time a combination of low exposure to pathogen biodiversity and decreased genetic variability in some animals increases susceptibility to new diseases? the investigation of such questions requires collaboration across disciplines and between countries. mixed infections, cryptic diversity, and vector-borne pathogens: evidence from polygenis fleas and bartonella species mhc diversity and differential 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biology of multihost pathogens temporal trends in the discovery of human viruses rna viral community in human feces: prevalence of plant pathogenic viruses genetic diversity and disease control in rice key: cord- - rit h authors: chinchio, eleonora; crotta, matteo; romeo, claudia; drewe, julian a.; guitian, javier; ferrari, nicola title: invasive alien species and disease risk: an open challenge in public and animal health date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: rit h nan protecting public and animal health. for example, empirical research on ias pathogens, which would be needed to assess the risk of infectious disease emergence, is skewed toward a few species (e.g., vector species like the tiger mosquito aedes albopictus) or toward selected pathogens known to harm biodiversity conservation, while a global vision of ias-associated health threats is still not available [ , [ ] [ ] [ ] . in this context, it is urgent to raise awareness in people working in the fields of animal and public health of the need to consider ias as a health threat. to this aim, we provide here an overview of how animal ias may affect local disease dynamics both directly and indirectly, i.e., acting as pathogen hosts or disrupting the recipient ecosystem structure, through real-case examples from the ecological literature, and, in the last paragraph, we propose future initiatives aimed at improving our capacity for targeted actions toward the ias most likely to threaten human and animal health, calling for an increased involvement of people working in the fields of animal and public health in a new invasion epidemiology field. ias may host pathogens that are absent in the area of release and cause their establishment and subsequent spillover to local species, possibly resulting in an increase of disease risk for humans, domestic animals, and native wildlife. the north-american raccoon procyon lotor, for example, introduced to central europe baylisascaris procyonis [ ] , a nematode causing larva migrans syndromes potentially inducing severe central nervous system disease in humans ( fig a) . introduction to europe of north-american crayfishes procambarus clarkii infected with the fungus aphanomyces astaci caused huge economic losses to fisheries, being the pathogen lethal to native crayfishes [ ] . similarly, squirrelpox virus, introduced to the united kingdom along with the american eastern gray squirrel sciurus carolinensis, is significantly contributing to the increased mortality of native red squirrels sciurus vulgaris [ ] . however, while pathogen co-introductions occur over a wide range of parasite and host taxa [ ] , some pathogens are lost during the invasion process [ ] : for example, there is no evidence for poxvirus in italian gray squirrel populations [ ] . pathogen loss may be due to the absence of the pathogen in the individuals of the founding populations or to its inability to survive to translocation or establish in the area of release. the outcome depends on several factors related to the ias (e.g., founding population origin), the pathogens (e.g., host specificity), and the area where the species is released (e.g., environmental conditions, presence, and density of local hosts) [ ] . as shown by a study on ectoparasites of introduced birds, factors related to transmission efficiency, such as the number of host introduced and host longevity, are likely to play a major role [ ] . an increase of local disease risk may also occur if the introduced ias is susceptible to, and able to transmit, local pathogens. pathogens acquired by ias may be amplified and possibly spill back to humans and local species [ ] . a case in point is the australian brushtail possum, trichosurus vulpecula, in new zealand ( fig b) . invasive possums probably became infected with mycobacterium bovis, the causal agent of tuberculosis in cattle, from wild deer, after the beginning of commercial deer hunting in . currently, they are the most important maintenance host for bovine tuberculosis, supporting higher transmission rates compared to local species and, being sympatric with cattle, providing interface for transmission between livestock and forest residents [ ] . another case is represented by invasive raccoon dogs nyctereutes procyonoides, which may amplify rabies circulation in eastern europe or cause its reemergence in currently rabies-free countries [ ] . ias competence for pathogen transmission plays a major role in defining the outcome of pathogen acquisition, and, as the possum-tuberculosis case exemplifies, it is the result of both ias-pathogen interaction (e.g., ias susceptibility, period of communicability, and pathogen excretion rate) and ias behavioral patterns (e.g., habitat, home range extension, and intraand interspecific contact rates). based on ias competence, the acquisition of a local pathogen may even lead to the reduction of disease risk (the so-called dilution effect [ ] ) or to no consequences at all. for example, in ireland, the invasive bank vole myodes glareolus has been found to divert fleas from the native wood mice apodemus sylvaticus, which is a more competent host for bartonella spp. [ ] . however, the identification of the contexts in which a dilution effect may occur is still highly debated in ecology, as it strongly depends on local host species diversity and on the interactions occurring between the species involved in the transmission cycle [ ] . introduced species may disrupt local infection dynamics also indirectly, i.e., nonacting as pathogen hosts but through competitive and trophic interactions with native species or modification of local habitats, thus altering the abundance and/or contact rates among local host species, parasite infective stages, or vectors. in southern florida, the invasive python python bivittatus caused the decrease of several mammal species, inducing the local mosquito vector of zoonotic everglades virus to feed almost exclusively on the virus' main reservoir host, the hispid cotton rat sigmodon hispidus, potentially leading to an increase in pathogen circulation (fig c) [ ] . an example of habitat alteration is given by the activity of invasive feral pigs sus scrofa on the island of hawaii: they create wallows and cavities in tree fern trunks improving habitat suitability for mosquito vectors for avian malaria plasmodium relictum [ ] , one of the main threats to native hawaiian forest birds' conservation. again, ias indirect effects on local infection dynamics are highly context dependent, and mechanisms presented so far may act in concert, producing unpredictable outcomes. in scotland and northern england, for example, the invasive gray squirrel has been found to harbor several local strains of borrelia burgdorferi [ ] . however, in those areas, gray squirrels are also causing the decline of another competent host for b. burgdorferi, the red squirrel, and the effect of these concurring mechanisms on human lyme disease risk remains unknown [ ] . during the last centuries, more than , ias introduction events have been recorded worldwide, and this number still presents an increasing trend [ ] . in such context, the identification of those species deserving priority attention, based on their actual and potential impacts, is essential to support decision-making [ ] . several tools to inform preventive and management actions on animal ias, including horizon scanning protocols, risk assessments, and impact assessments, have been developed in the last years (see [ ] for a recent review), but the majority of them focus on environmental impacts, not specifically considering disease emergence risks in humans and local animal populations [ , ] . some authors have called for a greater attention on the potential health risks posed by biological invasions [ ] [ ] [ ] ] , highlighting the need for a better integration between biological and health sciences, surveillance actions, and coordinated policies. we support their appeal, arguing that an increased awareness of people working in the fields of animal and public health on the risks concerning biological invasions and their consequent involvement in the invasion biology field is the first step toward a complementary invasion epidemiology field. such field would be integrated with invasion ecology, but more specifically aimed at the prevention of the emergence of diseases in human and animal populations consequent to ias introduction and establishment. to this aim, we propose some initiatives that should be addressed by future research work. a first major constraint in addressing the issue of disease emergence connected to ias is given by the lack of comprehensive data on pathogens affecting ias. in this sense, we recommend the gathering in ad hoc databases of all the available information on ias pathogens affecting human and animal health, including their geographical distribution and prevalence in ias populations, in both native and introduced ranges. it would also be advisable to improve our understanding of the key epidemiological events and factors driving the emergence of infectious diseases following ias establishment, for example, through ex-post analyses on the already established ias. in particular, as the emergence process of a disease is composed of several stages (introduction in a new area/host population, establishment, and spread) [ , , ] , the key factors involved in the process and related to ias biology, pathogenic features, and the biotic and abiotic components of the area of release should be identified for each of these stages. we also suggest urgently directing research efforts at developing transparent and flexible tools able to prioritize ias based on the risk of transmitting pathogens with the potential to impact the health of humans, production animals, and native wildlife. such tools could be based on the framework of the world organisation for animal health (oie)/international union for conservation of nature (iucn) for wildlife disease risk analysis and readapted to account for the main mechanisms through which alien species may affect local health, in particular the introduction of new pathogens and the acquisition and spread of local ones. the lack of data on ias pathogens is certainly an obstacle in underpinning in-depth risk assessments [ ] , in particular, quantitative ones. however, a simple and transparent qualitative disease risk assessment procedure would enable the prioritization of empirical research needed to cover these knowledge gaps, while at the same time guiding local health administrators in the allocation of resources for management and preventive actions toward ias. the issue related to irregular data availability could be partially overcome, as a first step, by eliciting opinions from experts. finally, awareness and action will be influenced by, and need to consider, the wider public perspective, not just researchers and institutions. initiatives aimed at sensitizing citizens about the health threats of ias will be needed to promote responsible behaviors when crossing borders and to improve the general public attitude toward ias control and eradication programs. all the suggested initiatives, to be successful, necessitate a stronger connection between ecologists, biologists, and other people working in the fields of animal and public health and beyond. only through wider collaboration and dialogue will the potential health impacts of biological invasions be fully appreciated and, perhaps, ameliorated. emerging infectious diseases of wildlife-threats to biodiversity and human health global trends in emerging infectious diseases alien species as a driver of recent extinctions invasive species database impact of the invasive brown marmorated stink bug in north america and europe: history, biology, ecology, and management aquatic invasive species and emerging infectious disease threats: a one health perspective the spread of invasive species and infectious disease as drivers of ecosystem change invasive species challenge the global response to emerging diseases invading with biological weapons: the importance of disease-mediated invasions is invasion success explained by the enemy release hypothesis? indirect effects of parasites in invasions parasites as drivers and passengers of human-mediated biological invasions co-invaders: the effects of alien parasites on native hosts parasites lost-do invaders miss the boat or drown on arrival? natural history of host-parasite interactions the effects of invasion on parasite dynamics and communities wildlife diseases: from individuals to ecosystems horizon scanning for invasive alien species with the potential to threaten biodiversity and human health on a mediterranean island alien species and public health impacts in europe: a literature review alien pathogens on the horizon: opportunities for predicting their threat to wildlife first detection of baylisascaris procyonis in wild raccoons (procyon lotor) from leipzig biological invaders in inland waters: profiles, distribution, and threats ecological replacement of native red squirrels by invasive greys driven by disease introduced species and their missing parasites invasions and conservation: no evidence of squirrelpox virus in grey squirrels introduced to italy parasite spillback: a neglected concept in invasion ecology? epidemiology and control of mycobacterium bovis infection in brushtail possums (trichosurus vulpecula), the primary wildlife host of bovine tuberculosis in new zealand rabies in northeastern europe-the threat from invasive raccoon dogs effects of species diversity on disease risk disruption of a host-parasite system following the introduction of an exotic host species mammal decline, linked to invasive burmese python, shifts host use of vector mosquito towards reservoir hosts of a zoonotic disease managing disease an invasive mammal (the gray squirrel, sciurus carolinensis) commonly hosts diverse and atypical genotypes of the zoonotic pathogen borrelia burgdorferi sensu lato no saturation in the accumulation of alien species worldwide prioritizing species, pathways, and sites to achieve conservation targets for biological invasion developing a framework of minimum standards for the risk assessment of alien species a comparison of impact and risk assessment methods based on the imo guidelines and eu invasive alien species risk assessment frameworks review of risk assessment systems of ias in europe and introducing the german-austrian black list information system (gablis) novel organisms: comparing invasive species, gmos, and emerging pathogens parasites and biological invasions: parallels, interactions, and control key: cord- -v spbo u authors: peterson, a. townsend title: biogeography of diseases: a framework for analysis date: - - journal: naturwissenschaften doi: . /s - - - sha: doc_id: cord_uid: v spbo u a growing body of literature offers a framework for understanding geographic and ecological distributions of species; a few applications of this framework have treated disease transmission systems and their geography. the general framework focuses on interactions among abiotic requirements, biotic constraints, and dispersal abilities of species as determinants of distributional areas. disease transmission systems have key differences from other sorts of biological phenomena: interactions among species are particularly important, interactions may be stable or unstable, abiotic conditions may be relatively less important in shaping disease distributions, and dispersal abilities may be quite variable. the ways in which these differences may influence disease transmission geography are complex; i illustrate their effects by means of worked examples regarding west nile virus, plague, filoviruses, and yellow fever. the past decade or so has seen considerable progress toward a general framework for understanding species' geographic distributions, essentially addressing the question, why is a species where it is and why is it not where it is not? key steps toward this understanding have included ( ) mathematical formulation and clarification of ecological niches of species and how they affect species' geographic distributions (pulliam ; hirzel et al. ; soberón ) , ( ) exploration of the role of historical events in shaping species' geographic distributions (wiens and graham ; martínez-meyer and peterson ; peterson and nyári ; waltari et al. ), and ( ) analysis of the time scale on which change in ecological niche characteristics is likely to occur (holt ; holt and gomulkiewicz ; peterson et al. ) . altogether, this body of work provides a basis for understanding the complexities of distributions of species in both geographic space and ecological dimensions. diseases are caused by pathogens-generally viruses, bacteria, fungi, and protozoa-that invade an individual's body and cause ill effects (for the purposes of this review, i distinguish between pathogens and parasites simply on the basis of size-pathogens being microorganisms and parasites being of larger size-and focus on pathogens). because disease processes are dynamic, taking place on extremely diverse scales of space (microscopic to continental) and time (minutes to centuries), and are the products of interactions among species (pathogens, reservoirs, vectors, etc.) , their ecological and distributional dynamics may differ from those of more "normal" species. these differences are the focus of this review, which i will illustrate via a series of examples. a recent concept paper presented a simple heuristic for understanding questions regarding species' distributions (soberón and peterson ) , which aims to provide a framework for thinking about species' distributional patterns, even if not a comprehensive summary of the phenomenon. this framework (fig. ) centered on a venn diagram showing interactions among three factors: abiotic conditions, biotic conditions, and accessibility considerations. the former two factors correspond roughly to the fundamental and realized niches as outlined by hutchinson ( ) ; although hutchinson focused more specifically on the role of competition among abiotic conditions, a more modern view would include other biotic interactions, such as predation, parasitism, and mutualism. accessibility considerations, including both current dispersal ability and opportunity for dispersal and colonization in the past (waltari et al. ) , were not integrated into hutchinson's ( ) framework. the heuristic diagram centers on requirements of particular abiotic conditions (i refer to this circle as "a" for abiotic conditions) and how they relate to biotic interactions (referred to as "b" for biotic conditions) and how they modify the abiotic requirements. for example, a species may have fairly broad tolerances of abiotic conditions such as temperature, solar radiation, rainfall, and soil chemistry. the interactions of this species with other species, however, may restrict it to only a subsector of the abiotically suitable areas-that is, some areas may not be suitable owing to the presence of a particular predator or pathogen or owing to the absence of a key mutualist or symbiont. as such, the potential distribution of the species can be seen as a∩b-or to put it into words, in these areas, both abiotic and biotic conditions are appropriate for the species to maintain populations. finally, the species may be limited from occupying the entirety of its distributional potential by accessibility considerations (referred to as "m" for mobility). that is, a hypothesis of the actual distribution of the species would be a∩b∩m, which are those areas that are simultaneously fig. diagrammatic representation of three important factors in determining species' geographic distributions. circle a summarizes abiotic variables that circumscribe species' geographic potential, circle b adds biotic considerations, and circle m indicates limitations on dispersal or movement ability. a∩ b is the potential geographic distribution of the species, and a∩b∩m is a hypothesis of the actual distribution of the species. three example disease systems are illustrated: west nile virus, the filoviruses (ebola and marburg viruses), and plague: changes are illustrated as the difference between broken (original) and entire (present) outlines of circles; particular geographic occurrences of the disease are labeled to illustrate points discussed in the text appropriate from both abiotic and biotic perspectives and that are accessible to the species in terms of dispersal. although this area cannot simply be assumed to hold populations of the species (consider, e.g., metapopulation dynamics, which may lead to absences in suitable, accessible areas), this area does provide an index to the likely distribution of the species. considerable exploration and testing has demonstrated both that a∩b∩m is an appropriate prediction of the actual distributions of species (lópez-cárdenas et al. ; elith et al. ) and that a∩ b offers highly accurate predictions of species' distributional potential when dispersal constraints are relaxed (peterson ; benedict et al. ). this framework (the "bam" diagram), although simple, offers considerable inferential power regarding distributions of species (soberón and peterson ) . i emphasize that this framework is only heuristic and cannot cover the full diversity and complexity of disease phenomena-a more complete and mathematical treatment has been developed elsewhere (soberón ) . a wide variety of biodiversity phenomena (including diseases) can be placed in this framework and understood with greater clarity-for example, species' invasions are simply the broadening of the m circle to include more of the potential distribution of the species (peterson ) . particular factors (e.g., abiotic conditions) can be visualized as more or less constraining by increasing or decreasing the size and overlap of the circle relative to the other circles. the bam framework encounters some intriguing challenges in applications to disease biogeography. for example, in coarse-scale biodiversity applications, the focus is generally on the role of abiotic conditions in constraining species' distributions (soberón )-in contrast, disease applications can emphasize biotic interactions as dominating the process. the following paragraphs highlight these differences. interactions rule a basic characteristic of a disease is its transmission cycle. generally, these cycles involve several species-one or more reservoirs, vector(s), incidental hosts, and the pathogen itself. in an autecological world, then, a disease transmission system could be seen as a suite of species, each distributed according to its own ecological needs (i.e., its own particular a). in this sense, we could consider each element in a disease transmission system (species , , ... i ... up to n) to have its own particular version of a (which we can denote a i )-disease transmission would then occur only where a ∩a ∩...∩ a i ∩...∩a n . from the perspective of any single species in the system, the combined intersections of the a's for all other species in the system compact into that species' b (peterson ) . put more simply, however, in disease transmission systems, interactions rule. much of the dynamics of these systems will be determined not simply by abiotic considerations but by the interactions among many species. examples are more than common across the world of diseases: malaria transmission occurs only when appropriate mosquito vector species are present (gu et al. ); plague transmission is most efficient when certain flea species are present (krasnov et al. ) ; much more complex examples can be found in any introductory parasitology text. this interactions-dominated landscape appears to stand in sharp contrast with the abiotic-dominated landscape in the biodiversity world (peterson ) . stable and unstable interactions beyond simply being dominant, interactions in the disease world can be stable or unstable (i.e., enzootic or epizootic). specifically, some disease systems show dramatic associations of pathogens and hosts (dragoo et al. ; field et al. )-in these cases, pathogens and hosts coevolve over evolutionary time and may establish stable relationships that may even involve evolution of resistance on the part of the host and/or avirulence on the part of the pathogen (lenski and may ) . these stable relationships can also lead to the evolution of parallel phylogenetic patterns, indicating a long period of shared evolutionary change (charleston and page ) . on the other hand, species interactions in disease transmission systems can also be extraordinarily unstable, particularly when interactions are relatively new. some disease systems have epizootic transmission phases in which hosts are 'burned through' at surprising rates: excellent examples include plague (yersinia pestis) transmission among prairie dogs (cynomys spp.; ubico et al. ; cully et al. ) , rabies (rabies virus, rhabdoviridae) transmission among mammals other than vampire bats (desmodus spp.; wandeler ; davis et al. ) , and ebola (ebolavirus spp., filoviridae) transmission among great apes (leroy et al. ) . in these (and other) cases, the host-pathogen interaction is so unstable that host mortality almost inevitably results, producing either unstable epizootic transmission systems or brief outbreaks that burn out after a few generations of transmission, although some instances of unstable transmission maintenance of diseases are known (e.g., bingham ) . a can be of minor importance another characteristic of disease systems that differs markedly from much of the remainder of biodiversity is the potential for a to be of relatively minor importance. that is, particular for microbial pathogens (viruses, bacteria) that may not have free-living stages, abiotic considerations may place few constraints on the distributional potential of species. some examples are obvious-influenza transmission on the international space station or at an antarctic research base-but more subtle examples should be considered more carefully, such as outbreaks of afrotropical diseases such as ebola virus in virginia and marburg virus (marburgvirus spp., filoviridae) in germany (murphy et al. ). more generally, some pathogens may not have free-living life stages or any other interface with the outside environmentas such, their "ecological niche" requirements may constitute simply that of having a live host. in other words, in many disease transmission systems, a may place only broad constraints on the potential geography of the system, and b and m may determine much more of the spatial dynamics of the system. m can vary dramatically pathogens and parasites are generally of small body size and often are not particularly well equipped for movement. as such, one might expect their dispersal abilities to be quite limited. however, because of the tight associations between pathogens and their much larger hosts, dispersal events of surprising magnitude become possible. that is, because pathogens can ride around with their much bigger and much more mobile hosts, dispersal events can yield surprising results, often termed disease "emergence" events, but really just constituting extreme dispersal events. disease dispersal ability can thus be surprising in its effects. for instance, bat-hosted coronaviruses related to that which causes severe acute respiratory syndrome (sars) had certainly been present in asia for what is effectively the entirety of human history in the region (guan et al. ; li et al. ) . although such viruses may, from time to time, have infected humans and caused disease events, the major recent sars outbreak not only had serious public health consequences in asia but also jumped rather dramatically to north america (mcdonald et al. ) . similarly, monkeypox (monkeypox virus, orthopoxviridae) is well known as a central african disease (arita and henderson ), but a seemingly innocent importation of african rodents for pets in the usa resulted in a small-scale monkeypox outbreak in north america (hutson et al. ). these dramatic variations in dispersal ability must be borne in mind in considering disease biogeography. in this section, i offer three brief case studies intended to illustrate the oddities of disease biogeography, as contrasted with the biogeography of most customary elements of biodiversity. the three studies are designed to illustrate different aspects of distributional biology and offer some intriguing insights into disease biogeography. these case studies also offer an illustration of the basics of tools for modeling species' ecological niches-i have used simple niche-modeling tools to develop some interesting coarsescale results about disease biogeography. in niche modeling, known occurrences of species (or diseases, in some cases) are related to raster geographic information system (gis) coverages summarizing relevant environmental parameters in an evolutionary computing environment; the result is a picture of the species' ecological distribution, which can be projected onto geography to identify a potential distribution for the species (peterson ; soberón ) . west nile virus west nile virus (wnv; west nile virus, flaviviridae) was first described and characterized from a patient in uganda in and came to be known as a cause of mildly serious encephalitis cases across eastern and southern africa (taylor et al. ; nir et al. ; mcintosh et al. ); eventually, its sporadic occurrence, occasionally in the form of major outbreaks, in the southern tier of europe was also documented (hubálek and halouzka ) . in , however, wnv appeared in new york, in the usa, and quickly spread west and south across essentially all of the americas (komar and clark ) . the disease is clearly now "endemic" to (i.e., established in) much of the americas as a permanent component of the pathogen landscape of the western hemisphere. figure , however, paints a picture that contrasts actual and potential distributions of wnv. the area shaded brown in the figure in africa, southern europe, and southwest asia represents the virus' known distribution as of ; the blue-shaded area represents areas globally that fit the same precipitation-temperature profile as the brown-shaded area, showing that the same climate regime was present across a much broader portion of the world. in , wnv managed to jump across the atlantic ocean and become established in new york city-in the ensuing - years, thanks probably to dispersal via migratory birds, the virus has spread west to the pacific ocean and south to argentina (komar and clark ) , fulfilling much of the spatial extent of its potential distribution in the americas. ebola and marburg viruses discovered only in (marburg) and (ebola), the two virus genera that make up the filoviridae family are exclusively african insofar as their known geographic distributions (peters et al. ) . what is more, however, ebola virus is restricted to humid lowland evergreen tropical forests in africa (congo basin and a small area along the liberia-ivory coast border), and marburg virus is restricted to less humid tropical forests in eastern and southern africa. as such, these two related virus genera occupy a predictable ecological niche space across african landscapes (peterson et al. a; peterson et al. ) . as an illustration of the volatility and unpredictability of disease geography given the association of pathogens with larger hosts, it is worthy of note the several times that ebola or marburg viruses have appeared outside the usual suite of conditions for filoviruses (feldmann et al. ) . several filovirus-caused disease outbreaks have appeared under conditions distinct from the norm for these viruses and in areas well outside the usual distributional area of these viruses: ( ) marburg virus in marburg, germany, in ; ( ) marburg virus in johannesburg, south africa, in ; and ( ) ebola (reston species) in virginia, texas, and the philippines at various points in the s (peters et al. ; miranda et al. ) . as can be seen in both geographic and ecological dimensions (fig. ) , these filovirus occurrences are outliers and clearly illustrate how pathogens can be carried around and into odd situations by their larger and more mobile vertebrate hosts. clearly, this example illustrates the point that the "normal," endemic transmission mode may lend itself much better to predictive modeling than the irruptive, epizootic phases. plague finally and the most complex, plague is a zoonotic disease that is held in a small mammal reservoir and vectored by several flea species (gage ) ; the particular species involved depend on the region in question. plague was originally-apparently-restricted to central asia and interior china (pavlovsky ) ; with the advent of largescale movements (i.e., silk route trade, intercontinental shipping), however, a series of pandemics began, in which plague became established in urban rattus populations (gage ) . particularly in the early twentieth century, however, these plague outbreaks could be extinguished by reducing the contact between humans and rattus; however, in a number of cases around the world (e.g., western north america, andean and northeastern south america, eastern and southern africa, madagascar), the plague "jumped" into native rodents and has established itself as an endemic zoonosis (levy and gage ; enscore et al. ) . viewed ecologically, the endemic (i.e., not epizootic) plague appears to occur under a consistent suite of environmental conditions (enscore et al. ) . as can be appreciated in fig. , the environmental conditions across its original range in asia are fairly restricted with respect to the extent to which it spread via global shipping (echenberg ) . then, however, plague retreated somewhat as it moved from epizootic to enzootic, for example, not being maintained on hawaii or in australia (fig. ) . overall, then, in the history of the plague, two shifts have been observed: ( ) broadened distributional potential thanks to improved dispersal abilities and ( ) reduced distributional potential thanks to reduction in urban rat populations. the above framework can be applied to understanding the geography of many biological phenomena, including diseases of various types (soberón and peterson ) . the distribution of disease occurrences can be seen as the joint spatial distribution of suitable ecological conditions for all of the biological species involved in the transmission fig. actual and potential distributions of wnv as of : brown shading indicates an approximate boundary around areas of known wnv occurrence prior to ; blue shading indicates areas matching the annual mean temperature×annual precipitation profile of the brown areas using the bioclim algorithm (nix ) . note that the dispersal event of allowed the colonization of essentially the entire american portion shaded blue in the course of just years cycle: pathogen, reservoir, vector, etc.-in essence, the geographic projection of the ecological distribution of the pathogen as limited by the ecological and geographic potential of each of its interacting species. this interplay among ecological and geographic spaces, among the ecological niche characteristics of various species and as constrained by the spatial configuration of suitable habitats and dispersal abilities of the species involved, provides a framework for understanding disease distributions (peterson ) . the discussions above emphasize the role of humans in increasing the geographic scope of disease transmission, yet many diseases have yet to expand distributions broadly beyond their original distributional areas. for example, chagas disease remains uniquely american, although it may be expanding northward . the filoviruses and monkeypox are endemic only to tropical africa. scrub typhus (orientia tsutsugamushi) and nipah virus (nipah virus, paramyxoviridae) are uniquely asian. these diseases either have yet to disperse successfully to other regions, or perhaps they have dispersed but have failed to find appropriate interacting species (reservoirs, vectors, etc.) upon arrival. the distributions of such endemic diseases will be governed by the joint ecological requirements outlined above. some enigmas remain, however, in understanding disease geography. for example, yellow fever (yellow fever virus, flaviviridae) was originally distributed across humid tropical africa; early shipping (probably via slave ships) transported it to south america, and it spread broadly across the humid tropical portions of that continent. curiously, however, yellow fever has never colonized humid tropical portions of asia, in spite of ample areas presenting appropriate climatic conditions (fig. ) . similarly, particular strains of malaria (e.g., falciparum malaria) have patchy and odd distributional patterns that would seem to challenge purely ecology-based explanations of distributions. this review may be seen as a nontraditional way of presenting disease geography. instead of a broad review of dark gray areas correspond to countries with current yellow fever transmission (who ) ; red areas indicate portions of asia that match yellow fever-endemic regions in terms of annual mean temperature and annual precipitation distributional patterns disease by disease (e.g., ackerknecht ) , i have instead attempted to present a general framework for understanding geographic distributions of species and to point out the ways in which disease transmission systems differ from other biological systems. my hope is that the generalities that emerge will assist in a new, more synthetic view of disease geography. many challenges remain to be addressed in this field. in particular, the way in which interactions among species act (and how these interactions can vary across geography) remains almost completely unexplored. that is, the a∩b framework described above is certainly an oversimplification-interactions among species may easily vary spatially and may even vary as a function of interactions with still other species. these considerations could produce complexity well beyond what has been appreciated in disease geography studies to date and indeed might explain some of the complexities and nonlinearities that confound the understanding of disease geography. another issue is the many diseases that have poorly known or unknown components to their transmission cycles. in such cases, niche modeling tools can only be applied to the overall cycle as a "black box"-human cases would be used as an integration over the entire a ∩a ∩...∩a i ∩...∩a n , even though the species involved are not 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epidemic-prone infectious diseases-yellow fever. world health organization niche conservatism: integrating evolution, ecology, and conservation biology acknowledgments i thank my many "disease" colleagues for their many kind hours spent educating me about their areas of expertise. i thank in particular d. carroll for his insightful comments on an earlier version of this manuscript. this work was supported in part by a grant from microsoft research. key: cord- -q fzxt r authors: conde, dalia a.; colchero, fernando; gusset, markus; pearce-kelly, paul; byers, onnie; flesness, nate; browne, robert k.; jones, owen r. title: zoos through the lens of the iucn red list: a global metapopulation approach to support conservation breeding programs date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: q fzxt r given current extinction trends, the number of species requiring conservation breeding programs (cbps) is likely to increase dramatically. to inform cbp policies for threatened terrestrial vertebrates, we evaluated the number and representation of threatened vertebrate species on the iucn red list held in the isis zoo network and estimated the complexity of their management as metapopulations. our results show that of the , ( %) terrestrial vertebrate species in isis zoos are threatened. only two of the taxonomic orders show a higher proportion of threatened species in isis zoos than would be expected if species were selected at random. in addition, for most taxa, the management of a zoo metapopulation of more than individuals will require the coordination of a cluster of to isis zoos within a radius of , km. thus, in the zoo network, the representation of species that may require cbps is currently low and the spatial distribution of these zoo populations makes management difficult. although the zoo community may have the will and the logistical potential to contribute to conservation actions, including cbps, to do so will require greater collaboration between zoos and other institutions, alongside the development of international agreements that facilitate cross-border movement of zoo animals. to maximize the effectiveness of integrated conservation actions that include cbps, it is fundamental that the non-zoo conservation community acknowledges and integrates the expertise and facilities of zoos where it can be helpful. the conservation status of known biodiversity has undergone a worrying decline in the last few decades [ , ] . if the present trends continue, the conservation community will be challenged with a large number of species for which there is no viable conservation outcome. as a result, conservation breeding programs (cbps) may offer the only feasible option to avoid the extinction of particular species until appropriate habitat can be found or restored [ ] [ ] [ ] . the need for well-managed cbps, including those supported by the zoo community, has never been greater, but the space and logistical demands for managing cbps could exceed the current capacity of the zoo community [ , ] . although we know that % of threatened vertebrate species are represented in zoos [ ] , we still do not have an overview of the number and distribution of these species within the global zoo network. therefore, to establish effective responses to species' cbps, it is important to determine the current representation of threatened species within zoos and have a general understanding of the complexity of managing them within the global zoo network. the development of cbps is challenging and has received criticism due to problems including hybridization risk, high costs and the diversion of resources away from habitat protection [ ] [ ] [ ] [ ] . because of these factors, cbps have usually been considered only as a last resort and, as a result, are normally only implemented when populations dwindle to fewer than individuals (e.g. whooping crane, grus americana [ ] ). still, cbps have played a major role in of the species that have shown a status improvement in iucn red list reassessments [ , ] . moreover, for mammals, captive breeding together with reintroduction programs and hunting restriction have been the most effective conservation actions, since ensuring protected areas alone has not been enough [ ] . as a result, in most cases effective conservation plans require the integration of a range of management options where cbps could be necessary. for example, martin et al. [ ] compared the failure of conservation intervention for the recently extinct christmas island pipistrelle (pipistrellus murrayi) with the potentially successful intervention in the case of the orange-bellied parrot (neophema chrysogaster), where conservation actions, including cbps, were implemented in a timely manner and coordinated among several institutions. as threats to biodiversity increase, the management of at-risk species requires a spectrum of interventions that can range from habitat protection to the establishment of cbps with the eventual aim of reintroduction into the wild [ , ] . therefore, differentiating between in situ and ex situ management is becoming irrelevant [ , ] . instead, a continuum of management practices exists, which ranges from truly wild and self-sustaining populations to managed populations dependent on a certain level of human care for persistence. for example, the iucn ssc conservation breeding specialist group has recently launched an ambitious conservation framework called the one plan approach, which promotes participation between different institutions and stakeholders with the aim to consider all populations of a particular species, both inside and outside their natural range, under a unified management plan [ ] . the zoo community is in an ideal position to support and further contribute to develop such conservation programs because they are involved in both captive breeding and field conservation programs. for example, zoos have extensive knowledge of the husbandry, behavior and veterinary procedures required to develop cbps [ ] , and members of the world association of zoos and aquariums (waza) are, collectively, the third largest financial supporter of species conservation in their natural habitats (providing us$ million/year), while also being actively involved in many of those projects [ ] . to understand the space devoted to threatened species and the potential complexity of managing cbps as zoo-held metapopulations, we evaluated the representation of the iucn red list threatened species held within the zoo network. we used data published from conde et al. [ ] , processed from the international species information system (isis) organization. isis is a network of more than zoos and aquariums that shares information about , . million individuals among the member institutions. this information system makes it possible to analyze the amount of space devoted to threatened species and its spatial distribution. therefore, isis is a key institution to assess the potential of zoos to develop cbps. to understand if the number of threatened species in zoos is the result of a sound prioritization or if it has been more an opportunistic process, we analyzed whether the representation of threatened species per taxonomic order is significantly higher to what would be expected if species were randomly collected (i.e. without collection planning). in addition, to appraise the approximate complexity of managing zoo-held species as metapopulations, we estimated the distance between zoos clusters at which the probability of reaching an average metapopulation size of , and individuals is maximized. with these analyses we can better infer how complex it is to reach increasing population sizes between isis zoos, assuming that cbps could be more successful when population sizes are larger and clustered in zoos at closer proximity. since the clustering of zoos could facilitate special treaties to move individuals across borders. based on our results, we discuss the potential of zoos to contribute towards cbps for terrestrial vertebrates and make some policy recommendations. to determine whether the number of threatened species in isis zoos is different from what would result from a random sample, we carried out the following analyses. for every taxonomic order i of terrestrial vertebrates there is a given number n i of species worldwide, out of which a fraction m i is known to be threatened. similarly, we know the number of species for every order z i that are represented in isis zoos and the subset w i of those that are threatened. we developed a monte carlo algorithm to understand which orders have a number of threatened species in isis zoos significantly different from a random sample of species in the wild. this algorithm was based on an iterative procedure that, at every step s, randomly sampled, without replacement, z i species within every order from the worldwide list. from these randomly sampled species, the algorithm counted the number of speciesŵ w i,s classified as threatened. the algorithm then calculated the indicator y i, j,s that assigned if a number j of threatened species were sampled and otherwise, such that j = , …, min(z i , m i ). the probability of randomly finding j threatened species is calculated as where s is the total number of iterations. since the algorithm ran for several thousand iterations, we were able to construct a distribution (i.e. empirical probability mass function, pmf) of the number of threatened species per order that could have been found in isis zoos if they had sampled species at random from the wild. the probability in the pmf that corresponds to the real number of threatened species in zoos ( p i,j : w i~j ) was used as an analogue to the p-value. those orders that had a number of threatened species that matched the lower bounds of the pmf (i.e. p-value of . ) were classified as being significantly underrepresented. similarly, those that matched upper bounds of the pmf (i.e. above the . quantile) were catalogued as being overrepresented. the remaining species could not be distinguished from a random sample. to understand the complexity of maintaining cbps of threatened species across the isis zoo network, we developed a second algorithm to find the optimal radial distance from any given zoo at which the probability of finding a metapopulation size of at least , or recruited living individuals was highest relative to that radial distance. the algorithm was based on a monte carlo procedure that, at each step s, a zoo j was selected at random, then found all zoos z j,r located at a distance r from zoo j, as well as the number of zoos z i j,r within that radial distance that held species i. we labeled the zoos included within that radius as a cluster, k j,r . the algorithm then counted how many individuals of a given threatened species i were included in the cluster, such that where n i,k is the population size for species i in zoo k. at each cluster, we assigned the indicator where m is a pre-established metapopulation threshold (i.e. , or individuals). we chose , and individual metapopulation thresholds because a population size of has been historically considered as a minimum viable population [ ] , and individuals is the threshold defined by the iucn red list as a ''very small wild population'' in the critically endangered category [ ] . moreover, very few threatened species in isis zoos have populations for which a higher threshold is possible. for instance, only species ( . %) have more than , individuals (see below). we considered individuals as an arbitrary intermediate between these two values. this procedure was repeated for , iterations and then the algorithm was repeated, increasing the radial distance by km until a maximum radius of , km was reached. from the , iterations for each radius, the algorithm calculated the probability of finding at least m individuals for species i and radius r as where s is the total number of steps ( , ) in the monte carlo procedure. here, we replaced the subscript j with s to indicate that zoos were chosen at random with replacement. similarly, we calculated the average number of zoos for each species at each distance interval as z z r~ s x s s~ z s,r : we repeated this procedure for radii ranging from to , km, using km increments. we excluded species for which the minimum population size was never reached, even when including all isis zoos. for each class c and metapopulation threshold m, we calculated the average ratio between probability p i,r,m and distance d r for radius r as where i c is the total number of threatened species in class c that was included in the analysis. finally, we found the optimal cluster, this is the optimal radius dà c,m and the optimal number of zoos zà c,m , where the ratio r c,r,m was highest for each class and population threshold m. these optimal clusters imply that, relative to that radius, the probability of reaching a metapopulation of at least m individuals is highest. for simplicity, we present the results pooled by taxonomic class. collectively, in , the isis zoos held , species of non-domestic terrestrial vertebrates (table ) . of these, more than half ( %) were birds, one quarter ( %) were mammals, % were reptiles and % were amphibians ( figure ). twenty-three percent ( / , ) of the species in isis zoos belong to a threatened category (table ) . within each class, the percentage of threatened species varies widely among each order, ranging between % for birds ( / , ) and % for mammals ( / ). isis zoos held a total of , individuals of non-domestic terrestrial vertebrates, of which % ( , / , ) belong to a threatened species (table s ). our analysis of the number of threatened species in isis zoos, broken down by order, shows that most collections are not distinguishable from what would be expected if the species were selected at random (figure , tables s , s , s , s ). exceptions occur in mammals in the order dasyuromorphia (australian carnivorous marsupials) and in reptiles for testudines (turtles): isis zoos hold % and %, respectively, of the order's threatened species. on the other hand, threatened species were under-represented in zoos for the mammalian orders eulipotyphyla (insectivores) and rodentia (rodents). for birds, threatened species were under-represented in nine of the orders held in zoos, whereas for amphibians this was only the case for caudata (salamanders). for the threatened amphibian species in isis zoos, % reach a metapopulation size threshold of . individuals. almost half of the threatened species of mammals, birds and reptiles in isis zoos reach the same threshold ( %, % and %, respectively), and % of the threatened mammals reach a threshold above individuals (table ) . however, many of those species are distributed among numerous zoos, thus if those species are not managed as a metapopulation, their conservation potential will be greatly reduced. as expected, the optimal distance radii and the number of zoos required to maximize the probability of reaching a given population increase with the metapopulation size threshold (. , . and . individuals) ( figure ) . however, the magnitude of the increase varies among classes; for birds and reptiles, the difference between optimal distances is just km and km, respectively, while for mammals and amphibians, the differences are up to km (table ) . furthermore, at the optimal radial distances, the probability that a cluster of zoos has more than , or individuals of a given threatened species ranges from . to . (table ) . however, only . % to . % of zoos within an optimized cluster have individuals of the species. moreover, these percentages vary considerably among the four classes of terrestrial vertebrates. birds and mammals make up the largest proportion of terrestrial vertebrates held in zoos. although the majority of zoo collection plans were not originally focused on holding threatened species [ ] , % of their collections are currently devoted to them. however, for most of the taxonomic orders, our results show that representation of threatened species is not different from what would be expected if species were selected at random. broken down by class, it is clear that threatened birds make up the lowest proportion in the zoo network. this may be explained by factors including: i) some species are difficult to breed in captivity (e.g. seabirds), ii) some species have specialized dietary requirements (e.g. insectivores), and iii) import/export restrictions, such as those added in the wake of the sars (severe acute respiratory syndrome) epidemic, which make it challenging to manage birds across borders and to import new species. mammalian and reptilian zoo collections include the highest proportion of threatened species; however, only the orders dasyromorphia and testudines are significantly over-represented. the dasyromorphia are a particular focus of cbp efforts in australian zoos [ ] . however, despite the interest in this group, six threatened dasyromorphia species are not yet represented in zoos. the high representation of threatened testudines is partly because many zoos serve as rescue centers for confiscated individuals. the turtle survival alliance has played a key role in promoting the conservation of turtles and has been working on linking zoos and governmental institutions to ensure the rescue of animals from the illegal trade [ ] . however, since many of those individuals come from the illegal trade, it is hard to include them as part of cbps because, in many cases, their origins are uncertain [ ] . for amphibians, the relatively low proportion of threatened caudata in zoos may reflect their cryptic behavior and small size [ ] , which makes them difficult to display. in general, the small number of amphibian species may also be due to practical issues such as the difficulty in obtaining permits to transport individuals. although amphibian collections in zoos and zoo-supported centers have significantly increased in the last years [ , ] , zoos still only hold % of the world's threatened amphibians [ ] . this emphasizes the need for zoos to increase their contribution towards amphibian cbps either as part of their collections or by further contributing to the development of breeding centers in their local areas [ ] . with this in mind, the amphibian ark emerged with a strong zoo component with the mission of ensuring the global survival of amphibians, focusing on those that cannot currently be safeguarded in nature and where zoos can play a key role [ ] . zoos hold , % of the world's threatened terrestrial vertebrates [ ] . however, in their collections, % of isis zoos species ( / , ) belong to a threatened category. nevertheless, most of their populations are small and are distributed across the zoo network. therefore, for the zoo community, one of the main challenges of managing their threatened species in cbps is the complexity of moving individuals across borders and the coordination of conservation efforts among zoos and other institutions at a global level. the enormity of this task is clear from our results. for example, the optimal radius for finding a metapopulation size of more than individuals for a given threatened species is , km for reptiles and , km for mammals. within these optimized clusters the number of zoos that hold a given threatened species is low compared with the number of zoos available within the clusters. for example, on average only % ( / ) of all the zoos within a cluster hold a given threatened reptile species. this implies that, under current conditions, to manage a metapopulation above individuals requires an optimized cluster of , zoos that could be up to , km apart. given this complexity, it is not surprising that most zoo populations of threatened species are not managed as metapopulations [ ] , nor that most are not yet sustainable in the long term [ ] . we found that, on average, for threatened species in the isis network, fewer than % of zoos within optimized clusters hold a particular species. therefore, it would be possible to improve the network within an optimized cluster by increasing the number of zoos that contain individuals of a focal species, managing these collections as a single metapopulation and potentially reducing the distances between zoos. this level of organization could result in zoos focusing on particular cbps for fewer taxa, rather than having a small number of individuals of many threatened species. this is particularly important since specialization has been shown to increase breeding success [ ] . this observation does not mean that zoos should shift their entire collections towards one or a few at-risk species, since responsible zoos have other conservation goals such being centers for education and research [ ] . rather, it means that zoos within a particular region can most efficiently increase their conservation contribution by developing collectively managed cbps devoted towards a smaller number of focal species. the proportion of threatened species that exceed a threshold metapopulation size of individuals is rather low, ranging from only % for birds to % for mammals. however, the percentages of species reaching the threshold of . individuals range from % for amphibians to , % for the other three classes. isis zoos have only a small number of threatened species for which population sizes are above , individuals. although this number has been suggested as an appropriate threshold over which genetic diversity should be maintained [ ] , most of the species that have been recovered from cbps come from populations below to individuals (see [ ] ). in this sense, it is important not to underestimate the potential of some of these isis populations. nonetheless, the zoo community should aim at providing populations to cbps that can ensure genetic and demographic sustainability [ ] . additionally, cbps should not be implemented only when species have reached dramatically low numbers, at which point their chances of success are lower [ ] . our results stress that, for many species, appropriate management and coordination within an optimized cluster of zoos can potentially increase their numbers to at least individuals. in table . number of species in isis zoos with population sizes within specific thresholds for the different red list categories (see table for red list categories definition). addition, it is expected that these population sizes will be larger if we include non-isis member institutions. however, it is important to stress that the successful management of cbps as metapopulations requires the collaboration and coordination of zoos within a global network such as isis. . the zoo community should identify potential zoo clusters for the conservation of prioritized species. particularly for smallbodied species, zoos could potentially hold a large number of individuals within a particular region. although the clusters should ideally be in close proximity to the species' natural habitat, most isis zoos are currently located away from major biodiversity hotspots [ ] and therefore, zoos' support of breeding centers within the native range may be an option. . cluster-level integrated management plans should be implemented to ensure the coordination of cbps with habitat conservation and other in situ efforts (one plan approach [ , ] . for this, the development of greater coordination among zoos using networks such as isis, together with conservation ngos, academic and governmental institutions, will be essential (see [ ] ). . species-specific clusters should ideally be replicated to minimize the potential impact of catastrophic events. . cross-border management policies for zoos should be modified. in this sense, the management of cbps by clusters can facilitate treaties for the management of particular focal species. to ensure sustainable metapopulations it will require more than the coordination and will of the zoo and conservation community. although we acknowledge the need for public health management and vigilance against illegal trade, the development of cross-border management policies will be key to achieving successful cbps. for example, cites could award special permits to facilitate movement of targeted individuals within cbp clusters. , average number of zoos where the species is found as a function of the radial distance (middle row) and ratio between the probability of finding the metapopulation and the radial distance (lower row) for each taxonomic class of terrestrial vertebrates. when the ratio is highest, we obtain the optimal radial distance between zoos and the optimal probability of finding a metapopulation size above the threshold. this is, at that ratio the probability of finding the metapopulation is highest with respect to the zoo cluster radial distance. for display purposes all ratios were multiplied by , . doi: . /journal.pone. .g global change represents an unprecedented challenge for the maintenance of biodiversity [ ] [ ] [ ] . it is expected that even under the most optimistic impact and adaptation scenarios, a great number of species may require the integration of a suite of conservation actions, including cbps. furthermore, species that have no likelihood of in situ persistence for the foreseeable future represent an additional conservation challenge. for example, under current global warming trends, most polar and some montane species are likely to fall into this category [ ] , in addition to species whose habitat will be lost by urbanization [ ] . as a result, deciding which species could be part of successful cbps and which institutions should modify their collections to become part of a particular cluster needs to be the result of a sound prioritization approach. furthermore, simply holding these species, even in numbers above the minimum individuals, is not sufficient [ ] , and cbps need to be integrated with other aspects of species conservation, such as habitat protection and restoration, eradication of invasive species and population management [ ] [ ] [ ] , ] . our results show that, in the zoo network, the representation of species that may require cbps is currently low for most taxa and the spatial distribution of these zoo populations makes management difficult. however, the zoo network already devotes % of its collections to threatened species; for mammals, % of those reach population sizes above individuals. zoos in collaboration with other institutions have already saved a number of species from extinction, but it has been mostly opportunistic rather than strategic. if zoos collectively focus on their strength as a global network, they have the potential for the development of integrated conservation programs that include cbps. to maximize effectiveness, the collaboration of the global zoo network with governmental institutions, regional and international trade authorities, ngos and academia should be fostered. such collaborations are already underway, and termed a one plan approach [ ] . however, it is essential to strengthen these institutions' contributions, include special international treaties and collaborations to help slow down current extinction trends. thanks to the iucn ssc conservation breeding specialist group for facilitating the discussions that set the direction of this paper; to isis member institutions for making their basic data available in the isis portal; to alexander scheuerlein and zjef pereboom for useful comments on the manuscript; and to robert wiese, william van lint, jenny gray and jeffrey bonner for sharing their perspectives and expertise on zoo collections. additionally, we would like to thank the editor and two external reviewers for helpful suggestions and detailed comments that significantly improved our manuscript. optimal radial distance between zoos, average probability of reaching the metapopulation size threshold within that radial distance, average number of zoos within radial distance and average number of zoos within radial distance that hold the threatened species. the optimal radial distance represents the distance needed to optimize the probability of reaching an average metapopulation size within the shortest possible distance between zoos (the metapopulation thresholds m are . , . and . individuals). the column for ''number of species in cluster'' indicates the number of threatened species for which all isis zoos have at least m individuals. doi: . /journal.pone. .t monitoring change in vertebrate abundance: the living planet index global biodiversity: indicators of recent declines an emerging role of zoos to conserve biodiversity bring the captive closer to the wild: redefining the role of ex situ conservation buying time for wild animals with zoos recent captive-breeding proposals and the return of the ark concept to global species conservation parks or arks: where to conserve threatened mammals? limitations of captive breeding in endangered species recovery captive breeding -a useful tool in the preservation of biodiversity? why we should aim for zero extinction the impact of conservation on the status of the world's vertebrates zoos and captive breeding -response using the iucn red list to determine effective conservation strategies acting fast helps avoid extinction integrating the captive and the wild the one plan approach: the philosophy and implementation of cbsg's approach to integrated species conservation planning research in zoos: a growth area in conservation building a future for wildlife?'' evaluating the contribution of the world zoo and aquarium community to in situ conservation minimum population sizes for species conservation conservation biology the application of zoos victoria's ''fighting extinction'' commitment to the conservation of leadbeater's possum gymnobelideus leadbeateri zoo-based amphibian research and conservation breeding programs the amphibian ark: a global community for ex situ conservation of amphibians identifying gaps and opportunities for inter-regional ex situ species management sustaining the ark: the challenges faced by zoos in maintaining viable populations minimum viable population size: a meta-analysis of years of published estimates achieving true sustainability of zoo populations synergies among extinction drivers under global change extinction risk from climate change impacts of climate change on the future of biodiversity climate change in the eastern himalayas: observed trends and model projections global forecasts of urban expansion to and direct impacts on biodiversity and carbon pools key: cord- -ghcwfcx authors: razanajatovo, norosoa h; nomenjanahary, lalaina a; wilkinson, david a; razafimanahaka, julie h; goodman, steven m; jenkins, richard k; jones, julia pg; heraud, jean-michel title: detection of new genetic variants of betacoronaviruses in endemic frugivorous bats of madagascar date: - - journal: virol j doi: . /s - - -y sha: doc_id: cord_uid: ghcwfcx background: bats are amongst the natural reservoirs of many coronaviruses (covs) of which some can lead to severe infection in human. african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks. a recent study in south africa isolated a genetic variant closely related to mers-cov from an insectivorous bat. though madagascar is home to bat species ( insectivorous and frugivorous) of which are endemic, no data exists concerning the circulation of covs in the island’s chiropteran fauna. certain malagasy bats can be frequently found in close contact with humans and frugivorous bats feed in the same trees where people collect and consume fruits and are hunted and consumed as bush meat. the purpose of our study is to detect and identify covs from frugivorous bats in madagascar to evaluate the risk of human infection from infected bats. methods: frugivorous bats belonging to three species were captured in four different regions of madagascar. we analyzed fecal and throat swabs to detect the presence of virus through amplification of the rna-dependent rna polymerase (rdrp) gene, which is highly conserved in all known coronaviruses. phylogenetic analyses were performed from positive specimens. results: from frugivorous bats, we detected coronaviruses from two endemic bats species, of which viruses were identified from pteropus rufus and one from eidolon dupreanum, giving an overall prevalence of . %. phylogenetic analysis revealed that the malagasy strains belong to the genus betacoronavirus but form three distinct clusters, which seem to represent previously undescribed genetic lineages. conclusions: our findings suggest that covs circulate in frugivorous bats of madagascar, demonstrating the needs to evaluate spillover risk to human populations especially for individuals that hunt and consume infected bats. possible dispersal mechanisms as to how coronaviruses arrived on madagascar are discussed. coronaviruses (covs) are enveloped viruses with singlestranded positive-sense rna belonging to the subfamily coronavirinae in the family coronaviridae (order nidovirales). genomes of covs range from to kb and show high genetic diversity [ ] . covs are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [ ] . in mammals and birds, covs are associated with upper and lower respiratory illnesses or gastroenteritis. in humans, covs infections are commonly caused by hcov- e and hcov-oc which generally cause mild respiratory illnesses [ ] . a new cov that causes severe acute respiratory syndrome (sars-cov) emerged in humans in - and infected more than , individuals with mortality rates estimated at around % [ ] . the emergence of sars-cov and its mortality rate have raised the risk of a new pandemic that could threaten public health. for this reason, the scientific community invested considerable interest in the identification and characterization of covs especially within mammal reservoirs. subsequently, two novel human covs were discovered: hcov-nl in [ ] and hcov-hku in [ ] . in june , a third novel coronavirus named hcov-emc/ (renamed mers-cov) was isolated from patients presenting with acute respiratory distress and pulmonary inflammation [ , ] . studies which aimed to identify potential reservoirs of emerging human covs have revealed that the betacoronavirus sars-cov was closely related to covs detected in bats, specifically members of the genus (rhinolophus), which brought the hypothesis of a spillover of this virus to several animal species (including civet cats and raccoons) sold in chinese markets as bushmeat for human consumption [ ] [ ] [ ] . bats have since become a particular focus and a number of alphacoronavirus and betacoronaviruses have been identified in many frugivorous and insectivorous bat species and in many countries worldwide in asia, the americas and europe (see review from drexler et al. ) [ ] . genomic characterization of the recently discovered mers-cov showed that this virus belongs to the genus betacoronavirus and seems to be closely related to bat coronaviruses hku and hku isolated from bats (tylonycteris and pipistrellus) [ ] . african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks [ ] [ ] [ ] . some authors have reported the detection of bat covs from mainland africa [ ] [ ] [ ] [ ] [ ] . a recent study in south africa detected a genetic variant closely related to mers-cov from neoromicia zuluensis, an insectivorous bat [ ] . the authors hypothesize that mers-cov may have a common ancestors with covs borne by bats from africa. though madagascar is home to bat species ( insectivorous and frugivorous) of which are endemic [ ] [ ] [ ] , no data exists concerning the circulation of covs in malagasy bats. certain bat species on the island can be frequently found in close contact with humans, particularly members of the family molossidae; these synanthropic species roost in human-occupied buildings, like houses, schools or hospitals [ ] , whilst frugivorous bats feed in the same fruit trees where people collect and consume fruits. moreover, hunting and consumption of bat bush meat, especially the larger frugivorous species of the family pteropodidae, is widespread on the island, bringing hunters, purveyors and consumers into contact with bats [ ] . in this study, we report the detection of covs amongst frugivorous bats in madagascar. our results demonstrated for the first time that covs belonging to the genus betacoronavirus are circulating amongst two endemic frugivorous bats species in madagascar. a total of bats belonging to endemic bat species of the family pteropodidae were captured and sampled: rousettus madagascariensis (n = ), pteropus rufus (n = ) and eidolon dupreanum (n = ) ( table ) . none of the throat swabs from any bat species (n = ) tested positive for cov, but . % ( / ) of fecal specimens tested positive for cov. prevalence within p. rufus, e. dupreanum and r. madagascariensis was respectively . % ( / ), . % ( / ) and % ( / ). all positive specimens originated from bats captured in the menabe region ( figure ). short amplicon sequences of bp in length of the rdrp gene were obtained for all pcr-positive animals, whereas larger fragment of bp sequences could only be obtained from seven of the pcr-positive animals. all amplicon sequences were aligned in-frame with a compilation of reference sequences from genbank for which collection-date data was available [ ] , giving final alignments containing different sequences of bp in length and different sequences of bp in length. gtr + i + g was identified as the optimal substitution model using jmodeltest v . . [ ] . multiple phylogenies were generated in beast using different combinations of model parameters, and the best models were selected using the tracer [ ] . bayes factor analysis employing marginal likelihoods, as detailed in [ ] . all parameter combinations produced identical, strongly supported tree topologies (data not shown). as has elsewhere been determined by lau et al. [ ] , bayesian skyline using a relaxed, exponentially distributed clock model was found to be the best fitting model for rdrp dated-tip phylogenies. the final phylogenetic analyses ( figure ) revealed that strains from madagascar are members of the betacoronavirus genus, rooting with hong kong strain btcov-hku (hm ) and kenyan strain ky (gu ), with posterior probabilities of . these lineages could be described as sars-like, and were uniquely affiliated with frugivorous bat hosts of the family pteripodidae. malagasy strains were sub-divided into three distinct clusters: two of which were closely related (clusters and ) and originating from p. rufus, and one more distantly related (cluster ) containing a strain detected from e. dupreanum and sequences previously detected from e. helvum in kenya [ ] . the malagasy covs were detected from bats captured in three different sites of the menabe region (west of madagascar). within cluster , strains were originated from bemanonga, mahabo and ankiliabo. within cluster , strains were originated from bemanonga and ankiliabo. the only virus detected belonging to cluster was detected in one bat captured in mahabo. overall, identities among malagasy covs ranged from to % at the nucleotide level and . to % at the amino acid level (data not shown). molecular dating estimates based on the bp fragment of the rdrp gene estimated the timescale of evolution of the coronavirus family to be thousands to tens of thousands of years, however dating estimates proved inaccurate, with broadly spanning hpd values at individual node positions. in the context of this study, we detected coronaviruses forming nine genetically distinct strains in two endemic malagasy frugivorous bat species. the overall prevalence ( . %) is consistent with those identified in studies elsewhere [ ] [ ] [ ] . thirteen viruses were detected from pteropus rufus and virus from eidolon dupreanum. we did not detect covs among the sampled r. madagascariensis. the detection of novel bat covs supports the observation that these viruses are diverse and have a nearly worldwide distribution [ , , , ] . we observed that all malagasy bat covs detected in the present study belong to a sars-related subgroup of the betacoronaviruses, with relatively close homology to btcov-hku [ ] . our strains displayed distinct clusters: clusters associated with p. rufus and cluster associated with e. dupreanum. it can be inferred from the results that multiple clusters of covs occurring within malagasy bat populations co-circulate and possibly in a syntopic manner. the high nucleotide and amino acid divergence between clusters and compared to the reference virus btcov-hku suggests previously undescribed genetic lineages. given the mobility of bats, and the especially long distances that can be travelled by colonies of fruit bats [ ] , these coronaviruses may be spread over a large region. however, host-genus-specific phylogenetic clustering (figure , inset) suggests likely host-specificity which may limit viral spill-over. thus, further molecular epidemiology studies would be required to fully understand the dispersal potential of covs amongst malagasy bats species. it is important to remember that, although all three of madagascar's fruit bat species were sampled, nothing is known of cov dynamics in tropical fruit bats, and many factors such as seasonality, bioclimate and the presence of other host species may have important influences on cov prevalence in these populations. more studies are needed in different locations including different species, particularly those with an insectivorous diet, to reveal a more comprehensive view of the diversity of these viruses in madagascar. since the strains of betacoronavirus identified from madagascar are closely related to those known from africa, some preliminary biogeographic considerations are in order. all three bat species analyzed herein are endemic to madagascar. the genus pteropus has a broad distribution from the australia-new guinea region west across the indian ocean to offshore islands of tanzania; it is unknown from the african continent. the genus eidolon is composed of two species: e. helvum occurring on the african mainland and offshore islands and e. dupreanum restricted to madagascar. based on a phylogeographical study, both species show broad panmictic population structure [ ] . further, these two taxa are estimated to have diverged from one another sometime in the late miocene or early pliocene [ ] . the genus rousettus is broadly distributed across africa and asia and the ancestral origin of the malagasy species, r. madagascariensis, is unresolved [ ] . as with the other two pteropodidae species occurring on madagascar, r. madagascariensis shows little genetic population structure and presumably broadly disperses across the island, which in turn has important epidemiological implications for these bats transmitting different zoonoses. although estimates of the most recent common ancestors (mrcas) proved inaccurate in our study, most likely as a result of a limited sequence availability from the identified viral strains, standard evolutionary analyses have estimated cov origins to date to somewhere between - , yrs. in the past [ ] [ ] [ ] . nevertheless, further investigations into the relevance of mrca prediction methodologies are required and a great level of caution must be employed in the interpretation of mrca data. . alternatively, viral lineages may have been imported to madagascar in recent history: while the vast majority of the island's bat fauna is endemic, a few species apparently disperse across the mozambique channel. probably the best example is the molossidae species mops midas, for which, based on genetic data, southern african animals are nested within malagasy populations [ ] . this bat makes its day roosts in rock crevices and may broadly occur synoptically at such sites with e. dupreanum and r. madagascariensis. these two fruit bat species are known to feed in the same fruiting trees with p. rufus [ ] , which would complete the cycle of how covs originated from africa mainland could be carried to madagascar and transmitted to different species of pteropodid bats. although we were not able to evaluate risk of human infection, the strains detected here may be considered as potential human pathogens, as bats are natural reservoirs of some pathogenic covs. isolation of malagasy covs using cell culture and molecular analysis of spike (s) gene could better evaluate risk of human infection. also, a longitudinal study amongst people who frequently handle live bats (e.g. bat hunters, bat bushmeat purveyors, and scientists), and who represent populations at higher risk of infection, would be interesting to establish possible cases of transmission to humans and public health risks. in our study, we confirm that covs are circulating in two species of endemic bats in madagascar. further work on cov diversity amongst the island's bat species, as well as aspects of the ecology and behavior of susceptible taxa, are needed to understand the origin, evolution and dispersal of these viruses across the island. to conclude, the results of our study demonstrate the need to develop research programs that aim at surveying viruses in the wild, especially in bats, in order to address possible emergence of zoonotic viruses within human populations. we sampled frugivorous bats in four different areas of madagascar: anjohibe, anosibe an' ala, menabe and toliara (table / figure ) based on known accessible colonies of roosting bats and sites where bats are frequently hunted and eaten by people. in the region of menabe, we selected different sites situated at a mean distance of km around mahabo to capture and collect specimens, while in the three other regions, sampling occurred at a single site. sampling was carried out under protocols approved and permitted by ministry of environment and forest (authorization # / , / , / , / and / ). fruit bats were captured by the use of mist-nets set near roosting sites (trees or caves) and with help of professional hunters [ ] . rectal and throat swabs were collected from each individual bat. all bats were identified according species specific morphological features well known by our field trained team (ecologist and veterinarian) and subsequently released. swabs were placed in viral transport media, almost immediately conserved in liquid nitrogen in the field and stored at − °c upon arrival at the laboratory in antananarivo. rna was extracted from specimens using the qiaamp viral rna minikit (qiagen, courtaboeuf, france) according to the manufacturer's protocol. briefly, total rna was extracted from μl of each sample and eluted in μl of qiagen ave elution buffer. the extracted rna was either immediately analyzed or stored at − °c until use. extracted rna was reverse transcribed to generate cdna by using the m-mlv reverse transriptase (invitrogen, california, usa) into a step reactions. first, μl of rna were mixed in a solution containing . μm of random hexamer primers (roche diagnostics, mannheim, germany), u/μl of rnase inhibitor ( units) and . μl of water, at °c for min, °c for min and °c for min. then, μl of rna issued from the first step was added to a mixture of . mm of each dntp (deoxynucleotide triphosphates), u/μl of reverse transcriptase m-mlv, x of buffer and . m of dtt (dithiothreitol), and incubated at °c for min and °c for min. pcr assay was performed to amplify the rna-dependent rna polymerase (rdrp) gene which is highly conserved in all known coronaviruses [ ] . the primers pair (forward ′-ggttgggactatcctaagtgtga- ′; reverse ′-c catcatcagatagaatcatcata- ′) was designed to amplify a bp fragment as described previously [ ] . reaction mixture was carried out using the gotaq/dntp mix, custom kit (promega corporation, madison, usa). briefly, μl of cdna was mixed with x of green gotaq flexi buffer, . mm of mgcl , . mm of each dntp, . μm of each primer, . u/μl of gotaq dna polymerase and . μl of water nuclease-free giving a final volume of μl. the thermocycling was performed under the following conditions: activation at °c for min and cycles of denaturation at °c for min, annealing at °c for min, extension at °c for min, and a final extension at °c for min. all negative samples were tested in a semi-nested pcr with the same pcr program and using the following pair of primers (forward ′-ggttg ggactatcctaagtgtga- ′; reverse ′-atcagata gaatcatcatagaga- ′). amplicons products were subsequently electrophorezed on a . % agarose gel and visualized using ethidium bromide under uv light. all specimens that showed a positive band at the expected size ( bp) were sequenced on both strands by beckman coulter genomics (essex, uk). from the sequences obtained from the bp, fragment, we designed new primers (reverse) that were strain specific for malagasy batcov ( ′-gatgacc tgtatattccca- ′ and ′-atgacctatacatacc catg- ). we then amplified a large fragment of the rdrp gene by using the consensus forward primer ′-gtgtacgctgctgatcctgctatgca- ′ [ ] . the following conditions were performed: °c for min and cycles of denaturation at °c for min, annealing °c for min, extension at °c for min, and a final extension at °c for min. the final size of sequences used for molecular dating was bp. sequences from the bp or bp fragments of the rdrp gene were cleaned and aligned with reference sequences collected from a literature search. alignment was performed using the translation alignment tool in geneious pro™ v. . . , created by biomatters (available from http:// www.geneious.com/), and the default clustalw cost matrix. the final alignment was respectively bp and bp in length for fragments bp and bp, and contained no free-end or internal gaps. from these alignments, the appropriate substitution model was identified in jmodeltest v. . . [ , ] . using the appropriate substitution model, * ^ iterations were run with or without the use of a base codon model, using different clock models, alternating between constant and bayesian skyline population size models, and seeding with uncorrelated log-normally distributed priors. trees were sampled every x ^ iterations, and analysis convergence was assessed in tracer v. . . [ ] (available from http://beast.bio.ed.ac.uk/tracer). all analyses converged after a % burn-in to give effective sample size values for all parameters superior to . bayes factor analyses were performed in tracer v . . , with bootstrap replicates to assess the relative performance of model selections on the generated phylogenies. after identification of an optimal model for phylogenetic classification and dating, two further independent analyses ( * ^ iterations, sampling every * ^ iterations) were run in beast, and all analyses were combined in logcombiner (beast package) with a burn-in of %, leaving only converged parameter estimates. the final phylogeny and mrca for fragments bp in length dates were extracted using treecombiner (beast package) and figtree v. . . 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methods for the study of bats identification of a novel coronavirus in bats genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was conducted in collaboration with the association madagasikara voakajy and the bangor university (darwin initiative project), a project examining aspects of emerging viruses in small wild mammals. the work in the toliara region was part of action concertée inter-pasteurienne (acip) research program. we would like to thank felicien herbert randrianandrianina of madagasikara voakajy and local hunters for their help in capturing bats. david a. wilkinson's post-doctoral fellowship was funded by "run-emerge", a european project funded by european commission under fp program. the authors declare that they have no competing interest.authors' contributions nhr and daw carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. lan coordinated the fieldwork, participated to the sampling collection, participated to molecular testing and drafted the manuscript. jhr participated in the design of the study and coordinated the fieldwork smg and rkj helped to draft the manuscript. jpgj participated in the design of the study and helped to draft the manuscript jmh: conceived, designed and coordinated the study and helped to draft the manuscript. all authors read and approved the final manuscript key: cord- -kd xvsg authors: zenetos, argyro; papathanassiou, evangelos; aartsen, jacobus j. van title: analysis of benthic communities in the cyclades plateau (aegean sea) using ecological and paleoecological data sets date: - - journal: mar ecol (berl) doi: . /j. - . .tb .x sha: doc_id: cord_uid: kd xvsg abstract. in the cyclades plateau (aegean sea), a qualitative and quantitative analysis of macro‐benthic fauna was carried out in . standard multivariate analysis techniques were applied to both ecological (living benthic fauna) and paleoecological data sets in order to distinguish distribution patterns. results showed that caution must prevail in drawing conclusions from a limited data set. the clearest classification was obtained using total living fauna, while the dead molluscan fauna gave a similar pattern; this indicates similar response to the environmental conditions of the area. in the analysis of the living molluscan fauna, the groups failed to show any clusters, probably as an effect of some impoverished sites. in the two groups delineated, depth seems to be the major factor in the distribution of species. the fact that two distinct data sets (subfossil assemblages and living communities), when treated separately, produce similar grouping indicates that the subfossil assemblages could be reliably used as a first approach for determination of the living communities' distribution patterns. similar pattern; this indicates similar response to the environmental conditions of the area. in the analysis of the living molluscan fauna, the groups failed to show any clusters, probably as an effect of some impoverished sites. in the two groups delineated, depth seems to be the major factor in the distribution of species. the fact that two distinct data sets (subfossil assemblages and living communities), when treated separately, produce similar grouping indicates that the subfossil assemblages could be reliably used as a first approach for determination of the living communities' distribution patterns. recognizing that our knowledge of the benthic communities of the aegean sea is poor (pbres & picard, ; jacquoite, ; vamvakas, ) , a largescale project was initiated by the national centre for marine research. it aimed at examining the structure of deeper benthic communities as well as mapping their distribution over the aegean sea. ecological surveys usually result in complex bodies of biotic and environmental data from which patterns and relationships need to be extracted. numerical taxonomy is "the numerical evaluation of the affinity or similarity between taxonomic units and the ordering of these units into taxa on the basis of their affinities" (sokal & sneath, ). in ecological studies the "taxonomic u. s. copyright clearance center code statement: - / / - $ . / units" are ecological units (stations) and the taxa are biotopes (q-mode analysis), or respectively the taxonomic units are species and the taxa are benthic communities or "biocoenosis" (r-mode analysis). in paleoecological studies the taxonomic units are species and the taxa are biofacies. a review of the multivariate analysis techniques applied to a variety of ecological data is given in clifford & stephenson ( ) , while an outline of the successfully employed methods for analysing multispecies distribution patterns is presented in field et al. ( ) and gray et al. ( ) . regarding macrobenthic fauna, the above-mentioned classification techniques have been mpst commonly used either taking into account the total number of species encountered in a survey (stephenson & williams, ; stephenson er al., ; reys, ; field el at., ; k n o~ et al., ; hruby, ; gray et at., ; weston, ) or a certain group only, e.g., molluscs (robert, ; coleman & cuff, ) , copepods (sarvala, ) . on the other hand, paleontologists have applied the same techniques in order to define biotopes and biofacies based on a single group. thus, we have biofacies and biotopes of ostracods (maddocks, ) , of foraminiferans (kaesler, ; michie, ), or molluscs (zenetos, . in this study an attempt is made to recognize biotopes using standard multivariate analysis techniques but based separately on a) ecological data and b) paleoecological data. the validity of the various data sets in delimiting biotopes is discussed. benthic samples were taken at stations in the cyclades plateau (fig. i ). with the r n "aigaio" in july . four replicate samples were collected at every station with a . m smith-mcintyre grab. all samples were washed on a mm mesh sieve and the animals removed and preserved in a % formalin solution with rose bengal stain. in the laboratory. macrofaunal organisms sorted from the samples were preserved in % isopropanol, identified to the species level, and counted. the four replicates from each station were lumped and the total number of individuals per m for each species was calculated. the dead molluscan fauna was identified to species level. the water depth at the sampling sites ranged from to . depth. sediment characteristics. and exact location of the sampling sites are given in table . three sets of data: a) total living macrofauna species (numerical abundance), b) living molluscan species (numerical abundance), and c) dead molluscan species (presenceabsence), were treated separately in order to define zones of faunal similarity (biotope analysis). from the species list of each station. margalef's species diversity index was calculated: margalef. ) in n where s = the number of species and n = the total number of individuals. correlations were sought between the biotic parameters (number of species, number of specimens, diversity index) with the abiotic ones (sampling depth. substrate type) using spearman's nonparametric rank correlation coefficient (siegel. ). , ) . in seeking the "best map of the results derived by classification" (field n ul.. ), ordination techniques (mds: multidimensional scaling) were applied. other non-parametric statistics were applied where appropriate using the software package statgraphics. finally, r-mode analysis was used in order to define which species are responsible for the grouping of the q-mode analysis. classification and ordination were carried out using the program primer of the plymouth marine laboratory. a total of specimens belonging to taxa were identified from the living macrofauna and molluscan species from the dead material. among the species of living macrofauna, were molfmca; here clustering was performed separately. the species richness, abundance, and diversity per station is given in table : the number of species ranged from (station a ) to (station a ), and the number of specimens from -m- (station a ) to .m- (station a the dendrograms can be truncated at any level, but the areal presentation derived with mds based on the total living fauna (fig. a) indicated that the more justified separation in terms of ecological sense was at the groups level ( % similarity) (two dimensional stress = . ). the same separation was evident when environmental parameters were superimposed (fig. b) . taking into account: a) in siru observations, b) the faunal composition of each site, and c) the type of the substrate it is clear that the best classification is obtained with the first data set ( taxonomic units). the grouping of the second set ( taxonomic units) was not well defined and thus not mappable. finally, the third data set led to a classification somewhat similar to the first one ( ' taxonomic units). two of the groups were single site groups (stations a and a ). the other two groups delineated with the dendrogram of fig. are presented in fig. . these are: group , composed of stations located in the middle of the study area with substrate ranging from coastal detritic mud to coarse gravel, rich in detritus. this corresponds to the biotope of the de (muddy detritic assemblages) as defined by picard ( ). stations of group were the most coarse grained in the study area, with coralligenous substrates in their typical aspect along with the soft algae peyssonnelia and numerous bryozoa on concretions produced by organisms. the coralligenous assemblage is very well defined by p q r~ ( ) . the groups delimited with the dendrogram of fig. b are roughly the same as those of fig. . the only difference is that station a m , otherwise clustered in group , was not clustered here but remained as a single site group. similarly, stations a and a , north and south of milos island, remained as single site groups in both cases. the r-mode analysis, when total living fauna was considered, produced clusters of species (table ) table . species groups distinguished by inverse (r-type) analysis. the groups are based on the dendrogram of fig. table shows the results of the spearman rank correlation coefficient between the biotic parameters (number of macrofaunal species, number of specimens, and species diversity) and depth and sediment type. the ranking of sediment type was arbitrary, with rank for the coarser sediments (coralligenous with peyssonnefia) and rank for the finer ones (mud with detritus). tied observations were taken into account and the appropriate formula (siegel, ) applied. fig. on the other hand the correlation is rather weak (not significant statistically), but still positive as might be expected due to the sediment type. the coarser mixed sediments (coralligenous substrate with peyssonneliu, coralligenous + detritus) had a higher species diversity than the fine sediments (e. g., mud with detritus), a correlation well known from the literature (gray, the mann-whitney u-test applied between groups and , for all biotic and abiotic parameters (table ) , indicated significant differences for most of the parameters tested. thus, the average number of species and species diversity were significantly greater in the coralligenous assemblage (group l), where the depth was significantly shallower and the sediment coarser. differences in the mean number of individuals between the groups were not significant. species distribution may be seen as a response to varying effects of certain primary gradients such as depth, latitude, and current speed (pearson & rosenberg, ) . further it is affected by another suite of factors dependent on the primary gradients (e. g., physical factors) or independent of these (e. g., vulcanicity, pollution, biotic interactions). o n the cyclades plateau, a strong negative correlation (p < . ) was found between the biotic parameters (species richness, abundance, and diversity) and sampling depth. unfortunately facilities for obtaining critical hydrographic data were not available, so the interpretation of results is based largely on personal observations on board. the substrate description is also subjective since no sediment grain size analyses were performed. a weak correlation exists between the biotic parameters and sediment type. multivariate classification methods have been widely used in the last decade in order to distinguish distribution patterns in benthic ecology and paleoecology (ormerod, ; warzocha, ) . the results of this study, where standard multivariate classification methods were applied to various data sets from the same area, showed that caution must prevail in drawing conclusions from a limited set of data. the clearest separation into station groups was obtained by using total living fauna ( taxonomic units), the least clear using the living molluscan fauna ( taxonomic units). the classification derived from the dead mollusca ( taxonomic units) also gave clear station groups. in the analysis of the living molluscan data the groups failed to show any clusters. this is probably due to the effect of the impoverished sites such as stations a and a with and living mollusc species, respectively. on the other hand, the dead molluscs gave a pattern similar to that of the total fauna and showed a similar response to the environmental conditions of the area. according to powell er al. ( ) , death assemblages provide two distinct types of data: a) data on recent recruitment and mortality and b) data on long-term events provided by the accumulation of remains buried beneath the surface zone and indefinitely preserved. the clusters in the dendrograms of figs. and can be converted into patterns on the locality map. these distinct non-overlapping areas are biotopes that can be described in physical terms. indeed, the primary division is into depth groups. stations of group are all in depths below loom, while group consists of the three shallower stations above (range: -wm). at the same time, considering the traditional p~r & ( ) classification, two main biotopes can be distinguished in the cyclades plateau area: group , corresponding to the biotope of the muddy detritic assemblages (de) and group , representing the biotope of the coralligenous assemblage (c). usually, station grouping can be interpreted largely by sedimentary characteristics such as median grain size. this information is missing in our case, but sampling depth is no doubt a major factor in the distribution of species. the species clusters (table ) responsible for the grouping of stations do not consist of species characteristic of the above biocoenoses. amphiura filiformis, hyalinoecia bilineara, and notomastus larericeus, all found in high densities, are cosmopolitan species (table ) . on the other hand, species with a fidelity to groups were all encountered in low numbers. such species for the group stations are: asychb biceps, golfngia vulgaris, arnpelisca tenuicornis, nephthys incisa, polymnia nesidensis, nucula nucleus, and cidaris cidaris, with an average density of indiv. * m . group occupied a relatively small geographical area. the species characteristic of the coralligenous assemblage are: catapaguroides timidus as well as the bryozoa setosella vuherata and several species of scrupocellaria. the fact that two distinct data sets (subfossil assemblages and living communities) produce similar groupings when treated separately, leads to the hypothesis that the death assemblages form directly from the living communities. however, in the cyclades plateau, the living mollusc data alone ( species), taken in one sampling cruise, represent a poor data set as indicated by the results of the classical (bray-curtis / group average) classification. given that post-mortem transportation is negligible, then the death assemblage is an important source of data on the living community prior to sampling or when facilities for sampling and analysing living fauna are not available. proper use of these data requires knowledge of how death assemblages form from living communities. a quantitative analysis of the benthic fauna was carried out at stations in the cyclades plateau (aegean sea). taxa were identified from the living fauna and molluscan species form the dead material. a biotic parameters (n, s , d) were strongly related to sampling depth (p < . ), yet only a weak correlation was calculated between the biotic parameters and sediment type. the coarser sediments had a higher species diversity than the fine ones. multivariate analysis techniques were applied separately for the ecological and paleoecological data sets. the dendrogram based on total living fauna using bray-curtk / group average led to a satisfactory classification at the two group level. the same group division was delineated with the dead molluscan fauna. the least clear classification was derived from the living molluscan fauna. the two groups distinguished correspond to depth groups, i . e., the primary division is into stations below m (group ) and stations shallower than m (group ). considering the faunal composition of the stations, group corresponds to the biotope of the de benthic assemblage and group to that of the coralligenous assemblage according to the pbrbs ( ) classification. an ordination of the upland forest communities of southern wisconsin an introduction to numerical classification the abundance distribution and diversity of the molluscs of : a practical strategy for analysing multispecies distribution patterns a similarity measure sensitive to the contribution of rare species and its use in investigation of variation in marine benthic communities analysis of community attributes of the benthic macrofauna of frierfjord langesundfjord, and in a mesocosm experiment using similarity measures in benthic impact assessments quantitative re-evaluation of ecology and distribution of recent foraminifera and ostracoda of todos santos bay macrobenthos of sandy beach and nearshore environments at murrells inlet : distribution patterns of living and subfossil podocopid ostracodes in the nosy be area data manipulation in cluster analysis and the concept of zero presence the influence of habitat and seasonal regimes on the ordination and classification of macroinvertebrate assemblages in the catchment of the river wye feast and famine: structuring factors in marine benthic communities. ih symp nouveau manuel de bionomie benthique de la mer mediterranee. recl. trav. stn. mar. endoume recherches qualitatives sur les biocoenoses marines des substrats meubles effect of a large larval settlement and catastrophic mortality on the ecological record of the community in the death assemblage les peuplements benthiques (zoobenthos) de la region marseillaire: un essai d'analyse multivarite benthic molluscan fauna of the st. lawrence estuary and its ecology as assessed by numerical methods patterns of benthic copepod assemblages in an oligotrophic lake principles of numerical taxonomy new guinea. using numerical analysis benthic macrofaunal assemblages of slope habitats in the southern california borderland. occasional papers of the allan hancock foundation peuplements benthiques des substrats meubles du sud dc la mer egee. tethys classification and community structure of the macrofauna in the southern baltic. ices council meeting (collected papers). ices. copenhaven (denmark) macrobenthos-sediment relationships on the continental shelf off cape hatteras animal-sediment relationships in intertidal marine benthic habitats: some determinants of deposit feeding species diversity molluscan populations of the eden estuary. fife and the usc of numerical taxonomy methods to determine their distribution patterns key: cord- -c saxwfj authors: guzy, jacquelyn c.; mccoy, earl d.; deyle, anna c.; gonzalez, shannon m.; halstead, neal; mushinsky, henry r. title: urbanization interferes with the use of amphibians as indicators of ecological integrity of wetlands date: - - journal: j appl ecol doi: . /j. - . . .x sha: doc_id: cord_uid: c saxwfj . wetlands are ecologically and economically important ecosystems but are threatened globally by many forms of human disturbance. understanding the responses of wetland species to human disturbance is essential for effective wetland management and conservation. . we undertook a study to determine (i) whether anurans can be used effectively to assess the ecological integrity of wetlands affected by groundwater withdrawal and, if so, (ii) what effect increasing urbanization might have on the utility of anurans as wetland indicators. we monitored the intensity of anuran calls at wetlands in south‐western florida throughout – and – . . we first validated the use of anurans to assess wetland integrity using a small group of wetlands by comparing anuran calling and subsequent tadpole development with an established index employing vegetation composition and structure. we then verified that the results could be expanded to a variety of sites throughout the region. finally, we focused on urbanized wetlands to determine whether urbanization could interfere with the use of anurans to assess wetland integrity. . we used presence to estimate occupancy and detection probabilities and to examine the relationship between occupancy and five covariates expected to influence individual species occurrence. we used fragstats to calculate the mean proximity index for urbanized wetlands, which assesses the size and distribution of land use types within a specified area. . our results showed that the group of species including oak toad anaxyrus quercicus, southern cricket frog acris gryllus, pinewoods treefrog hyla femoralis, barking treefrog hyla gratiosa, and little grass frog pseudacris ocularis is a reliable indicator of wetland integrity. however, this same group of species, which is sensitive to wetland health, is selectively excluded from urbanized wetlands. . synthesis and applications. although anurans are effective indicators of wetland health and complement vegetation surveys, the usefulness of this group for monitoring the ecological integrity of wetlands can be substantially reduced, or eliminated, as a consequence of urbanization. we urge for careful consideration of confounding factors in any studies examining the utility of indicator species. are essential to the effective management of wetlands. the composition of amphibian assemblages is influenced by changes in physical conditions of wetlands, such as the alteration of hydrologic regimes (vickers, harris & swindel ; skelly ) . alteration of hydrologic regimes by excessive groundwater withdrawal has been shown to affect anurans (guzy, campbell & campbell ; bunnell & ciraolo ) , and their responses might be immediately noticeable in some species on a local scale, especially regarding the breeding effort. while calling surveys in a given year may not reflect the actual population size of anurans, which can often survive years of catastrophic reproductive failure (petranka et al. ) , they can reflect wetland condition, as reproductive effort is completely dependent on water. the composition and structure of wetland vegetation are often used as indicators of wetland degradation from excessive groundwater withdrawal (sonenshein & hofstetter ; edwards & denton ; ormiston et al. ) . excessive withdrawal can lower the surface of the aquifer, which in turn can lower the surficial aquifer and finally the level of inundation in wetlands (cherry, stewart & mann ; stewart ; swfwmd ) . many places that have the potential to suffer from excessive groundwater withdrawal have regulations to forestall the problem. in south-western florida, a large area with thousands of wetlands, the ecological condition of wetlands within all well fields must be assessed annually (swfwmd ) , typically using a vegetative health rating (vhr), which incorporates several botanical and physicochemical measurements to rate wetlands according to the level of ecological damage caused by groundwater withdrawal (gonzalez ) . although using a measure of vegetation composition and structure as an indicator of wetland condition is relatively easy, changes in vegetation are gradual (especially for shrubs and trees which may survive several poor years) and thus vhrs are indicators of relatively long-term effects. while changes in amphibian populations can also show long-term effects, amphibian calling activity coupled with the presence of tadpoles and near-metamorphs can give a current measure of wetland condition: disturbed wetlands may not attract breeding adults, and hydrology that is too altered will not sustain larval development through metamorphosis. as the use of groundwater resources increases in many places, a more rapid assessment of wetland condition is required, and anurans may be a valuable suite of indicators. furthermore, if they are equal or more effective as indicators, anurans could provide an easier, more cost-effective and faster assessment than vegetation; this may be important given rapid human development of certain areas world-wide. urbanization currently threatens more than one-third of the world's known amphibian species; the main threats are from habitat loss, fragmentation and degradation (hamer & mcdonnell ) . although some anurans move relatively long distances ( - ae km; lemckert ) , amphibians in general tend to have poor dispersal abilities (semlitsch ) and dispersal distances typically are < ae km (gibbs ; semlitsch & bodie ) . as urban sprawl increases, dispersal corridors are likely to be disrupted, reducing amphibian richness because of increased fragmentation and degradation of wetlands (and the upland matrix between); this restricts the potential for migration among wetlands and limits recolonization of species to wetlands from which they have been extirpated. thus, wetlands in urban settings have reduced amphibian species richness (e.g. delis, mushinsky & mccoy ) . our objectives were to (i) conduct a long-term study of cypress domes in south-west florida to evaluate the potential utility of anurans in providing a quick and reliable assessment of wetland decline resulting from groundwater withdrawal and (ii) to determine whether urbanization interferes with the ability to use anurans as indicators for excessive groundwater withdrawal. throughout - and - , we monitored calling anurans at cypress-dome wetlands, grouped within seven sites, in the tampa bay region of south-western florida (fig. a) . during and (study ), we monitored ( 'blue', 'green', and 'red'; see below for definition of these vhr ratings) wetlands at starkey wellfield (fig. a) , to determine whether wetland assessment based on calling activity of anurans was correlated with wetland assessment based on the vhr (gonzalez ; swfwmd ) . a vhr rating of 'blue' is assigned to wetlands with vegetation, hydrology and soils indicative of a naturally functioning wetland (i.e. high ecological integrity); of 'green' to wetlands with moderate changes in vegetative composition and zonation, hydrologic indicators, and soil subsidence ('moderately impacted'); and of 'red' to wetlands with severe changes in vegetative composition or zonation, treefall or death, soil oxidation or subsidence, and other biological evidence of hydroperiod reduction ('highly impacted') (gonzalez ; fig. b-d) . a naturally functioning wetland is saturated by surface or groundwater at a frequency and duration sufficient to support a prevalence of vegetation adapted to saturated soils (epa ). 'moderate' vegetative changes occur when transitional species move from edges of a wetland into the deep zone and 'severe' changes occur when upland vegetation moves into the deep zone (swfwmd ). each wetland was monitored for calling anurans (described below) five to seven times each year, between may and september. because intensity of calling anurans does not necessarily indicate the quality of wetland habitats for amphibian reproduction, we also sampled tadpoles in a subset of wetlands ( 'blue', 'green' and 'red') to verify that wetlands had a sufficient hydroperiod to allow recruitment of tadpoles to the terrestrial stage. each wetland was sampled for tadpoles using d-frame dip nets; up to -m sweeps were conducted in each microhabitat (vegetation or open water) proportional to the fraction of the total area each microhabitat covered. passive sampling with traps ( · · cm) was applied at each site with water at least ae m deep. tadpoles were identified to species and classified in gosner stage categories (gosner ) ; fish species and invertebrates (potential predators) were also recorded during dip-netting and trapping. each wetland was monitored for tadpoles six to seven times each year, between july and december. during and (study ), to determine whether the results of the earlier monitoring were generally applicable, we monitored a subset of four wetlands at starkey wellfield plus four wetlands each at three other sites (cypress creek, cypress bridge, and morris bridge; fig. a ), all actively pumped for groundwater and with varying levels of nearby urbanization. we also monitored four wetlands at another site, green swamp (fig. a) , with no groundwater withdrawal or surrounding urbanization. each of the wetlands was monitored times each year, between may and september. during , and , we monitored the same wetlands plus three additional wetlands each at two sites, dale mabry and sheldon (fig. ) , surrounded by extensive urbanization. each of the wetlands was monitored for calling anurans approximately one evening every weeks, targeting rainfall events, from the end of may until the middle of september for a total of nine surveys each year. call surveys were performed after sunset between ae - ae h and timed to coincide with the onset of calls in late spring and the end of the calling period in early fall. during each calling anuran monitoring event throughout the study, simultaneous call surveys were conducted by two experienced researchers listening for min from the centre of each wetland. all species present were listed, water depth was recorded and any differences in species observed were reconciled before leaving the wetland. calling activity was measured in chorus size categories based on urbanization interferes with the use of amphibians north american amphibian monitoring program (naamp ) guidelines, but refined to reflect the following six categories of numbers calling males: - , - , - , - , - and > . subsequently, the length of time that each wetland contained surface water (hydroperiod) was determined from water-level data recorded during call surveys, supplemented with data obtained from regulatory agencies. the influence of hydroperiod on amphibian occupancy is well-documented (e.g. snodgrass et al. ; beja & alcazar ) , and highly ephemeral wetlands generally contain species with rapid development and conspicuous feeding, but wetlands with a relatively long hydroperiod contain more established predator populations, and thus slower growing, less conspicuous feeding species utilize these wetlands. we measured a series of variables for each wetland in study , by building a geographical information system in arcmap, v . . (esri ), based on georeferenced digital : usgs geological orthophoto quarter quadrangle maps of aerial photographs and national wetlands inventory shapefiles (swfwmd ). these variables included ( ) distance to nearest study wetland (indication of possible spatial dependence), ( ) distance to nearest natural wetland, ( - ) per cent forest cover within , and m, and ( - ) mean proximity index (mpx) of forest cover within , and m; other measured variables included ( ) average hydroperiod and ( ) water depth at deepest point and ( - ) time (month, year or month plus year) as calling activity of anurans is strongly seasonal. one variable, mpx within , and m of the perimeter of each wetland, reflected the possible importance of urbanization and its consequent fragmentation. mpx assesses not only the amount of forest cover within a specified search radius, but also the distribution from clumped to uniform by measuring the isolation of each forest patch within a complex of forest patches (gustafson & parker ) . other metrics calculated within the same search radius (e.g. road density, proportion of impervious surfaces) do not take into account the spatial distribution. in our study, a 'patch' is defined as each individual occurrence of a particular land cover type (e.g. forest) in the landscape; the mpx approaches zero if a patch has no neighbours of the same patch type and increases as the neighbourhood is increasingly occupied by patches of the same type and as those patches become closer and more contiguous (or less fragmented) in distribution (gustafson & parker ) . the upper limit of the mpx is affected by the search radius and the minimum distance between patches. habitat area and isolation affect amphibians, and the degree of importance is a species-specific property reflecting a combination of life-history and behavioural characteristics (cushman ) . we averaged mpx over all patches surrounding , and m of the perimeter of each wetland and scaled the resulting value between and . large mpx values represent large forest patches close together, and small mpx values represent small, widely distributed forest patches. buffer zones were chosen based on potential amphibian dispersal distances (e.g. semlitsch & bodie ; lemckert ) . we used fragstats, v . (mcgarigal & marks ) to calculate mpx. to determine whether calling anurans (calling male index data and average number of species) were selecting wetlands with sufficient hydroperiod length, we used linear regression with statistica . (statsoft ) to predict tadpole response (number of species and individuals). we examined calling index data gathered during studies and for patterns of species' co-occurrence. groupings of species (using average calling index) and wetlands were identified with two-way cluster analysis, as implemented in pcord, v (mccune & mefford ) . the bray-curtis index was used as the distance measure, and the group average method was used for linking groups. to test whether resulting groups of species and wetlands (not identified a priori) were significantly different from each other in multivariate patterns of co-occurrence across wetlands, we used the sim-prof routine in primer, v (clarke & gorley ) . co-occurrence patterns for individual species and also the relationship between individual species and vhr rating were identified by calculating spearman rank correlations in statistica . (statsoft ) . although detection of a species confirms its presence, lack of detection does not necessarily confirm absence. thus, when detection probabilities are less than one, the true proportion of wetlands occupied should be estimated (e.g. mackenzie & kendall ). we used a single season mark-recapture-like approach , as implemented in presence, v . (hines ) , to estimate true proportion of sites occupied by each species for study . we modelled detection probability and site occupancy as a function of different covariates according to the methods described by . because collinearity between predictor variables can confound their independent effects, we calculated spearman rank correlation coefficients for all pairwise combinations of independent variables (hair et al. ); five of the exploratory habitat variables (mpx, hydroperiod, water depth, time and distance to next study wetland) had correlation coefficients ranging between ) ae and ae , with p-values above ae , and were included individually in subsequent modelling. the remaining inter-correlated variables were eliminated. because mpx was strongly correlated between each buffer zone, we chose mpx at the -m buffer for subsequent modelling as the more biologically relevant variable when considering typical dispersal distances for most anurans. the resulting set of candidate models for each species were composed of one covariate each: ( ) mpx within m of the wetland, ( ) distance to next study wetland, ( ) average hydroperiod (each site specific predictors of occupancy), ( ) water depth (survey specific predictor of detection) and ( - ) timespecific effects on detection (month, year or month plus year) for each species. model selection was based on akaike information criteria (aic), which was adjusted for overdispersion and small sample sizes (i.e. qaicc) (mackenzie & bailey ) . we computed delta qaicc and akaike weights to determine the strength of evidence for each model (burnham & anderson ) . because it was impractical to summarize aic results for all possible models for each species, we present the top - models and weights to gauge importance of each factor for each species. all continuous variables were standardized (i.e. the mean was subtracted from each value and then divided by the sd) before analysis. we assumed models with higher weights, and lower aic values were better able to explain variation in data and selected the models with substantial empirical support which included models within d qaicc (burnham & anderson ) and parameter estimates with standard errors not overlapping zero. we eliminated models where b coefficients of covariates were not supported. linear regression analysis of study data indicated a significant (p = ae ) positive relationship between average calling male size category and the average effort-corrected number of late-stage tadpoles at sites with increasing vhr score, and the model accounted for much of the variation (r = ae ; fig. ) . similarly, linear regression analysis indicated a significant (p = ae ) positive relationship between average number of calling male species and the average number of tadpole species at sites with increasing vhr score and the model accounted for much of the variation (r = ae ; fig. ). cluster analysis of the study data revealed two significantly (simprof, p = ae , p < ae ) different groups of species (hereafter referred to as group and group ; fig. ). group is composed of five species: the oak toad anaxyrus quercicus, southern cricket frog acris gryllus, pinewoods treefrog hyla femoralis, barking treefrog hyla gratiosa and little grass frog pseudacris ocularis. all five species in the group positively co-occurred (r s = ae - ae , all p-values < ae ). group is composed of six species: the southern toad anaxyrus terrestris, squirrel treefrog hyla squirella, green treefrog hyla cinerea, eastern narrow-mouthed toad gastrophryne carolinensis, pig frog lithobates grylio and southern leopard frog lithobates sphenocephalus. all six species in the group also positively co-occurred (r s = ae - ae , all p-values £ ae ). the remaining four species (southern chorus frog pseudacris nigrita, greenhouse frog eleutherodactylus planirostris, gopher frog lithobates capito and bullfrog lithobates catesbeianus) had limited occurrences and are not considered further. the occurrence of species in group exhibited a positive relationship with wetlands having a 'blue' vhr rating (r s = ae - ae , all p-values £ ae ), but the occurrence of species in group did not exhibit any relationship to a particular vhr rating (r s = ae - ae , all p-values ‡ ae ) (fig. ) . precisely the same two groups were identified by cluster analysis of studies and data together (simprof, p = ae , p < ae ), and, once again, species within each group positively co-occurred (r s = ae - ae , all p-values £ ae , group ; r s = ae - ae , all p-values £ ae , group ). blue wetlands clustered tightly together (simprof, p = ae , p < ae ; fig. ), but green and red wetlands did not group out reliably (fig. ). environmental variables differed markedly among wetlands (table ) . with the exception of the green swamp (control site), all wetlands were located on sites with active groundwater withdrawal. all wetlands with high mpx values (within m of the wetland) were located on large undeveloped wellfields or wilderness preserves and all were buffered from urban areas (fig. ) ; at morris bridge, starkey wellfield, cypress creek wellfields and the green swamp, the nearest urban land cover ranged from ae to ae km away. wetlands with low mpx values were located in areas immediately adjacent to urban land cover. we present the results for the eight species with enough detections to allow occupancy models to converge and also provide standard errors not overlapping zero. top models showed that occupancy varied by the inclusion or omission of mpx or water depth. the best models selected for the four of the five species previously identified as group (a. quercicus, a. gryllus, h. femoralis and p. ocularis) were very similar, as their pattern of co-occurrence would suggest; for all species, mpx within m was identified as the best predictor for occupancy. positive b coefficients of covariates and occupancy estimates for each of these group species indicate that occupancy increases with increasing mpx (table , fig. ). the best model for detection for each of these group species included a constant probability of occupancy over time ( table ) . the best models selected for four of the six species previously identified as group (a. terrestris, h. cinerea, g. carolinensis and l. grylio) were also very similar. water depth (a sample specific covariate) in study wetlands was the best predictor of detection for each of these species; positive b coeffi- cients of covariates for each of these group species indicate that detection increases with increasing water depth (table ; fig. ). the best model for occupancy for each of these species indicated a constant probability of occupancy over time ( table ) . probability of occupancy differed greatly between groups and . species in group had a higher probability ( ae - ae ) of occupancy in sites with no or low levels of urbanization (mpx ‡ ae ) compared to that of sites with high levels of urbanization (mpx = - ae ; fig. ). for species in group , probability of occupancy was not different in low, moderate and highly urbanized areas (i.e. no pattern was observed at differing mpx levels; table ). we identified a group of positively co-occurring species of anurans in cypress-dome wetlands that can indicate wetland health more rapidly than the commonly employed vegetative health assessment. the calling index of species in this group was highest in wetlands with the highest vhr. coincidentally, these (a) (b) fig. . two-way cluster analysis of average calling index of species by site, for the wetlands in study . horizontal dendrogram is of species (coded by first two or three letters of genus and species names); vertical dendrogram is of sites (coded at the end by the first letter of the vegetative health rating score). intensity of shading is proportional to the strength of the calling index. significantly different species and site clusters are identified with asterisks and dotted lines. next study site = distance (m) from study wetland to the nearest on-site study wetland (n = sites); next natural wetland = distance (m) from a study wetland to the nearest natural wetland; % forest = percentage of forest within , or m radius of perimeter of each study wetland; mpx = mean proximity index, amount and distribution of forest cover within , , and m of the perimeter of each study wetland; hydroperiod = mean number of days each wetland held water during sampling season each year; water depth = depth of water in each wetland at every sampling occasion. 'healthy' wetlands supported the greatest species richness of anurans and were the only wetlands supporting the entire suite of species in the group. because the intensity of calling anurans does not necessarily indicate the quality of wetland habitats for amphibian reproduction, we verified that these same wetlands had hydroperiods long enough to support tadpole development and found that the presence of late-stage tadpoles was strongly related to calling index. thus, for all subsequent studies, we used data from calling anurans. our results suggest that certain calling anuran species can provide a superior method (compared with vegetation indexing), for the assessment of ecological integrity of wetlands that is accurate, rapid and can be applied at a large number of wetlands simultaneously. calling surveys also appear superior to tadpole surveys, because they are substantially cheaper and less time-consuming for areas with large numbers of wetlands, and perhaps more reliable when overflow from permanent water bodies could act as a source of predatory fish. during our study, all wetlands were ephemeral, and any overflow events that introduced fish did so after tadpoles began metamorphing. the high degree of co-occurrence and predictive ability of the species in the group was maintained as the number of wetlands studied was increased. thus, these species can be considered 'sensitive' to wetland degradation, in this case resulting from excessive groundwater withdrawal. while amphibians can be useful indicators, it can be difficult to separate natural population variability from true population decline and thus anuran studies should be designed as longerterm efforts at multiple locations, particularly during droughts. in addition, adults may be in wetlands for a short time; however, their reproductive windows are well-established, generally easily targeted, and occupancy statistics can accommodate imperfect detection. common problems that occur when using both vegetation and amphibians as indicators include misidentification and difficulties identifying dormant-season vegetation and anuran larvae. wetland plants have many characteristics suited to assessments of biological condition, including their relative immobility, well-developed sampling protocols and, for herbaceous species, moderate sensitivity to disturbance (doherty et al. ) . however, wetland plants are unreliable as sole indicators of change in hydrologic regime (tiner ) , and soil biogeochemistry should be used with vegetation to minimize lag times in plant response to hydrologic alteration; this is often cost prohibitive. therefore, amphibians can be used as indicators to supplement ongoing vegetative and hydroperiod monitoring to provide a rapid response measure. drastic alterations of wetland hydroperiod often are a direct consequence of urbanization (azous & horner ) . to ensure the effects of urbanization could be separated from those of excessive groundwater withdrawal, we carefully selected our study wetlands so the two variables were not correlated. occupancy modelling of a diverse set of wetlands indicated that species considered sensitive to wetland degradation also were highly responsive to the mpx of land within a m buffer surrounding a wetland. for all wetlands, land cover within - m was a mix of urban (including highways, roads, housing developments, and commercial and industrial areas) and natural (including forest, uplands, wetlands, creeks and streams), and mpx is a measure of the degree of habitat (specifically forest) fragmentation within a specified buffer, which is caused by urbanization in our study system. thus, the same group of species also can be considered 'sensitive' to urbanization. it is not particularly surprising that the species in this group should be sensitive to urbanization, as they depend upon upland forest areas (open-canopied oak and pine flatwoods) near cypress swamps and wet prairies characterized by short hydroperiods (harper ; wright & wright ; hamilton ; duellman & schwartz ; pechmann et al. ; marshall & camp ) . in a previous study conducted in the same general area, three of the species, a. quercicus, h. femoralis, and h. gratiosa, only occurred in wetlands surrounded by an upland matrix of natural pine flatwoods and were excluded from areas of urban development (delis, mushinsky & mccoy ) during the breeding season. some anurans are not as sensitive to urbanization as others, such as species that are fully aquatic or that depend on non-forested upland habitat (cushman ) , but, for the group of sensitive species we have identified, it appears that adverse effects of habitat fragmentation accompanying urbanization (reduced mpx values which equate to reduced patch size and increased patch isolation) are severe. in contrast, species we identified within a second positively cooccurring group are associated with wetland edges (wright ; carr ; wright & wright ; conant & collins ) and thus are less sensitive to the structure of the surrounding habitat. finding that a group of species is sensitive to wetland degradation and also simultaneously sensitive to urban sprawl means that our ability to use these species as reliable indicators of wetland health is severely hampered in urban areas. as urban wetlands gradually deteriorate from excessive groundwater withdrawal, the species sensitive to this degradation gradually disappear because of urbanization itself. to our knowledge, no study has shown that a useful indicator of one type of anthropogenic stressor is rendered useless because of another type. this is not likely to be local or restricted to urban wellfields, but potentially could occur anywhere that indicator species are used to monitor ecosystem health or provide an early warning signal of decline. examples of these indicators include great lakes coastal wetlands, where the spring peeper pseudacris crucifer has been shown to contribute to assessment of anthropogenic disturbance (price et al. ) ; in mediterranean wetlands, where amphibian richness relates positively to watershed habitat heterogeneity and negatively to the percentage of the watershed area devoted to olive cultivation (garcı´a-mun˜oz et al. ); in mexican cloud forests, where groups of frog species are sensitive to fragmentation, diminishing canopy cover, and declining patch area (pineda & halffter ) ; in california streams, where three species of amphibians are sensitive to sedimentation (welsh & ollivier ) ; and in kentucky streams, where juveniles of several anuran species are sensitive to pcb (polychlorinated biphenyl) contamination (degarady & halbrook ) . when amphibians are used to monitor the effects of increasing sedimentation rates, chemical pollutants and nutrient loading, for example, but are also affected by other stressors, their reliability as indicators may be compromised. dealing with this conundrum will not be easy. in some areas, we may be able to use anuran species that are relatively tolerant of urban conditions (e.g. fragmentation) to gauge wetland integrity (e.g. groundwater withdrawal). although most species that are sensitive to habitat degradation are likely to be excluded from urban wetlands, a few may use both natural and urban wetlands. however, using species tied to ephemeral wetlands is difficult in urban areas where there are often few ephemeral ponds (rubbo & kiesecker ) . additionally, urban tolerant species with either extremely rapid or slow larval development would not be good candidates as indicators because those with rapid development might still call at a monitored wetland, but metamorph quickly enough to escape pressures of increased groundwater withdrawal, and covariates: mpx, mean proximity index; amount and distribution of forest cover within m of wetland; water depth, depth of water in each wetland at every sampling occasion; time varying, parameter built into models to account for differences in detection which vary by month, year, or month and year; ave hp; average hydroperiod of the wetland over the course of the study. aic, akaike information criteria. *difference in qaicc relative to the top model. †qaicc weight. ‡number of parameters in the model. §proportion of sampling units where the species was detected. ¶constant probability of detection. **constant probability of occupancy. urbanization interferes with the use of amphibians those with considerably slower larval development could call at the wetland but tadpoles might not survive excessive withdrawal. to select a reliable set of anuran species to monitor wetland health, we should be aware of their potential responses to a variety of potential stressors, not just the stressor of immediate interest. critical thresholds in habitat alteration are species specific and related to reproductive potential, dispersal ability, home range size and habitat specificity (e.g. fahrig ) . even within the same taxonomic group or species guild, there is no assurance that habitat changes affecting one species will be the same for other species in the group or guild (block, brennan & gutie´rrez ) . we conclude that while anurans are effective indicators and are complementary to vegetation, the usefulness of this group in monitoring wetland health can be substantially reduced, or eliminated, because they are also simultaneously sensitive to urban sprawl. we urge for careful consideration of confounding factors in any studies examining the utility of indicator species. wetlands and urbanization: implications for the future conservation of mediterranean temporary ponds under agricultural intensification: an evaluation using amphibians. biological conservation evaluation of guild-indicator species for use in resource management the potential impact of simulated ground-water withdrawals on the oviposition, larval development, and metamorphosis of pond-breeding frogs model selection and multimodel inference: a practical information-theoretic approach jr ( ) a contribution to the herpetology of florida general hydrology of the middle gulf area user manual ⁄ tutorial. primer-e ltd a field guide to reptiles and amphibians: eastern and central north america effects of habitat loss and fragmentation on amphibians: a review and prospectus using anurans as bioindicators of pcb contaminated streams decline of some west-central florida anuran populations in response to habitat degradation biological criteria for inland freshwater wetlands in florida: a review of technical and scientific literature ( - ) amphibians and reptiles of southern florida cross bar ranch wellfield ecological monitoring report arcinfo user's manual, version . . . environmental systems research institute inc how much habitat is enough? anthropogenic correlates of species richness in southeastern ontario wetlands wetlands classification for amphibian conservation in mediterranean landscapes importance of small wetlands for the persistence of local populations of wetland-associated animals biological indicators of wetland health: comparing qualitative and quantitative vegetation measures with anuran measures a simplified table for staging anuran embryos and larvae with notes on identification relationships between land-cover proportion and indices of landscape spatial pattern effects of hydrological alterations on frog and toad populations at morris bridge wellfield, hillsborough county multivariate data analysis, th edn amphibian ecology and conservation in the urbanizing world: a review notes on the ecology of the oak toad in florida. herpetologica a voice from the pines presence-software to estimate patch occupancy and related parameters a probability-based indicator of ecological condition frog communities and wetland condition: relationships with grazing by domestic livestock along an australian floodplain river odonates as biological indicators of grazing effects on canadian prairie wetlands variations in anuran movements and habitat use: implications for conservation assessing fit of site occupancy models how should detection probability be incorporated into estimates of relative abundance? estimating site occupancy rates when detection probabilities are less than one aspects of the feeding ecology of the little grass frog, pseudacris ocularis (anura: hylidae) pc-ord-multivariate analysis of ecological data fragstats: spatial analysis program for quantifying landscape structure protocol description: index and code descriptions annual comprehensive report: ecological and hydrological monitoring of the cypress creek wellfield and vicinity influence of wetland and hydroperiod on diversity and abundance of metamorphosing juvenile amphibians long-term persistence of amphibian populations in a restored wetland complex species diversity and habitat fragmentation: frogs in a tropical montane landscape in mexico are anurans of great lakes coastal wetlands reliable indicators of ecological condition florida wetland condition index for depressional forested wetlands amphibian breeding distribution in an urbanized landscape principles for management of aquatic-breeding amphibians biological criteria for buffer zones around wetlands and riparian habitats pond drying, predators and the distribution of pseudacris tadpoles relationships among isolated wetland size, hydroperiod, and amphibian species richness: implications for wetlands regulations vegetative changes in a wetland in the vicinity of a well field statistica data analysis software system hydrologic effects of pumping from the floridan aquifer in northwest hillsborough, northeast pinellas, and southwest pasco counties swfwmd (southwest florida water management district) ( ) establishment of recovery levels in the northern tampa bay area. hydrologic evaluation section: resource conservation and development department swfwmd (southwest florida water management district) ( ) data and maps swfwmd (southwest florida water management district) ( ) wetland assessment procedure manual the concept of a hydrophyte for wetland identification changes in herpetofauna resulting from ditching of cypress ponds in coastal plains flatwoods stream amphibians as indicators of ecosystem stress: a case study from california's redwoods life histories of the frogs of the okefinokee swamp, georgia. north american salientia (anura) number handbook of frogs and toads of the united states and canada we thank the southwest florida water management district for funding this research, and in particular t. rochow. we thank m. barrett for assistance in implementing fragstats and also thank two anonymous reviewers for helpful comments. key: cord- -lcog authors: pimentel, david; pimentel, marcia; wilson, anne title: plant,animal, and microbe invasive species in the united states and world date: journal: biological invasions doi: . / - - - - _ sha: doc_id: cord_uid: lcog approximately , plant, animal, and microbe invasive species are present in the united states, and an estimated , plant, animal, and microbe invasive species have invaded other nations of the world. immediately, it should be pointed out that the us and world agriculture depend on introduced food crops and livestock.approximately % of all crops and livestock in all nations are intentionally introduced plants, animals, and microbes (pimentel ). worldwide, the value of agriculture (including beneficial non-indigenous species) is estimated to total $ trillion per year. other exotic species have been introduced for landscape restoration, biological pest control, sport, and food processing, also contributing significant benefits. calculating the negative economic impacts associated with the invasion of exotic species is difficult.for a few species, there are sufficient data to estimate some impacts on agriculture, forestry, fisheries, public health, and the natural ecosystem in the us and worldwide. in this article, we estimate the magnitude of the economic benefits, and environmental and economic costs associated with a variety of invasive species that exist in the united states and elsewhere in the world. some impacts on agriculture, forestry, fisheries, public health, and the natural ecosystem in the us and worldwide. in this article, we estimate the magnitude of the economic benefits, and environmental and economic costs associated with a variety of invasive species that exist in the united states and elsewhere in the world. the value of the us food system is more than $ billion per year (uscb (uscb - , and the value of the world food system is estimated at more than $ trillion per year. according to the world health organization (pimentel a) , the world's food system is not providing adequate amounts of food for all people on earth, more than . billion of the current population of . billion being malnourished. in addition, food production per capita has been declining each year for the past years (faostat (faostat - . this assessment is based on cereal grains, since cereal grains provide about % of the world's food. clearly, more needs to be done to increase food production per capita, at the same time significantly reducing the rate of growth of the world population . most plant and vertebrate animal introductions in the us and world have been intentional, whereas most invertebrate animal and microbe introductions have been accidental. during the past years, the total number of introductions of all species has nearly doubled in the world. the rate of introductions of exotic species has increased enormously because of high human population growth, rapid movement of people, and alteration of the environment everywhere in the world. in addition, significantly more goods and material are being exchanged among nations than ever before, creating greater opportunities for unintentional introductions (uscb (uscb - . some of the estimated , species of plants, animals, and microbes that have invaded the us, and , species of plants, animals, and microbes that have invaded the total world ecosystem provide significant benefits but also many types of damage to managed and natural ecosystems, as well as public health. most exotic plant species now established in the united states and elsewhere in the world were introduced for food, fiber, or ornamental purposes. an estimated , introduced plant species have escaped and now exist in us natural ecosystems (morse et al. ) , compared with a total of approximately , species of native plants (morin ) . in florida, of the approximate , alien plant species (mostly introduced ornamental species), more that have escaped and become established in neighboring natural ecosystems (frank et al. ; simberloff et al. ). more than , plant species have been introduced into california, and many of these have escaped into this natural ecosystem as well (dowell and krass ) . worldwide, an estimated , species of exotic plants have been intentionally introduced as crops, and have escaped to become established in various natural ecosystems. most of the non-indigenous plants that have escaped and become established have adapted well to the favorable living conditions characteristic of moist tropical regions in countries such as india, brazil, and australia. some of the invasive plants established in the us and world have displaced native plant species. in the united states, introduced plant species are spreading and invading approximately , ha of us natural ecosystems per year (babbitt ) . for instance, the european purple loosestrife (lythrum salicaria), which was introduced in the early th century as an ornamental plant (malecki et al. ) , has been spreading at a rate of , ha per year, strongly altering the basic structure of the wetlands that it has invaded (thompson et al. ) . stands of purple loosestrife have reduced the abundance of native plant species, and endangered many wildlife species, including turtles and ducks (gaudet and keddy ) . loosestrife is present in states, and about $ million are spent each year for control of the weed (attra ). many of these exotic species have become established in national parks. in the great smokey mountains national park, for example, of the , vascular plant species are exotic, and of these are currently displacing and threatening native plant species (hiebert and stubbendieck ) . the problem of introduced plants is particularly serious in hawaii, where of the total of , plant species on the island are non-indigenous (elredge and miller ) . in some cases, one exotic plant species may competitively overcome an entire ecosystem. in california, the yellow starthistle (centaurea solstitalis), for example, dominates more than million ha of northern grassland in the state, resulting in the total loss of this once productive forage system (campbell ) . in addition, the european cheat grass (bromus tectorum) is dramatically altering the vegetation and fauna of many natural ecosystems in the western us. cheat grass is an annual that has invaded and spread throughout the shrub-steppe habitat of the great basin in idaho and utah, predisposing the altered habitat to fires (kurdila ) . before the invasion of cheat grass, fire burned once every to years, and shrubs in the region had a chance to become reestablished. currently, fires occur once every to years, and this has led to a decrease in shrubs and other vegetation, and the occurrence of monocultures of cheat grass on more than million ha in idaho and utah. the reason that the alteration of original vegetation is so significant is that all the animals and microbes that were dependent on the original vegetation have been reduced or totally eliminated. insufficient information exists concerning invasive plants in the united states and other countries. this is true even in countries that are dominated by invasive plants, such as the british isles. for example, of the , total plant species on the british isles, only , species are considered native (crawley et al. ) . more than % of alien plant species in the british isles are established in disturbed habitats (clement and foster ; crawley et al. ) . one group of agriculturalists introduced species of plants as potential forage species in australia (lonsdale ). only species of this group of plant species turned out to be beneficial, many others had little impact, but several became serious pest weeds in australia. in india, weeds are estimated to cause a % loss in potential crop production each year (singh ) , amounting to about $ billion in reduced crop yields. assuming that % of the weeds in crops are alien (nandpuri et al. ), the total cost associated with the alien plants in india is about $ . billion per year. about mammal species have been intentionally introduced into the united states, including dogs, cats, horses, cattle, sheep, pigs, and goats (layne ). several of these mammal species escaped into the wild, and have become pests by preying on native animals, grazing on native vegetation, or intensifying soil erosion. goats (capra aegagrus hircus), for instance, introduced on san clemente island, california, have caused the extinction of endemic plant species and have endangered others (kurdila ) . several small mammal species, especially rodents, have been introduced into the united states. these include the european rat (rattus rattus), the asiatic rat (rattus norvegicus), the house mouse (mus musculus), and the european rabbit (oryctolagus cuniculus; layne ). some of the introduced rats and mice have become particularly abundant and destructive on farms. on poultry farms, there is about rat per chickens (smith ; d. pimentel, unpublished data) . using this ratio, it is estimated that the rat number is more than . billion on farms in the us. another million rats are estimated to be in homes and stores in cities and towns. if it is estimated that each rat causes $ in damages each year, then the damage per year would be about $ billion. although the cost of the impact of invasive mammals is relatively high, the percentage of alien mammals introduced into the united states is relatively low, or %; in the united kingdom, the percentage is relatively high, or % . the uk introduced mammals include those species recorded in the us, plus many others. australia is another nation that has a large number of alien mammals. in australia, pigs native to eurasia and north africa were introduced and now number from to million (emmerson and mcculloch ) . feral pigs cause soil erosion, damage agricultural crops, fences, native plants and animals, and are a threat to livestock and humans; they also spread various animal diseases, including tuberculosis, brucellosis, rabies, and foot-and-mouth disease (lever ) . the estimate of pig damage in australia is more than $ million per year (emmerson and mcculloch ) . rodents, including the european and asiatic rats and the house mouse, have invaded all countries in the world. in addition, domestic dogs, cats, and european rabbits have been introduced into all nations of the world. in australia, feral cats are a serious problem, killing native bird, mammal, marsupial, and amphibian populations. the estimate is that there are million pet cats, and million feral cats in australia (anon ) . the cats are considered responsible for having exterminated native australian species of animals (low ) . assuming that each bird has a minimum value of $ in the us (pimentel et al. ) , then the total impact from cats in australia is $ million per year. in the us, it is estimated that cats kill an estimated million birds per year, with an estimated damage of $ billion (pimentel et al. ) . of the , species of birds in the united states, nearly are exotic (temple ) . approximately % of the introduced birds are beneficial, such as the chicken. one of the bird pest species is the english sparrow (passer domesticus), introduced in into the us for the control of canker worm and other pest caterpillars (roots ) . by , english sparrows were reported to be a pest, consuming wheat, corn, and the buds of fruit trees (laycock ). in addition, they harass native birds, including robins, baltimore orioles, and the yellow-billed and black-billed cuckoos, and they displace bluebirds, wrens, purple martins, and cliff swallows (long ) . english sparrows are also associated with the spread of about human and livestock diseases (weber ) . one of the most serious bird pests is the common pigeon (columbia livia), which has been introduced to all cities in the world (robbins ) . pigeons present a nuisance because they foul buildings, statues, cars, and sometimes people, and they feed on grains (smith ) . it is estimated that pigeons cause an estimated $ . billion in damages per year in the united states. they also serve as reservoirs and vectors of more than human and livestock diseases, including parrot fever, ornithosis, histoplasmosis, and encephalitis (long ) . another serious bird pest in the us is the european starling (sturnus vulgaris), a species that in some cases occurs at densities of more than one per hectare in agricultural regions (moore ) . they are capable of destroying as much as $ , worth of cherries ha - in the spring (feare ). they also destroy large quantities of grain crops (feare ) . the estimate is that they are responsible for damages amounting to $ million per year (pimentel et al. ) . information on other bird species that have invaded other nations is not as abundant as one would expect. of the other nations, the uk has some of the best data. of the species of birds in the uk, are alien (gooders ) . pigeons in the uk are as serious a problem as they are in the us. in the uk, pigeons are estimated to cause more than $ million in damages each year (alexander and parsons ; bevan and bracewell ). about species of amphibian and reptile species have been introduced into the united states. these species invasions have all occurred in the warmer regions. for example, florida is host to species (lafferty and page ) . the negative impacts of these invasive species have been enormous. the brown tree snake (boiga irregularis) is one of the worst. it was introduced into the us territory of guam immediately after world war ii, when military equipment was transferred to the island (fritts and rodda ) . the snake population reached high densities of snakes ha - , and dramatically reduced populations of native bird species, small mammals, and lizards. a total of bird species and lizard species were exterminated from guam (rodda et al. ) . the brown tree snake also eats chickens, eggs, pet birds, and causes major problems to farmers. in some cases, the snake enters houses and bites small children in cribs and playpens (ota ) . another costly impact is that the snake is causing power failures by damaging electric transformers. the estimate is that the brown tree snake causes more than $ million in damages per year on guam.a major worry is that the snake will invade hawaii, and cause major extinctions of birds, mammals, and amphibians on the island. an estimated species of reptiles and amphibians exist in australia (fox ) . however, only two of these are exotic. one of the introduced species is the cane toad (bufo marinus), introduced from south america for insect con-trol in cane fields. however, it was soon reported to be a serious pest (fox ) . the cane toad is poisonous to dogs, cats, and other mammals (sabath et al. ) . in south africa, there have been species of reptiles and species of amphibians introduced (siegfried ). one of the invasive species is the red-eared slider (chrysemys scripta elegans) that was introduced from north america. this invasive turtle has become a major threat to the native turtle species (boycott and bourquin ) . a total of invasive fish species have been introduced into the united states (courtenay et al. ; courtenay ) . most of the invaders are found in the warmer regions such as florida, which has at least of these species (courtenay ) . introduced fish species frequently alter the ecology of aquatic ecosystems. in the great lakes, for instance, nearly invasive species are found, and these invaders are causing an estimated $ billion in damages to the fisheries per year (pimentel ) . in addition, most of the alien fish species in south africa are regarded as pests (bruton and van as ) . in total, alien fish species are responsible for the reduction or local extinction of at least species of fish in south africa (bruton and van as ). an estimated , arthropod species (more than , species in hawaii alone, and more than , in continental us) have been introduced into the united states (ota ) . approximately % of these introductions were accidental, the remainder being intentional for purposes of biological control and pollination. about , invasive species of insects and mites are crop pests in the us. introduced insects account for % of the crop insect pests in hawaii (beardsley ) . approximately % of the insect and mite pests in crops in continental us are pests of agricultural crops. the major group of pests consists of native insects and mites that switched from feeding on native vegetation to feeding on crops (pimentel et al. ) . pest insects are estimated to destroy $ billion worth of crops per year. one ant species, the red imported fire ant, is alone causing $ billion in damages and control costs (linn ) . of the species of invasive species in us forests, about % are now serious pests in these forests (liebold et al. ) , causing about $ billion in losses each year (hall and moody ) . a new introduction, the asian longhorn beetle, is threatening maple and ash trees in new york and illinois (hajek ) . of the , species of insects, and , species of spiders and numerous other arthropod species that exist in south africa, several invasive species are causing problems (south africa ). one of the most serious invaders is the argentine ant (linepithema humile), which is destroying native vegetation, including endangered plants (macdonald et al. ). this ant species is also negatively affecting native ants and other beneficial arthropod species. in addition, the argentine ant is a serious pest in agriculture. a total of about species of mollusks have been introduced and established in united states aquatic ecosystems (ota ). the two most serious pest species introduced are the zebra mussel, dreissena polymorpha, and the asian clam, corbicula fluminea (see also chaps. and ). the zebra mussel was introduced from europe, and probably gained entrance via ballast water released into the great lakes by ships traveling from europe (benson and boydstun ) . the mussel was first noted in lake st. clair, has spread into most of the great lakes and most aquatic ecosystems in the eastern united states, and is expected to invade most freshwater habitats throughout the nation. large mussel populations (up to , m ; griffiths et al. ) reduce food and oxygen for the native fauna. zebra mussels have been observed covering native mussel, clams, and snails, and threatening the survival of these and other species (benson and boydstun ; keniry and marsden ) . in addition to ecological effects on other aquatic organisms, the zebra mussel also invades and clogs water intake pipes in water infiltration and electric power plants. it is estimated that the mussels will cause $ billion in damages and associated control costs in the us. in the great lakes alone, they are reported to cause $ billion in damages and control costs (pimentel ) . although the asian clam grows and disperses less quickly than the zebra mussel, it also causes significant damage to native organisms and damage to water filtration plants and electric power plants. costs associated with this animal are estimated to be more than $ billion per year (ota ) . in various us coastal bay regions, the introduced shipworm (teredo navalis) is estimated to cause from $ million to $ million in damages per year (cohen and carlton ; d. and m. pimentel, unpublished data) . unfortunately, there are not data available on mollusk invaders in other nations. this is due to the general lack of knowledge concerning the ecology and systematics of mollusks in the world; they appear to be causing a relatively small amount of damage to aquatic ecosystems in other regions worldwide, and/or few biologists have investigated these organisms. for a start, it should be pointed out that the majority of livestock worldwide are introduced species. for example, in the united states more than % of the livestock species are introduced (pimentel b ). microbial and other parasitic organisms have generally been introduced when the livestock species have been introduced. in addition, to the more than species of pest microbes and other parasitic species that have already invaded the united states (pimentel ) , there are more than additional microbes and other parasitic species that could easily invade the united states and become serious pests of us livestock (pimentel ) . a conservative estimate of the losses to us livestock from exotic microbes and other parasitic species is more than $ billion per year. australia already has several species of alien diseases infecting and causing losses to livestock. in addition, there are an estimated exotic diseases in other regions of the world that could infect australian livestock, if they were introduced (meischke and geering ). at present, alien insect and mite species already cause $ million per year damage to the wool and sheep industry (slater et al. ) . in india, there are more than exotic species of disease and parasitic organisms that are causing major problems for the introduced livestock and native wildlife. already present in india is the serious foot-and-mouth disease. recently, it was reported that there were more than , cases of footand-mouth disease (foot-and-mouth disease leak ), treatment costs being about $ , per year. south africa also reports problems with introduced livestock pests. the exotic diseases include tuberculosis, brucellosis, east coast fever, anthrax, and rinderpest. estimates are that brucellosis alone is causing livestock losses of more than $ million per year (coetzer et al. ) . in brazil and other latin american countries, imported bovine tuberculosis has become a serious threat to the beef and dairy industry. these losses are estimated to be about $ million per year (cosivi et al. ). various influenza virus types, originating mostly in the far and near east, have quickly spread to the united states and other nations in the past. recent disease epidemics have been associated with sars, and now there is the major threat of bird flu that is infecting some people in the far and near east. the current influenza strains are responsible for nearly % of all human deaths in the us (uscb (uscb - . the costs of hospitalization for a single outbreak of influenza, such as type a, can exceed $ million per year. one of the most notorious of all alien human disease is hiv/aids. the pathogen is reported to have originated in east africa, probably from some species of monkey. the disease now occurs in all parts of the world. the costs of treatment of hiv/aids in the world today are estimated to be $ billion per year. in addition to influenza and hiv/aids, there are numerous other diseases infecting humans in various parts of the world. these include syphilis, lyme disease, and tuberculosis. these diseases are causing an estimated $ billion in losses and damages per year. new influenza strains in the uk are reported to cause from , to , deaths per year (kim ) . in total, both influenza and hiv/aids claim the lives of more than , people per year. the treatment costs are in excess of $ billion per year. influenza and tuberculosis in india are reported to cause more than million deaths per year (kim ) . several non-indigenous human diseases threaten people in south america. these diseases include hiv/aids, influenza, malaria, cholera, yellow fever, and dengue. more than million people are infected per year, associated with more than $ billion in damages and treatment costs per year. the number of invading species worldwide has been increasing rapidly, an estimated tenfold increase having been recorded in the past years. some countries with a rapidly increasing population, growing population movement, and increasing global trade, such as the united states, are suffering a greater problem from invaders than is the case for other nations. approximately , species of plants, animals, and microbes have invaded the nations of the world, with about , in the us alone. it must be pointed out that, for all nations combined, about % of all these species were intentionally introduced as crops and livestock. unfortunately, an estimated - % of the introduced species are, or have become, pests and are causing major environmental problems. although relatively few of these species become really serious pests, some species do inflict significant damage to natural and managed ecosystems, and cause serious public health problems. various ecological factors help exotic species become abundant and emerge as serious ecological threats in their new habitat. these factors include exotic plant and animal species being introduced without their natural enemies (e.g., purple loosestrife); the existence of favorable predator-prey conditions in the new habitat (e.g., for house cats); the development of new associations between alien parasites and hosts (e.g., hiv/aids and humans); the occurrence of disturbed habitats that promote invasion by some species (e.g., crop weeds); the occurrence of favorable, newly created artificial habitats for invasives (e.g., cheat grass); and the occurrence of species-specific traits promoting invasion by highly adaptable alien species (e.g., the water hyacinth and zebra mussel). this investigation reports on various economic damages associated with invasive species in various nations of the world that total more than $ . trillion per year (pimentel ) . this amounts to about % of the world gnp (uscb (uscb - . unfortunately, precise economic costs associated with some of the most ecologically damaging species of invasives are not available. for example, cats and pigs have been responsible for the extinction of various animals, and perhaps some plants. for these invasive animals, however, only minimal cost impact data are available. in addition, it is impossible to assess the value attached to various species that have been forced to extinction. if economic values could be assigned to species forced to extinction, then in terms of losses in biodiversity, ecosystem services, and esthetics, the costs of destructive invasive species would be extremely high. the value of $ . trillion cited above already suggests that exotic species are extracting major environmental and economic tolls worldwide. as mentioned above, - % of all crop and livestock are introduced species. these alien crops (e.g., corn and rice) and livestock (e.g., cattle and poultry) are vital to maintaining world agriculture and the food system. the food system has an estimated value of $ trillion worldwide. however, these benefits do not compensate for the enormous negative impacts of exotic pest species. a real challenge lies in preventing further damage from invading exotic species to natural and managed ecosystems of the world. this is especially true in view of rapid population growth and increasing global trade. the united states has taken a few steps to protect and prevent the invasion of exotic species into the nation. many governments of other nations have taken, and are taking, additional steps to combat non-indigenous species. evidently, it is being increasingly recognized that investing a few million dollars to prevent future introduced species from invading a country, where they might cause billions of dollars worth of damage and control costs, is worthwhile. specific laws are needed in all nations to diminish or prevent invasive species introductions. all introductions of exotic species of plants, animals, and microbes -for whatever purpose -should be strictly regulated. in addition, governments should make efforts to inform the public concerning the serious environmental and economic threats that are associated with the invasion of exotic species. introducing a new species into a nation for the control of a plant, animal, or microbe pest invasive species is sometimes criticized as being a hazardous technology. in the past, where vertebrate species such as mammals, amphibians, birds, and fishes were introduced for biological control, several became pests themselves (chaps. and ). for instance, the indian mongoose, introduced for rat control in the west indian islands and hawaiian islands, and the english sparrow, introduced into the us for caterpillar control, have both turned out to be disasters. however, introductions of insect species, such as the vedalia beetle rodolia cardinalis into the us, and of a virus species for the control of the european rabbit in australia, have been notable successes. controls of cacti in australia, knapweed in the us, and the cassava mealy bug in africa, all employing biocontrol insects, have also been successful. the first response after detecting an invasive pest in a country should be to immediately travel to the country of origin of the pest, and attempt to introduce natural enemies of the pest. this is sometimes successful, but not always. there have been almost as many successful biological controls employing new associated biocontrol agents. in new associated biocontrol, the biological control agents are sought from a related species of the pest invasive in another country. the new association biocontrol agent offers an ecological advantage because the biocontrol agent has never interacted with the invasive pest species, and often this advantage makes the new biocontrol agent highly pathogenic to the invasive pest species. the advantage of biological controls is that they reduce the invasive pest species without the need for using pesticides in the new ecosystem, and with minimal or no damage to the new ecosystem (hokkanen and pimentel ) . details on the pros and cons of biological control are given in chap. . pathogenicity for chickens of avian paramyxovirus type i isolates obtained from pigeons in great britain - rare plants protect cape's water supplies purple loosestrife: public enemy # on federal lands statement by secretary of the interior on invasive alien species introductions of arthropod pests into the hawaiian islands our living resources: a report to the nation on the distribution, abundance, and health of u.s. plants, animals, and ecosystems chlamydiosis in birds in great britain. . isolations of chlamydia psittaci from birds sampled between and faunal invasions of aquatic ecosystems in southern africa, with suggestions for their management killer pigs, vines, and fungi: alien species threaten native ecosystems alien plants of the british isles cape town cohen an, carlton jt ( ) nonindigenous aquatic species in a united states estuary: a case study of the biological invasions of the san francisco bay and delta zoonotic tuberculosis due to mycobacterium bovis in developing countries nonindigenous fishes appendix . exotic fishes of the united states and canada. in: robins cr (ed) a list of common and scientific names of fishes from the united state and canada comparative ecology of the native and alien floras of the british isles exotic pests pose growing problem for california numbers of hawaiian species: supplement , including a review of freshwater invertebrates queensland's introduced plants and animals. queensland parliamentary library, brisbane faostat ( - ) quarterly bulletin of statistics. fao, rome feare cj ( ) the economics of starling damage conserving biodiversity: impact 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united states: there goes the neighborhood predation of the endangered tidewater goby, eucyclogobius newberryi, by the introduced african clawed frog, xenopus laevis, with notes on the frog's parasites nonindigenous species naturalized animals: the ecology of successfully introduced species ant warfare: science fights fire with flies: natural enemy central to effort to limit spread of the insects. usa today (nation) introduced birds of the world: the worldwide history, distribution, and influence of birds introduced to new environments feral future: the untold story of australia's exotic invaders ecology and management of biological invasions in southern africa biodiversity: man is one among million species bird problems in agriculture. the british crop protection council our living resources: a report to the nation on the distribution, abundance, and health of us plants, animals, and ecosystems our living resources: a report to the nation on the distribution, abundance, and health of us plants, animals, and ecosystems harmful non-indigenous species in the united states biological invasions: economic and environmental costs of alien plant, animal, and microbe species agriculture: changing genes to feed the world livestock production and energy use aquatic nuisance species in the new york state canal and hudson river system and the great lakes basin: an economic and environmental assessment in: dorf r (ed) technology, humans and society: toward a sustainable world environmental and economic costs of non-indigenous species in the united states ecological and economic threat of alien plant, animal, and microbe invasions in the world update on the environmental and economic costs associated with alien-invasive species in the united states our living resources: a report to the nation on the distribution, abundance, and health of us plants, animals, and ecosystems the disappearance of guam's wildlife easteal s ( ) expansion of the range of the introduced toad bufo marinus in australia - preservation of species in southern africa nature reserves strangers in paradise years of crop science research in india. indian council of agricultural research an investigation into the effects of redlegged earth mite and lucerne flea on the performance of subterranean clover in annual pasture in s producers need not pay startling "rodent tax" losses books in soils, plants, and the environment: stored-grain ecosystems south africa government online exotic birds, a growing problem with no easy solution spread, impact, and control of purple loosestrife (lythrum salicaria) in north american wetland. us fish and wildlife service, and fish and wildlife research united states statistical abstracts health hazards from pigeons, starlings and english sparrow: disease and parasites associated with pigeons, starlings, and english sparrows key: cord- -axrv zfg authors: o'brien, stephen j. title: genomic prospecting date: journal: nat med doi: . /nm - sha: doc_id: cord_uid: axrv zfg the importahce of preserving biodiversity extends beyond the discovery of new drugs to understanding how other species have dealt with medical problems we currently face. there is no shortage of incurable diseases. cancer, autoimmune disease, emerging viruses and parasites, degenerative pathologies like arthritis and alzheimer's disease and over , hereditary diseases drive an us$ billion national institutes of health budget to resolve the physiologic bases, to clone the genes and to move beyond symptomatic treatment for fatal and debilitating conditions that fil our hospitals. the human genome project offers the prospect of identifying the genes affecting maladies from breast cancer and diabetes to werewolf-hair pattern and bipolar neuropathies. and another, largely untapped cache of genetic information has begun to emerge from an unexpected source: the genomes of free-ranging vertebrate populations that have acquired adaptations to abrogate or suppress the same diseases. over per cent of the species that ever existed have gone extinct or failed some evolutionary challenge. today's surviving species are descendants of the winners of ancient struggles who have evohred genetic defenses to infectious agents, cancers, and degenerative disease. included in the genomes of all species are the genetic footprints stephen j. o'brien (nci) maintains a large program that tests thousands of extracts of rare plants, fungi, microbes and marine life for anticancer and anti-hiv properties every products or their derivatives. of the aesthetic, ethical and practical justifications for species conservation, the preservation of heretofore undiscovered natural product medicinals is to many the most convincing. there are other potential medical benefits to species conservation in addition to pharmaceutical discovery. medically relevant information), recognizing that far more await discovery. the gene complex with the greatest extent of between-individual last april, a conference jointly sponsored by the smithsonian institution, the national science foundation and the national institutes of health met to explore the critical role of biological diversity on human health*. there was much discussion on the value of 'natural products,' the cornucopia of compounds discovered in the diverse biota with beneficial pharmaceutical properties. the national cancer institute *biodiversity and human health, april - , , washington, dc, usa. year'. this search for natural medicines, termed 'chemical prospecting' by thomas eisner of cornell university has led to identification of more than active compounds -from aeroplysinin to zthyhydroxy benzene -that have medicinal activity. the economic and human rationale of conserving earth's biodiversity for medical potential alone was emphasized by francesca grifo (american museum of natural history), who noted that nine of the ten top-selling pharmaceuticals on the market today are natural allelic variation in all mammals is the major histocompatibility complex (mhc), hla in humans'. stretched across two million base pairs on the short arm of human chromosome six, the hla region consists of a collection of about seventy coding genes, of which fifteen mediate immune recognition and clearing of infectious disease agents. three hla class i, ten class ii, and two tap genes encode over polymorphic alleles that provide the 'self' context or recognition signature for t cells to mount cellular and humoral (antibody) nature medicine, volume l, number , august • responses to invading pathogens. the purpose for this incredible genetic diversity had been a puzzle to immunologists and geneticists for many years. an important clue comes from a natural history study of the world's fastest land animal, the mrican cheetah ( fig. ). difficulty in breeding cheetahs in zoos prompted a multidisciplinary study that showed cheetahs as a species to be remarkably inbred, having a reduction of to per cent of overall genomic diversity based on molecular genetic markers•. perhaps most surprising was the demonstration that unrelated cheetahs would accept reciprocal skin grafts indicating immunological identity at the cheetah's mhc. the cheetah's genes were likely homogenized by a near extinction event followed by inbreeding in the species' ancestry. when cheetahs became exposed recently to feline infectious peritonitis virus, a coronavirus with low morbidity and mortality in domestic cats (about per cent affected), per cent of infected cheetahs developed symptoms (diarrhea, jaundice, depression, fever) and per cent died • the cheetah's homogeneous sensitivity to this agent in the face of genetic monomorphism at the mhc provided a rationale for the critical importance of mhc genetic variation in natural populations. when pathogens genetically circumnavigate the immune defence of an individual host, genetic diversity of immune response genes, particu- inbred individuals that teeters on the very edge of extinction in the big cypress swamp ecosystem in south florida•. the consequences of inbreeding are dramatic: ( ) an average of per cent developmentally abnormal sperm per ejaculate, ( ) an increase to per cent incidence of cryptorchidism (heritable defect causing one or two testicles to remain undescended) over the past years, ( ) per cent have heart murmurs, ( ) an abrupt appearance of an atrial septal heart defect (patent foramen larly the mhc in the population, provide a 'moving target' that would protect the species from widespread affliction. by contrast, a genetically uniform population like the cheetah loses this advantage and may suffer far more casualties. the cheetah example is frequently quoted as a natural verification of the adaptive value of high mhc and immune gene polymorphism. human demographic history has some dramatic parallels, particularly the loss of some million native americans (themselves genetically diminished) from infectious disease introduced by european contact•. a different moral for the consequences of a demographic contraction and inbreeding was demonstrated in the florida panther (f. concolor coryi, see fig. ) , a relict puma subspecies of to highly nature medicine, volume , number , august ovale, a common congenital human disorder) that has been fatal in at least three panthers and (s) an enormous microbial/ parasite load. the florida panther's genetic legacy proved so compelling that the u.s. department of interior decided to genetically augment the fragile population with a different puma subspecies, f. c. stanleyi. the florida panther experience illustrates rather graphically the critical role that rare genetic variants can have on reproductive characteristics and on congenital heart abnormalities in wildlife species and in humans. some fascinating insight about lethal infectious disease and natural genetic defences appeared from a wild mouse population in a squab farm near lake casitas, forty miles north of los angeles' • murray gardner (university of california at davis) discovered that the mice were suffering a raging epidemic with a retrovirus that caused fatal hindlimb paralysis. but only about per cent of the lake casitas mice became infected even though the virus caused per cent mortality when inoculated into laboratory mouse strains. the reason that per cent of the lake casitas mice were virus-free was the presence of a powerful trans-dominant restriction gene, fv- , that blocked infection of the retrovirus. fv- was subsequently shown to be a truncated version of the exogenous retrovirus. integrated into chromosome of resistant mice was a portion of retroviral pol, a full length env and '-ltr retroviral genes. in lymphoid cells of protected mice the env portion produced an env glycoprotein that bound and blocked the murine retroviral receptor, thereby conferring resistance to infection. the lc mice had acqu red a natural evolutionary solutio to a fatal paralytic retroviral disea e. in the presence of the retroviral pathogen, the fv- gene protected its carriers and their offspring, representing a remarkably effective genomic adaptation. chromosomally integrated endogenous retroviral genomes have been described in many mammal species including humans, and we have always suspected that they represent 'genomic fossils' of historic pathogenic outbreaks"· • at least . per cent of the human genome is related to retroviral genomes. it is likely that all of these were acquired by infection into the germ line of a developing embryo during an outbreak that conferred endogenous immunity to the carriers. the wild lake casitas mouse population and functional retroviral integrations seen in other species are now considered to be the paradigm for acquisition of endogenous retroviral sequences. expression of endogenous retroviral genes in humans is rare • any role they may play in disease resistance is only speculative until an interaction with pathogenic human if endogenous retroviral sequences originate as germline infections of naive populations during an outbreak of an exogenous counterpart, is the 'endogenization' process rapid or are there intermediate steps? a hint of an answer can be obtained by considering immunodeficiency viruses in free-ranging species. hiv- , the aetiologic agent for aids, causes a slow drop in cd -bearing tlymphocytes and has led to collapse of the immune system, opportunistic infections and death in over million people. the cosmopolitan hiv- and the less virulent west african hiv- strain erupted in human populations within the past few hundred years • hiv origins are likely to have derived from simian immunodeficiency virus (siv), a virus that is endemic in free-ranging sooty managabey, green monkey and other cercopithecus african monkey species. sly-infected african monkeys do not develop immunodeficiency disease; yet siv is quite virulent because it does induce aids when inoculated into asian macaques, species that have never seen siv in their native range". the simplest explanation for the balance between siv and african monkeys would be that they are descendants of an historic diseaseinducing outbreak that effectively selected genetically resistant survivors to repopulate subsequent generations. a nearly identical scenario has been uncovered with feline immunodeficiency virus (fiv) and wild cat species. fiv was discovered in an immunocompromised domestic cat and, like hiv, induces immune deficiency and death among domestic cats". fiv has been found in sixteen non-domestic cat species (including puma, leopards, lions, and cheetahs) sampled for antibody and genomic sequences••·". however, the fiv in wild cats, like the african monkeys, shows little evidence for immune suppression, associated disease or mortality. since viral genomic sequences are highly divergent within and between different cat species, we suspect that the virus is relatively old, perhaps coexisting with wild cat species for thousands of years. because retroviral sequence evolution is even less clocklike (in a molecular evolutionary sense) than gene sequences of host species, it is difficult to be precise about the timing. nevertheless, the pattern of fiv divergence is generally monophyletic within each species (as if virus in each species entered that species ·news & views only once and subsequently evolved), supporting the notion that fiv persistence in the felids is rather ancient. like the monkeys, the cat species may have achieved an evolutionary balance whereby disease is postponed at least until after the reproductive years. domestic cats, which do become ill, may simply represent a recent emergence of fiv in a heretofore naive species. is there an evolutionary explanation for the maintenance of a chronic exogenous fiv infection in wild cats? is there an adaptive advantage to carrying the virus once historic adaptation has muted its pathogenesis? the answer could involve a temporary acquisition of immune protection against pathogenic fiv which may be present in low frequency or be produced spontaneously by rapid leotiviral mutation. wild cats infected by an attenuated fiv strain would be immunized naturally against exposure to a virulent strain. an extended period of exogenous virus/host commensalism or symbiosis may provide the time period required for a genomic 'endogenization' event to occur. although primates and cats have abundant endogenous retroviral sequences, none of these are lentivirus-related. with passenger lentiviruses so common among mammals, it may be simply a matter of time before the birth of endogenous lentivirus sequences in the free-living species. finally, with the recent development of high resolution comparative gene maps of primate, cat, and other mammalian species, the genetic basis of lentivirus resistance can now be approached'". the prospect of genetic engineering to treat human pathologies will be a principal emphasis of twenty-first century medicine. the burgeoning biotechnology industry was born to accomplish this hope. harold varmus, director of nih, has moved to review gene therapy research and development as a high priority for medical research. in , david baltimore of the massachusetts institute of technology coined the term 'intracellular immunization' to describe the delivery of virus-restricting genes to cells of sensitive or infected patients as a genetic therapy to virus disease • attempts to apply this to aids are beginning, but to be honest we really do not know which genes to use. the answer may be lurking in wild monkeys, cats, gazelles, woodchucks or armadillos. molecular biologists and disease gene hunters need only to befriend a field biologist, a naturalist or a wildlife veterinarian. there is little question that timetested and selectively improved natural solutions to our own medical ills await discovery, and with the rapid advances in medical biotechnology the tools to recognize them are now available. the potential benefits of chemical and genomic prospecting are dazzling, because they promise both to reveal naturally tested solutions for difficult health conditions and to contribute to conservation of the earth's biodiversity. chemical prospecting: hope for van· ishing ecosystems chemical prospectors scour the seas for promising drugs a role for molecular genetics in biological conservation genetic and phylogenetic analyses of endangered species c:enomic structure and function in the mhc genetic basis for species vulnerability in the cheetah prevalence and implications of feline coronavirus infections of captive and freeranging cheetahs (acinonyx jubatus). f. virol why did they die? the consequences of demographic reduction and genetic depletion in the endangered florida panther the lake casitas wild mouse: evolving genetic resistance to retroviral disease the contributions of retroviruses to the study of mammalian evolution endogenous human retroviruses. in the retroviridae the simian immunodeficiency viruses pathogenic diversity of simian immunodeficiency viruses isolation of a t-lymphotropic virus from domestic cats with an immunodefi· clency-like syndrome worldwide prevalence of lentivlrus infection in wild feline species: epidemiologic and phylogenetic aspects a lion lentivlrus related to feline immunodeficiency virus: epidemiologic and phylogenetic aspects. f. virol anchored reference loci for comparative genome mapping in mammals intracellular immunization laboratory of viral carcinogenesis national cancer institute frederick key: cord- -a emso authors: coghlan, megan l.; white, nicole e.; parkinson, liza; haile, james; spencer, peter b.s.; bunce, michael title: egg forensics: an appraisal of dna sequencing to assist in species identification of illegally smuggled eggs date: - - journal: forensic sci int genet doi: . /j.fsigen. . . sha: doc_id: cord_uid: a emso psittaciformes (parrots and cockatoos) are charismatic birds, their plumage and capacity for learning make them highly sought after pets. the illegal trade in parrots and cockatoos poses a serious threat to the viability of native populations; in addition, species transported to non-endemic areas may potentially vector disease and genetically ‘pollute’ local native avifauna. to reduce the logistical difficulties associated with trafficking live birds, smugglers often transport eggs. this creates a problem for authorities in elucidating accurate species identification without the laborious task of incubation and hand rearing until a morphological identification can be made. here, we use avian eggs seized from carriers coming into and within australia, as a result of suspected illegal trade. we investigate and evaluate the use of mitochondrial dna (mtdna) to accurately identify eggs to family, genus or species level. however, identification of a species based on percentage mtdna similarities is difficult without good representations of the inter- and intra-levels of species variation. based on the available reference database, we were able to identify % of the eggs to species level. of those, species from eight genera were detected, all of which belong to the parrot (psittacidae) and cockatoo (cacatuidae) families. of the remaining %, a further % of eggs were identified to genus level, and % identified to family level using our assignment criteria. clearly the lack of validated dna reference sequences is hindering our ability to accurately assign a species identity, and accordingly, we advocate that more attention needs to be paid to establishing validated, multi locus mtdna reference databases for exotic birds that can both assist in genetic identifications and withstand legal scrutiny. psittaciformes (parrots and cockatoos) are charismatic birds, their plumage and capacity for learning make them highly sought after pets. the illegal trade in parrots and cockatoos poses a serious threat to the viability of native populations; in addition, species transported to non-endemic areas may potentially vector disease and genetically 'pollute' local native avifauna. to reduce the logistical difficulties associated with trafficking live birds, smugglers often transport eggs. this creates a problem for authorities in elucidating accurate species identification without the laborious task of incubation and hand rearing until a morphological identification can be made. here, we use avian eggs seized from carriers coming into and within australia, as a result of suspected illegal trade. we investigate and evaluate the use of mitochondrial dna (mtdna) to accurately identify eggs to family, genus or species level. however, identification of a species based on percentage mtdna similarities is difficult without good representations of the inter-and intra-levels of species variation. based on the available reference database, we were able to identify % of the eggs to species level. of those, species from eight genera were detected, all of which belong to the parrot (psittacidae) and cockatoo (cacatuidae) families. of the remaining %, a further % of eggs were identified to genus level, and % identified to family level using our assignment criteria. clearly the lack of validated dna reference sequences is hindering our ability to accurately assign a species identity, and accordingly, we advocate that more attention needs to be paid to establishing validated, multi locus mtdna reference databases for exotic birds that can both assist in genetic identifications and withstand legal scrutiny. ß elsevier ireland ltd. all rights reserved. [ , ] . another detrimental consequence is the threat to human and native species health, with the introduction of disease [ ] [ ] [ ] . the wildlife trade has already been associated with the spread of disease in humans, as in , with the outbreak of severe acute respiratory syndrome (sars), linked to a local wildlife market in china [ , ] . several other infectious diseases affecting avian species that are of concern include newcastle disease, psittacosis, and avian influenza virus. these diseases not only threaten the health of native avian species but also pose a threat to human public health [ , ] . in light of the detrimental ramifications of the illegal avian trade, it is imperative that global measures are taken to intercept, reduce and eliminate trafficking. many federal customs services play an active and extremely important role in the interruption of such trade at country borders. however, it is often difficult for authorities to detect all of those in possession of wildlife material owing to the sheer volume of travellers and to the fact that traffickers are skilled at concealing their cargo. it is common for illegal bird traffickers to transport eggs prior to hatching, as they are seemingly less cumbersome to transport than live animals [ ] . the use of purpose-built body vests to prevent damage to the bird eggs, maintain incubation, while also aiding concealment, has been observed recently in cases of illegal trafficking into australia (see fig. ) [ , ] . the seizure of eggs introduces one further challenge for detection authorities: correct species identification. a robust species identification is of critical importance in the success of wildlife forensic prosecutions [ ] . without the ability to identify the species of seized wildlife material, the likelihood of conviction and appropriate sentencing of traffickers may be greatly diminished [ , ] . correctly identifying avian species using eggshell morphology alone can be difficult. in these types of cases, genetic identification can be used as a tool when morphological features cannot be relied upon to accurately assign a species to a given wildlife sample [ ] . however, this method of species identification must rely on the successful dna extraction from eggshell or egg contents, including membrane, tissue or blood vessels where an embryo is/was present (see fig. ). recent work has shown that dna can be successfully extracted from eggshell membranes and from the eggshell itself, even in highly degraded samples. trimbos et al. [ ] determined that it was possible to obtain high quality dna from eggshell membranes of the black-tailed godwit. the results of the study showed that in % of eggshell membrane samples, nuclear dna could be successfully extracted and amplified for use in genotyping, and that the quality of the extracted dna was comparable to that extracted from blood samples [ ] . likewise oskam et al. [ ] successfully developed a method for extracting ancient dna from fossil eggshell $ , years old, illustrating the dna in the eggshell matrix is stable for extended periods of time. in wildlife forensics, dna species identification is commonly carried out by amplifying and sequencing fragments of the mitochondrial dna (mtdna) genes cytochrome oxidase i (coi), cytochrome b (cytb), or s ribosomal rna ( s) [ , , , ] . this method of species identification relies on the existence and availability of reference sequences having been deposited on a publicly accessible database, such as genbank [ ] , to enable a close match between the known sequence and that of the query sequence. in this study, bird eggs of unknown species identity were obtained from the australian customs and border protection service (acbps) ( eggs), and the department of water, heritage and the arts (dewha) ( eggs) between and . the aim of the study was to gain an insight into the target species being illegally smuggled into, or traded within, australia. lastly we feel there is a need to explore the viability of dna based species identification approaches, including the use of genbank and the barcode of life database (bold), and to develop criteria for cases involving egg seizures. ninety-nine eggs were received for this study through collaboration with the acbps and the dewha. sixty-two eggs were received from the acbps after being autoclaved for min at c ($ psi), as per the australian quarantine and inspection service (aqis) requirements. thirty-seven eggs were received from dewha, which had not been autoclaved. all samples received were stored in our secure and quarantine approved facility at murdoch university. samples in this study have been stripped of case details as they have been, or are being, used in legal proceedings. all egg samples were processed in a designated dna clean room for sample preparation and dna extraction. each sample was photographed, and a small piece of eggshell was cut out using sterilised scissors and forceps to expose the interior. depending on the stage of development of the egg, either a small piece of tissue from the embryo was cut away with scissors and placed into a ml eppendorf tube or, if no tissue could be accessed, a small piece of membrane or blood vessel was cut out with scissors and placed in a were then placed on a rotating oven and digested overnight ($ h) at c. all samples, post-overnight digestion, were centrifuged for min at , rpm. ml of supernatant was mixed with ml of qiagen pbi buffer (ca, usa) and transferred to a qiagen spin column and centrifuged for min at , rpm. two wash steps followed (qiagen awi buffer and awii buffer) prior to elution of dna from the spin column membrane with ml of mm tris (ph . ). all extracted dna was quantified via real-time quantitative pcr (qpcr; applied biosystems [abi]), using generic avian primers targeting the s rrna mitochondrial gene ( sa forward primer -ctgggattagataccccactat- , sh reverse primer -ccttgacctgtcttgttagc- ) [ ] . samples were assessed for quality and quantity of mitochondrial dna using three dna dilutions (neat, / , / ), not only to assess if successful isolation of dna was achieved, but also to identify the presence of pcr inhibition. the optimal dna concentration, free of inhibition [ ] was used for further analysis. briefly, pcr was conducted in ml reactions using abi power sybr master mix together with . mm of sa and sh primers and cycled at c genomic dna dilutions identified with cycle threshold (ct) values between and were determined to be ideal for subsequent pcr analysis. genomic samples with low dna yields (i.e. ct values greater than was used as an arbitrary cut-off) were excluded from further analysis in order to ensure the fidelity of the data and reduce the likelihood of both contamination and dna damage. the mitochondrial genes s ( sa/ sh primers, as above) [ ] , and cytb were pcr amplified (mcb cytb forward primer -taccatgaggacaaatatcattctg- and mcb cytb reverse primer -cctcctagtttgttagggattgatcg- ) [ ] , using the following pcr reagent concentrations in a ml total volume including ml of dna (as screened by qpcr): . mm magnesium chloride,  taq polymerase buffer, . mm dntps, . mg bsa, . mm of each primer, and . ml of amplitaq gold (abi). pcr conditions included: initial denaturation at c for min, followed by cycles of c for s, - c for s, c for s, and a final extension at c for min (corbett research, nsw, australia). pcr products were visualised on a % agarose gel to confirm the correct amplicon size. all successfully amplified pcr products were purified using a qiaquick purification kit (qiagen), and sequenced in both directions using abi big dye chemistry on a xl genetic analyser (abi, macrogen). cytb and s mtdna loci were selected in this casework primarily due to better database coverage of the psittaciformes (see discussion). due to the fact that the taxonomic identifications of these seized eggs are uncertain, we regard it as being counter-productive to upload them onto genbank. however, the sequence data can be requested directly from the authors. comparative sequence searches were conducted using the blastn portal within the national centre for biotechnology information (ncbi) database, to identify a matching sequence of high similarity. several factors were considered upon interpretation of the search results. firstly, the percentage similarity between the ncbi reference and query sequence were required to be high. a threshold value of > % was used for cytb and s genes, a similar value has been employed in dna barcoding approaches using coi [ ] . although the % cut-off is somewhat imprecise, such limits are required unless a detailed knowledge of the inter-specific and intra-specific variation exists for each mtdna gene. secondly, the number of reference sequences available for each gene ( s and cytb) was considered, together with a determination as to whether reference sequence(s) had arisen from published work (as opposed to direct submissions to genbank which has circumvented peer review). thirdly, the number of available reference sequences for other closely related species was also an important consideration, because if a group (e.g. cockatoos) had been genetically well characterised, the chance of misidentifying a sample as a closely related species decreased. the difficulty in accurate species assignments was confounded when only one reference sequence was available; in addition to only a few sequences for closely related species for comparative inter-species purposes. it would be difficult to argue, in a legal context, with any degree of certainty that a sample be assigned to a species without the knowledge of the genetic variability of closely related taxa. therefore, species level identification was only assigned to those samples when more than one published reference sequence was available for at least one of the two chosen genes for this study, or alternatively, when at least one published sequence was available for both s and cytb genes. we chose a sequence similarity value of > % for both genes as a requirement for assigning species identity, but acknowledge that more research needs to occur to generate gene specific cut-off values that are appropriate to the psittaciformes. in cases where only one species reference sequence was available on genbank, regardless of whether the sequence similarity was > %, species level identification was not assigned, due primarily to the effects that nuclear copies of mtdna (numts) might have. additionally, if the only reference sequence available was from an unpublished source, species identification was not assigned, as we believe they were not deemed fit for this purpose. genus and family level identification was allocated when search results showed close matches (< %) to more than one species, involving taxa that were classified within the same genus or family respectively. our criteria, through necessity, were conservative to minimise the chance of incorrect species assignment. however, it may be likely that each genetic identification needs to be handled on a case-bycase basis and may ultimately become a judgement call based on experience, available evidence, and the quality of reference collections, which are largely the same criteria that a morphological taxonomist might use to identify species. one final complicating factor is the ever-changing nature of the taxonomic framework in which the genus, species and subspecies status of avian taxa can change in response to new data. dna was successfully extracted from of the egg samples ( %). of the autoclaved eggs, only two failed to yield sufficient quantities of dna for further analysis. of the eggs that were not autoclaved, seven extracts were found to contain an insufficient quantity of dna to proceed with species identification. the autoclaving process deemed necessary by aqis, while likely to cause dna shearing was not identified as an impediment to dna amplification, at least for mtdna amplicons identified here. of the samples that underwent successful dna extraction, we sequenced two mitochondrial genes (cytb and s) that resulted in the identification of ( %) of the samples to species level (as judged by our criteria). of all of the samples identified to species level, one was a cites appendix i, critically endangered yellowcrested cockatoo (cacatua sulphurea; table ). a total of species were identified, with the majority of samples assigned to blackcapped lory (lorius lory) and eclectus parrot (eclectus roratus) ( table ) [ ] [ ] [ ] [ ] . all of our egg samples were identified to the order psittaciformes ('true' parrots, new zealand parrots, and cockatoos). the family level identification comprised % of the successfully extracted samples to psittacidae ('true' parrots), and cacatuidae (cockatoos; table ). approximately one third of egg samples ( %) could only be identified to genus level, and included the following genera: aprosmictus, lorius, eos, trichoglossus, pyrrhura, cacatua, and psittaculirostris. table shows the result of a survey of the number of currently available reference sequences for cytb, s, and coi mitochondrial genes that were found on genbank at the time of this study, for selected psittaciformes species. the percentages of the selected species that have reference sequences for these mtdna genes on genbank are listed. the species that were chosen in this survey are those that are popular in the international exotic pet trade [ ] . the lories and lorikeets appeared to have the least coverage of available reference sequences, while the neotropical parrots had the best sequence coverage. of note is the fact that the coi gene, while already widely known as the 'dna barcoding gene' [ ] , is currently not well represented in genbank (or the bold database) for many exotic parrot and cockatoo species, compared with other species that are often encountered in the wildlife trade. in this study we chose to employ the cytb and s mtdna genes for species identification over that of coi for three reasons. ( ) cytb has a long history of use in avian phylogenetics and as a result is one of the better represented genes for avian species on genbank [ ] (see tables and ). ( ) in a comprehensive analysis of mammals using kimura -parameter distances, cytb was shown to provide a greater ability than coi for separating species based on sequence data [ ] . ( ) . the s and cytb amplicons (approximately bp and bp respectively) were deemed a better choice than coi (amplicon of bp) for samples that may have been degraded during the autoclaving process. in light of the limited coverage of reference sequences for certain psittaciformes species on genbank, it was decided that using two mtdna genes, instead of just one, was more beneficial for the purpose of species identification. the benefit of using two mtdna genes in this study to adequately identify each sample was evident, as in some instances only one gene was able to provide species level identification that fit our criteria. another advantage of using two mtdna genes for species identification is that it is possible to encounter the problem of numts, in which case, the chances of correctly identifying the species of the sample are improved with the use of multiple genes [ , ] . in fact we recently detected a nuclear copy in genbank for the palm cockatoo [ ] where the published version of the cytb gene [ ] is completely inconsistent with our multi-locus phylogenetic analysis. the presence of numts is at times difficult to detect, but can be investigated by translation of the sequence to identify stop codons, indels in coding sequences and variation in rd codon positions [ ] . in our opinion the use of multiple genes also provides additional confirmation of correct species identification free of numts as similar tree topologies should be observed when the dna is modelled. in the field of wildlife forensics, species identifications of biological remains are one of the most commonly requested tests for dna labs [ ] , yet surprisingly, there are very few studies that systematically describe, and defend the criteria that were used to assign species. the recent publication by the international society for forensic genetics (isfg) presents a number of recommendations regarding dna testing of non-human samples [ ] . we would advocate that two further recommendations be added to this list: firstly, that dna quantification (qpcr) should be table samples identified to species level, conservation status, and available reference sequences [ , , [ ] [ ] [ ] . cytb: cytochrome b gene; s: mitochondrial s ribosomal rna gene; coi indicates the commonly used barcoding gene cytochrome oxidase [ ] ; bold, barcode of life database [ ] . eclectus parrot (eclectus roratus) ii (least concern) melanesian islands, indonesia, australia blue and yellow macaw (ara ararauna) ii (least concern) south america black-capped lory (lorius lory) ii (least concern) new guinea dusky lory (pseudeos fuscata) ii (least concern) indonesia and png ( a ) rainbow lorikeet (trichoglossus haematodus) ii (least concern) australasia ( a ) eastern rosella (platycercus eximius) ii (least concern) australia ( a ) black-capped conure (pyrrhura rupicola) ii (least concern) south america umbrella cockatoo (cacatua alba) ii (vulnerable) indonesian islands sulphur-crested cockatoo (cacatua galerita) ii (least concern) australia and new guinea ( performed as mtdna identifications are, like human microsatellite profiling, equally susceptible to effects of low copy number dna and inhibition. secondly, that more than one mtdna gene should be sequenced to reduce the chance of incorrect assignments due to the presence of numts. ultimately, the ability to make accurate species assignments based on mtdna sequences is underpinned by the coverage and quality of dna reference databases, together with the accuracy of the taxonomic framework. without a comprehensive knowledge of intra-and inter-species variation for the mtdna loci used for species identification, regardless of percentage sequence similarity, there still may be doubt as to the accuracy of taxonomic assignments [ ] . importantly, if mtdna sequence data are to be used in a legal context it is vital that reference sequences are fit for purpose and have passed the scrutiny of peer review. the use of 'unpublished data', or sequences without taxonomic provenance could compromise the fidelity of dna based species identifications. the peer-review process, while it may not always ensure data fidelity, is still widely regarded as the best available test of scientific rigour and has long been used to establish the credentials of expert witnesses. the alternative is to instigate an auditing procedure to verify both the specimen and the sequence data that it is derived from, in the same way that coi dna sequence data is scrutinised prior to addition to bold [ ] . this task does, however, require considerable resources. clearly there is a need for a diverse and highly reliable dna reference database to use in species identity testing for wildlife forensic casework. an ideal dna reference database should contain reference sequences that have been identified based on a solid taxonomic framework, and not where doubt still surrounds a species identity with that of closely related taxa [ ] . such a database should have a large number of individuals for each species and be available for more than one mtdna gene so that a true indication of intra-specific variation can be assessed. as clearly highlighted in table , there is an obvious deficiency of reference sequences in which all three mtdna genes ( s, cytb and coi) are present, both in genbank and in bold for the highly sought after avian species involved in the illegal trade. this disconcerting lack of reference samples for many parrot and cockatoo species may be in part due to the fact that psittaciformes comprise approximately species [ ] compared with other fauna of forensic importance, such as elephant (in regard to the ivory trade), of which there are only a small number of species [ , ] . this in itself presents a challenging task of sourcing adequate reference material, with provenance, for each species to build a comprehensive and robust dna database in which to facilitate the process of dna species identification. difficulties in sourcing new reference material can cause substantial delays to the development of reference sequence data as, somewhat ironically, our laboratory routinely struggles to obtain reference samples (e.g. feathers or dna) from overseas as cites legislation does not allow the movement of body parts or derivatives without extensive documentation. nevertheless, the task of compiling a 'fit for purpose' database should be systematically approached where prioritising commonly poached and traded species should be the logical first step. utilisation of pre-existing sequence data that has already been satisfactorily audited, as in bold, could be used in conjunction with new dna sequence data that is yet to be generated, for other mitochondrial genes. gaining information on which species are commonly targeted for the wildlife trade can, in some cases, be surveyed. recently a study was undertaken to survey the bird markets of indonesia over a -year period [ ] . it was found that much of the trade was conducted illegally, yet was carried out in open markets with the perception that the risk of being charged was minimal, despite there being legislation in place that prohibits this trade. interestingly, the list of surveyed parrot and cockatoo species seen in the market included many of the same species identified in this study (table ) , including the critically endangered yellowcrested cockatoo [ ] . with surveys such as this, information gained on which species are being targeted could be used to prioritise the acquisition of reference material for the development of a dna database, which could be utilised by wildlife forensic and conservation biologists with the aim of conserving avian species and more effectively prosecuting individuals involved in illegal wildlife trafficking. to enforce the highest penalties for illegal wildlife trafficking, accurate identification of cites listed wildlife material is essential. it is apparent that species identity testing from eggs using mtdna is a valuable tool and it is clear from the data presented here that caution needs to be exercised when making species identifications based solely on one mitochondrial locus. a focus on establishing dna reference databases for the most commonly traded wildlife species will assist in forensic casework. the ability for dna (mtdna, snp's and microsatellites) to assist in species identification and population assignment (as is currently practiced in monitoring the ivory trade) will not only help to prosecute wildlife traffickers, but may also help in the conservation of threatened and endangered species. we advocate that there is an urgent need to establish validated international databases for avian species of the parrot and cockatoo families, which are becoming increasingly threatened by illegal removal from the wild and by habitat destruction. international illegal trade in wildlife: threats and us policy dealing with the clandestine nature of wildlife-trade market surveys the international wildlife trade organised crime: a review of the evidence the role of the an overview of international wildlife trade from southeast asia wildlife across our borders: a review of the illegal trade in australia reducing the illicit trade in endangered wildlife: the market reduction approach heritage and the arts, wildlife trade and conservation: epbc act list of threatened fauna reducing the risks of the wildlife trade invasive species, environmental change and management, and health biological invasions at the gene level summarizing the evidence on the international trade in illegal wildlife wildlife trade and the emergence of infectious diseases black market for wildlife: combating transnational organized crime in the illegal wildlife trade animal origins of sars coronavirus: possible links with the international trade in small carnivores species identification using a small nuclear gene fragment: application to sympatric wild carnivores from south-western europe a novel strategy for avian species and gender identification using the chd gene a novel strategy for avian species identification by cytochrome b gene using eggshell membranes as a dna source for population genetic research fossil avian eggshell preserves ancient dna dna detective: a review of molecular approaches to wildlife forensics species identification using the cytochrome b gene of commercial turtle shells dna from museum specimens evaluation of real time pcr amplification efficiencies to detect pcr inhibitors novel universal primers establish identity of an enormous number of animal species for forensic application dna mini-barcoding: an approach for forensic identification of some endangered indian snake species geneious v iucn red list of threatened species psittacidae bold: the barcode of life data system the bird trade in medan, north sumatra: an overview validation of the barcoding gene coi for use in forensic genetic species identification validation of cytochrome b sequence analysis as a method of species identification reconstructing mammalian phylogenies: a detailed comparison of the cytochrome b and cytochrome oxidase subunit i mitochondrial genes many species in one: dna barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified the evolutionary history of cockatoos (aves: psittaciformes: cacatuidae) phylogenetic relationships within parrots (psittacidae) inferred from mitochondrial cytochrome-b gene sequences isfg: recommendations regarding the use of nonhuman (animal) dna in forensic genetic investigations an overview to the investigative approach to species testing in wildlife forensic science dna barcoding: error rates based on comprehensive sampling ivory identification by dna profiling of cytochrome b gene elephants-a conservation overview we gratefully acknowledge the support of the australian customs and border protection service (acbps), the department of environment, water, heritage and the arts (dewha) and the west australian department of environment and conservation (dec) in supplying samples for this study. we are grateful for funding from the arc future fellowship scheme (ft to mb), the robert hammond research studentship (to nw) and the animal research institute at murdoch university. key: cord- -yqc r ll authors: luis, angela d.; o'shea, thomas j.; hayman, david t. s.; wood, james l. n.; cunningham, andrew a.; gilbert, amy t.; mills, james n.; webb, colleen t. title: network analysis of host–virus communities in bats and rodents reveals determinants of cross‐species transmission date: - - journal: ecol lett doi: . /ele. sha: doc_id: cord_uid: yqc r ll bats are natural reservoirs of several important emerging viruses. cross‐species transmission appears to be quite common among bats, which may contribute to their unique reservoir potential. therefore, understanding the importance of bats as reservoirs requires examining them in a community context rather than concentrating on individual species. here, we use a network approach to identify ecological and biological correlates of cross‐species virus transmission in bats and rodents, another important host group. we show that given our current knowledge the bat viral sharing network is more connected than the rodent network, suggesting viruses may pass more easily between bat species. we identify host traits associated with important reservoir species: gregarious bats are more likely to share more viruses and bats which migrate regionally are important for spreading viruses through the network. we identify multiple communities of viral sharing within bats and rodents and highlight potential species traits that can help guide studies of novel pathogen emergence. most emerging infectious diseases are zoonotic (passed from animals to humans), with > % originating in wildlife (jones et al. ) . cross-species transmission is one of the most challenging and least studied aspects of disease ecology, yet it is the defining process in zoonotic disease emergence (lloyd-smith et al. ). bats are the reservoirs of a number of emerging viruses, including ebolaviruses, sars coronavirus and nipah and hendra paramyxoviruses (calisher et al. ). viral and malarial parasite diversity in bats is high (drexler et al. ; luis et al. ; quan et al. ; schaer et al. ; drexler et al. ) , and bats appear to be major and ancient natural reservoirs of several viral families, including hepaciviruses, pegiviruses, paramyxoviruses, coronaviruses and influenza a viruses ( drexler, et al. ( drexler, et al. ,, quan et al. ; tong et al. ) . since cross-species transmission can be common among bats (streicker et al. ; cui et al. ) , understanding their role as reservoir hosts requires examining them in a community context rather than concentrating on individual species. several traits have been hypothesised to make bats particularly suited to hosting and transmitting viruses, including life history traits such as relatively long lifespans for their body size (munshi-south & wilkinson ) , which may facilitate viral persistence for chronic infections; their reliance on torpor, which can decrease viral replication and immune function (dempster et al. ; prendergast et al. ) ; and flight, which can facilitate transmission of viruses to new areas. in addition, many bat species are gregarious, living in dense aggregations, and some roosting sites can house diverse assemblages of bat species (kunz ; kuzmin et al. ) , which could facilitate transmission of pathogens and sustain acute-immunising infections. in evolutionary terms, bats are ancient mammals, and it has been hypothesised that viruses that evolved in bats may use conserved cellular receptors enhancing their ability to transmit viruses to other mammals (calisher et al. ) . the diet of some species has been hypothesised to facilitate transmission of viruses; after feeding, frugivorous bats often leave partially eaten fruit behind which can be contaminated with viruses (chua et al. ; dobson ) . bats are the second most diverse mammalian order with over species, with many overlapping species distributions; multiple regions across the globe are home to more than bat species, allowing for species to interact and potentially spread viruses between them ( fig. ; calisher et al. ; luis et al. ) . here, we examine the structure of host-virus communities in bats and rodents and how host traits, such as those above, correlate with propensity to host and transmit viruses. rodents are a suitable group for comparison because they also host many important zoonotic viruses and share many of the characteristics hypothesised to make bats suitable as viral reservoirs. for example, rodents are also evolutionarily ancient; they are older than bats and more closely related to humans (dos reis et al. ) . rodents are the most diverse mammalian order with approximately twice the number of rodent species as bat species, many of which express torpor, and display a wide range of life history traits, including some long-lived species. networks have been used extensively in disease ecology to model the finite and heterogeneous number of social contacts between individuals. here, we use networks to describe sharing of viruses between different species, rather than individuals. this is akin to other ecological networks, such as food webs, mutualistic, or host-parasite networks (montoya et al. ) . however, we collapse the bipartite network (separate nodes for viruses and host species) to a unipartite projection, weighting contacts between host species by the number of viruses they share. in the same way social networks can highlight the disproportionate importance of specific individuals, such as 'super-spreaders', in population-level disease dynamics, our networks can highlight the importance of certain species in host-virus communities and disease emergence. in addition, we can use other network methods to look at substructures, or 'communities' in the network, highlighting sets of highly interconnected species (newman ) . here, we use databases of bat and rodent viruses spanning years of publications (luis et al. ) to create networks that connect host species via their shared viruses and examine cross-species transmission at several scales. a database of bat and rodent viruses compiled from the literature (luis et al. ) was used to create separate networks of viral sharing for bats and rodents, with each node representing a host species and edges between two nodes representing the presence of virus(es) in both species. edges were weighted by the number of viruses shared between the two species. see supplementary methods in supporting information for more details on network formulation. to account for sampling bias in the analyses, we used the number of citations on web of science (http://thomsonreuters.com/products_services/science /science_products/a-z/ web_of_science/) with the host species' latin species name for the search term (e.g. altizer et al. ) . the distribution was highly skewed, therefore we used the log of this number to normalise the distribution. we also performed the analyses using the species name and 'virus' as the search terms. all results were qualitatively similar. we calculated various node and network statistics ( table ). the node statistics that we calculated were degree (the number of links a node has), weighted degree (incorporating the number of viruses shared) and betweenness (the number of shortest paths that go through a node (weighted)). the network statistics that we calculated included the degree distribution, transitivity (if two nodes are connected, the probability that their adjacent nodes are also connected), degree assortativity (likelihood of high-degree nodes connecting to other high-degree degree nodes and low-degree nodes to other low-degree nodes) and connectance (links per species , or the proportion of links that are present out of all possible links). we also calculated quantitative connectance, which is the quantitative linkage density (which takes into account the weights of the edges; bersier et al. ) divided by the number of species in the network. to account for sampling bias, we additionally calculated the quantitative linkage densities and connectance for sampling effort-corrected networks. see supporting information for more details. as another test to account for bias, we calculated the quantitative connectance of the jaccard matrices (the intersection divided by the union of the viruses for each pair of host species). this gives the proportion of the viruses shared rather than the absolute number. the multiple regression on matrices and gls analyses described below use the original networks (absolute number of viruses not weighted by sampling effort) because for those, we take sampling effort into account as a covariate. to calculate p-values for the network statistics in table , we performed permutations of the networks, where we shuffled the edge weights. see supporting information for more details. species traits were compiled from online databases and the literature as described in luis et al. ( ) . we examined body mass, number of litters per year, litter size, maximum longevity, torpor use, international union for conservation of nature (iucn) conservation status, geographical distribution area, latitude of the midpoint of the species distribution, number of other species in the same taxonomic order that are sympatric, and for bats only, migratory classification, diet, gregariousness and propensity to roost in caves. see supplemental figures s -s in luis et al. ( ) for plots of the raw data. bat species traits that were new to these analyses were gregariousness, propensity to roost in caves and diet (see supporting information for more details). virus and host trait data and r code are provided online as supporting information. multiple regression on distance matrices (mrm) (lichstein ) was used to detect significant correlations between viral sharing, phylogeny, sympatry and similarity in host traits using 'mrm' in the r package 'ecodist' (goslee & urban ; r core team ). the response variable was the viral sharing matrix (adjacency matrix of the network), with each entry giving the number of viruses shared between each pair of host species. the 'ape' package in r (paradis et al. ) was used to calculate a phylogenetic correlation matrix based on species' shared branch lengths of the most complete mammalian phylogenetic supertree available containing bats and rodents (bininda-emonds et al. ). see figs s and s in supporting information. using the species distribution shape files from the iucn website (http://www.iucnredlist.org/technical-documents/spatial-data) and the r packages 'sp' and 'rgeos' (pebesma & bivand ), a sympatry matrix was created. each entry of the matrix was either a if the two species' distributions overlap or a if they do not. matrices of sampling effort were created by multiplying the logged number of citations for each species together. matrices showing similarity in host traits were also calculated. for example for each pair of species, the difference in the number of litters per year was calculated. this matrix was standardised so values ranged between and , and the matrix used in the multiple regression models was minus this matrix (so it would be a correlation matrix similar to the phylogenetic and sympatry matrices). host species were grouped into 'communities', partitions of highly interconnected nodes, by using the community detection algorithm described in blondel et al. ( ) , which maximises the modularity between groups of the weighted networks. see supporting information for more details. as an alternative to using modularity to assign nodes to communities, we also used clique percolation theory (palla et al. ) . in this method, nodes can belong to multiple communities. see supporting information for more details. ‡quantitative connectance, which takes into account edge weights. §quantitative connectance, with weights adjusted according to sampling intensity. ¶quantitative connectance, where the weights are the proportion of viruses shared rather than the absolute number (see methods). generalised least squares (gls) were used to examine correlates of viral richness per host species, degree and betweenness, while allowing for phylogenetic correlation to be incorporated into the error structure as described below. because many life history traits are correlated, we performed principal components analyses (pca) on the life history traits: logged body mass, maximum longevity, number of litters per year and litter size (separately for bats and rodents). the variables were rescaled to have unit variance before analysis in r using the 'prcomp' function (r core team ), and these principal components were then used in subsequent analyses. see fig. s for pca plots and table s for loading values and variance explained by each principal component. because closely related species share traits, we tested for phylogenetic dependence by setting the error term for the gls to the phylogenetic correlation matrix multiplied by an additional parameter, pagel's k, that was estimated to determine the strength of phylogenetic dependence (pagel ; freckleton et al. ) . a k estimate of indicates that the error structure of the model was directly proportional to the species shared branch lengths. a k of indicates the error structure of the model was not related to the species shared branch lengths (i.e., phylogeny does not explain any additional variation). note that phylogeny is not a covariate in this analysis, and may not be significant if the model covariates themselves have phylogenetic dependence. models were ranked by their aicc (akaike information criterion adjusting for finite sample sizes) values. correlation coefficients (r) were pearson's product moment correlation comparing observed values to model predictions. we also ran these models using mcmc generalised linear mixed effects models (hadfield, ) . see the supporting information for more details. maps were made using the r packages 'maptools ', 'maps', 'sp' and 'pbsmapping' (pebesma & bivand ; becker et al., ; schnute et al. ; bivand & lewin-koh ) . viruses were mapped by the union of their hosts' distributions using the command 'gunioncascaded' from the 'rgeos' package (pebesma & bivand ) . the assumption was made that the virus exists in the entire distribution of its host(s), analogous to the fundamental niche concept (hutchinson ; harris & dunn ) . to visually assess whether sampling effort had a strong effect on the virus distributions, we also plotted these distributions discounting by sampling effort of the host species (figs s and s ). the bat network had host species connected by viruses, and the rodent network had host species connected by viruses (figs a, a, s and s ). a species' degree is the number of other host species it is connected to by shared viruses. degree in bats ranged from to with a mean of . and in rodents ranged from to with a mean of . (table ; see fig. s for degree distributions). the mean degree and mean weighted degree were significantly higher in bats than in rodents. there was significant transitivity (the probability that the adjacent nodes of a node are also connected) in both bats and rodents. assortativity in bats was significant, suggesting that bat species with high degree tend to interact with other bat species of high degree (and low degree with low degree), but there is no evidence of this in rodents (table ) . the bat network was . times more connected (connectance = links/species ) than the rodent network (table ) . this was statistically significantnot once in permutations was the difference between connectance for the bat and rodent networks at least the observed difference. the quantitative connectance adjusted for sampling effort and the quantitative connectance on the jaccard matrices were also higher in bats (table ) . next, we examined the connections between species, using multiple regression on matrices (lichstein ) to determine the correlates of the number of viruses that two host species share. both phylogeny (relatedness) and sympatry (geographic range overlap) were important predictors of viral sharing (table ) . species with overlapping distributions and closer phylogenetic relationships shared more viruses. sympatry was more important than phylogeny for viral sharing in both orders, but both explained more variation in bats. sampling effort (by number of citations on web of science) was also important. well-studied pairs of species were found to have more viruses shared between them. the amount of variation explained by sympatry, phylogeny and citations together was % in bats and . % in rodents. similarity in life history traits explained a small amount of additional variation (table s ) . although bats generally have received attention as disease reservoirs, some species are more important for disease emergence. we determined which species have the most viruses, the highest degree (the most connections) and the highest betweenness in the network (a measure of network centrality the number of shortest paths between any two nodes that go through the node of interest) and host traits associated with these network metrics. for all analyses of the bat and rodent networks, sampling effort was importantthe more a species was studied, the more viruses, higher degree and higher betweenness. consequently, we adjusted for sampling effort by number of citations on web of science. the most important variable associated with hosting more viruses in bats was diet (fig. a, table s and s ). host traits that correlated with the highest degree within the bat network (the most connections or viruses shared), in order of importance, were gregariousness and sympatry; diet was marginally important (fig. b, table s and s ). migration was the most important host trait associated with betweenness in bats (fig. c, table s and s ). in these analyses, phylogeny is not a covariate, but incorporated into the error structure, and an additional phylogenetic correlation parameter (k; pagel ) is estimated, to determine if phylogeny can explain variation unexplained by the model covariates. for bats, k, was for the best models for number of viruses and degree (table s and s ), indicating that phylogeny did not explain any additional variation. a likely explanation is that the best models included diet, which is strongly related to phylogeny. however, k for the best model for betweenness was . (table s ), indicating that phylogeny explained additional variation. for rodents, sympatry was the most important host trait; species whose distributions overlapped with a greater number of other rodent species had more viruses and higher degree and betweenness (fig. d-f and table s -s ). in addition, rodent species that are larger, longer lived and with higher reproductive rates (e.g. sigmodon hispidus, rattus norvegicus) had more viruses (pc ; table s , s -s ). species at higher latitudes had marginally higher degree (after controlling for sympatry: table s -s ). the results from the mcmc generalised linear mixed effects models were largely similar to these results (see tables s , s , s , s and s ). the only significant difference was the best model for the number of viruses in bats included only citations and phylogeny (table s ). see the supporting information for more details. although and % of all possible pair-wise combinations of species are connected by < degrees of separation in the bat and rodent networks, respectively, the influence of a single species may be more localised. we detected distinct 'communities' of viral sharing within orders (figs a and a) , by maximising the modularity (blondel et al. ) . modularity can attain values in the range from À . to . if positive, there are more within-group connections than would be expected at random, and in practice, values > . indicate significant community structure (newman ) . modularity scores were . and . for the bat and rodent networks respectively. in the bat network, there were viral sharing communities. each species had distributions that on average overlapped with % of the species within its community vs. % of species outside (t = . , d.f. = . , p = . À ; fig. b-f ). the largest and most densely connected community (fig. b) consisted of bat species in the americas, largely connected by vector-borne viruses. the remaining communities were connected by a mixture of vector-borne and non-vector-borne viruses. the green community (fig. c ) was broadly distributed from europe to australia, and approximately half of the species in this community also shared viruses with species in other communities that overlapped spatially. there were two globally distributed communities of rodents with and species (fig. b,c) and two communities that were more spatially restricted. each rodent species on average overlapped with % of the species within its community vs. % outside of its community (t = . , d.f. = . , p = . ; fig. b-e) . the house mouse, mus musculus, has spread across the globe along with its viruses (fig. b) . removing this species results in largely the same community structure, indicating this species has already facilitated the transmission of viruses to multiple sympatric species across the globe. the black rat, rattus rattus, and the brown rat, r. norvegicus, are also widely distributed and are the main species holding the green community together (fig. c) . when either rattus species is removed from this community, it splits into several smaller communities, suggesting less secondary viral spread to sympatric rodent species. overall properties of the bat and rodent networks provide a global view of viral sharing within each order, to our current knowledge. the number of viruses per host species, the number of hosts per virus, the mean degree and connectance were higher in the bat network than the rodent network. this suggests that viruses may pass more easily between bat species than between rodent species. our analyses indicate that characteristics unique to bats, such as gregariousness and migration, may facilitate this increased transmission. previous studies hypothesised frugivory as an important viral transmission mechanism among bats and from bats to other species, because virus has been isolated from partially eaten fruit, which is often shared and dropped to the ground (chua et al. ; dobson ) . our study is consistent with that hypothesis, with frugivores hosting more viruses than nectivores, insectivores and sanguivores. however, diet is strongly correlated with phylogeny, and the best model using mcmc glmm only included phylogeny. therefore, these results could indicate the importance of diet or, alternatively, some other factor correlated with phylogeny, such as immunological functioning, for example. the most important host characteristics associated with degree in bats were gregariousness and sympatry. these characteristics have been hypothesised as important mechanisms for viral maintenance and spread (calisher et al. ; luis et al. )bat species with distributions that overlap with a greater number of other bat species, and particularly those which are gregarious, will have greater interspecific contacts and chance for cross-species transmission. at some roosting sites, bat densities can be as high as bats per square metre, and some roosts house diverse assemblages of species (constantine ; kunz ) . high densities can lead to high contact rates, facilitating pathogen transmission and persistence. these dense roosting sites are often caves, therefore we had hypothesised that propensity to roost in caves would be an important predictor of viral sharing in bats. however, colony size (gregariousness) was more important than where the colony roosts. large colony sizes do not occur on the same scale in rodents and may have led to the higher mean degree and connectance in bats compared to rodents. species with high betweenness might not have or share the most viruses, but could play a key role in disseminating viruses because they can connect disparate regions of the network. migration was the most important host trait associated with betweenness in bats. long-distance animal movement has been hypothesised to enhance geographic spread of infectious disease, however, evidence in most cases is lacking (altizer et al. ). in the bat network, many of the shortest network paths went through regional migrants (moving - km annually), which were more important than long-distance migrants (> km). this is consistent with the idea of 'migratory culling' (bradley & altizer ) in which the physiological stress of long-distance migration can increase mortality of infected individuals. for rodents, sympatry was the most important covariate, again emphasising the importance of spatial overlap in cross-species transmission. we found that species that had higher reproductive rates and body mass had more viruses (pc of the pca), which is in accordance with the findings of han et al. for all rodent pathogens and parasites ( ). this is the first analysis we are aware of that attempts to identify communities of pathogen sharing at a global scale. these communities reveal which species are most highly connected, providing insight into virus maintenance and spread. they may also be useful in guiding public health strategies. for example if a new zoonotic virus is identified in hipposideros speoris, it may be useful to look for it in other species of the green community (fig. c ) as well. the links in our networks represent real connections, the sharing of viruses, and may be evidence of cross-species transmission, coevolution of host and virus lineages, or indirect links of cross-species transmission through an intermediate host or vector. the importance of phylogeny for viral sharing could indicate the virus originated in a common ancestor and was maintained or coevolved in separate lineages, or the greater ability of viruses to transmit between closely related species. however, the greater importance of sympatry in viral sharing indicates that cross-species transmission is at least as important if not more important than coevolution in establishing the network. for cross-species transmission to take place, there must be contact, direct or indirect, between two host species, and therefore, it is not surprising that sympatry is important in the sharing of viruses. the importance of sympatry is reflected in similarities between viral and host species richness maps (fig. ). locations where host species diversity is highest generally have more viruses. however, bats in europe and india have greater viral richness than predicted by the host species richness map (fig. a,c) , and this does not appear to be due to sampling bias (fig. s ) . the community context may provide additional insight. there are two host-virus communities connecting europe to east asia where species richness is high (fig. c,f) , and these communities may provide a bridge. higher viral richness than expected from host species richness in india may also reflect connections with areas of high species richness in s.e. asia (fig. c) . for the viral richness maps, we assumed that the virus exists throughout the distribution of its host(s) analogous to the fundamental niche concept (hutchinson ; harris & dunn ) and representing the broadest possible distribution. however, there may be barriers that restrict the realised niche of a virus. for example rhinolophus ferrumequinum hosts sars-like coronavirus and japanese encephalitis virus, and its range extends from e. asia to w. europe, but the populations in e. asia and europe do not appear to mix (flanders et al. ). thirteen viruses were present in both bats and rodents. ten of these were vector transmitted. therefore, direct transmission between bats and rodents appears rare. of the three directly transmitted viruses, only ebola could have been transmitted from bats via fruit. the other viruses shared between bats and rodents in this study, hantaan and puumala, were found in insectivorous bats, and transmission may have occurred from rodents, as they are currently thought to be the reservoirs of these hantaviruses. however, a recent study indicates bats may be important and ancient reservoirs of hantaviruses more generally (guo et al. ) . a recent study examining the sharing of parasites in primates using similar network methods used an overall metric for network centrality which included weighted degree, betweenness and several other network measures of the importance of a node (g omez et al. ) . in this study, the authors found that hosts with denser populations living in larger groups and having broad distributions had higher centrality. although overall, degree and betweenness were correlated in our study, the metrics were different enough to be best predicted by different covariates in batsgregariousness for degree, and migration for betweenness. however, in rodents, the most important factor was the same for the different metricssympatry best predicted the number of viruses, degree and betweenness. for our analysis we used a database covering years of publications . subsequent analyses, particularly focusing on bats as reservoirs of viruses, have been published (e.g. drexler et al. ; quan et al. ). studies such as drexler et al.'s ( ) report short genome fragments that are yet to be categorised by the ictv, which makes it difficult to discern what sequences should be considered the same virus for classification of viral sharing, and thus we have not included them in our analyses. however, molecular studies increase our ability to distinguish viruses and understand virus-host relationships. as interest in reservoirs of emerging infectious diseases increases, especially in bats, we predict that results such as these will further strengthen our findings and allow for more refined analyses at the community level. indeed, several recent studies support our conclusions, showing a preponderance of host switching between bats and from bats to other mammals (drexler et al. ; guo et al. ; drexler et al. ). this study is based on a large literature search, and therefore there are necessarily constraints on inference, given different motivations for, and methods used during studies of both rodent and bat viruses through time. we were concerned that the recent attention on bats as reservoirs may have biased the sampling and artificially increased the apparent connectance of the bat network; therefore as a secondary analysis, we also created networks using only viral accounts before the year . we found that although both the bat and rodent networks were smaller (the bat network was reduced to species and links; and the rodent network to species and links), the connectance remained relatively steady ( . % in bats and . % in rodents), suggesting that the apparent greater viral sharing in bats compared to rodents is not merely a function of recent attention on bats. however, the importance of citations in our analyses for both orders highlights the importance of sampling effort overall. although we were able to account for sampling effort in the analyses examining species traits associated with viral sharing, it is difficult to account for sampling effort in creating the networks themselves. one approach which we implemented was creating networks using weighted edges based on sampling effort; this gave us confidence that among the species in our database, the connectance is higher in bats than in rodents. although our database only has % of extant rodent species and % of extant bat species, the rodents in our database have been more studied overall and more studied for viruses than bats even when removing the most highly studied, lab rodents -mus musculus and rattus norvegicus (significant by t-test on log number of citations for the species name, means = . & . , p = . ; and the species name + virus, means = . & . , p < . ). we would expect more of the total viral community to have been identified in the more highly studied group of species (rodents), thus the sampling bias should increase the apparent connectance in rodents compared to bats. however, this does not account for species not in our database. if the rodent species that have not been sampled share more viruses than the bat species that have not been sampled, then these results may not hold. similarly, the species trait data was only available for roughly % of the bat and rodent species in our networks, which corresponds to and % of known extant bat and rodent species respectively; therefore, the species traits associated with important hosts may also change as our knowledge increases. new viruses are being discovered frequently, suggesting that there are many more viruses yet undetected in both bats and rodents. therefore, our conclusions and the maps of viral diversity for both orders (fig. ) may change significantly as our knowledge increases. we hope that this first, broad examination of viral sharing will help motivate future efforts including more directed data collection. another potential source of bias may be reports of spillover or incidental hosts that are not important reservoirs but are treated with equal weight in these analyses. we took care to distinguish the hantaviruses and arenaviruses, for which there is relatively good knowledge of host-virus relationships. however, we found that this did not impact the qualitative conclusionswhen using ictv classifications the rodent network still had lower mean degree ( . ), mean weighted degree ( . ), connectance ( . %) and transitivity ( . ) than the bat network. however, for most of the reported viruses in bats and rodents, there is little knowledge of the ecology and epidemiology in these hosts. this again highlights the importance of further study of both bats and rodents as reservoirs of viruses. our networks connect hosts by their shared viruses. for a given pair of connected species, we do not know in which direction the cross-species transmission may have taken place, or if transmission even occurred between these species. there may have been a third species that transmitted the virus to both species. as more viral sequences become available, molecular methods, such as those used by streicker et al. ( ) , may be used to infer directionality in cross-species transmission, and help resolve these issues. our study highlights the interconnectedness of species with respect to viruses and shows the benefits of examining pathogens in a community ecology context at several scales. our analyses suggest that unique bat characteristics figure importantly in sharing and spreading viruses, lending quantitative support for bats' special status in zoonotic virus emergence and demonstrating which characteristics affect reservoir potential. practically, our analyses may help guide future surveillance for optimal prevention of emerging zoonoses. this could help campaigns aimed at preventing spillover between bats and humans to benefit human health while conserving bats' important roles in ecosystem services such as pest management, plant pollination and seed dispersal (boyles et al. ) . animal migration and infectious disease risk & r version by ray brownrigg quantitative descriptors of food-web matrices the delayed rise of present-day mammals maptools: tools for reading and handling spatial objects fast unfolding of communities in large networks economic importance of bats in agriculture parasites hinder monarch butterfly flight: implications for disease spread in migratory hosts bats: important reservoir hosts of emerging viruses isolation of nipah virus from malaysian island flying-foxes activity patterns of the mexican free-tailed bat discovery of retroviral homologs in bats: implications for the origin of mammalian gammaretroviruses experimental coxsackie b- virus infection in citellus lateralis what links bats to emerging infectious diseases? bats host major mammalian paramyxoviruses ecology, evolution and classification of bat coronaviruses in the aftermath of sars phylogeography of the greater horseshoe bat, rhinolophus ferrumequinum: contrasting results from mitochondrial and microsatellite data phylogenetic analysis and comparative data: a test and review of evidence centrality in primateparasite networks reveals the potential for the transmission of emerging infectious diseases to humans the ecodist package for dissimilaritybased analysis of ecological data phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents mcmc methods for multi-response generalized linear mixed models: the mcmcglmm r package rodent reservoirs of future zoonotic diseases using host associations to predict spatial patterns in the species richness of the parasites of north american carnivores concluding remarks. cold spring harbor symp iucn red list of threatened species global trends in emerging infectious diseases ecology of bats shimoni bat virus, a new representative of the lyssavirus genus multiple regression on distance matrices: a multivariate spatial analysis tool epidemic dynamics at the human-animal interface a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? ecological networks and their fragility bats and birds: exceptional longevity despite high metabolic rates fast algorithm for detecting community structure in networks inferring the historical patterns of biological evolution uncovering the overlapping community structure of complex networks in nature and society ape: analyses of phylogenetics and evolution in r language periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels bats are a major natural reservoir for hepaciviruses and pegiviruses r: a language and environment for statistical computing. r foundation for statistical computing phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny high diversity of west african bat malaria parasites and a tight link with rodent plasmodium taxa pbsmapping: mapping fisheries data and spatial analysis tools. r package version last accessed host phylogeny constrains crossspecies emergence and establishment of rabies virus in bats new world bats harbor diverse influenza a viruses adl, ctw, jlnw and aac designed study. adl, tjo, dtsh, atg and jnm compiled data. adl performed analyses and wrote the manuscript, and all other authors contributed substantially to editing. key: cord- -sxh mq q authors: milne, d. j.; armstrong, m.; fisher, a.; flores, t.; pavey, c. r. title: structure and environmental relationships of insectivorous bat assemblages in tropical australian savannas date: - - journal: austral ecol doi: . /j. - . . .x sha: doc_id: cord_uid: sxh mq q abstract patterns in the composition of assemblages of microbat species sampled during the late dry season (the ‘build‐up’) in north australian savannas were assessed against a range of environmental factors as well as four a priori defined habitat types (riparian, escarpments, coastal and woodlands). distinct species assemblages were most strongly associated with topographic and climatic variables. there were also limited associations with vegetation structure, fire and local roost potential but no associations with insects or water availability. total species diversity at sample sites was associated with distance to rivers and rainfall. in general, species assemblages were not clearly defined and the number of significant environmental associations was relatively few. we compare these associations with those reported for bat assemblages elsewhere in australia. understanding of the diversity and evolutionary ecology of australia's mammal fauna has not been uniform across orders. in particular, most detailed tests of evolutionary hypotheses (e.g. johnson ; fisher et al . ) omit bats (order chiroptera). assessments of population trends and extinction proneness have also excluded chiroptera (e.g. johnson ) . this is a significant shortcoming as bats represent over % of australia's mammal species, many of which are endemic. although australian mammal diversity peaks in the tropical forests of eastern queensland including cape york peninsula, significant diversity also occurs in the savannas of north-western australia where species are known ). an assessment of the response of mammals within this region to environmental variables revealed that a single environmental gradient (of substrate and disturbance) described the distribution of all species, excluding bats . rock-inhabiting mammals are a significant component of this fauna, however, diversity of this assemblage decreases with decreasing outcrop size and increasing isolation. identified three other trends. first, that the mammal fauna of eucalypt open forest/woodland habitats of north-western australia is characterized by extensive distributions of its component species. sec-ond, that monsoon forests support a depauperate mammal fauna. last, that the mammal fauna of this region undergoes substantial latitudinal change associated with a steep north-south rainfall gradient. did not include systematic sampling of bats, preventing a rigorous examination of the response of the bat fauna to environmental measures. however, data from captures (mist netting, harp trapping, roost searches) indicated that most bat species were present across the environmental range sampled ). here we revisit the issue of the response of bats to environmental variables in the tropical savanna of the northern territory and north-west queensland using a more rigorous data set. our data collection incorporated the use of ultrasonic detectors to sample bats and geographic information system (gis) derived variables to represent environmental conditions. the study region supports a rich microbat fauna ( of australia's species, of australia's genera), including one endemic species ( taphozous kapalgensis ), and both of australia's monotypic genera ( rhinonicteris , macroderma ). we assessed environmental factors at two levels, first at the landscape scale, using data available from a gis, and second at a local scale where information was collected on the physical environment and food resource availability (insects) at individual sampling sites. we predicted that the high vagility of bats would result in species responding broadly to environmental variables. however, specific responses to a number of environmental variables were expected. in particular, we predicted that the distribution, composition and segregation of bat assemblages would respond to geographical patterns in annual rainfall, presence of rocky escarpments, water bodies and canopy cover. although a relationship with insect abundance and composition was examined, we predicted that this relationship would not be significant given the generalist feeding ecology of most insect-eating bats (fenton ) . the bat assemblages of tropical australian savannas are also compared with assemblages elsewhere in australia. specifically, we compared our results with community composition and environmental association studies in north queensland rainforest (crome & richards ) , mangroves in north-western western australia (mckenzie & muir ) , and open forest/ woodland in victoria (kutt ; lumsden & bennett ; herr ) , south-east new south wales (nsw) (law et al . ) and tasmania (taylor & o'neill ) . the study area, called the top end of australia, included the tropical savanna of the northern terri-tory and north-west queensland, north of ° s, but excluding offshore islands (fig. ) . across this area, maximum mean weekly temperature ranges between ° c and ° c and mean annual rainfall between mm and mm (houlder ; fig. ). rainfall is highly seasonal with almost all precipitation occurring from november to april. topographic relief is relatively low. the maximum elevation is m on the arnhem land plateau, with the main areas of topographic relief being the kakadu escarpment and the eastern edge of the kimberley region in the southwest of the study area. eucalypt woodlands and forests dominate % of the study area (fox et al . ) . other notable environments include monsoon rainforests and floodplains dominated by sedgelands and grasslands. on average, over half ( %) of the top end is burnt every year (a. edwards, pers. comm. ) . a total of sampling sites were located across the top end (fig. ) . map of the study area showing the location of sampling sites and site labels as well as average annual rainfall isohyets (in millimetres). sites are symbolized according to bat assemblage (diamond = group , square = group , cross = group , triangle = group , circle = group ). sonal variation on species composition. each site was a circular plot of m radius. plots were primarily selected to cover a large geographical area and to sample four broad habitat types: . riparian -adjacent to perennial rivers, creeks or permanent waterholes ( sites). . escarpments -sandstone cliffs ( sites). . coastal -coastal and near coastal environments (excluding estuaries and mangroves) (eight sites). . woodlands -continuous areas of eucalypt woodlands or open forests not associated with the other habitats types ( sites). habitat types were chosen a priori and were based on information gleaned from species' distribution maps and descriptions of microbat habitat preferences (strahan ; churchill ) that suggested these habitats may contain distinctive species assemblages. two sites were usually sampled at a time on the same nights. with one exception, no two sites within a sampling pair sampled the same habitat type. distances between sampling pairs ranged between km and km (mean km). at each site we used a range of sampling techniques to maximize the likelihood of obtaining a full inventory of bat species (kuenzi & morrison ; murray et al . ; o'farrell & gannon ) . bats were sampled using two ( sites) or three ( sites) harp traps over two consecutive nights as well as one night of shot sampling for a -h period after dusk. harp traps were usually placed across 'flyways' (tracks, streams or other gaps within the vegetation where bats are more likely to be trapped) and were either positioned side by side or spaced between and m apart. we also conducted active searches of caves, road culverts and any other features potentially used as diurnal roosts by bats within m of the centre of the sampling site. in addition, bat calls were recorded at every site with ultrasonic bat-detectors (anabat ii, titley electronics, ballina, australia) using two methods. the first method involved placing a detector on the ground, elevated to approximately ° , and operated from dusk for a cumulative total of at least six recording hours over two consecutive nights (maximum h, mean h). this time period has been shown to sample % of species calls at a given site (milne et al . ) . detectors were connected to either an anabat ii delay switch with output recorded to -min cassette tape (sony chrome ux, tokyo, japan) via tape recorder (optimus ctr- d, fort worth, usa) ( sites) or an anabat v zcaim and computer (toshiba portégé ct or toshiba tecra ct d, tokyo, japan) running anabat software in monitor mode ( sites). there are no differences in the species detected between these two recording techniques (milne et al . ) . for the second method, an anabat detector was held in the hand and manually activated on detection of a bat-call and actively pointed in the direction of the call. calls were recorded via tape-recorder and cassette tape, for h after dusk for one night. all recorded calls were identified according to milne ( ) . at several sites ( ), shot sampling was not permitted. instead we trapped bats using mist-nets at these sites. it is likely therefore, that some 'high-flying' bat species that are readily detected using shot sampling, may not have been trapped at these sites. however, we expect this will have a negligible effect on our results as shot sampling at all other sites, used in conjunction with anabat detectors, enabled us to collect an extensive reference call library for 'high-flying' bat species for the entire study area (milne ) . anabat detectors were systematically used at all sites and will normally detect 'high-flying' species that are not readily trapped (o'farrell & gannon ) . we collated environmental data for each site from field habitat measurements, analysis of spatial data and insect sampling. at the centre of each site we measured tree basal area, canopy cover and stem count, m either side of a -m transect ( . ha) in an area of undisturbed vegetation usually adjacent and parallel to flyways where harp traps were set. on escarpment sites, the transects either traversed the escarpment or were situated at the base of the escarpment. basal area and stem counts were derived by measuring diameter at breast height (d.b.h.) of every tree along the transect, whereas canopy cover was measured using a spherical densiometer at m, m and m along the transect. for the entire site ( . ha), we measured slope, maximum canopy height, crown cover, rock cover, distance to water and local roost potential. crown cover in three height classes ( - m, - m and > m) was estimated using crown separation ratios (mcdonald et al . -mostly large trees and rock outcrop with large cracks and holes we chose to assess whole trees and rock outcrops rather than count individual hollows because small microbats ( < g) can roost in hollows equivalent to their own body diameter (pers. obs. ). entrances to these hollows are very small and would regularly be overlooked if we attempted to count hollows directly. large trees have been shown to contain more tree hollows than smaller trees (whitford ) and are preferred roost sites for many bat species (lunney et al . ; herr & klomp ; law & anderson ; lumsden et al . ) . several variables were derived using gis from a s ( c . m) digital elevation model (dem, provided by the department of defence) including elevation, ruggedness index (the range in cell values of the dem within a × cell neighbourhood), and distance to and m 'escarpments' (defined here as any adjacent dem cells having an altitude difference of or m). climate variables (annual mean temperature, minimum monthly temperature and annual rainfall) were derived using bioclim (houlder ) . other gis data included fire frequency (number of years in which the site was burnt over the preceding years) and years since last fire (data sets provided by the bushfires council of the northern territory), distance to perennial rivers, and ndvi (normalized difference vegetation index, which is a measure of vegetation 'greenness' derived from satellite imagery) and projective foliage cover (meakin et al . ) . at each site we trapped flying nocturnal insects for one night concurrently with bat sampling. the insect trap was constructed from a white cotton sheet ( . m × . m), suspended off the ground by strings tied to the corners to form a funnel, one end higher than the other. at the bottom of the funnel a hole was cut in the sheet and a plastic jar ( mm diameter × mm depth) partially filled with % ethanol was attached to hang underneath. a -v fluorescent light ('col-light' brand col-lite, maleny, australia) was hung from the higher end of the sheet to attract insects. the trap was positioned approximately m from the anabat detector so as not to disturb bats from natural flight habits in the vicinity of the detector, and left unattended for the entire night. insects that fell into the jar were collected the following morning. in the laboratory, insect samples were filtered through a -mm sieve to remove the smallest insects (mostly < mm in length) and then identified to order and assigned to four size (head-body length) classes: < mm, - mm, - mm and > mm. the choice of size classes was based on the range of body sizes found to be prey items of bats in tasmania (o'neill & taylor ) . analysis of bat communities was based on species presence-absence at each site derived from the combination of all sampling methods. anabat calls for the following combinations cannot be reliably separated in the top end: (i) chalinolobus nigrogriseus , scotorepens greyii and scotorepens sanborni ; (ii) miniopterus schreibersii and pipistrellus westralis ; and (iii) nyctophilus arnhemensis , nyctophilus bifax and nyctophilus geoffroyi (milne ) . anabat call sequences that were attributed to these species combinations were therefore excluded from the analysis, although species within these combinations were included if identified using one of the physical sampling methods. the one exception was s. greyii and s. sanborni which cannot be readily separated in the field (churchill ) and were treated here as a single species although in some areas their distributions are allopatric (mckenzie & muir ) . species assemblages were assessed using patn software (belbin ) . similarities in species composition between sites were calculated using the bray-curtis association measure. cluster analysis (unweighted pair group mean) was used to define assemblages (groups of sites) following visual inspection of the dendrogram. anosim (clarke & green ) was used to test whether bat species composition differed significantly between the defined assemblages as well as the four a priori habitat types. the relationship between sites was also portrayed by ordination (multidimensional scaling) of sites by their bat species composition. in both analyses, only sites with at least three species were included. all environmental variables (table ) were continuous or rank ordered. variables were initially compared using the spearman rank correlation test. where pairs of variables had a correlation coefficient greater than . , one of the pair was excluded from further analysis. the mean of each environmental variable was calculated for each group of sites derived from the cluster analysis and the significance of differences between bat assemblage groups was tested using kruskal-wallis anova . the relationship between environmental variables and the arrangement of sites in the ordination space was also tested using vector fitting (kantvilas & minchin ) . finally, generalized linear modelling (glm; crawley ) was used to develop a predictive habitat model for total site species richness. a poisson error distribution and log link function was used and a backward stepwise procedure was adopted to generate the minimum adequate model with only those variables having a significant correlation in the vector fitting included in the model development. a total of microbat species were identified from the sites, representing over % of the species recorded from north-western australia . two species known to occur in the top end , macroderma gigas (ghost bat) and saccolaimus saccolaimus were not detected in this study. we identified five groups from the classification of all sites by their species composition (fig. ) . the initial classification divided the sites into four groups. we subdivided the largest of these groups into two and assigned two outlying sites to group based on the relative position of these sites in the ordination. anosim analysis confirmed that the groups differed significantly in composition ( r = . , p < . ) and that there was a significant difference between each pair of groups ( p < . or better). the occurrence of bat species within the derived groups and habitat types is summarized in table and the geographical distribution of sites (classified according to group) is shown in fig. . four species were ubiquitous throughout the groups and habitats ( chaerephon jobensis , pipistrellus adamsi, mormopterus loriae and saccolaimus flaviventris ) while three species were each detected at single sites only ( hipposideros diadema , hipposideros stenotis and m. schreibersii ). the distribution of sites in ordination space and the relationship with environmental vectors is shown in fig. . a total of environmental variables were significantly correlated with variation in species composition between sites (table ) . a summary of mean values for these variables for each group is provided in table . a description of species composition and the environmental characteristics for each group is provided below. species that were detected most often in this group include chalinolobus gouldii (present in all sites), c. jobensis, s. flaviventris, s. greyii/s. sanborni and p. adamsi. chalinolobus gouldii was strongly associated with this group (i.e. tended to occur in group more than the other four groups). group had the highest total species richness and mean site species richness of all groups. sites were characterized by high percent-age canopy cover, frequent burning and high annual rainfall and were located in the north and west of the top end. the greatest number of sites occurred in this group ( ) . species that were detected most often include vespadelus caurinus (present at all sites), s. flaviventris , taphozous georgianus and c. jobensis . v. caurinus was strongly associated with this group. sites were characterized by rugged, steep rocky slopes, high elevations and short distances to escarpments and rivers. minimum temperatures were cool and annual rainfall low. sites were widely distributed across the top end, except the coastal zone. species detected most often in this group include s. flaviventris, n. arnhemensis, myotis macropus and s. greyii/s. sanborni. n. arnhemensis was strongly associated with this group. group was not clearly associated with any of the environmental variables measured and occupied an intermediate value on most environmental gradients. however, this group displayed the highest mean values for minimum temperature. figures represent percentage of sites within each group or habitat in which each species was detected. differences in proportions were tested using χ statistic and differences in mean species richness were tested using kruskal-wallis anova (ns, not significant; *p < . ; **p < . ; ***p < . ). this group had equal fewest sites (five) and had the lowest total and mean site species richness. species detected most often include c. jobensis (present at all sites) s. flaviventris, m. loriae and c. nigrogriseus. there were no strong species associations, although c. nigrogriseus occurred at proportionately more sites in this group than any other. sites were characterized by lower mean annual temperatures, long distances to rivers and no rock cover. species detected most often include s. flaviventris, p. westralis (both present at all sites), p. adamsi, m. loriae and t. kapalgensis. p. westralis and t. kapalgensis were strongly associated with this group. group also had relatively few sites and low species richness, but was associated with the minima or maxima of several environmental variables including long distances to escarpments, flat terrain at low elevations with no rock, low local roost potential, high annual temperatures and low fire frequency. all five sites were located near the coast (fig. ) . there was a significant difference in species composition between habitat types (anosim, r = . , p < . ) as well as between all pairwise combinations of habitats except between 'woodland' and 'riverine' (r = . , p = . ). v. caurinus and t. georgianus were detected most often in 'escarpment' habitat. both of these species, as well as rhinonicteris aurantius and c. nigrogriseus, were absent from 'coastal' habitat. p. westralis was strongly associated with 'coastal' habitat and absent from both 'escarpment' and 'riparian' habitat. n. arnhemensis was also absent from 'escarpment' habitat. all habitats had similar total and site species richness, with slightly lower species richness in the 'coastal' habitat. the relationship between groups and habitats is summarized in table . the habitat type of each site was not independent of group classification (χ = . , p < . ). most of the 'escarpment' sites occurred in group (steep, rocky, rugged sites) with two further sites in group . 'coastal' sites mainly occurred in group (few environmental correlates) and group (flat, low elevation), whereas 'riparian' sites occurred across four of the groups and 'woodland' sites were evenly represented across all five groups. we found no significant associations between bat species assemblages and various measures of insect availability including total number of insects, total number of insect orders, total number of insects in various size classes, proportion of insects in various size classes or total number of insects in each order. values are the mean for sites in each group, h-values refer to the kruskal-wallis statistic (ns; not significant; *p < . ; **p < . ; ***p < . ). habitat modelling identified distance to perennial rivers and annual rainfall as the major predictors for site species richness (table ). the minimum adequate model was only moderately robust with % of the deviance captured. this suggests that there was considerable 'noise' in the data or that some important explanatory variables were not quantified. as predicted, the insectivorous bat fauna of northwestern australia responded broadly to most environmental variables. the main environmental feature associated with the distribution of microbat assemblages in the study area was topography (variation in elevation, slope, topographic ruggedness and distance to escarpments). not surprisingly, therefore, species considered to be obligate cave roosters (hipposideros ater, h. diadema, h. stenotis, m. macropus, m. schreibersii, r. aurantius, t. georgianus, v. caurinus, vespadelus finlaysoni) , mainly occurred in (but were not restricted to) groups and which were associated with high values for the topographic variables. although we expected a relationship between microbat assemblages and distance to escarpments, the significant effect of elevation was not predicted. elevation generally increased away from the coast and was auto-correlated with 'distance to coastlines', making it unclear which of these features was most important in influencing bat composition. the second factor influencing bat composition was climate, specifically annual rainfall (corrolated with maximum temperature and latitude), mean temperature and minimum temperature (corrolated with temperature range; refer table ). the influence of annual rainfall was expected given the relationship between rainfall and species composition exhibited by the entire mammal fauna of north-western australia . a similar pattern was shown by the vegetation (bowman et al. ) and birds (whitehead et al. ) of north-western australia. in contrast to the significant relationship identified between bat assemblages and mean climatic variables, there was no significant relationship between ambient temperature (measured at pm each sampling night) at each site and species composition. at the time of year that we sampled, temperature was unlikely to limit the number of bat species that were active. however, during the dry season, low inland temperatures may reduce insect activity, restrict bat activity to the earlier, warmer times of the night and/or induce some species to enter torpor. therefore, restricting our sampling to one period of the year may have affected our results but sampling at different times of the year would have required a much greater sampling effort. between years, there was no observable difference in general weather patterns during each sampling period, therefore inter-year variations were unlikely to have affected our results. there were significant associations between bat species assemblages and fire frequency (corrolated with time since fire). the effects of fire on landscapes in northern australia can depend on the number of times an area is burnt and on the time since last fire (assessed here), fire intensity, seasonal timing of fires and spatial extent of burning (dyer et al. ; andersen et al. ) . the link between fire and bat species composition is likely to be an indirect one. it is also possible that characteristics of the landscape such as fuel loads, geography and habitat type are actually the primary influence for species assemblages and fire frequency is a secondary consequence of these landscape characteristics. therefore, our results should be viewed with caution and further investigation into the effects of fire on bat species assemblages is required before conclusions can be drawn. we assessed several variables involving insect availability at each site. none of the variables showed any significant relationship with microbat assemblages. this suggests that, in the top end at least, available food resources do not influence the composition of bat communities. this conclusion was consistent with previous research on insect-eating bats that indicated most species capture prey opportunistically (fenton minimum adequate models and explanatory power (per cent of deviance captured) are shown. probability levels *p < . , **p < . . ). specific research on tasmanian bats also concluded that bat assemblages were generally opportunistic foragers (o'neill & taylor ) . four aspects of our sampling strategy may have influenced our analysis. first, we did not sample non-volant insects and other arthropods that are eaten by some bat species in the top end (e.g. spiders, c.r. pavey, unpubl. data ) . second, high flying insects that are preyed on by bats such as taphozous spp. were probably not attracted to our light trap. third, bats may only show a response to insects at certain times of the year. it is likely that at the time of sampling (late dry season), insects were abundant and food resources did not affect the activity of bats. fourth, insect sampling was limited to one night per site, which may not have been sufficient to provide an adequate representation of overall insect availability. therefore, we suggest that a combination of insect sampling methods should be used in future assessments of prey availability and bat assemblages, particularly when the diversity of bats is high. these methods should aim to sample volant and non-volant invertebrates. did our study adequately sample a cross section of the major environmental gradients in the top end? compared with much of australia, the environment of the top end is relatively uniform. landscape relief is low, woodlands dominate most of the landscape, temperature varies little throughout the year and the climatic gradients are gradual. therefore, environmental variation is relatively small, and fewer sampling sites should be required compared with areas with greater topographic, climatic and vegetative variation. however, there may have been two significant deficiencies in our sampling. first, the highly seasonal rainfall in the monsoon tropics results in starkly contrasting 'wet' and 'dry' seasons. from our study, we were unable to say how bat composition may vary seasonally and there are no data available to assess seasonal patterns. second, the chosen study area was huge ( km ) and most of the north-east of the top end (arnhem land) was unsampled. therefore, clearer patterns of bat assemblages may have emerged if we had sampled more comprehensively, both spatially and temporally. most sites ( ) were sampled in pairs and the minimum distance between any two sites was km (mean km). bats can travel long distances during the night. foraging distances for a selection of species range between km and km (herr & klomp ; law & anderson ; lumsden et al. ) , therefore some of our results were potentially autocorrelated due to the same bats being sampled at both sites within a pair. therefore, we assessed the similarity in site species composition using the bray-curtis index. this index was calculated by dividing the number of shared species between pairs of sites by the total number of species of both sites. the resulting value was plotted against the distance between each pair of sites (fig. ) . the scatter of points was highly variable, however, the slope of the regression line was shallow. this pattern indicated that the relative change in species composition as a result of geographical separation was small. one of the environments largely neglected during sampling was monsoon rainforest, although 'riverine' sites did sample components of monsoon rainforest environments. we considered this had little effect on our results because monsoon rainforests occupy just . % of the landscape (based on mapping by fox et al. ) and usually occur in patches less than ha (russell-smith ) . in addition, menkhorst and woinarski ( ) found no bat species that were tightly associated with monsoon rainforests in the top end and these forests support a depauperate mammal fauna in general . some of the environmental variables that we found to be significantly correlated with bat assemblages in the top end differed from those related to bat community variation in other areas of australia. waterbodies have been found to support high species diversity and some species are strictly associated with them (law et al. ; south-west slopes of nsw.; young & ford ; central western queensland) . in the top end, glm analysis suggested bat species richness increases with decreasing distance to perennial rivers. however, species richness was not exceptionally high at our 'riparian' sites. further, group , which was on average closest to rivers, did not have the highest species diversity. also, we found no significant difference in species assemblages between 'riverine' and 'wood- fig. . similarity in bat species composition between pairs of sites (using the bray curtis similarity index, refer text) plotted against the distance between each pair. graph also displays the fitted linear regression line. land' habitats and there was no relationship with distance to available surface water. given that sampling was carried out during the driest time of the year (late dry season september-november) the likelihood of detecting significant associations with waterbodies was maximized. a relationship between vegetation structural complexity and microbat diversity has been established in studies in western australia (mckenzie & muir ) and nsw (law et al. ) . by contrast, we found significant correlations of bat species diversity with canopy cover but no associations with structural complexity. compared with the vegetation in the areas sampled by mckenzie and muir ( ) and law et al. ( ) , the vegetation of the top end is usually shorter and contains fewer understorey layers (d. lewis pers. comm. ). this limits the degree of vegetation structural complexity in the top end that likely accounts for the lack of correlation between structural complexity and bat communities. although we identified significant differences between the species assemblages within classification groups and habitat types, the assemblages were not clearly defined. most species occurred in more than one group and some were present in all groups. in addition, there were no associations between insect variables and bat assemblages and relatively few associations with environmental variables. this pattern is not restricted to microbats. birds, reptiles and nonvolant mammals also exhibit 'loose' patterns of species composition (woinarski & fisher ; woinarski et al. ) and limited associations with particular environments and environmental gradients woinarski et al. ) in the top end. woinarski et al. ( ) suggested this trend was a consequence of the homogeneity of eucalypt woodlands and forests that dominate the top end landscape. this relatively uniform environment militates against highly specialized and habitat-specific faunas. however, there were exceptions. specifically, some microbat species had a clear association with rugged rocky areas, particularly escarpments and adjacent areas. these areas provided a complex mix of habitats that contained foraging and roosting sites suitable for both cave and tree roosting species. this pattern extended to other vertebrate species as well. rocky escarpment regions in the top end support high species diversity as well as a number of endemic or habitat restricted species (woinarski & gambold ; . vegetation corridors beside rivers and surrounding areas (but not the waterbodies themselves) appeared to be important environments as they supported high bat species richness. bats are regularly characterized by the foraging strategy they employ within their immediate environment (mckenzie & rolfe ; neuweiler ; schnitzler & kalko ) . rivers are often associated with environments with tall dense vegetation. these areas do not appear to be of conservation significance because we did not observe high species richness at our 'riparian' sample sites. however, riverine environments usually have a distinct outer 'edge' and vegetation surrounding these areas is usually shorter and relatively open. we propose that these areas had greater species richness as they provide a diversity of environments for bats that employ different foraging strategies. we recommend that further research be conducted to examine the relationship between rivers and bats in the top end. our study did not take into account longer-term bat population dynamics. bats in the top end are poorly surveyed and, with few exceptions, surveys have been unstructured and unsystematic. therefore, attempting to identify and compare historical trends in bat populations is very difficult. given the top end environment is (currently) relatively unmodified, it could be assumed that mammal populations will remain stable and secure over the short to medium term. unfortunately, this is not the case. woinarski et al. ( ) described a case of decline in terrestrial small mammals in a conservation reserve in the top end that could not be confidently attributed to any clear environmental factor(s). further cases have also emerged (e.g. pardon et al. ; watson & woinarski ) . therefore, we recommend establishing longterm monitoring programs to track changes in bat populations so that changes may be quickly identified, assessed and appropriately managed. this is a highly challenging task that can only be achieved through a considerable commitment of time and resources. fire in tropical savannas: the kapalga experiment patn. pattern analysis package. csiro division of wildlife & ecology preliminary biogeographic analysis of the northern territory vascular flora australian bats statistical design and analysis for a 'biological effects' study glim for ecologists bats and gaps: microchiropteran community structure in a queensland rain forest the foraging behaviour and ecology of animal-eating bats the ecological basis of life history variation in marsupials the vegetation of the australian tropical savannas. environmental protection agency aspects of the ecology of insectivorous forestdwelling bats (microchiroptera) in the western slopes of the australian alps preliminary investigation of roosting habitat preferences of the large forest bat vespadelus darlingtoni (chiroptera, vespertilionidae) anuclim version . . centre for resource and environmental studies rarity in the tropics: latitudinal gradients in distribution and abundance in australian mammals determinants of loss of mammal species during the late quaternary 'megafauna' extinctions: life history and ecology, but not body size an analysis of epiphytic lichen communities in tasmanian cool temperate rainforest detection of bats by mist-nets and ultrasonic sensors activity and stratification of microchiropteran bat communities in thinned, unthinned and old lowland regrowth forest, east gippsland roost preferences and foraging 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patterns, environmental correlates and conservation of avifauna in the northern territory hollows in jarrah (eucalyptus marginata) and marri (corymbia calophilla) trees. i. hollow sizes, tree attributes and ages gradient analysis of a tropical herpetofauna: distribution patterns of terrestrial reptiles and amphibians in stage iii of kakadu national park wildlife of lancewood, acacia shirleyi thickets and woodlands in northern australia. . variation in vertebrate species composition across the environmental range occupied by lancewood vegetation in the northern territory gradient analysis of the distribution of mammals in stage iii of kakadu national park, with a review of the distribution patterns of mammals across north-western australia distribution patterns of vertebrates in relation to an extensive rainfall gradient and variation in soil texture in the tropical savannas of the northern territory bird distribution in riparian vegetation in the extensive natural landscape of australia's tropical savanna: a broad-scale survey and analysis of a distributional data base changes in mammal populations in relatively intact landscapes of kakadu national park bat fauna of a semi-arid environment in central western queensland our thanks go to bryan baker, chaz delacoeur, chris grant, david holland, marieke lettink, tony mitchell, steve owen, terry reardon, nathalia velez and felicity watt for their help with field work. greg connors, andrew edwards and peter brocklehurst assisted with various aspects of the analysis. john sheppard tracked down many of the references. thanks to irene rainey and peta jones who did a wonderful job with data entry. many parks and wildlife rangers and landholders assisted in planning surveys and locating suitable sampling sites. john woinarski, chris johnson, brad law, michael bull and an anonymous referee provided valuable comments on the manuscript. d.m. also thanks john woinarski for his support throughout this project. the tropical savannas crc provided funding for this project. animal sampling procedures were approved by the n.t. animal ethics committee (ref: a ). key: cord- -a oy sz authors: yang, shenshu; lian, gaojian title: ros and diseases: role in metabolism and energy supply date: - - journal: mol cell biochem doi: . /s - - - sha: doc_id: cord_uid: a oy sz researches dedicated to reactive oxygen species (ros) had been performed for decades, yet the outcomes remain controversial. with the relentless effort of studies, researchers have explored the role of ros in biosystem and various diseases. ros are beneficial for biosystem presenting as signalling molecules and enhancing immunologic defence. however, they also have harmful effects such as causing tissue and organ damages. the results are controversial in studies focusing on ros and ros-related diseases by regulating ros with inhibitors or promotors. these competing results hindered the process for further investigation of the specific mechanisms lying behind. the opinions presented in this review interpret the researches of ros from a different dimension that might explain the competing results of ros introduced so far from a broader perspective. this review brings a different thinking to researchers, with the neglected features and potentials of ros, to relate their works with ros and to explore the mechanisms between their subject and ros. ros are a set of unstable molecules including hydrogen peroxide (h o ), hydroxyl radical (oh − ), singlet oxygen ( o ) and superoxide (o − ) that are produced by all kinds of cells [ ] . the comprehensive distribution of ros may grant them with a fundamental role in biosystem. although ros play an important role in pathogen resistance and cellular signalling, they are also broadly recognized as harmful reactive particles to cell as they damage intracellular proteins, lipids and nucleic acids. it usually appears in pathological processes when they are not scavenged on time [ ] . the essence that ros are produced in energy demanding conditions where vigorous metabolism is in demand shall not be neglected. the pathogenic role of ros in self-damage and the beneficial role in the immune system may be due to the requirement of energy supply. in these conditions, excessively produced ros bring about oxidative damage to body and pathogens. ros are widely involved in basic mechanisms and pathways. they not only impair cells and tissues with oxidative damage, but also play an important role in many homeostasis processes involving metabolism, immunity, growth and differentiation [ ] . researchers have been regarding ros as byproducts and exploring their effects on organisms, but the fundamental features of ros might illuminate their role in pathologies and biomechanisms. mitochondrial respiratory chain is one of the major sources of cellular ros. atp synthesis produces ros during normal oxygen metabolism. thus, ros are regarded as byproducts during energy perfusion to cell activities in most cases. the primary function of nadph oxidase (nox) enzymes is the generation of ros [ ] . belonging to the nox family, activated nox could promote ros production through ryanodine receptors and thus trigger ca + sparks [ ] . the involvement of nadph and nadh in repiratory chain and cellular metabolisms makes ros produce in all kinds of cells. toll-like receptors (tlr) tlr , tlr and tlr can enhance ros production by recruiting mitochondria to macrophage phagosomes and translocating tumour necrosis factor receptor-associated factor (traf ) to mitochondria to engage in evolutionarily conserved signalling intermediate in toll pathways (ecsit) [ ] . although ros are mostly produced in mitochondria, the detailed mechanisms of the production are still not fully understood. however, the major factors responsible for ros production are respiratory chain complexes (fig. ). nadh-ubiquinone oxidoreductase (complex i) is the major source of mitochondrial ros production in varying diseases. the components responsible for ros production of complex i include ubisemiquinone, flavin mononucleotide, fe-s cluster and nad [ ] . however, ros production by complex i in healthy state is humble presenting little oxidative damage. the major production of ros in this state comes mainly from complex ii through tca cycle. nadh gene mutation which causes deficiency in respiratory complex i could end up in the overproduction of ros and enhance metastasis of tumour cells [ ] . succinate dehydrogenase (sdh) or succinate-coenzyme q reductase (sqr) is composed of sdha, sdhb, sdhc and sdhd. sdha and sdhb are hydrophilic proteins. sdhc and sdhd are hydrophobic proteins that bind to ubiquinone. this oxidoreductase is also known as complex ii that plays an important role in tca cycle and respiratory chain. succinate is the intermediate of tca cycle and also a metabolic signature of ischaemia-reperfusion. it is responsible for ros generation when accumulated from fumarate overproduction and malate/aspartate shuttle during reperfusion. ischaemia injury can be ameliorated by the inhibition of succinate or ros. complex ii turns succinate into fumarate through oxidation in mitochondria with reduced ubiquinone in the membrane [ ] , and succinate could be re-oxidized by sdh, thus increasing ros generation through reverse electron transport in mitochondria. [ ] . ubiquinol-cytochrome c oxidoreductase (complex iii) is encoded by uqcrc (ubiquinol-cytochrome c reductase core protein ) gene and could receive reducing equivalents from complex i and complex ii. the received reducing equivalents are proceeded with ubiquinol and produces semiquinone for further proton transfer. p shc (src homologous-collagen homologue adaptor protein) generates mitochondrial ros as apoptosis signal through oxidation of cytochrome c in mitochondrial electron transfer chain. p mutants could lose the ability to generate ros and induce mitochondrial apoptosis [ ] , but genetic mutation may also contribute to increased generation of ros. isp- and nuo- encode complex iii subunit rieske and complex i subunit ndufb (nadh dehydrogenase [ubiquinone] beta subcomplex subunit ), respectively. mutants in isp- and nuo- are all related with enhanced ros level that leads to lengthened lifespan. ros promotor treatments can lengthen the wild-type lifespan while having no effect on those longevity mutants. and the enhanced ros induces apoptosis pathway fig. generation of ros in mitochondria triggered by ced- that changed the gene expression to protect mitochondrial dysfunction [ ] . tumour necrosis factor alpha (tnfα ) could regulate cell proliferation and death, and the inhibition of nuclear factor kappa-b (nf-κb) makes tnfα bias to cell death. tnfα-induced ros could support c-jun n-terminal kinase (jnk) activation during nf-κb inhibition. the sustained jnk activation enables cytochrome c release and leads to necrotic cell death [ ] . the homeostasis of ros plays an important role in reducing oxidative damage and fulfil energy demand. ros present as signalling molecules in multiple pathways and mechanisms. thus, they are inevitably influenced by proteins and genes involved within. apart from that, other environmental complexes and antioxidants could contribute to ros production according to their redox potential. it is also noted that different mtdna haplotypes may have distinct respiration capacity triggered by varying production of ros [ ] ( table ) . relatively high levels of ros may cause oxidative damage or induce apoptosis during immunological defences or pathological conditions. the mechanisms to survive under such environment are essential for body cells or tumour cells and bacteria. hypoxia inducible factor- alpha (hif- α ) encoded by endothelial pas domain protein (epas ) gene could control ros level in mitochondria through antioxidant enzymes and maintain ros homeostasis [ ] . pparγ coactivator α (pgc- α) is required for antioxidative enzymes including glutathione peroxidase (gpx ) and superoxide dismutase (sod ) [ ] . ros level also could be controlled through degradation of nox on endoplasmic reticulum by protein negative regulator of ros (nrros). this reduces tissue damage and maintains its function upon immunological defence [ , ] . however, ros themselves could activate extracellular signal-regulated kinase (erk) by targeting proteins gαi and gα and protect cardiac cell from oxidative damage [ ] . these proteins present protective effect on body cells and redox balance. it is also noted that the opened potassium channels may reduce ros level [ ] . ros tolerance may be partly involved in the mechanisms behind the tumour cells avoiding immunological defence. cancer cells could produce enough nadph to support vigorous proliferation while maintaining ros homeostasis through gsh (glutathione). enhanced ros in lung cancer cells could inhibit glycolytic enzyme pyruvate kinase m (pkm ). this also allows them to survive under acute oxidative stress and still supports their proliferation [ ] . nuclear factor erythroid- -related factor (nrf ) transcription is increased in tumour cells to suppress ros generation by nrf -keap (kelch-like echassociated protein ) interaction. oncogenic alleles of k-ras, b-raf and myc could increase nrf antioxidant activity and reduce ros level [ ] . researches also indicate that gene ucp (uncoupling protein ) could limit ros production and inflammation in macrophage [ ] . apart from that, antioxidants also include organics like vitamin e, vitamin c and complexes like fhc (ferritin heavy chain). they reduce apoptosis induced by tnfα and jnk activity through suppression of ros accumulation and iron sequestration as a downstream product of nf-κb pathway [ , ] . change cells from proliferation into differentiation ros are important particles involved in immunological defence. overexpressed tnf induces ros in mitochondria through rip -rip -dependent (receptor-interacting protein kinase) pathways. the increased ros leads to both enhanced macrophages killing and necroptosis. this necroptosis relies on mitochondrial cyclophilin d and ceramide [ ] . tlr / / could enhance ros by recruiting mitochondria to macrophage phagosomes and translocating traf to mitochondria to engage ecsit. this further increases the bacterial killing [ ] . ros induces oxidative damage and apoptosis which may contribute to the control of lifespan. p shc generates mitochondrial ros as apoptosis signal through oxidation of cytochrome c in mitochondrial electron transfer chain [ ] . other factors such as matrix metalloproteinase- (mmp- ) could increase cellular ros and stimulate transcription factor snail and epithelial-mesenchymal transition (emt). this process causes dna oxidative damage and genomic instability in breast cancer and turns normal cells into cancer cells [ ] . heart cell stretch could activate nox to produce ros through ryanodine receptors and trigger ca + sparks [ ] . the deficiency of nox inhibitor nrros could lead to elevated oxidative damage. [ ] erythroblastosis virus transcription factor- (ets- ) requires ros to regulate p phox expression. however, this also could contribute to nadph oxidase and ros generation and become an ets-ros positive feedback [ ] . the ros-induced ros-release circle could lead to elevated ros generation as well [ ] . transcription factor upbeat could regulate the balance between cellular proliferation and differentiation through ros. vigorous changes in metabolism may occur during the shift from cell elongation to differentiation to fulfil metabolic demands. upbeat enhances ros level through the repression of peroxidases which could change the pattern of cell from proliferation into differentiation. [ ] . researchers have been trying to elucidate the mechanisms and the role that ros plays in diseases since they were identified. ros influences diseases basically with its function as signalling molecules and oxidants that influence cell survival and oxidative damage. ros could also drive immunity through immunological defence and maintain metabolic balance or heat dissolving. the multiple functions of ros in biosystem may influence each pathema from different aspects ( table ). abnormal cell proliferation and metastasis are common features of cancer. vigorous proliferation demands substantial nadph to produce energy. this process also abundantly increases ros. high levels of ros could induce apoptosis of tumour cells. however, they also protect cells from oxidative damage by suppressing glycolytic enzyme pkm through gsh in cancer [ ] . tumour cells exhibit enhanced nrf transcription. nrf present as antioxidants that control ros level in cancer. the inhibition of nrf could impair tumourigenesis with increased ros level [ ] . oncogenic alleles k-ras, b-raf and myc could contribute to nrf -keap interaction. ros also regulate tumour suppressor protein p and mediate apoptosis in cancer [ ] . stem cells tend to contain lower ros than regularly differentiated cells. cancer stem cells also maintain low levels of ros to avoid apoptosis induced by ros. it also makes them suffer less dna damage from radiation with enhanced ros scavenging systems [ ] . cancer cells resistant to braf and mek inhibitors develop vulnerability to high levels of ros [ ] . thus, the strategy to enhance ros level may seem to present as an important way for cancer chemotherapy. however, researchers also indicated that tumour cells with high metastasis contain nadh gene mutation. the mutation causes deficiency in respiratory complex i and ended up in overproduction of ros, and the metastatic activity could be suppressed with ros scavenger [ ] . increased ros generation could trigger enhanced epidermal growth factor receptor (egfr) signalling and promote tumour progression [ ] . snail and emt stimulated by mmp- could increase ros generation. the elevated ros level could turn normal cells into cancer cells with dna damage and genomic variation. [ ] . ros could also mediate the tumour microenvironment through epithelial-mesenchymal transition that contributes to radioresistance and therapeutic failure [ ] . although suppressing ros signalling to inhibit tumour growth with ros scavenger is not ideal, the process of inhibition impairs ros-mediated oxidative damage and apoptosis [ ] . the uninhibited ros generation and uncontrolled ros level could also promote cancer cell metastasis and the process of canceration. these controversial results bring about hindered exploration of ros-mediated treatments against cancer. rather than focusing on symptoms, the fundamental role of ros and its comprehensive distribution among biosystem may explain these competing results from a broader perspective. ros are highly related with energy production rather than just byproducts. cancer development and tumour metastasis demand larger amounts of energy than normal cells. this energy-acquiring process also produces high levels of ros. the enhancement of ros may also increase energy production to facilitate tumourigenesis. rather than a regulator of the cancer pathology, ros are more likely the representative of energy consumption. it is easy to induce cancer cells death in vitro with oxidative damage. however, the failure to apply oxidative damage to cancer cells clinically seems to be the result of ros homeostasis system in vivo. being part of the mechanisms involved in innate immunity, inflammation eliminates pathogenic factors while causing tissue damage. ros play a similar role in immunity by enhancing immunological defence and causing oxidative damage. nlr family, pyrin domain-containing (nlrp ) inflammasome could enhance inflammation by activating caspase and promoting secretion of il- β and il- . ros are crucial for nlrp activation [ ] . drosophila multipotent haematopoietic progenitors present relatively high levels of ros in in vivo physiological conditions and become low during differentiation. the enhanced ros could promote the differentiation through jnk and the forkhead box o (foxo) pathway, but ros inhibition disabled its differentiation [ ] . although the differentiation prefers low levels of ros, they are still essential for the process. different t-cell subsets also have distinct sensitivity to ros level that may influence their development and function. th cells are involved in autoimmune diseases and inflammatory diseases. experimental autoimmune encephalomyelitis (eae) is a th -mediated autoimmune disease. regulating th cell differentiation by interfering ros level through glutathione metabolism could prevent eae development [ ] . influencing chromatin structure with gls inhibition also enhances ros level and prevents th differentiation [ ] . discovery of brand new t-cell subsets also endows deeper understanding on immune system and provides aspects for the exploration of t-cellregulated autoimmune diseases [ ] . ros could support immune system, but they become cytotoxic while overload [ ] . ros play a role in both activation-induced t-cell death and activated t-cell autonomous death [ ] . oxidative damage leads to cellular damage on dna, protein and lipids. the damage-induced apoptosis plays an important role in inflammatory bowel diseases [ ] . ros also stimulate parasite growth and cause tissue damage to host's organs [ ] . the expression of nadph oxidase is elevated in phagocytic leukocytes upon stimuli. [ ] the ros-mediated autophagy could promote periodontitis and tendinopathy as well [ ] . apart from oxidative damage, ros also serve as signalling molecules and play an important role in homeostasis, metabolism, growth and differentiation [ ] . ucp could limit ros production and inflammation in macrophage and reduce parasitic cysts [ ] . however, ucp relies on ros level required for heat dissipation through thermogenic respiration in brown adipose tissues. the depletion of ros inhibits ucp and heat generation [ ] . cigarette smoking induces oxidative stress in bronchitis and emphysema. inflammation also occurs in these chronic obstructive pulmonary diseases [ ] . the role of ros in bacterial killing appears to be inconsistent among different studies. some research state that increased ros level in bacteria can enhance the killing ability of antibiotics and oxidants [ ] . enhanced ros by excess tnf through rip -rip -dependent pathways in mitochondria lead to both enhanced macrophages killing and necroptosis that relies on mitochondrial cyclophilin d and ceramide [ ] . tnf-α is indicated to contribute to ros production in rheumatoid arthritis [ ] . however, other studies indicate that ros response during bacterial antibiotic killing is dispensable [ ] . ros scavenger and hydroxyl radical inhibitor could suppress antibiotic bacterial killing. antibiotic bacterial killing does not strictly depend on ros [ ] . it is also noted that the level of ros does not influence antibiotics' activity on killing bacteria at all [ ] . antibiotic killing of escherichia coli does not rely on ros [ ] . the enhanced bacterial killing with increased ros level may due to increased metabolism and energy supply that support oxidation and immunity system. however, it applies little effect when they reaches saturation. but moderated metabolism with lower levels of ros surely decreases the ability of bacterial killing. neurons are important cells that control sensory organs and muscle system. the injury of these cells may lead to neuropathy and movement disorder. the relatively low antioxidant activity makes them vulnerable to oxidative damage. the defects in mitochondria may enhance ros generation and thus promote jnk and sterol-regulatory element binding proteins (srebp) activation in neurons that results in neurodegeneration through the accumulation of lipid droplets [ ] . the adipogenesis could also be influenced by ros via signal transducers and activators of transcription (stat ) [ ] . however, antioxidants could rescue the apoptosis [ ] . fhc could suppress ros accumulation and jnk activity through iron sequestration that inhibits tnf-α-dependent apoptosis [ ] . pgc- α could protect neural cells from oxidative damage by reducing ros level via antioxidative enzymes gpx and sod [ ] . methylmercury and manganese could induce neurotoxicity with enhanced ros level [ , ] . and the increased level of ros in the substantia nigra pars compacta leads to neuronal apoptosis of dopaminergic neurons in down syndrome and parkinson's disease. this process may ultimately lead to retardation [ ] . nrros could protect central nervous system from eae by reducing oxidative damage through nox degradation on endoplasmic reticulum [ ] . nevertheless, ros still play an important role in neuronal development and are essential for synaptic plasticity and memory formation with its fundamental role in energy perfusion. the essence that neurons are differentiated cells that lack the potential to proliferate explained these competing results of antioxidative strategies. they maintain a relatively low demand in energy and metabolism. in the heart, angiotensin ii, norepinephrine and tnf-α mediated ros are related with cardiac hypertrophy, myocardial infarction and heart failure. myocardial ischaemia is the most common cause of heart failure. the ischaemia-reperfusion injury leads to apoptosis of cardiomyocytes that is associated with high levels of ros [ ] . the shortage of atp during ischaemia impairs ion pump and causes calcium accumulation. calcium overload and increased ros could rupture plasma membrane and lead to cell death [ ] . cardiac hypertrophy is a compensating process that enables heart to maintain sufficient function. the increased ros during the process is responsive to energy demand caused by insufficient heart function. thioredoxin could reduce cardiac hypertrophy through heat shock protein and class ii histone deacetylases, the latter being a master negative regulator of cardiac hypertrophy [ ] . and it is also noted that ros increased via d-amino acid oxidase in the hearts of rats could directly lead to systolic heart failure without cardiac hypertrophy [ ] . the oxidative damage-mediated apoptosis is the major cause of heart failure as well. the method to fulfil energy demand by using nox to protect heart from failure with improved myocardial energetics via fatty acid oxidation is also proved to be successful [ ] . to reduce oxidative damage, ubia prenyltransferase domain-containing protein presents cardiovascular protective function via antioxidant coenzyme q [ ] . however, the inability to recover from cardiac damage and pathology is also critical for heart failure. postnatal cardiomyocyte cell-cycle arrest is mediated by ros through dna damage response [ ] . heart cell stretch could cause arrhythmogenic ca + sparks based on microtubules [ ] . although the oxidative damage caused by ros is the major reason for heart failure, the role of ros in energy supply is rather important that protect heart from an even sudden failure of insufficient function. ros regulate vascular cell proliferation and apoptosis with their fundamental role in metabolism. oxidative stress could lead to hypertension and promote its pathological process. however, ros are also needed for the relaxation of cerebral arteries [ ] . ets- and angiotensin ii-generated ros play an important role in vascular changes and injury, and no could regulate blood flow homeostasis in blood vessels [ ] . these outcomes seem to be confusing to tell whether ros are beneficial or harmful. the role of ros in biosystem is rather neutral that they mainly respond to energy demand. nox family could influence the neovascularity of tumour and physiological vascular processes [ ] . similar results also presented in ros-mediated wound repair [ ] . ischaemia-reperfusion (ir) causes oxidative damage with increased generation of ros in mitochondria. succinate is the metabolic signature of ischaemia and responsible for ros generation during reperfusion. the reperfusion injury also leads to retinal dysfunction-associated ros production when the blood pressure is low [ ] . succinate accumulates during reperfusion from fumarate overproduction and malate/aspartate shuttle and then re-oxidized by succinate dehydrogenase. this process increases ros generation through reverse electron transport in mitochondria. the inhibition of succinate or ros could ameliorate ir injury [ ] . pre-conditioning protocols could reduce ischaemia-reperfusion injury by regulating ros level [ ] . atherosclerosis could be regulated by ros interacting with transcription factors related with lipid peroxidation and macrophage [ ] . ros induces dna damage and lipid peroxidation in pneumoconiosis and carcinogenesis as well [ ] . increased ros promote thrombus formation in artery and influence other cardiovascular diseases as well [ , ] . the pulmonary vascular lesions and inflammation are broadly recognized pathological changes in acute respiratory distress syndrome (ards) caused by oxidative damage [ ] . taken together, researchers revealed the position of ros in metabolism and energy supply. ros are needed for basic energy demand and vigorous metabolism rather than simply affecting cellular signalling and organism damages. the continuous oxidative damage applied on cell and tissue may lead to severe organic injuries and eventually cause organ failure. the ros level leading to organ failures far exceeds the extent to maintain basic metabolism and thus the balance between energy supply and oxidative damage is tilted. increasing ros grants little beneficial effect in this situation. inhibition of ros could reduce tnf-α-mediated fulminant liver failure. tnfα regulates cell proliferation and death and the inhibition of nf-κb makes tnfα bias to cell death. tnfα-induced ros supports jnk activation during nf-κb inhibition. sustained jnk activation enables cytochrome c release and leads to necrotic cell death [ ] . fhc is a downstream product of nf-κb. they could reduce apoptosis induced by tnfα through suppression of ros accumulation and jnk activity. the suppression of ros is achieved by iron sequestration [ ] . ros are produced by glomerular cells as autacoids [ ] . ros-mediated glomerular basement membrane degradation and altered cell function may contribute to ischaemic renal failure as well [ ] . and the inhibition of ros could decrease caox stone in kidney [ ] . hypoxia-induced requirement of energy supply and metabolism could lead to increased ros response through ca + influx pathway. this mechanism results in physiological, biochemical and molecular changes. the hypoxia-induced ros production is important for respiratory plasticity and sensory plasticity. ros-mediated apoptosis and cellular dysfunction are associated with heart failure [ ] . the arrhythmias caused by elevated ros and altered mitochondrial function may lead to sudden cardiac death [ ] . the comprehensive distribution of ros intrigues researchers to explore the relationship between their subject and ros. the reduced ros level could lower insulin resistance and improve insulin sensitivity in diabetes ii [ ] , and the glucose-stimulated insulin relies on ros signalling [ ] . however, the cellular death owing to ros-mediated oxidative damage also brings about diabetic complications [ ] . mmp activity and transcription factor-β (tgf-β )-induced excessive deposition of extracellular matrix mediated by ros could lead to renal fibrosis [ ] . the process of ageing caused by oxidative damage and muscle dysfunction could lead to sarcopenia. however, ros are also essential for muscle cell development as signalling molecules [ ] . generation of ros in skeletal muscle is enhanced during contractile activity [ ] . ros are increased in the early stage of muscular dystrophy development [ ] . the elevated ros may reduce muscle mass and bring about frailty [ ] . however, ros also plays an important role in muscle remodelling as signalling molecules [ ] . overproduced ros released through mitochondrial permeability transition pore will damage dna and accelerate ageing by reducing cellular nad [ ] . apart from suppressing tumour, p also plays a role in premature ageing by causing reactive damage to dna [ ] . mushroom-contained antioxidants may protect against oxidative damage and ageing [ ] . ros overproduction may contribute to reproductivity issue and infertility through oxidative damage and disturbed hormone balance. the excessive ros may damage spermatogenesis, sperm lipid/protein layer and dna structure. the ros scavenging system to reduce ovarian toxicity is important for follicular development [ ] . the role of ros seems to be similar in different diseases that they are essential for cellular metabolism and become pathogenic while overload. researches of ros have been carried out since last century, the cognition of which varies along elapse of time. ros promote macrophage bacterial killing through oxidative damage and apoptosis. they also engage in multiple cellular pathways as signalling molecules. however, they also have negative effects like inflammation and cytotoxicity. ros can function as intermediates in varying pathways, but they are also widely regarded as etiologic factors for diseases including cancer, inflammation and organ injuries. evidence suggests that the scavenging ros in pathological condition may reduce cell damage and control the pathological process. however, other researches also indicate the positive side of enhancing ros in diseases. thus, the mechanisms of how ros influences diseases remain obscure. europeans have dedicated to the study of ros and made a comprehensive exploration [ ] . yet, it remains controversial in results of ros-targeted strategies applied to clinical research. with the advancement in technique, ros can be measured in living cells of its transient generation with y . eu . vo nanoparticles by illuminating under oxidative conditions [ ] . specific chemical probes and low-temperature electron paramagnetic resonance (epr) technique could monitor ros level of tumour cells in vitro and in vivo [ ] . other environmental factors like ph and ion concentration are also suggested in ros regulation and generation [ ] . also magnetic fields could influence cellular ros level according to its intensity, frequency and exposure time [ ] . the fundamental understandings of ros remain basically the same in the past years. ros are generally regarded as signalling molecules and harmful particles. researchers always focus on ros levels and their results and analyses them from a single perspective. they should be illuminated from different perspectives and with extensive sight. based upon the characteristics of ros discovered by previous researches, i provide a hypothesis here that may explain the competing results so far. ros thrives in conditions that abundant energy is in demand for vigorous metabolism, either in cancer cells' proliferation and inflammatory necrosis, or immunological defence required immune system functioning. thus, ros do not just act as signalling molecules. they may present as basic energy particles like other acknowledged basic nutrient particles including proteins and carbohydrates. and they provide a much more fundamental impact on cellular metabolism. it is more likely that ros respond to elevated metabolism to fulfil energy demand, rather than directly bending itself to oxidative stress, which interprets why different researches demonstrate controversial outcome in regulating ros. the treatment of both inhibition and enhancement of ros in cancer in vitro may due to exhausted energy supply for metabolism and overload of energy supply-induced oxidative damage, rather than just the regulation of a byproduct. of course, it is easier to suppress ros generation with antioxidants or genetic depletion in experimental animal models. but the inability of clinically gene modulation in vivo and the multiple functions of ros in metabolism may lead to limited potrential of direct ros modulation in diseases with ros and metabolic imbalance. the fundamental role of ros grants them with the potential in metabolic regulation. from another aspect, the essence that ros are common particles with comprehensive distribution endows them with more fundamental mechanisms to be explored. the nox family of ros-generating nadph oxidases: 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therapeutic approach? nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? reactive oxygen species prime drosophila haematopoietic progenitors for differentiation glutathione de novo synthesis but not recycling process coordinates with glutamine catabolism to control redox homeostasis and directs murine t cell differentiation distinct regulation of th and th cell differentiation by glutaminase-dependent metabolism a cd (+) t cell population expanded in lupus blood provides b cell help through interleukin- and succinate t cell apoptosis and reactive oxygen species reactive oxygen species and antioxidant defense in human gastrointestinal diseases ros and trypanosoma cruzi: fuel to infection, poison to the heart reactive oxygen species in phagocytic leukocytes sudden death risk in older athletes: increasing the denominator mitochondrial ros regulate thermogenic energy expenditure and sulfenylation of ucp reactive oxygen species in chronic obstructive pulmonary disease potentiating antibacterial activity by predictably enhancing endogenous microbial ros production the role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis fe-s cluster biosynthesis controls uptake of aminoglycosides in a ros-less death pathway antibiotic and ros linkage questioned killing by bactericidal antibiotics does not depend on reactive oygen species cell death from antibiotics without the involvement of reactive oxygen species glial lipid droplets and ros induced by mitochondrial defects promote neurodegeneration mitochondrial stat and reactive oxygen species: a fulcrum of adipogenesis? jak-stat apoptosis and increased generation of reactive oxygen species in down's syndrome neurons in vitro involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity manganese neurotoxicity and the role of reactive oxygen species nitric oxide and reactive oxygen species in parkinson's disease reactive oxygen species, mitochondria, and nad(p)h oxidases in the development and progression of heart failure a redox-dependent pathway for regulating class ii hdacs and cardiac hypertrophy chemogenetic generation of hydrogen peroxide in the heart induces severe cardiac dysfunction cardiac-targeted nadph oxidase in the adaptive cardiac remodelling of the murine heart the oxygen-rich postnatal environment induces cardiomyocyte cellcycle arrest through dna damage response mechanisms underlying acute protection from cardiac ischemia-reperfusion injury ubiad is an antioxidant enzyme that regulates enos activity by coq synthesis reactive oxygen species: influence on cerebral vascular tone sources of vascular nitric oxide and reactive oxygen species and their regulation nadph oxidase(s): new source(s) of reactive oxygen species in the vascular system? reactive oxygen species and nox enzymes are emerging as key players in cutaneous wound repair reactive oxygen species, oxidative stress, glaucoma and hyperbaric oxygen therapy ros-mediated nlrp inflammasome activation in brain, heart, kidney, and testis ischemia/ reperfusion injury free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis reactive oxygen species: their relation to pneumoconiosis and carcinogenesis the role of reactive oxygen species in the pathophysiology of cardiovascular diseases and the clinical significance of myocardial redox regulation of platelet activation and thrombus formation by reactive oxygen species reactive oxygen species in acute lung injury reactive oxygen species as glomerular autacoids reactive oxygen species: production and role in the kidney reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations metabolic stress, reactive oxygen species, and arrhythmia reactive oxygen species have a causal role in multiple forms of insulin resistance ros signaling, oxidative stress and nrf in pancreatic beta-cell function the role of reactive oxygen species in apoptosis of the diabetic kidney reactive oxygen species and matrix remodeling in diabetic kidney reactive oxygen species as mediators of cellular senescence reactive oxygen species and redox-regulation of skeletal muscle adaptations to exercise absence of dystrophin disrupts skeletal muscle signaling: roles of ca + , reactive oxygen species, and nitric oxide in the development of muscular dystrophy control of reactive oxygen species production in contracting skeletal muscle reactive oxygen species are signalling molecules for skeletal muscle adaptation role of reactive oxygen species-mediated signaling in aging ros and senescence in the control of aging reactive oxygen species and antioxidant properties from mushrooms reactive oxygen species and sperm cells single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells teaching the basics of reactive oxygen species and their relevance to cancer biology: mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies extra-cellular but extraordinarily important for cells: apoplastic reactive oxygen species metabolism magnetic fields and reactive oxygen species publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord- -jy l zm authors: havel, john e.; kovalenko, katya e.; thomaz, sidinei magela; amalfitano, stefano; kats, lee b. title: aquatic invasive species: challenges for the future date: - - journal: hydrobiologia doi: . /s - - - sha: doc_id: cord_uid: jy l zm humans have effectively transported thousands of species around the globe and, with accelerated trade; the rate of introductions has increased over time. aquatic ecosystems seem at particular risk from invasive species because of threats to biodiversity and human needs for water resources. here, we review some known aspects of aquatic invasive species (ais) and explore several new questions. we describe impacts of ais, factors limiting their dispersal, and the role that humans play in transporting ais. we also review the characteristics of species that should be the greatest threat for future invasions, including those that pave the way for invasions by other species (“invasional meltdown”). susceptible aquatic communities, such as reservoirs, may serve as stepping stones for invasions of new landscapes. some microbes disperse long distance, infect new hosts and grow in the external aquatic medium, a process that has consequences for human health. we also discuss the interaction between species invasions and other human impacts (climate change, landscape conversion), as well as the possible connection of invasions with regime shifts in lakes. since many invaders become permanent features of the environment, we discuss how humans live with invasive species, and conclude with questions for future research. humans have had enormous impacts on earth and its biodiversity and many of these effects are global. lakes and streams are particularly prone to species loss (ricciardi & rasmussen, ) , with the greatest threats coming from land use changes and exotic invasive species (''biotic exchange'', sala et al., ) . humans have been particularly effective in breaking down biogeographic barriers through long-distance trade, intentionally introducing some species and carrying others as hitchhikers (kolar & lodge, ) . the result has been a translocation of numerous freshwater species (hulme, ) . although most introduced species fail to establish and spread (williamson, ) , many freshwater species have become invasive (see definition in box ) and some have caused widespread environmental effects and economic harm (pimentel et al., ) . freshwater ecosystems have greater biodiversity per surface area than marine and terrestrial ecosystems (dudgeon et al., ; balian et al., ) . freshwater ecosystems also play an active role in nutrient and water cycling (wetzel, ) , which translate into goods and services for human societies. at the same time, freshwater ecosystems have been deeply transformed by invasive species from a wide variety of taxonomic groups (strayer, ; simberloff et al., ) . it is thus vital to understand the factors that govern the introduction, spread, and subsequent impacts of invasive species in these ecosystems. among several important syntheses relevant to the topic of invasive species in aquatic environments are reviews of climate change (rahel & olden, ) , epidemiology and control (mack et al., ) , assessments of invasion potential (ibanez et al., ) , invader traits and impacts (strayer, ; pyšek & richardson, ) , vectors (carlton, ) , and functional homogenization (olden et al., ) . the purpose of this paper is to explore and review the littlestudied interactions of aquatic invasive species (ais) with natural and anthropogenic environmental changes in freshwater ecosystems and the challenges they present to humans in the st century. in particular, we examine impacts of invasive species; the role of humans in accelerating transport of aquatic invasive species beyond ordinary dispersal barriers; the characteristics of successful invaders; the susceptibility of communities to invasion and the positive feedbacks for future invasion (''invasional meltdown''); microbial invasions and their hosts; the interaction of invasion with other human impacts; and living with invasive species as a permanent fixture of the environment. biological invasions of freshwater ecosystems have a large number of known and potential impacts on community structure and ecosystem function. the establishment rates of ais are also high, as has been shown for several types of aquatic organisms and ecosystems (ricciardi, ; garcía-berthou et al., ; bobeldyk et al., ) . even though biotic interchanges and paleo-invasions contributed to biodiversity as we know it (pascal & lorvelec, ; gillson et al., ) , the pace of current invasions far exceeds that of the previous events that took place over geologic time scales (ricciardi, ) . in view of the great biodiversity and vulnerability of freshwater communities to biotic exchange (sala et al., ) , impacts caused by invasions are certainly a cause of concern. furthermore, the change itself is often perceived as undesirable even if an invasion is a delayed response to previous climatic events (gillson et al., ) or has seemingly minor effects on the overall ecosystem function. although some invasive species have had little or no detectable effects (e.g., daphnia lumholtzi, havel et al., a, b) , many ais have had damaging effects on the environment and on human interests. case histories of their effects are widely reported in primary literature (e.g., zaret & paine, ; goldschmidt et al., ) , the popular press (zinsser, ; bright, ) , and scholarly books (elton, ; simberloff, ) . here we focus on the impacts of ais on communities and ecosystems, since perturbations to these levels cause other unsuspected effects . for example, when invasive species behave as ecosystem engineers they may rapidly transform entire ecosystems (strayer, ). an illustration of this scenario is the transformation of the hudson river following zebra mussel invasion, from a pelagic-based food web to a littoral-based food web (strayer, ) . the high densities of zebra mussels in places like lake erie and the hudson river can filter most of the algae from the water column, leaving little food for pelagic zooplankton (strayer, ) and increasing light for vascular plants. increased growth of these plants, in turn, increases habitat structure and has numerous other effects (schefler, ) . another example of an ecosystem transformation is the effect of invasive plants in australia, where high densities of african poaceae altered stream hydrology by trapping sediments in the channel (bunn et al., ) . hydrologic changes, in turn, have large impacts on nutrient dynamics (allan & castillo, ) . overall, ecosystem effects of ais are still greatly under-studied and a challenge for future research. invasive species are well known to restructure freshwater food webs (vander zanden et al., ) . because predatory fish often control community structure of lakes and streams (brooks & dodson, ; power, ) and game fish have been widely introduced by fisheries agencies, much attention has been placed on exploring the community impacts of fish. some impacts have been huge. the introduction of nile perch (lates niloticus) to lake victoria, east africa, annihilated the native community of haplochromine cichlid fishes, driving many species to extinction (goldschmidt et al., ) . the introduction of peacock bass (cichla spp.) to lake gatun, panama, greatly simplified the food web, extending to zooplankton and insects (zaret & paine, ) . by reducing mosquitofish (gambusia affinis), this introduction had the further effect of increasing mosquito populations and mosquito-borne illness (zaret & paine, ) . omnivores can also have large impacts on aquatic communities. lodge et al. ( ) describe how non-native rusty crayfish (orconectes rusticus) not only consume macroinvertebrates, but also aggressively out-compete native crayfishes and destroy macrophyte cover, indirectly affecting the fish community. invasive plants have community level effects as well. for example, eurasian water-milfoil (myriophyllum spicatum) forms dense surface mats, and reduces light and diversity of native aquatic plants (madsen et al., ) . the simplified plant community is a poorer food resource for macroinvertebrates and higher-level consumers. similarly, experiments conducted in ponds showed that dense monotypic stands of hydrilla (hydrilla verticillata) changed the taxa composition of invertebrate assemblages compared with macrophyte multi-species treatments, although both treatments did not differ in regard to invertebrate taxa richness (theel et al., ) . hydrilla likely impacts microbes as well, with shifts of metabolic profiles and phylogenetic structure of bacterial communities as density of hydrilla increases (gordon-bradley et al., ) . although a half century of research has provided a wealth of knowledge about the spread and impacts of particular invasive species, aquatic ecologists still have a number of unanswered questions about the general nature of invasive species impacts on communities and ecosystems. at what densities do invasive species begin to impact native communities? this question has to do with density dependence of impacts from invasive species, which increase with their abundance. some studies have failed to identify impacts from invasive species that were in low or similar abundance to native species (dougas & Ó connor, ; aday, ) . for example, surveys in the river paraná showed that fish species richness was no different in the invasive hydrilla than in native egeria najas, and this finding was attributed in part to similar biomass of both macrophytes (cunha et al., ) . investigation of impacts along a gradient of invasive density is necessary to identify abundance thresholds above which impacts would occur. do invasive species differ in their impacts from that of native species if they were to achieve similar densities? this question deals with the comparison of the change in an ecological property caused by the invasive species with the change of the same property caused by a native one. in this case, the characteristics of the native species should be as similar as possible to the invasive and the changes caused by the native would be considered the ''control'' (e.g., mormul et al., ) . for instance, the invasive new zealand mud snail (potamopyrgus antipodarum) reaches very high densities in streams of the western united states, where it dominates nutrient cycling (hall et al., ) . if native hydrobiid snails achieved a similar density, would they also dominate nutrient cycling? what environmental factors contribute to time lags in impacts from invasions? in several ecosystems, spread, and/or impacts of non-native species occur several years or even decades following their introduction (pelicice & agostinho, ; downing et al., ) . such time lags create difficulties for predicting their impacts. ''legacy effects'' are effects of invasive species that persist after their removal (corbin & d'antonio, ) . such lagged impacts are mediated via changes in resource pools, habitat structure, or persistent feedback loops. legacy effects are virtually unstudied in aquatic systems, but have been demonstrated to have lasting effects in some terrestrial ecosystems (yelenik & d'antonio, ) . invasional meltdown may also lead to lags in impacts caused by invasive species, because a given non-native species may have its spread and impacts enhanced after it has been facilitated by the arrival of other non-native species (invasional meltdown section below). although all species disperse to some degree, the rate of moving species across the planet has been greatly accelerated by humans (kolar & lodge, ) . once established in a new habitat, most invasive species are nearly impossible to eradicate and some of the major pests are extremely expensive to control (pimentel et al., ) . for these reasons, human-mediated colonization is generally best avoided in the first place. one key to understanding the process of invasion is the different ways in which humans transport species. invasion of an exotic species involves long-distance transport to a suitable habitat and establishment (colonization), followed by spread into other suitable habitats in the region (fig. ) . propagule pressure has an important role in the first steps of invasion and in latter spread. it is related to the number of individuals and/or propagules (e.g., seeds) arriving in a region and the number of release events (duncan, ) . colonization of each new population requires tolerance of the physical and chemical conditions of the new habitat, food acquisition, predator avoidance, and reproduction within the new habitat. ais must find another water body in the ''terrestrial matrix''. a crayfish that is dumped on land will quickly dry out and fail to survive more than a day. introduced to a lake too low in dissolved calcium, the crayfish may live for a while but fail to molt. introduced to a lake with sufficient calcium but lacking habitat structure as protection, the crayfish may succumb to predation by fish. finally, a single crayfish that has passed through these ''physiological and biotic filters'' (rahel, ) may fail to reproduce if only a single gender of a bisexual species is introduced or if potential mates are too rare to find (allee effect). the widespread occurrence of asexual reproduction (bell, ) provides many species with the capability to establish a population from a single colonist. in this respect, macrophytes and cladocerans have an advantage over crayfish. once established in one lake, ais can more-easily spread to other nearby lakes. the chance of colonization is much greater for nearby populations than those far away. (this invasion probability-with-distance function is called a ''dispersal kernel''.) for instance, logistic regression models using multiple years of daphnia lumholtzi distributions, together with environmental data for ca. reservoirs, revealed that the chance of new invasions declined rapidly for reservoirs more than km away from source populations (havel et al., ) . such spread within a region is important for longer-term invasion success. metapopulation theory predicts that the chance of regional persistence is greatly enhanced by increasing the number of populations (greater ''patch occupancy'', hanski, ) . for instance, invading crayfish that have successfully colonized one lake and subsequently spread to several other lakes in the region will be more likely to persist in the region than will a single population, because a single population is more prone to become extinct by some localized catastrophe (e.g., pollution event, invasion of largemouth bass, etc.). many freshwater species have adaptations that allow persistence in temporary environments and these characteristics are also useful for remaining viable during overland dispersal. examples include seeds of aquatic plants and their counterpart (resting eggs) in zooplankton (hairston & caćeres, ) , cryptobiosis of rotifers (wallace & snell, ) , resistance of freshwater snails to extended periods of drying (havel et al., ) , and dormant stages and spores of prokaryotes (fazi et al., ) . survival is only half of the story; the propagule must move from one habitat to the other. with the exception of flying insects and mobile vertebrates, most aquatic species lack the means for active dispersal into isolated drainages and instead use a variety of transport vectors for passive dispersal (havel & shurin, ) . numerous vascular plants produce wind-dispersed seeds, using a variety of different structures to increase drag and remain aloft (vogel, ; raven et al., ) . despite its apparent potential for long-distance movement, wind transports the dried eggs of freshwater invertebrates to colonize water bodies for only short distances from the source (jenkins & buikema, ; brendonck & riddock, ) . transport by birds and other vertebrates is made possible by attachment to fur and feathers and viable germination or hatching after passing through the digestive tract (bilton et al., ) . nevertheless, the fact that most plant and animal species are not cosmopolitan (brown & lomolino, ) implies that dispersal rate is limited at larger geographic scales. the increase in global travel and commerce has provided many more avenues for human-mediated transport of exotic species across great distances (kolar & lodge, ; padilla & williams, ) , and the increased speed and efficiency of travel should increase the chance that individuals arrive to their new habitats alive. humans move aquatic species by intentional stocking, accidental releases, and through hitchhiking with other carriers (vectors). resource agencies deliberately translocate and stock game fish in lakes worldwide (kolar & lodge, ; rahel ; pelicice et al., ) . anglers introduce other species by dumping bait. the aquarium and ornamentals trades import a stunning variety of fishes, invertebrates, and aquatic plants (padilla & williams, ) , with very little regulatory oversight. aquarium releases, either on purpose by naïve (though well-meaning) people or by accident, are commonplace events. worldwide, these releases have led to establishment of over species, many on the ''worst invasive species'' list (padilla & williams, ) . hitchhiking plants and small animals are carried by a variety of human vectors (carlton, ; havel & shurin, ) . large ships carrying ballast water have been documented to carry hundreds of species (carlton & geller, ) , and such ships are the likely source for % of the species introduced to the laurentian great lakes since (ricciardi & macisaac, ) . these ballast-vector species include zebra mussels (dreissena polymorpha), which had been previously released from the ponto-caspian region through european shipping canals (bij de vaate et al., ) , allowing their transport to north america from the baltic sea (ricciardi & macisaac, ) . similarly, ballast water has been the source of asian bivalves (corbicula fluminea) that invaded south american freshwater ecosystems in the last two decades (boltovskoy et al., ) , as well as freeliving microorganisms, including pathogenic bacteria, viruses, and toxic algae (drake et al., ) . water transported for fish stocking carries exotic plants and invertebrates, and is the likely source for introducing eurasian water-milfoil to north america (couch & nelson, ) . subsequent spread of invasive plants and invertebrates into new drainages have been linked to recreational boats (johnstone et al., ; johnson et al., ) . the importance of these vectors provides a clue for how regulatory agencies and volunteers can help reduce the rate of spread. interrupting the chain of transmission although some invasions that are quickly recognized and contained may be eradicated (see living with aquatic invasive species below), many ais achieve high population sizes before they are detected. this is particularly true for invasive plants that reproduce by fragmentation and plants and invertebrates that form resting eggs and seeds. avoiding introduction in the first place seems particularly important. understanding the vectors for transmitting invasive species (above) allows the chance to control their spread through influencing human behavior. some methods seem more feasible than others. for instance, rules on ballast water release from large ships have been legislated in a number of countries (e.g., united states: nonindigenous aquatic nuisance prevention and control act of ) and invasion models predict that mid-ocean exchange of water should reduce the load of propagules by a factor of - (macisaac et al., ) . on a regional scale, recreational boats are a target for control. the spread of nuisance aquatic plants and the invertebrates may be reduced by state laws, public education (e.g., signs at boat landings and information brochures), and coordinated volunteer groups (e.g., wisconsin: clean boats, clean waters; http://www.dnr.wi.gov/lakes/ cbcw/). in such lake-rich regions where tourism is a billion-dollar industry, many people are motivated by a common goal of preserving lakes. nevertheless, protecting lakes visited by many boaters is a challenge, since most boaters visit multiple lakes over short periods of time (buchan & padilla, ; bossenbroek et al., ; b. beardmore, university of wisconsin, pers. com., december , ) . more problematic is the aquarium and water garden industry, where the business relies on demand of people for exotic plants and animals (padilla & williams, ) . this industry successfully lobbies against regulation and people are able to easily circumvent specific outlawed species (e.g., water hyacinth, eichhornia crassipes) through purchases over the internet. similarly, the live bait industry resists efforts to restrict selling live crayfish in many states, despite the problem of widespread bait dumping. in both the aquarium and bait industries, public education through signs and brochures could reduce the rate of dumping, despite the natural instincts of most people to treat animals humanely. only a small percentage of those ais that arrive in a place will establish successfully. establishment success is explained both by differences in species traits (below), as well as by differences in the recipient communities (invasibility section below). like many ecological phenomena, early studies on invasive species consisted primarily of elegant descriptions of the successful invaders. these studies described in detail how non-native species arrived and their detrimental effects on local communities (elton, ) . more recently, ecologists have analyzed common features that separate successful invaders from those that fail (ehrlich, ; simberloff, ) . although each successful invasion has some unique properties, generalizations on invasion traits allow better prediction of species that are most likely to invade. invasion success actually depends upon two groups of characteristics. the first includes traits that allow non-native species to reach new habitats (dispersal section above). the second suite of characteristics applies to the ability of a colonizing species to succeed in its new aquatic habitat (ehrlich, ) . a variety of life history characteristics can be used to predict invasion success. one of the most important is asexual reproduction, common in many aquatic plants and invertebrates. vegetative reproduction allows single viable macrophyte fragments to give rise to an entire population. similarly, resistant resting eggs of rotifers and cladocerans, after overland dispersal and hatching, can initiate new populations or clonal lineages through asexual reproduction (apomictic parthenogenesis) (dodson & frey, ) . among terrestrial plants, species that are invasive tend to be larger and have higher growth rate and shoot allocation than do species that are not invasive (van kleunen et al., ) . we expect that similar traits would increase invasive potential of aquatic plants. for fish, a study from the colorado river basin found that invasive species were more likely to be characterized by early maturation and production of smaller eggs ). yet many successful invaders are large-bodied fishes with large eggs and/ or parental care (e.g., nile perch; peacock bass; brown trout, salmo trutta). in the laurentian great lakes, fast growth was associated with establishment success in non-native fish, but not with the rate of spread, demonstrating the importance of different traits at different stages of invasion (kolar & lodge, ) . trophic interactions are also an important feature for predicting invaders. invasive species are often represented by different ratios of functional feeding groups than native species. in freshwater macroinvertebrates, invaders were much more frequently represented by collector-filterers and less likely to be collector-gatherers, predators, and scrapers, than were members of the native assemblage (karatayev et al., ) . invasive fish were more likely to be represented by piscivores, planktivores, and omnivores than were native fish (moyle & light, ; olden et al., ) . in simulated food webs, models combining both network structure and non-linear population dynamics demonstrated that being a trophic generalist was one of the best predictors of invasion success (romanuk et al., ) . the greater prevalence of generalist traits, shifts to different functional feeding groups, and increasing dominance of invaders are likely to accelerate the loss of functional diversity in native communities (olden et al., ) . geographic range and environmental tolerance are also useful predictors of invasion success. for freshwater and marine invertebrates and fish, species with greater geographic range tend to be over-represented among invasive species, relative to those not invading (kolar & lodge, ; bates et al., ) . furthermore, species with greater heat tolerance tend to also be over-represented in the invaders (bates et al., ) , a trend that has important implications for interactions between invasive species and climate change (interaction with other processes section below). increased pollution tolerance has also been noted in some invaders as compared to native species (e.g., karatayev et al., ; früh et al., b) . some invasive mollusks use rapid growth and reproduction for rapid population recovery following disturbance (mcmahon, ) , which enhances their role as colonizers. in summary, we would predict that the most common successful invaders will include those that are both likely to be carried into new freshwater environments and also those that have some combination of asexual breeding, high reproductive output, generalist feeding, and broad environmental tolerance. using information about species biogeography and ecology, the australian weed risk assessment was shown to be effective in classifying plant invaders across several islands and continents (gordon et al., ) . this example shows that increasing our understanding of species traits as predictors of future invasions, likely specific for each assemblage, may also hold promise for aquatic invasions as well (see also kolar & lodge, ) . nevertheless, predicting invasions also depends on characteristics of the community at risk. biotic resistance, together with abiotic environmental factors, can often explain the failure of non-native species to invade a novel ecosystem (catford et al., ). the biotic resistance hypothesis was proposed by elton ( ) , who considered that the species diversity of the recipient community is the main determinant of invasion success. in its essence, resistance of a native community to invasion by nonnative species is related to competition, predation, and parasitism (elton, ; simberloff, ) . the intensity of these negative interactions generally increases with the number of native species. thus, tests on the role of biotic resistance often use diversity as a surrogate for the resistance of native (e.g., fridley et al., ; jeschke et al., and references therein) . however, a meta-analysis showed that although such biotic resistance reduces invader establishment and performance, it does not completely repel plant invasions (levine et al., ) . the role of biotic resistance to invasions has been widely investigated in freshwater ecosystems. the importance of competition in biotic resistance has been shown for macrophytes, whose growth is reduced with increasing density and diversity of native species (peter and burdick, ; michelan et al., ) . herbivory also plays a role in reducing invasive macrophytes abundance, as evidenced by the impacts of beavers on one species of invasive macrophyte in wetlands (parker et al., ) . native predators can impact invasive species as well. crabs and other predators increase the mortality of zebra mussels in rivers (carlsson et al., ) and native fish depress non-native crayfish abundance in lakes (tetzlaff et al., ) . predators such as crayfish, fish, and turtles resist invasion by apple snails in rivers (yamanishi et al., ) . although less studied, parasitism also reduces invasion success. for example, an oomycete parasite reduced the abundance of an invasive amphipod and allowed coexistence with another native species of amphipod in a canadian river (kestrup et al., ) . most of these studies support the role of biotic resistance in reducing, and sometimes excluding, invasive species in their novel habitats. this decrease may be important if an invader is thus unable to reach a critical density necessary for maintaining a viable population. furthermore, higher native diversity can make a freshwater community more resilient to invasion, by increasing the threshold to community collapse (downing et al., ) . biotic resistance depends on spatial scale. invasion success is usually negatively related to native diversity on fine scales (at the scale of plots or experimental units; xu et al., ; michelan et al., ) , but positively related to diversity at coarse scales (plant patches or entire ecosystems) (stohlgren et al., ; capers et al., ) . this pattern became known as the ''invasion paradox'' (fridley et al., ) , and it may be explained by the more important role of biotic interactions (e.g., competition and predation) on fine scales and of abiotic influence (e.g., resources availability and habitat heterogeneity) on coarse scales (fridley et al., ) . as an example from field studies of aquatic communities, positive associations between invasive establishment and native macrophyte diversity have been shown at coarse scales in both natural lakes (capers et al., ) and reservoirs (thomaz et al., (thomaz et al., , . in contrast, experiments conducted at fine spatial scales showed an opposite pattern, with a negative association between growth rates of invasive species and native diversity (xu et al., ; michelan et al., ) . because different factors likely influence invasions at different spatial scales, aquatic ecologists should carry out more studies that encompass different scales simultaneously (e.g., see pintor & sih, ) . invasional meltdown (im) can be defined as a ''community-level phenomenon in which the net effect of facilitations would lead to an increasing rate of establishment of introduced species and/or an accelerating impact'' (simberloff, ) . the ''meltdown'' implies a positive feedback among invasions over time, leading to an increase in the cumulative number of successful invaders. unless native species are replaced by invaders, the im hypothesis predicts an increase in species richness. the im hypothesis thus appears to contradict the biotic resistance hypothesis, which predicts enhanced resistance to invasion with increased species richness (ricciardi, ) . in a recent review of the guiding principles of invasion biology, im was one of the few hypotheses with good empirical support (jeschki et al., ) . however, most of this support consists of descriptive case studies of facilitation between non-native species. however, the im prediction that precedent invasions result in increasing rates of invasions and of impact caused by invasive species has not yet been conclusively demonstrated (simberloff, ) . this conclusion by simberloff also applies to freshwater ecosystems. while a variety of observational and experimental investigations have demonstrated facilitation between non-native species (adams et al., ; devin et al., ; cucherousset et al., ; chucholl, ; michelan et al., ; thiébaut & martinez, ) , the data are insufficient to show the existence of a true meltdown through increasing invasion rates and/or impacts over time. one of the best examples for the possible occurrence of im in freshwater ecosystems is the case of the laurentian great lakes, for which a good history of multiple invasions and their consequences are known (mills et al., ) . in these lakes, both direct and indirect facilitation between non-native species was more common than negative interactions and invasions increased over time, results consistent with the im model (ricciardi, ) . nevertheless, this conclusion is based primarily on correlative observations from field surveys (simberloff, ) , which may be influenced by other confounding variables. for example, increased propagule pressure (from increased vector traffic) has also increased the opportunities for new invasions over time (ricciardi, ) . indeed, the conclusion about the occurrence of a true im in the great lakes has been questioned by de vanna et al. ( ) , who stated that indiscriminate facilitation of both native and non-native species by the ecosystem engineer dreissena (zebra mussels and quagga mussels) is a better explanation for what is occurring in those lakes. future research to demonstrate im would benefit from carefully designed experiments involving multiple species. such experiments require a test of two questions. first, are invasive species facilitating each other? and, second, does facilitation between nonnatives propagate in such a way as to impact the community level and enhance invasion rates? these kinds of experiments require careful attention to controls and understanding what exactly are the response variables (e.g., invasion rate) and treatments (e.g., presence or absence of prior invader). to conclude about a true meltdown, both the individual performance of non-natives and their impacts would have to be greater when they interact than when they are alone. in summary, based on the evidence to date, we cannot conclude whether im in freshwater ecosystems is common but poorly documented or if im is rare in nature. the challenge for future research is to refine experiments, using a variety of different aquatic communities, which produce clear-cut conclusions about the impact of invasion on future invasion rates. reservoirs are now a significant proportion of freshwater ecosystems (rosenberg et al., ) , and their representation is increasing with the rapid hydroelectric development in south america and asia (dudgeon et al., ) . reservoirs act as stepping stones for establishing invaders in new watersheds (havel et al., a, b) . reservoirs serve this role by providing a propagule source close to uninvaded water bodies and simultaneously altering the habitat, making it more prone to invasions. recent studies suggest that reservoirs are indeed more susceptible to invasion than natural systems (johnson et al., ; banks & duggan, ). the high invasibility of reservoirs was first noted in dammed mountain streams (moyle & light, ) and was later supported in a comparison of reservoirs with natural lakes in northern wisconsin (johnson et al., ) . the latter study demonstrated that a number of ais (eurasian water-milfoil, zebra mussels, spiny water fleas [bythotrephes longimanus], rainbow smelt [osmerus mordax], and rusty crayfish) are much more likely to occur in reservoirs than in natural lentic systems, and this effect was robust to inclusion of potentially confounding environmental factors (johnson et al., ) . this study has also clearly shown the role of reservoirs in increasing connectivity by reducing the distance to other aquatic ecosystems and facilitating further spread of invasive species (johnson et al., ) . nevertheless, the relative importance of biotic resistance and disturbance or habitat degradation in reservoirs has not yet been elucidated. as young, early successional and often species-poor systems, reservoirs are likely to have decreased biotic resistance to invasion. a recent study in mediterranean reservoirs demonstrated that richness of non-native fish was negatively related to richness of the native fish community, indicating the importance of biotic resistance, whereas native fish were mostly affected by the abiotic factors (clavero et al., ) . the role of biotic resistance on invasibility of constructed systems has also been demonstrated with zooplankton (shurin, ; taylor & duggan, ) . for instance, in a mesocosm experiment (viewed as a model of a reservoir in the its earlier phase), seeding newly constructed tanks with native species eggs decreased establishment success of invasive zooplankton one year later, relative to tanks without a native assemblage (taylor & duggan, ) . abiotic conditions could also play an important role in colonization of reservoirs by invasive species. such abiotic factors include water quality degradation and increased variability in water levels from regulation for flood control and hydropower, which reduces macrophytes and creates sub-optimal habitats for native species (havel et al., a, b) . mainstem reservoirs lack the riffle-pool structure characteristic of the streams they impound. reservoirs also lack the flood pulses characteristic of large rivers and their floodplains, and dams reduce the migratory opportunities of fish (agostinho et al., (agostinho et al., , . many shallow water reservoirs are exceedingly prone to sediment resuspension and eutrophication . physically and chemically degraded habitats are more likely to harbor invasive species (früh et al., a) . reservoirs create conditions more suitable for the invasive and more heat-tolerant species (bates et al., ) , as well as fauna tolerant of degraded ecosystems (karatayev et al., ; früh et al., b) . therefore, reservoirs can promote establishment of invasive species via habitat degradation and also allow for the selection for taxa different from the native assemblage in functional attributes. the large-scale environmental changes due to reservoir construction have important consequences to freshwater ecosystems. reservoirs greatly contribute to worldwide homogenization of freshwater faunas as endemic riverine species are replaced by cosmopolitan lake taxa by removing biogeographic barriers and causing habitat alteration (rahel, ; gido et al., ; vitule et al., ) . native species declines in response to invasion in reservoirs have been shown for a number of assemblages (e.g., fish: agostinho et al., ; macrophytes: michelan et al., ) . with that, it is important to understand how taxonomic changes and homogenization translate into shifting balance among functional groups and ecosystem-level changes. invaders are not a random collection of species and can be functionally distinct from the native assemblage (karatayev et al., ; gido et al., ) . the resulting functional homogenization can in turn lead to further food web disruption, decrease in resilience to anthropogenic disturbance and increase in invasibility (olden et al., ) . indepth comparisons of invasive and native species functional attributes in impoundments vs. natural similar-sized lentic systems in the same geographic area are needed to understand the role of reservoirs in functional homogenization of freshwater faunas. the aquatic microbes comprise microscopic eukaryotes, two prokaryotic domains (bacteria and archaea), and viruses. most of those also retain a high potential for long-range dispersal given their minute sizes, high densities of individuals, and ability to rapidly form resting stages (fontaneto, ; lennon & jones, ) . using many molecular tools, microbial ecologists can now describe the great genetic and metabolic diversity of eukaryotic and prokaryotic microorganisms in their natural environments, allowing comparisons of species and genotypes across the globe. a large body of research demonstrates that aquatic microorganisms can exhibit distinctive biogeographic patterns (fontaneto, ; incagnone et al., , and references therein). such distributions of microbial assemblages suggest that some microbial taxa disperse globally (e.g., in oceans; pommier et al. ; schauer et al., ; ladau et al., ) , while others disperse only over short distances martiny et al., ; ramette & tiedje, ; lindstrom & langenheder, ) . although such conclusions are tentative at this time (jenkins et al., ; fontaneto, ) , the biogeographic patterns of freshwater microbes offer the potential for future range expansion and invasion. microbial population traits (e.g., individual density, genotypic diversity) and community characteristics (e.g., taxonomic richness, interspecific interactions, community evenness, functional redundancy) may affect the capability of native microbial communities to resist or accept microbial invaders (shade et al., ) . though important, such features of microbial communities have not yet been clearly linked to invasion mechanisms. climate-related changes in the environment (e.g., increases in atmospheric temperature, changes in precipitation patterns) will likely affect both the dispersal of microbes (e.g., through flooding) and the conditions of lakes and streams that favor particular species and their hosts. we have much to learn about whether microbial invasions follow patterns and processes that are already known for plants and animals (ehrlich, ; amalfitano et al., ) . to date, there have been few comparative studies based on a set of samples of aquatic micro-and macro-organisms from the same set of sites, which would enable a direct comparison of the relative dispersal and colonization mechanisms. in one example, beisner et al. ( ) evaluated the community composition of bacteria, phytoplankton, crustacean zooplankton, and fish in canadian lakes and reported that larger and less motile species (zooplankton and fish) were more strongly influenced by spatial factors than by local environmental factors. from the perspective of this review on biological invasion, we have two primary questions about microbes, both based on the idea that freshwaters could represent a ''melting pot'' for microbial invasive species. in particular, does the freshwater medium offer an environment where infectious microbes can meet and interact prior to colonizing their hosts? second, do natural and human-assisted movements of waters and organisms transport microbes to new environments? clearly, freshwater provides a medium for infectious microbes. a large body of literature is dedicated to waterborne diseases and invasive microbial pathogens transmitted through contact with infected waters or infected hosts having aquatic phases in their life cycles. reported cases include a long list of microbial eukaryotes (e.g., entamoeba histolytica, cryptosporidium parvum, cyclospora cayetanensis, giardia lamblia, microsporidia), bacteria (e.g., vibrio cholera, escherichia coli, campylobacter jejuni, legionella spp., salmonella spp., leptospira spp.), and viruses (e.g., coronavirus, hepatitis a virus, poliovirus, polyomavirus, norovirus) (see leclerc et al., for a review). thus, aquatic microbes may infect an introduced host species, with the aquatic environment acting itself as a reservoir. the aquatic environment can also mediate polymicrobial interactions, particularly within the context of infections. in the case of fecal bacteria, e. coli and enterococci are known to acquire new determinants of virulence and resistance by gene transfer mechanisms during their stay in the secondary habitat (the freshwater environment outside the host) (luna et al., ) . movement of non-native plants and animals also transports aquatic pathogens. for example, endozoic and epizoic microbes find new opportunities to proliferate within or on individual plants and animals of native communities. guts and teguments of fishes and crustaceans are important pathogen transmission routes in aquatic systems (blokesch & schoolnik, ; de schryver & vadstein, ) . considering that opportunistic pathogens preferentially infect young and stressed individuals (skjermo & vadstein, ; hajek, ) , host-associated invaders may play a role so far underestimated within the ecological interactions of invaded systems. although controlling contamination and proliferation of potential pathogens in freshwater is extremely challenging (cabral, ) , their control could be achieved partly at the level of the host, such as by infection tracking in human populations. for example, gatto et al. ( ) observed that hydrological network, human mobility, and landscape complexity mediate the spread of diarrheal infections from vibrio cholera that could be potentially modeled to predict which communities will be hit hardest during the epidemic. so far, studies exploring microbial ais and emerging infectious threats in freshwaters have oriented research efforts toward relevant human health issues and ecosystem services (e.g., morens et al., ; conn, ) . while less attention has been placed on the invasion of aquatic non-pathogenic microbes (''invisible invaders'', litchman, ) , these microbes offer an interesting challenge for distinguishing native from non-native microbial species. such studies require a greater understanding of bacterial taxonomy and historical patterns of microbial community composition. for bacteria, such historical studies are few, but offer interesting possibilities using methods from paleolimnology (romero et al., ; smol, ) . such studies require easily detectable microscopic, biochemical, and genetic traits. invasion of non-native cyanobacteria into freshwater has several important consequences for native food webs and human uses of water. in a summary of recently published records on the invasion of cyanobacteria into subtropical and temperate lakes and reservoirs, sukenik et al. ( ) analyzed the dynamics of two genera of invasive nostocales (cylindrospermopsis and aphanizomenon). the immediate threat of the invasion by cyanobacteria is that numerous strains, belonging to a variety of genera (e.g., microcystis, nostocales, oscillatoria, anabaena), produce harmful substances toxic to humans, animals, and other eukaryotes (codd et al., ) . such secondary metabolites function as allelochemicals that may inhibit the growth of other phytoplankton species and grazers, thus affecting the entire food web (ger et al., ; paerl & paul, ) . moreover, invasive cyanobacteria can potentially alter the nitrogen budgets in invaded aquatic systems by nitrogenfixation (litchman, ) . to our knowledge, there are no published examples on the global impact of invasive microbes on major biogeochemical cycles. thus, this topic is a fertile area for future research. microbial invasion ecology has been tested experimentally using artificially assembled microbial communities. hornak and corno ( ) reported the effects of the invasion by the opportunistic bacterium limnohabitans planktonicus on growth of pure cultures and mixtures of freshwater bacteria (arthrobacter agilis, aeromonas hydrophila, brevundimonas sp., flavobacterium sp.). in most experimental conditions, the invader had a strong detrimental effect on the abundance of the dominant species, owing to competition for nutrients and possibly by allelopathic interactions. as obligate intracellular life forms, viruses deserve further consideration as an important component of microbial communities. since many viruses are transported by aquatic animals and plants (kurath & winton, ; roossinck, ) , they also may play an important role in biological invasions of freshwater communities. on the one hand, introduced hosts commonly convey viruses from their native ranges, which may allow the hosts to achieve greater fitness in their introduced ranges. on the other hand, viruses that do infect introduced hosts may reduce the fitness of invasive hosts (rua et al., ) . however, methodological limitations and a paucity of environmental data prevent us from describing the distribution of most viruses and hence differentiating native and nonnative viruses. now that we are able to survey viral diversity in the environment using metagenomic tools (fierer, ) , we can begin to investigate the spatial structure and dynamics of viruses and their transport vectors. in conclusion, we have much to learn about microbial communities and the mechanisms that control microbial invasions. first, information is needed on the geographic distribution of species and factors related to their spatial structure. such knowledge will allow distinguishing native from non-native species. second, experiments on the ecological mechanisms that control microbial assembly in aquatic systems would provide the understanding necessary for identifying and managing the effects of disturbed conditions, including anthropogenic impacts, on natural microbial communities. because of the important role that humans play in spreading invasive species, agencies have begun to develop procedures and policies that attempt to prevent further introductions (hulme, ) . nevertheless, humans may affect non-native species distributions and impacts in the future by other indirect means, including global climate change, land use conversion, and pollution (dullinger et al., ) . as climate and global environmental conditions continue to change in response to anthropogenic disturbances, many non-native aquatic species are expected to flourish (rahel & olden, ; sorte et al., ) . since many non-native species are tolerant to a wide range of environmental conditions (kolar & lodge, ; bates et al., ; sorte et al., ) , we expect changing climate should facilitate the establishment of non-native species. in contrast, many native species seem less likely to adapt to their changing environment . thus, native aquatic species may be buffeted by both changing environmental conditions and rapidly expanding invasive species (dukes, ) . as an example of what changes may happen in aquatic habitats, loyola et al. ( ) modeled the range for one of the most widespread non-native aquatic vertebrates, the american bullfrog (lithobates catesbeianus) and how it may spread in the future. taking into account the current distribution of l. catesbianus, its known ability to tolerate wide environmental conditions, and current climate models, the authors predict that the american bullfrog will invade reserves associated with the atlantic forest biodiversity hotspot (brazil). this could very well be the pattern we see for many successfully introduced aquatic species. in addition, climate change may facilitate the dispersal of potential invaders that were previously held in place by past environmental conditions. changes in weather patterns may also modify species interactions in favor of the non-native species. increasingly, biologists are reporting that historic weather patterns are promoting coexistence between invasive and native species, with certain weather extremes limiting the negative effects of invasive species. luja and rodriguez-estrella ( ) found that tropical cyclones produce enough heavy rainfall to wash invasive american bullfrogs downstream. native baja california treefrogs (pseudacris hypochondriaca curta), which would otherwise be eliminated with rising bullfrog populations, are well adapted to the cyclones and persist through the heavy rainfalls. similarly, kats et al. ( ) reported that invasive crayfish (procambarus clarkii) and native amphibians likely coexist because of periodic heavy rainfall that washes crayfish from streams. procambarus clarkii is not well adapted to the rapid flows of post-storm southern california mountain streams. native frogs (pseudacris regilla and p. hypochondriaca) and newts (taricha torosa) persist through the periodic spates. during a -year study, newts had significantly more reproduction in years with above average rainfall than in years with average or lower rainfall. in both of these instances, if climate change results in decreased rainfall, which is predicted for parts of western north america, then conditions will become increasingly favorable for such invasive species. as a result, local amphibians are in danger to becoming extirpated. a different potential consequence of climate change is the increase in humic substances in aquatic ecosystems in response to rainfall, and consequent reduction in light transmission through the stained water column (''brownification''). such circumstances favor species that thrive in low light environments. for example, mormul et al. ( ) showed that increases in humic substances in freshwater north-temperate ecosystems allows the invasive macrophyte elodea nuttalli to out-compete native macrophytes, facilitating its spread and potential impacts in european freshwater ecosystems. this example highlights that an indirect outcome from global warming (in this case, brownification) may be more important than temperature itself to magnify impacts caused by invasive species. increases of disturbances (e.g., extreme drought and rainy periods) are also predicted to occur in response to global changes in some regions. the effects of these disturbances on ais success are still to be identified. an indirect, but nonetheless, important consequence of climate change could be human activities that attempt to counteract drying conditions accompanying climate change. rahel and olden ( ) suggest that as humans experience increasingly dry conditions, more water reservoirs will be built. in addition to the reservoirs, ditches, aqueducts, and canals will likely be constructed to transport water to areas where it is needed. these new water bodies have the potential to facilitate the spread of ais (bij de vaat et al., ; havel et al., a, b) , as well as provide them permanent habitat. land use intensification affects freshwater communities via increased sedimentation, altered hydrology, pollution, and nearshore habitat destruction (allan, ) . by altering aquatic habitats, such disturbance reduces species richness and hence biotic resistance to invasion. watershed development and urbanization cause non-linear changes in fish, macroinvertebrate, and diatom assemblages (hilderbrand et al., ; king et al., ; kovalenko et al., ) , which are also correlated with the presence of invasive species (riley et al., ) . notably, increasing anthropogenic development resulted in simultaneous non-linear decreases in abundance of sensitive native species and increases in invasive species (riley et al., ) . several invasive macrophytes were associated with increase in watershed agriculture, which was in turn correlated with increased turbidity and nutrient levels (trebitz & taylor, ) . similarly, both development and agriculture were important predictors of invasive fish species richness in california watersheds (marchetti et al., ) . analysis of a large number of european streams (früh et al., a) revealed that habitats that were physically and chemically degraded were also more prone to invasion by a variety of macroinvertebrates. decreased dissolved oxygen and increased temperature and chloride concentrations were the most important predictors of the presence of invasive amphipods, isopods, and mollusks, and invasive species displayed greater tolerance of degraded conditions than their native counterparts (früh et al., b) . however, this association between disturbed landscapes and increased prevalence of invasive species is difficult to interpret, since increased boater traffic, and thus propagule pressure, is also expected in the more developed areas. this problem with confounding factors may explain why the mostly intuitive assumption that invasive species would interact with eutrophication, habitat destruction, and other anthropogenic stressors has only been tested in a handful of studies (reviewed in strayer, ) . invasive species are likely to complicate management of other issues facing freshwater ecosystems (strayer, ) , so understanding the interaction between the effects of invaders and landscape change on the native assemblages is of great interest for identifying vulnerable ecosystems and applying adaptive management to address a variety of stressors (thomas et al., ) . regime shifts, or large changes in the state of the system in response to a small change in the driver, are a common phenomenon, particularly in shallow lakes. regime shifts have an intriguing connection with invasive species. invasive floating plants are a classic example of an alternative stable state: a shift from submerged to floating plants is induced by nutrient enrichment and maintained by floating plants shading out the submerged (scheffer, ) . invasive species can induce a regime shift via several, often complementary, pathways. the shift can be triggered directly by changes in assemblage composition or water quality due to invader dominance (witte et al., ; strayer et al., ) . regime shifts can also be triggered by invasive species increasing the system's vulnerability to other stressors, such as eutrophication, which shifts or widens the basin of attraction of an alternative stable state. invasive species may also cause a regime shift by tipping the state of the system via self-reinforcing mechanisms, such as alteration of fire regimes in terrestrial systems (d'antonio et al., ) or light attenuation by floating macrophytes (scheffer et al., ) . invasive species that directly affect the state of the system are often species that have functional traits distinctive from those present in the native community. classic examples of this effect are herbivores (carlsson et al., ; strayer et al., ; nicholls et al., ) and predators (zaret & paine, ; witte et al., ) that invade communities with low herbivore or predator biomass and cause considerable shifts in the native assemblages via direct consumption. new growth forms of aquatic plants are another example of such distinctive traits (yarrow et al., ) . however, even invaders that are similar in their traits to members of the native community can contribute to a regime shift via indirect mechanisms. for example, invasive species often cause decreased diversity at all trophic levels, and decreased diversity has been shown to reduce reticulation (number of food web pathways) and decrease food web stability, pushing the system away from equilibrium and increasing the amplitude of oscillations in abundance (rooney & mccann, ) . even small declines in native species abundance, such as those commonly occurring with increasing invader dominance, could lead to functional extinctions and result in food web reorganization (säterberg et al., ) . furthermore, spatially heterogeneous systems (e.g., species-rich littoral zones) are more likely to exhibit a gradual response to stress due to decreased spatial coupling between nearby patches (van nes & scheffer, ) . monocultures of invasive plants often result in decreased heterogeneity in habitat structure, assemblage composition and environmental conditions compared with diverse native assemblages, thus potentially unraveling the role of greater heterogeneity in preventing abrupt regime shifts. invasive species may also be playing a large role in the so-called slow regime shifts with transitions that are more gradual but even more difficult to reverse (hughes et al., ) . based on a recent meta-analysis of terrestrial invasive plants, gaertner et al. ( ) showed that invasive species that are most likely to cause regime shifts are those that can enhance their own persistence by changing internal feedback mechanisms. the most common reinforcing feedbacks identified in this study were associated with the ability of invasive species to produce large numbers of seeds and litter, changing the fire frequency or intensity, accumulating soil nitrogen, and altering soil biota (gaertner et al., ) . a comparable meta-analysis and overview has not been attempted for aquatic invasive plants; however, potential pathways for disruption by invasive aquatic plants include their ability to increase sediment loads (bunn et al., ) , reduce sediment resuspension, and uptake nutrients from water (yarrow et al., ) , and reduce underwater light (farrel et al., ) . a systematic analysis of regime shift-associated traits of aquatic species may be increasingly important, given the high susceptibility of aquatic ecosystems to negative impacts from invasive species and to regime shifts and possibly unique mechanisms of self-reinforcement by aquatic invaders. the vast number of ais has resulted in freshwater communities that are quite different in composition from those that existed before human introductions began. impacts caused by non-native species depend a bit on human perception. although an impact occurs when an invasive species causes a net change of any ecological property simberloff et al., ) , interpretation of these impacts depends on the type of ecological attribute measured . a given invasive species may cause a neutral or positive effect on one ecological property but a negative effect on another. for example, the invasive macrophyte hydrilla verticillata did not significantly impact the native invertebrate richness but did impact invertebrate assemblage composition (theel et al., ; mormul et al., ) . how the effects caused by invasive species on native communities and ecosystems are interpreted (''good'' or ''bad'') depends on human values. for example, introduction of generalist herbivores such as sterile triploid grass carp requires careful attention to density, in order to avoid destroying all the vegetation and creating a turbid, algae-choked lake (mccomas, , dibble & kovalenko, ). the reduction of macrophytes biomass is perceived by lake managers and society as a positive impact of this procedure; however, its side effects (elimination of native vegetation and increasing turbidity by resuspending sediments) are understood as negative impacts. a tradeoff between these two types of effects and on their perception by managers and ecosystem users will determine whether or not carp are introduced. quantifying the cost of impacts biological invasions can cause tremendous economic impacts. an estimate carried out for aquatic ecosystems of the us showed an approximate annual cost of . billion usd associated with damages caused by alien species and their control (pimentel et al., ) . these costs are associated mainly with introduced fish (ca. . billion), mollusks ( . billion), and aquatic weeds ( . billion). as reviewed in pimentel et al. ( ) , these cost estimates are based both on direct losses and damage and on control costs to prevent a variety of impacts. introduced fish cause extinctions of native species and, for some species like carp, a reduction of water quality. invasive mussels, such as dreissena sp., reduce food and oxygen for native fauna and they clog pipes and filters for humans (drinking water, industry). invasive aquatic plants block light to native plants, choke waterways, alter nutrient cycles, and reduce recreational use of rivers and lakes. control of invasive aquatic weeds involves spending considerable time and effort each year on reducing densities to levels that are tolerable to recreation, navigation, and other uses of lakes and streams. a secondary effect of nuisance plant control may be restoration of a more diverse native plant community (pedlow et al., ) . similar efforts are required for control of biofouling animals, such as dreisenna sp., which overgrow natural and man-made structures. further estimates of the economic cost from invasive species are a challenge for the future. better metrics to quantify impacts are required by costbenefit analyses for management and risk assessment . there is a need to shift focus from exclusively biodiversity-related impacts to assessment of functional changes, as the biodiversity loss is a poor and inconsistent metric to reflect the dramatic turnover and reorganization experienced by many biological communities (dornelas et al., ; pandolfi & lovelock, ) . biodiversity alone often does not reflect the more consequential underlying functional changes (säterberg et al., ) . nevertheless, additional work is needed to quantify and interpret such functional changes in freshwater ecosystems. one reflection of human values is the perspective on invasive versus native species. there is currently a debate about whether invasive species should be treated in the same way as natives, and even a call for the end of invasion biology has been proposed (valéry et al., ) . countering this idea, simberloff & vitule ( ) provide compelling argument that invasive species are fundamentally different from most natives. invasive species may create impacts only after time lags and impacts caused by invasive species have been shown to be more severe and more frequent than those caused by excessive growth of natives (simberloff et al., ; hassan & ricciardi, ) . is extirpation of aquatic invasive species possible? since non-native species have invaded many aquatic habitats, natural resource managers must deal with them now and in the future. some have suggested that we need to focus increasing efforts on the early detection and rapid eradication response toward invasive species (westbrooks and eplee, ) . this approach has proven effective with some terrestrial and aquatic plants (westbrooks, ) . we are seeing an increasing emphasis placed on eradication and restoration of habitats to conditions they were in before the non-native species invaded. for instance, a combination of flooding and removal of two invasive plants allowed quick reestablishment of native plants and enhanced richness of native fishes in a tropical australian floodplain (perna et al., ) . where restoration proves difficult or impossible, ecologists, and managers have focused on strategies that minimize the potential impacts of the invader. eradication of non-native invasive species has occasionally been successful in both terrestrial and aquatic communities (simberloff, ) . in such locations, the invasive species could be isolated and the habitat either treated chemically or drained. in streams or more open waterways, such approaches will likely fail. physical removal, such as by exhaustive trapping (e.g., crayfish: hansen et al., ) or hand pulling (e.g., nuisance plant control), can greatly reduce densities but fail to completely eradicate the population. a few studies have reported successful transitions back to native species dominance after non-native species removal (e.g., aquatic macrophytes: perna & burrows, ; perna et al., ) , although recovery may take a longer time in other ecosystems. other studies (e.g., fish: gaeta et al., ) indicate greater resilience of the invasive species to return again to higher population densities once control efforts stop, particularly when top-down control from native predators is weak. some native populations recover following extirpation of introduced species. knapp et al. ( ) found that a small population of rare native frogs began to recover quickly after introduced trout (oncorhynchus spp. and salvelinus fontinalis) were removed from high elevation mountain lakes. zooplankton recovery was slower; some species of zooplankton did not recolonize lakes at all if trout had been present for more than years (knapp & sarnelle, ) . just as there is frequently a time lag between the colonization of an introduced species and the subsequent impacts on the aquatic community, there evidently is also a time lag in native species recovery after the successful removal of an invasive species. a comprehensive review of successful invasive eradications in europe showed that the majority of projects involved either mammal removals or removal of specific terrestrial species from islands (genovesi, ). there has not been an in-depth overview of eradication effort success for ais. even when there has been a well-coordinated and systematic strategy to remove a high profile invasive mammal, the task has been daunting (bryce et al., ) . for example, in order to protect native small mammal populations in north east scotland, teams of volunteers were trained to trap american mink in a systematic approach beginning in high mountain lakes and continuing down rivers. scientists and project managers are confident that mink can be eradicated from northern scotland, but this will be a multi-year, volunteer-based effort taking tremendous effort (bryce et al., ) . this example reminds us that policies surrounding invasion ecology should focus on prevention given that restoration can be difficult if not impossible. concluding remarks: challenges of invasive species and directions for future research invasive species provide challenges to management, as well as an opportunity to explore basic ecological questions. how are communities assembled and how do these communities respond when new species come in? how commonly do niche shifts occur (guisan et al., ) ? what processes regulate dispersal and what is the relative importance of such regional processes with local community interactions? how does community succession proceed following disturbance? how redundant are biological communities across the planet? what are the main impacts of invasive species and how do these impacts change ecosystem goods and services? this review touches upon some persistent challenges in aquatic invasive species, such as the limited ability to predict establishment success and generalize invasive species impacts across systems. this review also considers the role of invasional meltdown in invasibility and the complex interactions of invasive species with other stressors affecting aquatic ecosystems. these areas, as well as our limited knowledge of the density-dependence, time lags, and legacy effects of invasive species, suggest several emerging issues and fruitful avenues for future research. in addition, our current review of literature on ais points out some important biases in the amount of research conducted in different ecosystems. first, freshwater ecosystems have received much less attention than terrestrial ecosystems. for instance, prospective studies of invasions in progress have many examples from terrestrial communities (reviewed in elton, ; jeschke et al., ) , whereas such studies are rather few in freshwater (e.g., zebra mussels: johnson & padilla, ; daphnia lumholtzi: havel et al., ) . second, far more research has been conducted in temperature regions (north america, europe, and australia) than in the tropics . part of this bias is a consequence of our poorer understanding of species ranges in the tropics. nevertheless, because of the higher diversity in the tropics, particularly of plants and fishes (brown & lomolino, ) , tropical freshwater communities are threatened to lose a higher number of species in response to invasions than are temperate systems. invasion biologists thus have numerous opportunities for important contributions from research investigated in the tropics. another challenge to aquatic biologists studying ais is to examine the combined impacts of multiple invasive species on freshwater communities. multiple species invasions are common in lakes (e.g., mills et al., ) . are their effects additives, synergistic, or antagonistic? such interaction effects can be explored with factorial design experiments, conducted at a large enough scale to capture the key responses. finally, we see a great challenge in management strategies to reduce introductions, such as through better legislation over the transportation of species across countries and between basins. management and eradication actions are poorly coordinated for freshwater ecosystems. in addition, there is a need of adequate distributional databases to track invasive species and help prioritize management actions (bobeldyk et al., ) . if predictions about climate change and invasive species are correct, aquatic systems will experience increasing pressures from invasive species. countries and regions will need to redouble efforts toward the prevention of invasive species and toward eradication of newly invaded species. historic ''wait and see'' approaches toward invasive species will not be viable and a more aggressive approach toward prevention and eradication will be necessary to preserve current ecosystems. indirect facilitation of an anuran invasion by non-native fishes the presence of an invasive macrophyte (phragmites australis) does not influence juvenile fish habitat use in a freshwater estuary flood regime, dam regulation and fish in the upper paraná river: effects on assemblage attributes, reproduction and recruitment dams and the fish fauna of the neotropical region: impacts and management related to diversity and fisheries landscapes and riverscapes: the influence of land use on stream ecosystems stream ecology: structure and function of running waters a microbial perspective on biological invasions in aquatic ecosystems the freshwater animal diversity assessment: an overview of the results lake construction has facilitated calanoid copepod invasions in new zealand geographical range, heat tolerance and invasion success in aquatic species the role of environmental and spatial processes in structuring lake communities from bacteria to fish the masterpiece of nature: the evolution and genetics of sexuality geographical patterns in range extension of ponto-caspian macroinvertebrate species in europe dispersal in freshwater invertebrates serogroup conversion of vibrio cholerae in aquatic reservoirs freshwater hotspots of biological invasion are a function of speciespathway interactions dispersion and ecological impact of the invasive freshwater bivalve limnoperna fortunei in the rio de la plata watershed and beyond prediction of long-distance dispersal using gravity models: zebra mussel invasion of inland lakes wind-borne short-range egg dispersal in anostracans (crustacea: branchiopoda) life out of bounds: bioinvasion in a borderless world predation, body size, and composition of plankton turning back the tide of american mink invasion at an unprecedented scale through community participation and adaptive management estimating the probability of long-distance overland dispersal of invading aquatic species influence of invasive macrophytes on channel morphology and hydrology in an open tropical lowland stream, and potential control by riparian shading water microbiology. bacterial pathogens and water aquatic plant community invasibility and scale-dependent patterns in native and invasive species richness invading herbivory: the golden apple snail alters ecosystem functioning in asian wetlands biotic resistance on the increase: native predators structure invasive zebra mussel populations dispersal of living organisms into aquatic ecosystems as mediated by aquaculture and fisheries activities ecological roulette: the global transport of nonindigenous marine organisms reducing redundancy in invasion ecology by integrating hypotheses into a single theoretical framework feeding ecology and ecological impact of an alien 'warm-water' omnivore in cold lakes biodiversity in heavily modified waterbodies: native and introduced fish in iberian reservoirs cyanobacterial toxins: risk management for health protection aquatic invasive species and emerging infectious disease threats: a one health perspective gone but not forgotten? 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a review on passive dispersal and colonization processes with a special focus on temporary ponds do similar communities develop in similar sites? a test with zooplankton structure and function does size matter for dispersal distance? support for major hypotheses in invasion biology is uneven and declining geographic spread of exotic species: ecological lessons and opportunities from the invasion of the zebra mussel dreissena polymorpha patterns and pathways in the post-establishment spread of nonindigenous aquatic species: the slowing invasion of north american inland lakes by the zebra mussel dam invaders: impoundments facilitate biological invasions into freshwaters the role of recreational boat traffic in interlake dispersal of macrophytes: a new zealand case study invaders are not a random selection of species effects of natural flooding and manual trapping on the facilitation of invasive crayfish-native amphibian coexistence in a semi-arid perennial stream how novel is too novel? 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observations on the abundance and behavior of frogs by extreme events in baja california peninsula extraintestinal escherichia coli carrying virulence genes in coastal marine sediments modeling ships' ballast water as invasion threats to the great lakes biotic invasions: causes, epidemiology, global consequences and control the decline of native vegetation under dense eurasian watermilfoil canopies fish invasions in california watersheds: testing hypotheses using landscape patterns microbial biogeography: putting microorganisms on the map lake smarts: the first lake maintenance handbook evolutionary and physiological adaptations of aquatic invasive animals: r selection versus resistance effects of an exotic invasive macrophyte (tropical signalgrass) on native plant community composition, species richness and functional diversity native macrophyte density and richness matter for invasiveness of a tropical poaceae the invasive aquatic macrophyte hydrilla verticillata facilitates the establishment of the invasive mussel limnoperna fortunei in neotropical reservoirs exotic species in the great lakes: a history of biotic crises and anthropogenic introductions the challenge of emerging and re-emerging infectious diseases ostracod (crustacea) colonization of a native and a nonnative macrophyte species of hydrocharitaceae in the upper paraná floodplain (brazil): an experimental evaluation water brownification may increase the invasibility of a sub-merged non-native macrophyte biological invasions of freshwater: empirical rules and assembly theory a biological regime shift in the bay of quinte ecosystem (lake ontario) associated with the establishment of invasive dreissenid mussels experimental evidence of the dynamic effect of free-floating plants on phytoplankton ecology ecological and evolutionary consequences of biotic homogenization life-history strategies predict fish invasions and extirpations in the colorado river basin beyond ballast water: aquarium and ornamental trades as sources of invasive species in aquatic ecosystems climate change: links to global expansion of harmful cyanobacteria novelty trumps loss in global biodiversity beaver herbivory on aquatic plants holocene turnover of the french vertebrate fauna littoral habitat heterogeneity and shifts in plant composition relative to a fall whole-lake fluridone application in perch lake fish fauna destruction after the introduction of a non-native predator (cichla kelberi) in a neotropical reservoir a serious new threat to brazilian freshwater ecosystems: the naturalization of nonnative fish by decree improved dissolved oxygen status following removal of exotic weed mats in important fish habitat lagoons of the tropical burdekin river floodplain removal of aquatic weeds greatly enhances fish community richness and diversity: an example from the burdekin river floodplain can plant competition and diversity reduce the growth and survival of exotic phragmites australis invading a tidal marsh? update on the environmental and economic costs associated with alieninvasive species in the united states scale dependent effects of native prey diversity, prey biomass and natural disturbance on the invasion success of an exotic predator global patterns of diversity and community structure in marine bacterioplankton effects of fish in river food webs invasive species, environmental change and management, and health a global assessment of invasive plant impacts on resident species, communities and ecosystems: the interaction of impact measures, invading species' traits and environment homogenization of freshwater faunas assessing the effects of climate change on aquatic invasive species biogeography: an emerging cornerstone for understanding prokaryotic diversity, ecology, and evolution facilitative interactions among aquatic invaders: is an ''invasvional meltdown'' occurring in the great lakes? are modern biological invasions an unprecedented form of global change? recent mass invasion of the north american great lakes by ponto-caspian species extinction rates of north american freshwater fauna effects of urbanization on the distribution and abundance of amphibians and invasive species in southern california streams predicting invasion success in complex ecological networks sedimentation patterns of photosynthetic bacteria based on pigment markers in meromictic lake la cruz (spain): paleolimnological implications integrating food web diversity, structure and stability the big unknown: plant virus biodiversity globalscale environmental effects of hydrological alterations: introduction the role of viruses in biological invasions: friend or foe? global biodiversity scenarios for the year high frequency of functional extinctions in ecological networks bacterial diversity and biogeography in deep-sea surface sediments of the south atlantic ocean critical transitions in nature and society floating plant dominance as a stable state fundamentals of microbial community resistance and resilience dispersal limitation, invasion resistance, and the structure of pond zooplankton communities invasional meltdown years later: important phenomenon, unfortunate metaphor, or both? encyclopedia of biological invasions invasive species: what everyone needs to know a call for an end to calls for the end of invasion biology the natives are restless, but not often and mostly when disturbed impacts of biological invasions: what's what and the way forward techniques for microbial control in the intensive rearing of marine larvae pollution of lakes and rivers: a paleolimnological perspective poised to prosper? a cross-system comparison of climate change effects on native and non-native species performance scale and plant invasions: a theory of biotic acceptance alien species in fresh waters: ecological effects, interactions with other stressors, and prospects for the future eight questions about invasions and ecosystem functioning hydrology and grazing jointly control a large-river food web invasion of nostocales (cyanobacteria) to subtropical and temperate freshwater lakes-physiological, regional, and global driving forces can biotic resistance be utilized to reduce establishment rates of non-indigenous species in constructed waters? predation by native sunfishes (centrarchidae) on the invasive crayfish orconectes rusticus in four northern wisconsin lakes differential influence of a monotypic and diverse native aquatic plant bed on a macroinvertebrate assemblage; an experimental implication of exotic plant induced habitat an exotic macrophyte bed may facilitate the anchorage of exotic propagules during the first stage of invasion effects of climate change on aquatic invasive species and implications for management and research temporal trends and effects of diversity on occurrence of exotic macrophytes in a large reservoir the colonization success of an exotic poaceae is related to native macrophyte richness, wind disturbance and riparian vegetation propagule pressure, invasibility of freshwater ecosystems by macrophytes and their ecological impacts: a review of tropical freshwater ecosystems exotic and invasive aquatic plants in great lakes coastal wetlands: distribution and relation to watershed land use and plant richness and cover another call for the end of invasion biology a meta-analysis of trait differences between invasive and non-invasive plant species implications of spatial heterogeneity for catastrophic regime shifts in ecosystems stable isotope evidence for the food web consequences of species invasions in lakes homogenization of freshwater faunas after the elimination of a natural barrier by a dam in neotropics life in moving fluids rotifera new approaches for early detection and rapid response to invasive plants in the united states early detection and rapid response limnology: lake and river ecosystems biological invasions aquatic invasive species the destruction of an endemic species flock: quantitative data on the decline of the haplochromine cichlids of lake victoria the role of diversity and functional traits of species in community invasibility predator-driven biotic resistance and propagule pressure regulate the invasive apple snail pomacea canaliculata in japan the ecology of egeria densa planchon (liliopsida: alismatales): a wetland ecosystem engineer? self-reinforcing impacts of plant invasions change over time species introduction in a tropical lake: a newly introduced piscivore can produce population changes in a wide range of trophic levels rats, lice and history. little, brown and company acknowledgments we thank k. martens for organizing this symposium volume and two anonymous reviewers for their insightful comments on an earlier version of this manuscript. smt is especially thankful to the brazilian national council for scientific and technological development (cnpq) for their continuous funding through a research productivity grant. key: cord- -rnt ivcy authors: turc̄ek, f. j. title: the bird succession in the conifer plantations on mat‐grass land in slovakia (csr). date: - - journal: ibis (lond ) doi: . /j. - x. .tb .x sha: doc_id: cord_uid: rnt ivcy . the species‐composition, number of individuals and biomass per one‐hectare are given. the three groups of areas studied were: ( ) bare pastures, with an intermediate number of bird species, few individuals and high biomass of mainly carnivorous and insectivorous birds; ( ) conifer plantations to years old, with the smallest number of bird species and the smallest biomass, but an intermediate number of individuals; ( ) plantations six to twenty years old, with the highest number of both species and individuals of birds, but only intermediate biomass. . the change from pasture to forest occurs about six to ten years after planting. the last forest birds come in about years after planting. . birds have an important role in establishing the shrub layer and mixing some tree‐species into the plantation, by dissemination. . throughout the entire successional series the bird population seems to have a kernel of species, while other species come or go and/or change in density with the stages. the area studied consists of several hundred hectares around banskp stiavnica in central slovakia, between about and m. a d . a detailed description of the plant communities concerned, both treeless and afforested, was given by korenek ( ). in the present study the area is divided into three groups, namely, ( ) unafforested pastures, ( ) plantations one to five years old, ( ) plantations six to twenty years old. this division, especially between groups ( ) and ( ) is artificial, but in the development of the bird community a marked change can be observed about the fifth or sixth year after planting. the bird (and also mammal) population has been studied since but mainly qualitatively and not regularly (tureek (tureek , . quantitative studies were carried out in the years , and from march to october over a total of days. in group ( ) areas birds have been counted on nearly square -hectare sample plots and on line transects a total of birds species has been found. ( ) pasture ( ) plantations to years results. the bird species found, their density and biomass per hectare in each group of areas are given in table . the birds are expressed as mean number per hectare and their masses calculated according to the weight-data used in earlier work (turtiek ). the nesting species in each area are marked n, the dominant species by an asterisk. the succession of the bird community in respect of species is shown in table . while group ( ) has no species peculiar to it, group ( ) has nine such species and group ( ) has . table gives the distribution of species according to their utilization of the respective areas. this division is similar to one given by kendeigh ( ). in table it is of particular interest that the mean weight of the birds found in each group (obtained by dividing the total biomass of the respective group by the total number of individuals of the same group) diminishes with the age of the plantation. in the plantations studied, mainly of norway spruce and scots pine mixed with some european larch and a few deciduous trees, we were able to study the development of the bird community from bare pasture up to a forest of about years of age. although some german ecologists (cf. tischler ) are of opinion that such a study of succession is not dynamic, this comparison of areas at different stages of development is the only possible way; for-as yapp ( ) states-it would be indeed impossible for one man to follow through the complete succession in a single wood. the first group, bare pastures, is characterized by the dominance of the mat-grass, with here and there crippled spruce and juniper or hawthorn. only small patches-usually the moist ones-are utilized as meadows for hay, the others being intensively grazed and in most parts burned off in early spring. the solitary trees and shrubs are suitable for the nesting, singing or shelter of some birds while the rocks and groups of stones are suitable for others. the ground breeders are not abundant, for the grass is short and overgrazed, the surface constantly disturbed and burned. the rock breeders and the birds nesting among stones are more protected and more abundant. small rodents are abundant, firstly the ground squirrel citellus citellus, with an average summer population of about individuals (over kg. biomass) per hectare (turkk ), secondly the field vole microtus arvalis. this abundance is responsible for the high density of raptorial birds, but it is only temporary, as the squirrels are below ground october-march and the voles below the snow in winter. a second seasonal food-supply consists of saltatoria from about mid-july to october, and a minor one of spiders march to may. owing to these nutritional circumstances most of the bird species are carnivorous and omnivorous, while the herbivorous species are almost absent or forage outside the pastures. thus, the goldfinch carduelis carduelis forages there for thistle seeds in late summer, while the linnet c. cannabina forages in relatively distant fields. while most of the mamma species are herbivorous there, most of the bird species are carnivorous and insectivorous, owing to the abundance of herbivoroug mammals and insects, respectively. as shown in table , most of the bird species utilize the bare pastures for foraging only, as e.g. the thrushes, starling sturnus vulgaris, crows and magpie pica pica as well as birds of prey. the density of the bird population is low and similar to those given in the literature from other countries (lack , kendeigh ), but the biomass is relatively high, owing to the presence of large species, e.g. raptors and crows. i n this group no occasional species are mentioned, such as the turtle dove streptopelk turtur collecting grit there, or the nutcracker nucifraga caryocatuctes visiting groups of hazel shrubs for nuts in late summer and autumn. similarly, the participation of the owls could not be numerically evaluated. the second group is characterized by a dense cover of grasses and other ground vegetation after the elimination of grazing, burning, disturbance and hay utilization. the young conifers are from . to about m. high, but their crowns do not form any layer. the species of birds are fewer, none are specific to this group, and most are shared with group ( ). there are only two new species, the whitethroats sylvia communis and s. curruca. the raptor population falls rapidly, apparently owing to the decrease, almost the absence, of the ground squirrel and to the favourable cover for other small mammals, voles in particular. but the absence of grazing, hay utilization and disturbance is favourable to ground-nesting birds. the most abundant bird is the linnet, nesting in young spruces and juniper-shrubs; none was found in pines. in general, group ( ) areas are transitional so far as the birds are concerned, with nine of group ( ) species lost and only two species gained. in all, the number of birds per unit area is higher than in group (l), though their biomass is lower. in group ( ) there is a canopy of trees about . to m. high, that may or may not touch. grass disappears except on open patches, the ground is covered with fallen needles and seedlings of shrubs or trees, mainly broadleaved, begin to appear owing to dissemination by animals, mainly birds. sixteen new species of forest-birds come in. the change from group ( ), which is ecologically a shrubby pasture, into forest both physiognomically and ecologically, occurs in about six to ten years after planting. the forest species of birds, such as tits, cuckoo, turtle dove (nesting) now appear; but it is only after ten years, when the first cones are formed, that the first seedeating species, such as the great spotted woodpecker dendrocopus major (and the squirrel sciurus m~uis) colonize. the last newcomers-after about to years-are the hazel grouse tetrastes bonasia (seeking cover) and chaffinch fringilla coehbs (for nesting). since for tits there is no suitable nesting site, only the coal tit parus ater is present, nesting in the ground. i n this late group of birds belongs also the greenfinch chloris chloris, which prefers, too, spruce. as the grass disappears from the floor, the roosting and burying birds, namely the thrushes and the jay garrutusglandmius, have ibis an important role at this time. blackbirds twdus merukz, in particular, regularly roost at this group of plantations during the non-breeding season and in their faeces they disseminate many plants, such as taxus, sambunrs, rosa, cratuegus, jutriperus, rubrrs, ribes, pnmus. on the other hand, jays bury there many acorns of oaks (mainly qwrcus sesdzk) and beech-nuts in september and october. on three sample plots, each m. square, i found in autumn from to seedlings and saplings of treespecies, carried there by birds. thus, the forming of a shrub layer or stratum is in this'case the almost exclusive r le of birds, so that they are of great importance in seral stages. group ( ) is the highest in number of bird species and individuals, but the biomass of birds is slightly lower than in the first group. the distribution of bird species according to their ecological requirements (table ) is more regular, without the extremes of group here again, (cf. tureek ) . the species dominant numerically are not those dominant in biomass and each group is dominated to some degree by bird species common to all three groups, i.e. by the species ecologically the most valent. as the bird population has not been distinguished according to the kind of plantation, for example pine and spruce, or mixed plantations, a direct comparison with lack's ( ) data is hardly possible, the population in the slovakian areas is, however, in both density and number of species higher than those given by lack. apart from any differences in method and calculation of results, the reason for this may be that the areas studied are no more than to km. from forests, so that birds can easily colonize from there. for the same reason the surrounding areas will influence the populationsuccession of birds (and other animals, as well as plants) during the seral stages. the most striking difference between the english and slovakian plantations is the absence of the willow warbler phyzloscopus irochizus from the plantations described here. this may be due to geographical and ecological causes. in the area studied (and southwards to the danube) the willow warbler is abundant only during the migrations (less in autumn than spring), and mainly in localities with flowering willows and poplars. as a nesting species it is absent in the south and in the area studied it is confined to moist young forest stands, mainly mixed, its density rising with the altitude, up to about m. a.s. . (the limit of beech and most deciduous trees). now, the areas in our three groups are extremely dry, the plantations up to about ten years after planting having a heavy grass floor, and are not dense enough to be favourable for this warbler. probably still other factora are responsible. it might be concluded that the wiliow warbler is in this case compensated for by the two whitethroats, as is the nightingale lust m.a megmhynchar in the heavily inundated parts of bottomland woods around the ( ) and ( ). the dominance of species in the three groups is of interest. danube by the garden warbler sylvia borin and to some degree by the dunnwk. in the present succession of the bird community it is evident that the entire series has a kernel of species, that persists through all the sera stages, while the remaining species change in abundance or come or go with the stages. . the s ecies-composition, number of individuals and biomass per one-hectare are given. h e three groups of areas studied were : ( ) bare pastures, with an intumediate number of bird species, few individuals and high biomass of mainly carnivorous and insectivorous birds; ( ) conifer plantations to years old, with the smallest number of bird species and the smallest biomass, but an intermediate number of individuals; ( ) plantations six to twenty years old, with the highest number of both species and individuals of birds, but only intermediate biomass. the change from pasture to forest occurs about six to ten years after planting. the last forest birds come in about years after planting. . birds have an important role in establishing the shrub layer and mixing some t r e tions by birds synokologie der landtiere birds and mammals of the mat-grass community in the vicinity of banskh &avnica on the distribution of the ground squirrel on the bird population of the spruce-forest community in slovakia the succession of birds in developing qumcctum petracac zur frage der borstengrasweidenaufforstung in der slowakei. further changes in bird life caused by afforestation species into the plantation, by dissemination. throughout the entire successional series the bird population seems to have a kernel of species, while other species come or go and/or change in density with the stages. key: cord- - nxsua authors: paul-pierre, pastoret title: emerging diseases, zoonoses and vaccines to control them date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nxsua abstract vaccination, when available, is undoubtedly the most cost-effective means of preventing and controlling, and even eradicating, infectious diseases. in recent years vaccination has also been used for other purposes in animal health, production and welfare, e.g. immunocastration. vaccination of animals serves many different purposes, such as controlling animal infections and infestations, thus improving animal health and welfare; controlling anthropozoonoses and food poisoning in humans, thereby protecting public health; solving problems associated with antibiotic and anthelmintic resistance; helping to leave food-producing animals free of chemical residues; protecting the environment and biodiversity and ensuring animal farming sustainability. the problem is nevertheless more complex when facing emerging or re-emerging infections particularly zoonotic ones. vaccination, when available, is undoubtedly the most costeffective means of preventing and controlling, and even eradicating, infectious diseases. unfortunately the problem is more complex when facing emerging or re-emerging infections particularly zoonotic ones. for instance canine parvovirosis was a real emergence in animal health [ , ] ; the first step was to vaccinate dogs with a vaccine directed against feline panleukopaenia since the two causative viruses are antigenically nearly identical. this first step was rapidly followed by the development of vaccines, either inactivated or attenuated, specifically directed against canine parvovirosis. some situations are more problematic when facing the outbreaks of diseases caused by viruses showing broad antigenic diversity such as foot-and-mouth disease virus or bluetongue virus; in this latter case it is even more difficult due to the fact that the infection is transmitted by a culicoides vector from the family ceratopogonidae (biting midges). it took two years in northern europe before inactivated vaccines against serotype of bluetongue virus were available [ ] . in northern america, the spectacular spread of west nile virus infection, another vector transmitted disease, in humans and horses, was rapidly followed by the development of several vaccines, including a dna-based vaccine for horses. one solution to be ready to vaccinate in face of an outbreak of a re-emerging infection is to stockpile vaccines as exemplified by foot-and-mouth virus vaccines as concentrated antigens. stockpiling is also envisaged for the possible pandemic of avian influenza h n in humans [ ] (pandemic pre- * tel.: + ; fax: + . e-mail address: pp.pastoret@oie.int. paredness) or to mitigate the risk of bio-agro-terrorisms. for time being it seems more appropriate to combat the h n influenza infection at the animal source to reduce human exposure. to prevent nipah virus (henipavirus) infection in pigs a vaccine has recently been developed but, unfortunately, in countries like bangladesh, humans are directly infected by the reservoir, a fruit bat species. animals may be vaccinated against certain infections not for their own sake, but to prevent human contamination. one of the best example being wildlife vaccination against terrestrial rabies by the oral route using baits [ ] . animal vaccination may also be used to prevent food poisoning in humans; a vaccine against escherichia coli :h has for example recently been conditionally approved for cattle in the united states. the changes following globalisation, climatic change [ , ] , and the opening of previously closed ecosystems, have considerably modified the pattern of endemic (or enzootic) infections/diseases, and contributed to the emergence of new agents that are pathogenic for humans and domestic animals. emerging infections is a collective name for infections that have been identified and taxonomically classified recently. in humans, in the final quarter of the twentieth century, more than such conditions were recognised [ ] . zoonoses are defined as infectious diseases that can be transmitted naturally between humans and wild or domestic animals. these infections are particularly important in the context of emerging infectious diseases of humans as the majority of these are of zoonotic origin; a comprehensive review by cleaveland et al. [ ] identified species of infectious organisms known to be pathogenic to humans, including viruses and prions, bacteria and rickettsia, fungi, protozoa and helminths. out of these, ( %) were classified as zoonotic and pathogenic species were considered to be associated with emerging diseases. of emerging pathogens of this group, ( %) were zoonotic [ ] , the vast majority of which coming from wildlife. wildlife obviously constitutes an important potential of new pathogenic agents for humans and domestic animals [ ] . this paper will mainly focus on viruses, mammals and birds. nowadays, the total number of viruses identified reaches approximately species [ ] , but the likely number could exceed , according to the first estimates. even this number is most probably an underestimate (e.g. a well-known mammal species like human beings harbours at least different herpesviruses, and different herpesviruses have been identified in cattle until now), but if one takes into account the estimated number of , , only % of viruses have already been identified. moreover, it does not take into account their extreme variability, particularly rna viruses, leading to populations of quasi-species. if one considers that there are recognised mammal species and that, for instance, herpesviruses have been isolated from all classes of vertebrates and even from oysters, one must admit that the world of viruses is huge. the use of viral metagenomics will help to identify more viruses [ ] . for mammalians, different species have already been recognised, whereas the expected number of species is estimated to be around ; for mammalian species we are, therefore, nearly at the end of the inventory, since % of the species are already known. the inventory of mammalian species was first established in , when only species were recognised; the same inventory established in contained different species. in , as already mentioned, the complete list of mammal species consisted of species [ ] . this increase in number seems to be paradoxical and even contradictory if one takes into account the extinction of some species during the same period of time. this increase in number can be accounted for when one considers that each phenotype of newly discovered species is listed separately and, more importantly, that the advent of modern molecular technology allows for the discrimination of species according to their genotypes and increasingly detailed comparisons of species limits and evolutionary relationships (taxonomic revision). among mammals, there are species of rodents pertaining to genera. since , new rodent species have been recognised. the rodents therefore compose % of recognised mammal species. this number is particularly important if one takes into account the fact that the order of rodents harbours, and is the reservoir of numerous zoonotic infections. among the most spectacular are hantaviroses [ ] ; some african sciuridae species (funisciurus spp., heliosciurus spp.), are the reservoir of monkeypox [ ] . recently, the introduction of one of these species into the united states nearly provoked an ecological disaster, due to the transmission of the virus to indigenous rodent species (prairie dogs) [ ] . to date, the order of chiroptera contains species, pertaining to genera; new species have been identified since . bats make up therefore . % of the total number of mammal species. this is worrying, since bats have been the source of many emerging diseases, many of them being previously unknown. for instance, insectivorous and frugivorous bats are the reservoir of the archeolyssaviruses, from which all lyssavirus strains derive, even the strains responsible for terrestrial rabies. frugivorous bats are the reservoir of newly discovered viruses such as nipah and hendra (henipavirus), responsible for numerous human fatalities, of the coronavirus responsible for the epidemics of the severe acute res-piratory syndrome (sars) and, most probably, of filoviridae such as the virus responsible for ebola disease in africa. there are approximately , species of birds. in , species were recognised, pertaining to genera [ ] , of which are passerines ( genera) and are non-passerines ( genera). birds, previously dinobirds, descend from dinosaurs and are therefore further removed from us than mammals but they are, nevertheless, reservoirs of zoonotic infections. the recent epidemics of west nile virus infection in the americas is a good example. the problem arising from highly pathogenic avian influenza, particularly its strain h n , is equally worrying, since avian infection, which is already zoonotic, may be responsible for a new human pandemic, similar to the one following the first world war. the virus responsible for this pandemic, which caused more human fatalities than the war itself, was recently reconstituted [ ] [ ] [ ] and is highly virulent when inoculated to non-human primates. it is noteworthy that wildlife biodiversity hot-spots are mainly found in tropical and sub-tropical regions, such as sub-saharan africa, indonesia and south america. through selection, man has created a number of different breeds of domestic animals, e.g. there are approximately recognised breeds of cattle worldwide [ ] , but many of these are on the verge of extinction (less than breeding cows). there is therefore currently a swift erosion of genetic variability in cattle that is really worrying. there are more than recognised dog breeds, showing a remarkable phenotypic and genotypic variability. for instance a survey of the adaptive humoral response of different dog breeds following vaccination against rabies within the british pet scheme showed that there was a significant variation in response between breeds after vaccination [ ] . there obviously exists a large variation of responses between breeds after vaccination which could be used for the selection, marker assisted or not, of good or bad responders to vaccination. poultry lines selected according to their humoral adaptive immune response have already been obtained [ ] . as a matter of fact, the differences in the susceptibility of breeds to some infections or infestations has been well observed by breeders, for instance the "resistance" of the n'dama cattle breed to trypanomosis. the mechanisms allowing emergence or re-emergence of infections are numerous [ ] . a key factor is the extreme variability of viruses (particularly rna viruses) leading to generation of populations of quasi-species, which allows them to easily cross the species barrier. viruses evolve far quicker than their hosts, by several mechanisms: point mutations, deletions, recombination, reassortment and acquisition of cellular genes. moreover, viruses have co-evolved with their natural hosts, often leading to unapparent infections. in animal health, the spectacular emergence of canine parvovirosis in , which resulted in a disastrous epizooty within the dog population worldwide, was the result of a mutation of another parvovirus which is responsible for feline panleukopaenia [ , ] , whereas dogs and cats were living peacefully before, without inter-specific transmission. another source of emergence is the opening of previously closed ecosystems, which leads to new contacts between unrelated species and shows that a species previously unknown to be susceptible to an infection (because of the lack of opportunity to be infected) is in fact fully susceptible. a recent illustration is given by the emergence of bluetongue serotype , in northern europe, transmitted by a culicoides vector culicoides dewulfi previously unknown to be susceptible to the infection [ ] . invasive species or migratory species may also be responsible for emergence, as could be the deliberate or accidental release of foreign species in a new environment, as exemplified by the introduction of monkeypox virus in the united states. other possible sources of emergence include veterinary biologicals, climate change and globalisation with its ts (trade, transport, travel, tourism and terrorism). in animal health, in face of an emergency, there still exist two possibilities, either mass slaughtering of animals or vaccination. unfortunately vaccines are not always available; for instance in face of an outbreak of african swine fever and in the absence of a vaccine, the only solution is to kill the infected animals as quickly as possible, and to destroy the carcasses, in order to avoid the transmission of infection to uninfected premises. anyway, the pigs will die from a disease which provokes nearly % mortality. it is even more true when facing a really emerging disease that moreover is zoonotic such as nipah virus infection [ ] for which no vaccine was available yet, because the causative agent was previously unknown; the only solution is once again to kill and destroy the infected and in-contact animals. a vaccine has recently been developed to prevent nipah virus infection in pigs [ ] ; unfortunately in countries like bangladesh, man is mainly at risk due to direct contact with the reservoir, a fruit bat, or contact with its secretions. in other cases, when re-emergence of a previously well-known infection and when a vaccine is already available one may have the choice; either slaughtering or vaccination [ ] . foot-and-mouth virus infection gives an excellent example. footand-mouth virus is represented by seven serotypes, further divided into numerous sub-types. preventive vaccines are available as highly purified concentrated antigens stockpiled in liquid nitrogen [ ] ; being highly purified, they allow the differentiation between vaccinated or infected animals (even if previously vaccinated) thanks to a companion diagnostic test based on the detection of antibodies directed against non-structural proteins. unfortunately this technology only allows certification of freedom at the herd level. in the two recent outbreaks in united kingdom, the choice was to slaughter the animals. following the dramatic outbreak of foot-and-mouth disease in united kingdom, and to a lesser extend in france and in the netherlands, the european union lightened its regulation and is nowadays more prone to consider emergency vaccination as an alternative to slaughtering. if preventive vaccines are used in an emergency situation they could still be improved by conferring an early onset of protection. whenever viruses are represented by several serotypes, an infection can re-emerge in a previously vaccinated population against another serotype than the wild circulating one. for instance, an influenza pandemic can emerge in a human population with a herd immunity against seasonal flu. in case of highly pathogenic avian influenza (particularly strain h n ), one may choose to kill the birds or to vaccinate depending on the situation [ ] . there are some instances where vaccination is the only reasonable option, particularly when facing arthropod-borne virus infections. among arthropod-borne infections, there are infections caused by viruses represented only by one serotype, such as west nile virus or rift valley fever virus and others caused by viruses presenting multiple serotypes such as bluetongue virus. the majority of animal infections involve only two partners: the pathogen and the host, which has at its disposal its genetic background (natural resistance to infection) [ ] and its immune system. besides these infections or infestations, some vectorial infections are arthropod-borne and mainly transmitted by biting arthropod. these infectious systems are therefore more complex, because the vectors must be competent and able to multiply the pathogenic agent. in northern america there was the spectacular spread of west nile virus infection in humans and horses, rapidly followed by the development of several vaccines for horses, including a dna-based one. vaccination seems to be the only option since the reservoir is to find among birds and the infection is transmitted by mosquitoes. it is therefore nearly impossible to control the infection by other mean than vaccination without highly detrimental effect on the environment [ ] . rift valley fever is expanding its range in africa [ ] . until it was introduced into saudi arabia and yemen in ; rift valley fever tended to be confined to sub-saharan africa. the disease has recently occurred in madagascar. as of july , at least people reportedly died as a result of the infection, and the disease has claimed the lives of thousands of animals since the beginning of the year . since the disease is transmitted by mosquitoes, extreme weather events might create the necessary conditions for rift valley fever to expand its geographical range northwards and cross the mediterranean and arabian seas, with an unexpected impact on the animal and human health of newly affected countries [ ] . once again, vaccination is the best way to prevent the disease; an attenuated vaccine exists for sheep but is still abortigenic and should be improved. an arthropod-borne disease caused by a virus with multiple serotypes is typically bluetongue ( serotypes) and perhaps a new one [ ] . until , bluetongue was only observed in the mediterranean regions of europe and only serotypes , , , , and were involved. the disease appeared unexpectedly in northern europe in and serotype was involved, which is typically a sub-saharan serotype. cattle and sheep herds were fully susceptible to the infection; moreover, the strain involved was particularly virulent in cattle. bluetongue is transmitted by a biting midge, a member of the family ceratopogonidae, the culicoides. in the mediterranean region, the main species involved in the transmission is culicoides imicola, which originated in africa and asia and extended its range towards the north of its previous distribution, probably due to climate change. however climatic change does not seem to be responsible for the extension of the infection in northern europe, since the main culicoides species involved is culicoides dewulfi, a typically nordic species, whose transmission competence was unknown until bluetongue appeared in northern regions; in fact, this potential vector competence had not previously had the opportunity to be expressed due to the lack of bluetongue virus. the biology of the larval stage of culicoides impedes the control of the vector without damaging the environment. the most sensible option is vaccination of domestic ruminants with an inactivated vaccine containing serotype . in developed countries, partly as a result of overproduction, public concern for food security has been replaced by a major concern about food safety [ ] . this concern has increased following the bse (bovine spongiform encephalopathy) crisis. people are concerned about food-borne infections, the presence of drug residues following treatment of food-producing animals and the possible transfer of antibiotic resistance from bacteria causing disease in livestock to those which affect man [ ] . veterinary vaccines may help to solve some of these problems. the best example of a veterinary vaccine used for public health purposes is the vaccination of wildlife against rabies; the primary goal was not to protect wildlife species from rabies but to prevent human exposure and the disease in human populations [ , ] . being considered as products working by natural mechanisms, vaccines, except for some of their excipients, do not need to have an mrl (maximum residue limit) determination associated with a withdrawal period. in fact, since vaccine protection works after a lag period, the use of vaccines intrinsically contains a withdrawal period. veterinary vaccines can be used to prevent food poisoning as demonstrated by the "in ovo" vaccination of poultry against salmonellosis, in order to decrease carcass contamination. more recently a vaccine against escherichia coli :h has been conditionally approved for cattle in the united states. a vaccine against sheep cysticercosis has been developed experimentally and may lead to the development of similar vaccines to control bovine cysticercosis and thus taenia saginata infestation in humans. bacterial resistance to antibiotics is an emerging problem for both the animal and public health sector. several antibacterial vaccines used in veterinary medicine disappeared after the second world war, and were replaced by the use of antibiotics. the resistance to antibiotics in the animal health sector with possible implications for human health, as well as the resistance of several parasites to anthelmintics may lead to the reappearance or the appearance of antibacterial and antiparasitic vaccines. even if other pathways such as the selection of food-producing animals for genetic resistance to diseases are followed, the story of marek's disease in chickens demonstrates that vaccines are often more economical to procure an animal's resistance to pathogens. canine haemorrhagic enteritis: detection of viral particles by electron microscopy fréquence en belgique de l'infection à parvovirus chez le chien, avant et après l'observation des premiers cas cliniques bluetongue in northern europe. world animal health publication stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans largescale eradication of rabies using recombinant vaccinia-rabies vaccine climate change and biodiversity how the biodiversity sciences may aid biological tools and ecological engineering to assess the impact of climatic changes all creatures great and minute: a public policy primer for companion animal zoonoses diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergency infectious diseases: preparing for the future virus taxonomy; eight report of the international committee on taxonomy of viruses. virology division international union of microbiological societies viral metagenomics mammal species of the world-a taxonomic and geographic reference monkeypox outbreak traced to wisconsin pet dealer distribution and taxonomy of birds of the world characterization of the reconstructed spanish influenza pandemic virus the flu virus is resurrected aberrant innate immune response in lethal infection of macaques with the influenza virus cattle breeds, an encyclopedia. doetinchem: misset uitgeverij factors influencing the antibody response of dogs vaccinated against rabies genetic and phenotypic correlation between antibody response to escherichia coli, infectious bursa disease (ibdv), and newcastle disease virus (ndv), in broiler lines selected on antibody response to escherichia coli la faune sauvage et les maladies émergentes the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses regulatory issues surrounding the temporary authorisation of animal vaccination in emergency situations: the example of bluetongue in nipah virus infection of pigs in peninsular malaysia recombinant nipah virus vaccines protect pigs against challenge antigen and vaccine bank: technical requirements and the role of the european antigen bank in emergency foot and mouth disease vaccination control strategies for highly pathogenic avian influenza: a global perspective animal genomics for animal health west nile virus and north america: an unfolding story rift valley fever the impact of climate change on the epidemiology and control of rift valley fever genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats in switzerland. emerging infectious diseases, personal communication veterinary vaccines for animal and public health initiative aims to merge animal and human health science to benefit both the development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies: a link between jenner and pasteur risk factors for nipah virus encephalitis in bangladesh ecological sources of zoonotic diseases key: cord- -xvc wx authors: wink, michael title: chapter allelochemical properties or the raison d'être of alkaloids date: - - journal: nan doi: . /s - ( ) - sha: doc_id: cord_uid: xvc wx this chapter provides evidence that alkaloids are not waste products or functionless molecules as formerly assumed, but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells, which are seen in molecules that mimic endogenous neurotransmitters. the chapter discusses that microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. many herbivores have potent mechanisms to detoxify xenobiotics, which allow the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. it needs more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. organisms. we must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients. how do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? because plants do rather well in nature, this question has often been overlooked. we are well aware of the defensive strategies of higher animals against microbes and predators ( , , , , , , ) . the complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. against predating animals, nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called "poisons") ( ). it is evident that most of these possibilities are not available for plants with their sessile and "passive" life-style. what then is their evolutionary solution? we can distinguish the following defense mechanisms in plants ( , , , , ) ; the mechanisms are not independent and may act cooperatively and synergistically. we should be aware that many species have additionally evolved specialized traits in this context. . mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). . formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. b. cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. callose and lignin are often accumulated at the site of infection or wounding ( , ) and form a penetration barrier. c. synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. these proteins are either stored in the vacuole . allelochemical properties of alkaloids or are secreted as exoenzymes into the cell wall or the extracellular space ( , ) . these compounds are thus positioned at an "advanced and strategically important defense position." in addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. as a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced ( - , - ) . these allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides, glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~o u o synthesis may be induced by elicitors (so-called phytoalexins), infection, or herbivory ( , , [ ] [ ] [ ] . these products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection ( , )] or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots ( , , ) ]. in animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals ( , , , , , - , [ ] [ ] [ ] ) . in many instances, the animals have obtained the toxins from their host plants ( , , , , - ). hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ''toxins" in this context) in animals is that of defense against predators or microorganisms ( , , , [ ] [ ] [ ] . these defense compounds are better known as natural products or secondary metabolites. the latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites ( , , ) . unfortunately the term "secondary" has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. as discussed in this chapter, just the opposite is true. more than , natural products have been reported from plants so far ( , , ) . owing to the sophistication in phytochemical methods, such as chromatography (hplc, glc) and spectroscopy (nmr, ms) , new products are reported at rapid intervals. because only - % of all higher plants, which consist of over , species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large. it is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. within a taxon secondary metabolites often share a common distribution pattern and are therefore of some importance for phytochemical systematics. classic taxonomy, however, has taken little account of alkaloid distribution: if the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated ( ). for many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of nature ( , ) . in and , errera and stahl ( , , ) published the idea that natural products are used by plants for chemical defense against herbivores. since the leading plant physiologists of that time were mostly anti-darwinian, they were not willing to accept the defense argument, which was too much in line with the darwinian concept. therefore, this early defense concept was negated and remained forgotten for nearly years. in , fraenkel( ) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. during the next three decades this concept was improved experimentally, and we can summarize the present situation as follows although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century ( , ) . in many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy). in some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes ( , ) . physiological roles, such as uv protection [by flavonoids or coumarins ( , )], nitrogen transport or storage ( , , ) , or photosynthesis (carotenoids), may be an additional function. allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and antimicrobial). thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). to understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach. it might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. however, we have to understand and accept that chemical defense is not an absolute process. rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. during evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them ( , , ) . this is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. the number of these "specialists" is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. we can compare this situation with our immune system: it works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. nobody would call the immune system and antibodies useless because of these few adapted specialists! we should adopt the same argument when we consider plants' defenses by secondary metabolites ( ) . since secondary metabolites have evolved in nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. in addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds. about - % of higher plants accumulate alkaloids ( , ). the incidence of alkaloid production varies between taxa to some degree; for example, about - % of species of the solanaceae and apocynaceae are michael wink alkaloidal, whereas other families contain few alkaloid-producing species. some alkaloids have a wide distribution in nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. alkaloids are not restricted to higher plants (although they are here most numerous); they are also present in club mosses (lycopodium), horsetails (equisetum), fungi, and animals such as marine worms (e.g., nereidae), bryozoans, insects (e.g., coccinellidae, solenopsidae), amphibians (toads, frogs, salamanders), and fishes. alkaloids thus represent one of the largest groups of natural products, with over , known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan ( ) ( ) ( ) . in addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates ( [ ] [ ] [ ] . whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. this is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals. alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as w. . james and kurt mothes ( , , )l. because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. the waste product argument probably came from animal physiology: carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. a similar situation or need, however, is not applicable for plants. in fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves ( , , ). if alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. on the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals ( , , ) . thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. this idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and homo sapiens. as a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. ( ) the alkaloid should have significant effects against microbes and/or animals in bioassays. ( ) the compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. ( ) the compound should be present in the plant at the right time and the right place. ( ) evidence should be provided that a particular compound is indeed important for the fitness of a plant. although more than , alkaloids are known, only few (- - %) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. in most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities. furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. these objections have to be kept in mind, because an alkaloid is sometimes termed "inactive" in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). in many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. if the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. however, if a compound is active against escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. nevertheless, most of the data obtained in these studies (tables i-viii ) provide important information which at present permits extrapolation to the function of alkaloids in plants. in this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. in the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail. it is nearly impossible to cover the literature exhaustively. therefore, an overview of the allelochemical properties of alkaloids is presented. because of the large amount of data (literature up to is included), the selection of examples must remain subjective to some degree. nevertheless, the author would be grateful to receive information or publications about relevant omissions. because homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (tables i through v) and alkaloid-containing plants. previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products ( , ) . in the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates. among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. entomologists estimate that the number of insects is at least million, but tropical rain forests may harbor up to - million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists. most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( i , , , , , - , , ) . whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. in the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). at the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds. the close association between plants, especially the angiosperms, and insects evolved during the last million years. some scientists have called this phenomenon a "coevolutionary" process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved ( , , ). applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. times can also be much longer. if plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. for example, lupinus polyphyllus from north america has a number of specialized herbivores, but is rarely attacked by herbivores in europe. this lupine left its enemies behind when it was transferred to europe three centuries ago. about years ago, however, the north american lupine aphid (macrosiphum albifrons) was introduced to europe accidentally. this aphid is specialized to alkaloid-rich lupines with lupanine as a major alkaloid. at present, this aphid has spread over most of europe and is now colonizing its former host, l. polyphyllus ( , ) . insect herbivores can be divided into two large groups whose strategies with respect to the plant's defense chemistry differ substantially ( ). the polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagous insects are often specialized on one or a small number of (often systematically related) hosts. polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors ( ) that allow the distinction between plants with high or low amounts of "toxins." the receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates ( ). these "generalists," as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see table xi ). alternatively, they change host plants rapidly and thus avoid intoxication. in addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products ( , , , ). in contrast, mono-and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. for these "specialists" the originally noxious defense compounds are often attractive feeding and oviposition stimulants. these insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes ( , , - , , , , , - ). these observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as mothes ( ) and robinson ( ). however, these specialized insects are exceptions to the general rule. for these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants ( ) . as compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species. quite a number of alkaloids have been tested toward herbivorous insects (table i ). in general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>i%, w/w). given the choice, insects tend to select a diet with no or only a small dose of alkaloids. also, specialists avoid most "toxins" except those of their host plants. these data indicate that under natural conditions plants with a high content of alkaloids should be safe from most herbivorous insects, with the exception of particular monophagous species or a few very potent polyphagous ones. if insects have no choice or if they are very hungry, the deterrency threshold value is much reduced, and they often feed on a diet with alkaloids that they would normally avoid ( , ). in this case we have the chance to test the toxicity of an ingested alkaloid. if insects do not take up alkaloid-containing food, alkaloid toxicity can be assessed to some degree by topical application or by injection ( table i) . as can be seen from table i a substantial number of alkaloids display significant insect toxicity, including nicotine, piperine, lupine alkaloids, caffeine, gramine, strychnine, berberine, ephedrine, and steroidal alkaloids. only the specialists can tolerate the respective alkaloids. the tobacco hornworm (manduca sexta), for example, can grow on a diet with more than % nicotine without any adverse effects. most of the nicotine is either degraded or directly eliminated via the malpighian tubules and in feces ( ). because nicotine binds to the acetylcholine (ach) receptor, it is likely that in manduca this receptor has been modified in such a way that ach can still bind, but not nicotine (so-called target site modification). the toxic effects of alkaloids in insects (table i) can be caused by their interference with diverse cellular and intracellular targets. since most mechanisms have not yet been elucidated for insects, this issue is discussed below in the section on vertebrate toxicity (see table iv ). with some caution we can extrapolate to insect toxicity. because homo sapiens and domestic animals are largely herbivores, a voluminous body of information on the adverse effects of secondary metabolites has accumulated over the centuries. many allelochemicals and alkaloids are feeding deterrents for vertebrates, owing to their bitter or pungent taste or bad smell, and instinctively a foul-smelling, bitter, or pungent diet is normally avoided. examples of bitter alkaloids (at least for man) are quinine, strychnine, brucine, and sparteine, and for pungent alkaloids are capsaicin, and piperine. it should be recalled that these taste properties are not identical for all animals. for example, geese, which are obligate herbivores, hardly avoid food with alkaloids or smelly compounds (amines, mercaptoethanol) that man would hardly touch ( ). conversely, fragrances that are attractive to us are highly repellent to geese ( ). even within a given population taste can differ significantly. it has been observed that a substantial proportion of homo sapiens cannot detect the smell of hcn, whereas others are highly sensitive. furthermore, olfactory sensitivity can differ with age, sex, and hormonal cycles. bitterness varies with the chemical structure of an alkaloid. with the quinolizidine alkaloids (qas) the following scale was assessed for man: mean detection levels are . % for sparteine, . % for lupanine, and . % for hydroxylupanine ( ). whereas we know a few parameters of olfactory qualities in homo sapiens, often much less or hardly anything is known for most other vertebrates. alkaloids are famous for their toxic properties in vertebrates, and plants that produce alkaloids are often classified by man as poisonous or toxic plants. for a number of alkaloids the respective ld,, values have been determined with laboratory animals, especially mice, but also rats, guinea pigs, cats, rabbits, dogs, or pigeons. table i presents an overview for alkaloids, including the very poisonous alkaloids aconitine, coniine, atropine, brucine, curarine, ergocornine, physostigmine, strychnine, colchicine, germerine, veratridine, cytisine, delphinidine, and nicotine. toxicity is usually highest if the alkaloids are applied parenterally [intravenously (i.v.), intraperitoneally (i.p.), and subcutaneously (s.c.)] as compared to oral application [per s (p.o.)]. also, some of the alkaloids which are made or stored by animals are strong vertebrate poisons, including batrachotoxin, batrachotoxinin a, anabasine, glomerine, maitotoxin, nereistoxin, palytoxin, saxitoxin, and tetrodotoxin ( , , , ) . although the general toxicity of alkaloids differs from species to species, the data in table i generally show that many alkaloids are more or less toxic to vertebrates. the toxic effects observed with intact animals has its counterpart in the cytotoxic effect, which has been recorded for nearly alkaloids (table ). these data have been obtained by screening many natural products for anticancer activity. however, an alkaloid that can kill a cancer cell is usually also toxic for "normal" cells. therefore, the data shown in table i are another indication of the general toxicity of alkaloids toward animals. because this toxicity applies also for herbivores, the production of alkaloids by plants can certainly be interpreted as a potent antiherbivore mechanism. for a number of alkaloids the mechanisms underlying the toxic effects have already been elucidated in some detail. we can distinguish molecular targets and processes that are important for all cells, such as synthesis of dna, rna, and proteins, replication, transcription, translation, membrane assembly and stability, electron chains, or metabolically important enzymes or proteins including receptors, hormones, and signal compounds (table iv ). in the following we discuss some of these toxic effects. a. cellular targets nucleic acids. dna, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. it is not surprising that a number of secondary metabolites have been selected during evolution which interact with dna or dnaprocessing enzymes. some alkaloids bind to or intercalate with dna/rna (table iv) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (table v) : -methoxyellipticine, dictamnine, ellipticine, harmane alkaloids, melinone f, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (pas), cycasin, olivacine, etc. many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of at and gc base pairs. other alkaloids act at the level of dna and rna polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (table v) . besides basic data obtained in salmonella or drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. anagyrine, anabasine, and coniine cause "crooked calf disease" if pregnant cows or sheep feed on these alkaloids during the first period of gestation ( , , , , , ) . the offspring born show strong malformation of the legs. some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by veratrum species, cause the formation of a central large cyclopean eye ( - , an observation that was probably made by the ancient greeks and thus led to the mythical figure of the cyclops. it is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. in effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores. another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (tables iv and v) . the alkaloid aristolochic acid, which is produced by plants of the genus aristolochia, is carcinogenic. the mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine ( , ) , because of its no, group. pyrrolizidine alkaloids and their n-oxides, which are abundantly produced by members of the asteraceae and boraginaceae but also occur in the families apocynaceae, celestraceae, elaeocarpaceae, euphorbiaceae, fabaceae, orchidaceae, poaceae, ranunculaceae, rhizo- esterified ( , ) . after oral intake, the n-oxides are reduced by bacteria in the gut. the lipophilic alkaloid base is resorbed and transported to the liver, where it is "detoxified" by microsomal enzymes. as a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. the alkaloid can then cross-link dna and rna and thus cause mutagenic or carcinogenic effects (especially in the liver) ( ). thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. the pa story is very intriguing, since it shows how ingenious nature was in the "arms race." the herbivores invented detoxifying enzymes, and nature the compound which is activated by this process. a herbivore feeding on pa-containing plants will eventually die, usually without reproducing properly. only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. the protection due to pa can easily be seen on meadows, where senecio and other pa-containing plants are usually not taken by cows and sheep, at least as long other food is available. protein biosynthesis is essential for all cells and thus another important target. indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (table iv) , such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. for lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyl-trna with amino acids, as well as the elongation step. the inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected ( ). electron chains. the respiratory chain and atp synthesis in mitochondria demand the controlled flux of electrons. this target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, ch,-(ch ),,- , -methyl-piperidines, capsaicin, the hydroxamic acid dimboa, and solenopsine. as mentioned before, however, only a few alkaloids have been evaluated in this context (table v) . biomembranes and transport processes. a cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmenta- tion that provides separated reaction chambers. because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. the controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems. these complex systems are also targets of many natural products (table iv) . disturbance of membrane stability is achieved by -methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. steroidal alkaloids, such as solanine and tomatine, which are present in many members of the solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky. cells carefully control the homeostasis of their ion concentrations by the action of ion channels (na+,k+, ca + channels) and through na+,k+-atpase and ca +-atpase. these channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. inhibition of transport processes (ion channels, carriers) is achieved by (table iv) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin. a special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (na), serotonin, dopamine, glycine, and acetylcholine (ach). we can distinguish two types. type is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ach-receptor. in type the receptor is an integral protein. when a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent gprotein molecule, which consists of three subunits. the a subunit then activates the enzyme adenylate cyclase, which in turn produces camp from atp. the camp molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ach receptor). a number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. alkaloids relevant here include (table iv) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, ga- lanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine, anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. a number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly. cytoskeleton. many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. a number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (table iv) , namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. colchicine, the major alkaloid of colchicum autumnale (liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. as a consequence, chromosomes are no longer separated, leading to polyploidy . whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. the derivative colcemide is less toxic and can be employed in the treatment of certain cancers ( ). also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. for normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity . another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from catharanthus roseus (apocynaceae). these alkaloids also bind to tubulin ( ). both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias. from taxus baccata (taxaceae) the alkaloid taxol has been isolated. taxol also affects the architecture of microtubules in inhibiting their disassembly ( ). nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin ( ). in conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule. enzyme inhibition. the inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see table iv ). briefly, inhibition of camp metabolism (which is important for signal transduction and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by -ethyl-p-carboline, p-carboline- -propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (table iv) b. action at organ level. whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too. central nervous system and neuromuscular junction. a remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (table iv) . the cellular interactions have been discussed above. disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects. a herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. some individuals of homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically. the activity of muscles is controlled by ach and na. it is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. in the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis. alkaloids which activate neuromuscular action (so-called parasympathomimetics) include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) ( ) . skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. the compounds are usually considered to be strong poisons, and it is obvious that they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare ld,, values in table ). inhibition of digestive processes. food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. the next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. the ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms ( ). another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (table iv) . nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. in the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. the liver is also the main site for detoxification of xenobiotics. lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids ( ). these conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination. both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (tables iv and v) . the alkaloids are activated during the detoxification process, and this can lead to liver cancer. also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins. many alkaloids and other allelochemicals are known for their diuretic activity ( ). for an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. since na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially. disturbance of reproduction. quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. antihormonal effects could be achieved by mimicking the structure of sexual hormones. these effects are not known for alkaloids yet, but have been confirmed for other natural products. estrogenic properties have been reported for coumarins, which di-merize to dicoumarols, and isoflavones ( , ) . insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern polypodium uulgare, or azadirachtin from the neem tree ( , ) . juvenile hormone is mimicked by a number of terpenes, present in some coniferae. spermatogenesis is reduced by gossypol from cottonseed oil ( ) . the next target is the gestation process itself. as outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo ( table v) . the last step would be the premature abortion of the embryo. this dramatic activity has been reported for a number of allelochemicals, such as mono-and sesquiterpenes and alkaloids. some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids ( ) . the antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. nevertheless, they are defense strategies with long-term consequences. blood and circulatory system. all animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. inhibitors of the driving force for this process, the heart muscle, have already been discussed. however, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine ( ) . some allelochemicals have hemolytic properties, such as saponins. if resorbed, these compounds complex membrane sterols and make the cells leaky. steroidal alkaloids from solanum or veratrum species display this sort of activity as well as influencing ion channels (table iv) . allergenic effects. a number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds ( , , ) . activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. the function of alkaloids in this context is hardly known. this selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. however, most of the potential targets for the , alkaloids known at present remain to be established. if no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property. summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly. dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay ( ) . how is this achieved? the aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products ( , ) . this cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. in perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues. the only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. at the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas ( , ) . in addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. these studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. it may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. in the pharmaceutical context we search for very active compounds which can be employed at low concentrations. therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. these precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids. secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono-and disesquiterpenes, and last but not least, alkaloids ( , , , , ) . in table vi alkaloids are tabulated for which antibacterial activities have been detected. the alkaloids usually affect more gram-positive than gram-negative bacteria. especially well represented are alkaloids which '-hydroxytabernamine hydroxytetrahydrosecamine tetrandrine thalicarpine thalicerbine thalidasine thalidezine thaliglucinone thalistine thalistyline thalmelatine thalmirabine thalphenine thalrugosaminine thalrugosidine thalrugosine tubocurarine ( i +. active; -, no activity observed in the concentration range tested (many alkaloids were only assayed in low concentrations as microbial antibiotics); ad, agar diffusion, al, agar dilution; bg, biogram; ld, liquid culture; mic, minimal inhibitory concentration; pd, paper disk; sp, suspension; tlc, tlc disk test according to wolters and eilert ( ) . if more than one value is given, the data refer to different bacterial species tested. derive from tryptophan (indole alkaloids) and phenylalaninehyrosine, which may be due to the fact that these alkaloids have obtained considerable scientific attention since the discovery of many medicinally important compounds within these groups ( , , , , , , - ) . some of these alkaloids are highly antibiotic, with similar activities as fungal antibiotics, namely, cinchophylline ( ), dictamnine ( , fagarine ( ), stemmadine ( ), yuehchukene ( ), liriodenine ( , lysicamine ( ), oxonantenine ( ), sanguinarine ( ), solacasine ( , ) , rutacridone epoxide ( ), tryptanthrine (i@#), and tuberin ( , ) (table vi) . in many instances, when alkaloids are assessed for their antibacterial activity, they are often also tested for antifungal properties. usually yeasts and candidu are used as test organisms (table vii) . table vii lists ( i , ), thaliglucinone ( ), demissidine ( , ), solacasine ( ), soladulcidine ( , ), solasodine ( , )tidine ( , ), tomatine ( , ) , verazine ( ), cryptopleurine ( ) hydroxyrutacridone epoxide ( , tryptanthrine ( ), and tuberin ( ) . whereas the mode of action and targets of antibiotics of fungal and bacterial origin have been elucidated in many instances (see table iv ), relevant information for plant-derived compounds is scant. however, the molecular targets of some alkaloids have been determined at the general level, but not specifically for bacterial or fungal systems (table iv) that may be responsible for the antibiotic effects observed. the following interactions of alkaloids having antimicrobial properties with molecular targets of bacterial or fungal cells are likely (compare tables vi and vii with tables iv and v) . protein biosynthesis in ribosomes is affected by sparteine ( , , lupanine, angustifoline, -tigloyloxylupanine, and hydroxylupanine ( , , , , , ) . intercalation or binding to dna is influenced by fagaronine, dictamnine ( ), harman alkaloids ( , ) [binding to dna is light dependent ( )], berberine ( - , chelerythrine ( ), and sanguinarine ( , ) ; these compounds may thus inhibit important processes such as dna replication and rna transcription that are also vital for microorganisms. the stability of biomembranes may be disturbed by cepharanthine, tetrandrine, and steroidal alkaloids such as solamargine ( , solanine ( , , ), and solasonine ( ) , thus leading to an uncontrolled flux of metabolites and ions into microbial cells. inhibition of metabolically important enzymes is affected by berber- ine ( ), chelerythrine ( , ), chelidonine ( ), palmatine ( ), sanguinarine ( , ), solacongestidine ( ) , and papaverine. in contrast to antibiotics of microbial origin that could be classified as alkaloids from a chemical point of view in many instances, and which often interfere with the biosynthesis or maintenance of the cell wall (murein) (table iv) , such an interaction has not been described for plantderived compounds. since this topic has not been studied in detail it remains open whether this complex is another target for alkaloids. we can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de now. inducing agents, which have been termed "elicitors" by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents ( , , - ) . the induced compounds are called "phytoalexins," which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. in another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below). many of the antimicrobial alkaloids found are constitutively expressed and accumulated, that is, they are already present before an infection. using plant cell cultures, it was observed that some cultures start to produce new secondary metabolites when challenged with bacterial or fungal cell walls, culture fluids, or other chemical factors ( , , - ) . among the compounds found to be inducible are alkaloids such as sanguinarine and hydroxyrutacridone epoxide (see table xi ). quinolizidine alkaloids display some antimicrobial properties, besides their main role in antiherbivore defense ( ) (see table i ). on wounding, qa production is enhanced, thus increasing the already high alkaloid concentration in the plant; in other words, the antimicrobial and herbivoral effect is further amplified (table xi) ( , , ) . the reactions leading to the induction and accumulation of phytoalexins with phenolic structures have been studied in molecular detail ( , , - ) . these studies revealed that plants can detect and react rapidly to environmental problems, such as wounding or infection: within min of elicitation, mrnas coding for enzymes that catalyze the reactions leading to the respective defense compounds are increasingly generated, leading to the accumulation of the respective enzymes and consequently the production of the secondary metabolites ( , , - ) . similar processes are likely for alkaloids, but so far the mechanisms have not been elucidated. we assume that a substantial number of the , alkaloids have antimicrobial properties (which remain to be tested in most cases) that are directed against the ubiquitous and generalist microbes which have not table vi . if a range is given, the first value gives a % inhibition, the second value a % inhibition. specialized on a particular host plant. however, alkaloid production does not necessarily have to be involved with antimicrobial defense. for example, phytophthora or fusarium will attack alkaloid-rich plants of nicotiana, solanum esculentum, and s . tuberosum. cladosporium and fusarium can develop in nutrient-containing media enriched with alkaloids, and aspergillus niger can utilize alkaloids as a nitrogen source ( ). in addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. in these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. we suggest that these specialists have adapted to the chemistry of their host plants. mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). these mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. some associations between plants and fungi are symbiotic in nature, such as rhizobia in root nodules of legumes or microrhizal fungi in many species. in lupines, nitrogen-fixing rhizobia are present both in alkaloid-rich and alkaloid-free plants. they must therefore be able to tolerate the alkaloids, which are also present in the root. alkaloid production in lupines is more or less unaffected whether or not the plants harbor rhizobia ( , ) . an ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi ( ). a similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity. from the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (tables i and ). since many alkaloids are antibacterial and antifungal (tables vi and vii) and are present in plants at relatively high concentrations (section iila), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. it should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes ( - , ) . a cooperative, or even synergistic, process could thus be operating. c. antiviral properties plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (heteroptera). resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place. the assessment of antiviral activity is relatively difficult. as a result, only a few investigators have studied the influence of alkaloids on virus multiplication. nevertheless, at least alkaloids have been reported with antiviral properties (table viii) . only sparteine ( ) and cinchonidine ( ) have been tested for antiviral activities against a plant virus, the potato x virus. all other evidence for antiviral activities (table viii) table viii are difficult to interpret at present. polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum-and golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins. because alkaloids often deter the feeding of insects, such as aphids and bugs (table i ), viral infection rates may be reduced in alkaloid-rich plants. such a correlation exists for alkaloid-rich lupines (so-called bitter lupines) and low-alkaloid varieties (the so-called sweet lupines) (see table xii) . plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. this phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense ( , , - ) . a number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated. it was observed by molisch in ( ) that plants can also influence each other by their constituent natural products, and he coined the term "allelopathy" for this process. secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain ( , ) . both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments ( , , , - ) , but how it operates in ecosystems is still often a matter of controversy. it is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. thus allelochemicals might never reach concentrations high enough to be allelopathic. allelopathic natural products have been recorded in all classes of secondary metabolites. few research groups have studied the effect of alkaloids in this context, but at least alkaloids have been reported with allelopathic properties (table ix) . as can be seen from table ix , allelopathic activities can be found within nearly all structural types of alkaloids. at higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. more sensitive, however, is the growth of the radicle and hypocotyl. they respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. in species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in colchicum autumnale ( , ) . it is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms. alkaloids ( , , ), berberine ( - ), sanguinarine ( , ) and veratrum alkaloids]; inhibition of protein biosynthesis [e.g., emetine ( ) and quinolizidine alkaloids ( , , - , ) tables iv and ix) . the inhibitory action of quinolizidine alkaloids should be explained in this context ( , ) . they are very abundant in lupine seeds (up to - % dry weight). during germination, -hydroxylupanine is converted to ester alkaloids, such as -tigloyloxylupanine. the latter compound is predominantly excreted via the roots of young seedlings and in germination assays proved to be the most allelopathic qa. these alkaloids influence only heterologous systems, not the germination of lupine seeds themselves. when lupine and lepidium seeds were grown together in the same pot, growth of the lepidium seedlings was much reduced and inhibited, indicating that qas may also be relevant in the ecological context ( ) . although the number of alkaloids with known allelopathic properties is not large, owing to the limited number of studies conducted, it is clear from table ix that alkaloids can be toxic to plants, probably by interfering with basic metabolic or molecular processes. although comparably few alkaloids have been studied for their biological activities in detail, and considering that our data collection (tables i-ix) is far from complete, we can safely state that alkaloids have potent deterrent or poisonous properties in herbivorous animals, and also affect bacteria, fungi, viruses, and plants. the next question will be whether all the adverse activities of alkaloids, which are often assayed in in uitro systems only, are meaningful in nature. because most of the allelochemical activities are dose dependent (others may be synergistic, additive, etc.), the question is whether the amounts of alkaloids produced and stored in plants are high enough to be ecologically meaningful. it is difficult, and also dangerous, to make a general statement concerning alkaloid levels in plants. we must remember that alkaloid composition and levels are often tissue or organ specific ( , , ) . they may vary during the day [a diurnal cycle has been observed for qas and tropane alkaloids ( , , )l or during the vegetation period ( . , ) . furthermore, as in all biological systems, there are differences at the level of individual plants and between populations and subspecies. unfortunately, many phytochemical reports do not contain any quantitative information, or these data are given for the whole plant without realizing the above-mentioned variables. in addition, concentrations are usually given on a dry weight basis, which is appropriate in the chemical or pharmaceutical context. however, herbivores or pathogens do not feed on the dry plant in general, but on the "wet" fresh material. in the context of chemical ecology we urgently need data on a fresh weight basis. as an approximation, in this chapter we use a conversion factor of to convert dry weight to fresh weight data if only the dry weight data are available. summarizing the relevant phytochemical literature, we find that alkaloid levels are between . and % (dry weight), which is equivalent to o.oi-ls%fresh weight, or . - mg/gfresh weight. for plantscontaining quinolizidine alkaloids, actual alkaloid contents are given for a number organs or parts (table x ) , which fall in the range deduced before. we have evaluated the situation for quinolizidine alkaloids and found that the actual concentrations of alkaloids in the plant are usually much higher than the concentrations needed to inhibit, deter, or poison a microorganism or herbivore ( , , , ) . this means that plants obviously play safe and have stored more defense chemicals than actually needed. if we look at the ed,, and ld,, values given in tables through ix, it is likely that the situation is similar for other alkaloid-producing plants, but these correlations need to be experimentally established in most instances. it seems trivial that plants not only synthesize but also store their secondary products, which makes sense only in view of their ecological functions as defense compounds, since they can fulfil these functions only if the amounts stored are appropriate. achieving and maintaining the high levels of a defense compound are very demanding from the point of view of physiology and biochemistry. most allelochemicals would probably interfere with the metabolism of the producing plant if they would accumulate in the compartments where they are made ( ). whereas biosynthesis takes place in the cytoplasm, or in vesicles (berberine) or organelles such as chloroplasts (qas, coniine), the site of accumulation of water-soluble alkaloids is the central vacuole, and that of lipophilic compounds includes latex, resin ducts, or glandular hairs (e.g., nicotine) ( , ) . in this context it should be recalled that many alkaloids are charged molecules at cellular ph and do not diffuse across biomembranes easily. during recent years, evidence has been obtained that at least some alkaloids pass the tonoplast with the aid of a carrier system. the next problem is determining how the uphill transport, that is, the accumulation against a concentration gradient, is achieved. proton-alkaloid antiport mechanisms and ion trap and chemical trap mechanisms have been postulated and partially proved experimentally ( , , ) . thus, the sequestration of high amounts of alkaloids in the vacuole is a complex and energy-requiring task, which would certainly have been lost during evolution were it not important for fitness. as a rule of thumb, we can assume that all parts of an alkaloidal plant contain alkaloids, although the site of synthesis is often restricted to a particular organ, such as the roots or leaves. translocation via the phloem, xylem, or apoplastically must have therefore occurred. phloem transport has been demonstrated for quinolizidine, pyrrolizidine, and indolizidine alkaloids, and xylem transport for nicotine and tropane alkaloids ( , , ) . if the plant relies on alkaloids as a defense compound, these molecules have to be present at the right place and at the right time. alkaloids are often stored in specific cell layers, which can differ from the site of biosynthesis ( , , ) . in lupines, but also in other species ( , , alkaloids are preferentially accumulated in epidermal and subepidermal cell layers, reaching local concentrations between and mm (table x) , which seems advantageous from the point of view of chemical ecology, since a pathogen or small herbivore encounters a high alkaloid barrier when trying to invade a lupine. the accumulation of many alkaloids in the root or stem bark, such as berberine, cinchonine, and quinine, can be interpreted in a similar way. a number of plants produce laticifers filled with latex. for example, isoquinoline alkaloids in the family papaveraceae are abundant in the latex ( ), where they are sequestered in many small latex vesicles. in latex vesicles of chelidonium mujus the concentration of protoberberine and benzophenanthridine alkaloids can be in the range of . - . m, which is achieved by their complexation with equal amounts of chelidonic acid ( ). if a herbivore wounds such a plant, the latex spills out immediately. besides gluing the mandibles of an insect, the high concentration of deterrent and toxic alkaloids will usually do the rest, and, indeed, chelidonium plants are hardly attacked by herbivores. in addition, as these alkaloids are also highly antimicrobial (table iv) , the site of wounding is quickly sealed and impregnated with natural antibiotics. other well-known plants that have biologically active alkaloids in their latex belong to the families papaveraceae (genera papauer, macleya, and sanguinaria) and campanulaceae (genus lobelia) ( ) . it is intuitively plausible that a valuable plant organ must be more protected than others. alkaloid levels are usually highest during the time of flowering and fruit/seed formation. in annual species actively growing young tissue, leaves, flowers, and seeds are often alkaloid-rich, whereas in perennial ones, like shrubs and trees, we find alkaloid-rich stem and root barks in addition. all these plant parts and organs have in common that they are important for the actual fitness or for the reproduction and thus the long-term survival of the species. spiny species, which invest in mechanical defense, accumulate fewer alkaloids than soft-bodied ones ( ); examples are isoquinoline alkaloids in cacti or qas in legumes ( ) . if a plant produces few and large seeds, their alkaloid levels tend to be higher than in species with many and small seeds ( , ); thus. a plant with few and big seeds is generally a rich source of alkaloids, which makes sense in view of the defense hypothesis. these few examples show that accumulation and storage of alkaloids have been optimized in such a way that they are present at strategically important sites where they can ward off an intruder at the first instance of attack. thus, specialized locations must be regarded as adaptive. alkaloid concentrations can fluctuate during the vegetation period, or even during a day ( , ). but in biochemical terms their biosynthesis and accumulation are constitutive processes. this ensures that a certain level of defensive compounds is present at any time. furthermore, continuous turnover is a common theme for molecules of the cells whose integrity is important, such as proteins, nucleic acids, and signal molecules. the same seems to be true for a defense compound. an alkaloid which mimics a neurotransmitter, such as hyoscyamine, nicotine, or sparteine, could be oxidized or hydrolyzed in the cell by chance, and thus would be automatically inactivated. only by replacing these molecules continuously can the presence of the active compounds be guaranteed. for example, it was suggested that nicotine has a half-life of hr in nicotiana plants, and that more than % of the co, fixed passes through this alkaloid ( ). in other groups of natural products it was possible to show that plants can react to infection by microbes or to wounding by herbivores by inducing the production of new defense compounds. these compounds are termed "phytoalexins" in phytopathology ( ) ( ) ( ) . classic examples of phytoalexins include isoflavones, phenolics, terpenes. protease inhibitors, coumarins, and furanocoumarins. using plant cell cultures it could be shown that a similar process can be observed with some alkaloidal plants, which start to produce alkaloids with antimicrobial properties (e.g., sanguinarine, canthin- -one, rutacridone alkaloids) when challenged with elicitors from bacterial or fungal cell walls (table xi) . but what is the situation after herbivory? when plants are eaten by large herbivores, a de nouo synthesis would be almost useless for a plant (except maybe trees), since this would not be quick enough. the situation is different, however for small herbivores such as insects or worms, which may feed on a particular plant for days or weeks. here the de nouo production of an allelochemical would be worthwhile. there are indeed some preliminary experimental data that support this view. in liriodendron rirlipifera several aporphine alkaloids accumulate after wounding, which are otherwise not present ( ). in tobacco the produc- " cc, cell culture; pl, plant tion of nicotine, in lupines that of qas, and in atropci belleidonnu that of hyoscyamine are induced by wounding, thus increasing the already high levels of alkaloids by up to a factor of . whereas the response was seen after - hr in lupines, it took days in nicotiunu and in atropei (table xi) . we suggest that the wound-induced stimulation of alkaloid formation is not an isolated phenomenon, but rather an integral part of the chemical defense system. the induced antimicrobial and antiherbivoral responses show that plants can detect environmental stress and that secondary metabolism is flexible and incorporated in the overall defense reactions. many details on how a plant perceives and transmits information remain to be disclosed, but this will surely be a stimulating area of research in the future. although the physiology and metabolism of most alkaloids are extremely intricate ( ) and often not known, the available data suggest that they are organized and regulated in such a way that alkaloids can fulfill their ecological defense function. in other words, the alkaloids are present at the right time, the right place, and the right concentration. the aforementioned arguments strongly support the hypothesis that alkaloids serve as defense compounds for plants. besides circumstantial evidence, we would welcome critical experiments which clearly prove that alkaloids are indeed important for the fitness and survival of the plants producing them. we suggest that if a plant species which normally produces alkaloids is rendered alkaloid-free, it should have a reduced fitness because it is much more molested by microorganims and herbivores than its alkaloid-producing counterpart. for one group of alkaloids, the quinolizidine alkaloids, these experiments have already been performed ( , , , , ) . as mentioned before, qas constitute the main secondary products of many members of the leguminosae, especially in the genera lirpinus, genistu, cyfisiis, bccptisiu, thrrmopsis, sophoru, ormosici, and others ( ). lupines have relatively large seeds which contain up to - % protein, up to % lipids, and - % alkaloids. to use lupine seed for animal or human nutrition, homo scipiens, for several thousand years, used to cook the seeds and leach out the alkaloids in running water. this habit has been reported for the egyptians and greeks in the old world, and for the indians and incas of the new world. the resulting seeds taste sweet, in contrast to the alkaloid-rich ones which are very bitter. in mediterranean countries people still process lupines in the old way, and sometimes the seeds are salted afterward and served as an appetizer, comparable to peanuts. at the turn of the twentieth century, german plant breeders set out to grow alkaloid-free lupines, the so-called sweet lupines. although sweet lupines are extremely rare in nature ( in > . ), the efforts were largely successful, and at present, sweet varieties with an alkaloid content lower than . % exist for lupinus albus, l. mutabilis, l . luteus, l. angustifolius, and l . polyphyllus. as far as we know, the sweet varieties differ from the original bitter wild forms only in the degree of alkaloid accumulation. this offers the chance to test experimentally whether bitter lupines have a higher fitness than sweet ones with regard to microorganisms and herbivores. the results of these experiments were clearcut ( , , , ) (table xu). in the greenhouse, where plants are protected from herbivores or pathogens, no clear advantage was seen. when lupines were planted in the field, without being fenced in and without man-made chemical protection, however, a dramatic effect was regularly encountered, especially with regard to herbivores ( , , , ) . rabbits (cuniculus europaeus) and hares (lepus europaeus) clearly prefer the sweet plants and leave the bitter plants almost untouched, at least as long as there was an alternative food source. before dying rabbits will certainly try to eat bitter lupines. a similar picture was seen for a number of insect species, such as aphids, beetles, thrips, and leaf-mining flies (table xii) , namely, the sweet forms were attacked, whereas the alkaloid-rich ones were largely protected. the alkaloid-poor variety of l . luteus also became a host of acyrthosiphon pisii ( ). in poland, where the sweet yellow lupine is one of the more important fodder plants, the invasion of the aphids became a serious problem not only because the aphid enfeebles the plants by sucking its phloem sap, but also because it transfers a viral disease. the disease, known as lupine narrow leafness, decreases seed production in infected plants, and the infection takes place early, that is, prior to the plants' blossoming. thus, a mixed population of sweet and bitter lupines can, after a few generations, lose all sweet forms. infestation by the aphid and the following viral infection accelerate the elimination of alkaloid-poor plants, which, even without infection, are already inferior in seed production ( ). this observation again stresses the importance of alkaloids for the fitness of lupines. plant breeders have also observed that bacterial, fungal, and viral diseases are more abundant in the sweet forms, but this effect has not been documented in necessary detail. these experiments and observations clearly prove the importance of qas for lupines, but it should not be forgotten that other secondary metabolites, such as phenolics, isoflavones, terpenes, saponins, stachyose, erucic acid, and phytic acid, are also present in lupines and may exert additional or even synergistic effects. the lupine example also tells us about the standard philosophy and problems of plant breeding. with our present knowledge on the ecological importance of qas for the fitness of lupines, it seems doubtful whether the selection of sweet lupines was a wise decision. in order to grow them we have had to build fences and, worse, to employ man-made chemical pesticides, which have a number of well-documented disadvantages. it can be assumed that similar strategies, namely, breeding away unwanted chemical traits, have been followed with our other agricultural crops, with the consequence that the overall fitness was much reduced ( ). we can easily observe the reduced fitness by trying to leave crop species to themselves in the wild: they will quickly disappear and not colonize new habitats. there are, however, alternatives. taking lupines as an example, we could devise large-scale technological procedures to remove alkaloids from the seeds after harvest (similar to sugar raffination from sugar beets). at present a few companies are actively exploring these possibilities. one idea is to produce pure protein, lipids, dietary fibers from bitter seeds. a spin-off product would be alkaloids, which could be used either in medicine (sparteine is exploited as a drug to treat heart arrhythmia) or in agriculture as a natural plant protective, that is, as an insecticide ( , ) . it is evident, however, that each plant has developed its own strategy for survival. if all plants would follow the same strategy, it would be an easy life for herbivores and pathogens, since being adapted to one species would mean adapted to all species. this specialization becomes evident if we analyze the qualitative patterns of secondary metabolite profiles present in the plant. we regularly see one to five main alkaloids in a plant, but also several (up to ) minor alkaloids. this qualitative pattern is not constant, but differs among organs, developmental stages, individuals, populations, and species. normally, we classify the compounds as belonging to one or two chemical groups. this does not mean, however, that their biological activities are identical. on the contrary, the addition of a lipophilic side chain to a molecule seems to be a small and insignificant variation from the chemical point of view, but this may render the compound more lipophilic, and thus more resorbable. in consequence, its toxicity may be higher (see qas in table i ). thus, a herbivore or pathogen has to adapt not only to one group of chemicals but to the individual compounds present. as the composition of these chemicals changes, it is even more difficult for them to cope. therefore, we suggest that structural diversity and continuous variation are means by which nature counteracts the adaptation of specialists. in medicine, we do a similar thing if we want to control microbial diseases. to overcome or to prevent resistance of bacteria toward a particular antibiotic, very often mixtures of structurally different antibiotics are applied, whose molecular targets often differ. if only one antibiotic were given to all patients, the development of resistance would be much favored. it has been argued that alkaloids cannot have a significant role in plants because not all plant species produce alkaloids (only % of all plants do). these authors, such as robinson ( , have overlooked the fact that if all plants would produce one single alkaloid, even a very toxic alkaloid such as colchicine, it could be certain that nearly all herbivores would have developed a resistance toward this alkaloid. only the variation of secondary metabolites, and thus of the targets which they affect, provides a means to develop efficient defense compounds. the arguments of robinson would be correct if there were higher plants without any secondary metabolites, which, nevertheless, would thrive in nature; however, these plants are not known. from an evolutionary perspective it is not important whether the defense chemical is an alkaloid or a terpene; it is only essential that it affect certain and important targets in herbivores or pathogens. although the biological activities of many alkaloids have not yet been studied and their ecological functions remain to be elucidated or proved, we can nevertheless safely say that alkaloids are neither waste nor functionless molecules, but rather they are important fitness factors, probably mostly antiherbivore compounds. since nature obviously favored multitasking, additional activities, such as allelopathic or antimicrobial activities, are plausible. for quinolizidine and pyrrolizidine alkaloids, these multiple functions are already well documented (tables i-x) . plants that defend themselves effectively constitute an ecological niche almost devoid of herbivores and pathogens. it is not surprising that during evolution a number of organisms evolved which have specialized on a particular host plant species and found ways to tolerate, or even to exploit, the defense chemistry of their hosts ( , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as compared to the huge number of potential enemies, the number of adapted specialists is usually small, and in general a "status quo" or equilibrium can be observed between the specialists (or parasites) and their hosts. a specialist is not well advised to kill its host, since this would destroy its own resources; a mutualism is more productive for survival. host plant-specific specialists occur within bacteria, fungi, and herbivores. the interaction of the former two groups is a central topic for plant pathologists. they often find that susceptible and nonsusceptible microbe strains exist. in most cases, it is not known how these microbial specialists achieved a relationship with the host plant chemistry, for example, whether they degrade secondary metabolites or whether they simply toler-ate them. many phytopathogenic bacteria and fungi produce their own secondary metabolites, which are often toxic to plants. it is assumed that these phytotoxins serve to weaken the host plants' defense, but may be this is not the whole story. many grasses are infected with fungi that produce ergot alkaloids. it has been assumed that these fungi (e.g., clauiceps) are proper parasites. in recent years, however, experimental evidence suggests that the relationship between grasses and ergot may be of a symbiotic nature ( ). ergot alkaloids are strong vertebrate toxins (tables i-iv) ; they mimic the activity of several neurotransmitters, such as dopamine, serotonin, and noradrenaline (table iv) . in fact, the impact of herbivores on populations which were highly infected by fungi was more reduced than those without. this means that the fungi exploit the nutrients of their host plants and supply them with strong poisons, which are not produced by the plants themselves. since the fungi do not kill their hosts, this close interrelationship seems to be of mutual interest. we expect that similar relationships are likely to be detected in the future. as mentioned earlier, a large number of mono-and oligophagous insects exist which have adapted to their host plants and the respective defense chemistry in complex fashions. in general, we can see the following main schemes ( , , , , , ) . in type adaptations, a species "learns" (or, as we should say, during evolution variants have been selected by natural selection which can tolerate a noxious defense compound) (a) by finding a way to avoid its resorption in the gut; (b) if resorption cannot prevented, by eliminating the toxin quickly via the malpighian tubules or degrading it by detoxifying microsomal and other enzymes; and (c) by developing a target site that is resistant to the toxin, such as a receptor which no longer bind the exogenous ligand. alternatively, in type strategies a species not only tolerates a plants' defense compound, but exploits it for its own defense or for other purposes, such as pheromones i , , ) . examples of type include manduca sexra, whose larvae live on nicoriana and other solanaceous plants. the alkaloids present in these plants, such as nicotine or hyoscyamine, are not stored but are degraded or directly eliminated with the feces ( ). in addition, it has been postulated that nicotine may either not diffuse into nerve cells or that the acetylcholine recpetor no longer binds nicotine as in "normal" animals ( ). the potato beetle (leptinotarsa decernlineata) lives on solanurn species containing steroid alkaloids, which are tolerated, but not stored, by this species, the bruchid beetle callosohruchus fasciarus predates seeds of qa-rich plants, such as laburnum anagyroides; this beetle eliminates most of the dietary cytisine with the feces ( ). examples of type are to some degree more interesting. in a number of plants alkaloids are translocated via the phloem ( ). when aphids live on these plants they are in direct contact with the alkaloids present. a number of examples are known at present which show that adapted aphids can store the dietary alkaloids. examples are the quinolizidines in aphis cytisorum, a. genistae, and macrosiphum albifrons, the pyrrolizidines in aphis jacobaea, a . cacaliaster, and aconitine in aphis aconiti ( , ) . for alkaloid-storing m . albifrons it was shown experimentally that the qas stored provide protection against carnivorous beetles, such as carabus problematicus or coccinella septempunctata ( , ) . acyrthosiphon spartii prefers sparteine-rich cytisus scoparius plants ( ); although it is likely that this species also stores qas, it has not been demonstrated to do so. larvae of the pyralid moth uresiphita reversalis live on qa-producing plants, such as teline monspessulana. the larvae store some of the dietary alkaloids, especially in the integument and also the silk glands. the uptake is both specific and selective and is achieved by a carrier mechanism. whereas alkaloids of the -oxosparteine type dominate in the plant, it is the more toxic cytisine that is accumulated by the larvae, with the oxosparteines being eliminated with the feces ( , ) . the larvae gain some protection from storing qas, as was shown in experiments with predatory ants and wasps. when the larvae pupate, most of the alkaloids stored are used to impregnate the silk of the cocoon, thereby providing defense for this critical developmental stage ( , ). the emerging moth lives cryptically, has no aposematic coloring, and does not contain alkaloids. in contrast the alkaloid-rich larvae are aposematically colored and live openly on the plants ( , ) . the larvae of the blue butterfly (plebejus icaroides) feed only on lupines, rich in alkaloids. as far as we know, the larvae do not sequester or store the dietary alkaloids ( ). helopeltis feeds on cinchona bark, which is rich in cinchonine-like alkaloids; it stores and uses them for its own defense ( ). larvae of the butterflies pachlioptera aristolochiae, zerynthia polyxena, ornithoptera priamus, and battus philenor live on arisrolochia plants and were shown to take up and sequester aristolochic acid, a carcinogenic alkaloid discussed earlier, as an effective defense compound ( , , ) . the best-studied group of acquired alkaloids are the pyrrolizidines, which are produced by plants, especially in the families asteraceae and boraginaceae ( ). some arctiid larvae of tyria jacobaea, cycnia mendica, amphicallia bellafrix, arginia cribaria, and arctia caja were shown to store the dietary pas and exploit them for their own defense ( , , , , - , ) . in tyria jacobaea, arctia caja, diacrisia sannio, phragmatobia fuligonosa, and callimorpha dominula pas are taken up and stored in the integument ( ). monarch butterflies (e.g., danaus plexipus) combine two sets of natural compounds. larvae feed on plants rich in cardiac glycosides and use them as chemical defense compounds. adult butterflies visit plants with pas, where they collect pas that are converted to pheromones or transferred to their eggs ( ,f , , , f, ) . a similar pa utilization scheme was observed with larvae of the moth utetheisa ornatrix ( , ) , where the compounds were shown to be deterrent for spiders and birds ( , ) . the chrysomelid beetle oreina feeds on pa-containing plants, such as adenostyles, and stores the dietary pas in the defense fluid ( , ). in the arctiid creatonotos transiens was observed an advanced exploitation of pas ( , , , - ) . the alkaloids are phagostimulants for larvae, which are endowed with specific alkaloid receptors. dietary pyrrolizidine n-oxides are resorbed by carrier-mediated transport. after resorption, free pas are converted to the respective n-oxides and ( s)-heliotrine to ( r)-heliotrine. the latter form is later converted to a male pheromone, ( r)-hydroxydanaidal. pas are stored in the integument, where they serve as defense compounds and are not lost during metamorphosis. in the adult moth, however, the pas are mobilized. in the female adult, pas are translocated into the ovary and subsequently into the eggs. in the male, pas are necessary for the induction of abdominal scent organs and concomitantly for the biosynthesis of pa-derived pheromones, which are dissipated from these coremata. in addition, pas are transferred into the spermatophore and thus donated to the female. a significant amount of pas is further transferred to the eggs, which thus obtain chemical protection from the pas previously acquired by both male and female larvae. marine dinoflagellates produce a number of toxins, such as saxitoxin, surugatoxin, tetrodotoxin, and gonyautoxin, that affect ion channels (table iv). these algae are eaten by some copepods, fish, and molluscs that also store these neurotoxins ( , , , , , ) . as a consequence, these animals have acquired chemical defense compounds, which they can use against predators. this discussion is not meant to be complete, but should illustrate that a number of insect herbivores exploit the chemistry of their food plants. these insects are adapted and have evolved a number of molecular and biochemical traits that can be considered as prerequisites. however, many of the respective plant-insect interactions have not yet been studied, and it is therefore likely that the acquisition of dietary defense compounds is even more widely distributed in nature than anticipated. whereas insect herbivores are often highly host plant specific, vertebrate herbivores tend to be more of the polyphagous type, although some specialization may occur. for example, grouse (lagopus lagopus) or capercaillies (tetra urogallus) prefer plants of the families of ericaceae or coniferae, and crossbills seeds of picea and abies species, which are rich in terpenes. the australian koala is oligophagous and prefers terpene-rich species of the genus eucalyptus. for approximately million years, the only true herbivorous vertebrates have been the mammals. the mesozoic reptiles disappeared following the mesophytic flora. birds, though a few species feed on seeds and berries, seldom eat leaves (except geese and grouse), and they frequently use insects, in addition to plant parts, as a food source ( ). although a single plant can be a host for hundreds of insect larvae, hundreds of plants comprise a daily menu for a larger mammal. the strategies of the polyphagous species include the following. avoidance of plants with very toxic vertebrate poisons (these species are usually labeled toxic or poisonous by man) by olfaction or taste discrimination. often such compounds may be described as bitter, pungent, bad smelling, or in some other way repellent. . sampling of food from a wide variety of sources and thus minimizing the ingestion of high amounts of a single toxin. . detoxification of dietary alleochemicals, which can be achieved by symbiotic bacteria or protozoa living in the rumen or intestines, or by liver enzymes which are specialized for the chemical modification of xenobiotics. this evolutionary trait is very helpful for homo sapiens, since it endowed us with a means to cope with our man-made chemicals which pollute the environment. carnivorous animals, such as cats, are known to be much more sensitive toward plant poisons ( ). it was suggested that these animals, which d o not face the problem of toxic food normally, are thus not adapted to the handling of allelochemicals. some animals, such as monkeys, parrots, or geese, ingest soil. for geese ( ) it was shown that the ingested soil binds dietary allelochemicals, especially alkaloids ( ). this procedure would reduce the allelochemical content available for resorption. . animals are intelligent and can learn. the role of learning in food and toxin avoidance should not be underestimated, but it has not been studied in most species. for most vertebrate herbivores, the ways they manage to avoid, tolerate, or detoxify their dietary allelochemicals have not been explored. sometimes, only domesticated animals were used in experiments, but they tend to make more mistakes in food choice than the wild animals. more evidence on this subject is available for homo sapiens, who has evolved a number of "tricks," some of them obviously not anticipated by evolution. first, man tends to avoid food with bitter, pungent, or strongly scented ingredients. as a prerequisite he needs corresponding receptors in the nose or on the tongue which evolved during the long run of evolution as a means to avoid intoxication. second, our liver still contains a set of detoxifying enzymes which can handle most xenobiotics. furthermore, some of these enzymes, such as cytochrome p. oxidase, is inducible by dietary xenobiotics. third, besides these biological adaptations, man has also used his brain to avoid plant allelochemicals. (a) many fruits or vegetables are peeled. as many alkaloids and other compounds are stored in the epidermis, for example, steroid alkaloids in potato tubers or cucurbitacins in cucurbits, peeling eliminates some of these compounds from consumption. (b) most food is boiled in water. this leads to the thermal destruction of a number of toxic allelochemicals, such as phytohaemagglutinins, protease inhibitors, and some esters and glycosides. many watersoluble compounds are leached out into the cooking water and are discarded after cooking (e.g., lupines or potatoes). (c) south american indians ingest clay when alkaloid-rich potato tubers are on the menu. since clay binds steroidal alkaloids, geophagy is thus an ingenious way to detoxify potential toxins in the diet ( ) . (d) man has modified the composition of allelochemicals in his crop plants, in that unpleasant taste components have been reduced by plant breeding. from the point of view of avoidance, this strategy is plausible, but, as was discussed earlier, it is deleterious from the point of view of chemical ecology. these plants often lose their resistance against herbivores and pathogens, which then has to be replaced by man-made pesticides. in general, only a few plants are exploited by man as food, as compared to the , species present on our planet. this means that even homo sapiens with all his ingenuity has achieved only a rather small success, indicating the importance and power of chemical plant defenses. in this context, it is worth recalling that a number of animals are able to synthesize their own defense compounds, among them several alkaloids ( , , , - ) . these animals have the common feature that they are usually slow-moving, soft-bodied organisms. marine animals, such as mol-luscs, sponges, zooanthids, and fishes, have been shown to contain a variety of alkaloids, such as acrylcholine, neosaxitoxin, murexin, pahutoxin, palytoxin, petrosine, and tetramine, that are toxic to other animals ( . , , , , , , , , , ) . a number of nemertine worms, such as amphiporus or nereis, produce alkaloids such as , -bipyridyl, anabaseine, nemertelline, or nereistoxin, which are toxic to predators such as crayfish ( , , , ,) . arthropod-made alkaloids include glomerine and homoglomerine in glomerus ( ) , adaline in adalia ( ), coccinelline, euphococcinine, and derivatives in coccinella, epilachna, and other coccinellid beetles ( , , , ) , and stenusine in stenus ( ) , which are considered to be antipredatory compounds ( , , , - ) . solenopsis ants produce piperidine alkaloids which resemble the plant alkaloid coniine. these alkaloids are strong deterrents and inhibit several cellular processes, such as electron transport chains (table iv) ( , ) . many insects indicate the content of toxic natural products by warning colors (aposematism) or by the production of malodorous pyrazines ( , , , ) . not only are lower animals able to synthesize alkaloids, but also vertebrates, especially in the class amphibia. tree frogs of the genus dendrobates accumulate steroidal alkaloids, such as batrachotoxin, pumiliotoxins a-c, gephyrotoxin, and histrionicotoxin, in their skin, which are strong neurotoxins (table iv) ( , , ) . natives have used the alkaloids as arrow poisons. similar alkaloids (i.e., homobatrachotoxin) have recently been detected in passerine birds of the genus pitohui ( ) . salamanders, salamandra maculosa, which are aposematically colored, produce the toxic salamandrine and derivatives, alkaloids of the steroidal group ( , , ). salamandrine is both an animal toxic (paralytic) and an antibiotic. toads (bufonidae) produce in their skin cardiac glycosides of the bufadienolide type, but also a set of alkaloids, such adrenaline, noradrenaline, adenine, bufotenine, or bufotoxin ( , , ). except for bufotoxin, the other chemicals are, or mimic, neurotransmitters. these examples show that alkaloids found in animals can either be derived from dietary sources (see section ,d, ) or be made endogenously. common to both origins is their use as chemical defense compounds, analogous to the situation found in plants. in animals we can observe the trend that sessile species, such as sponges and bryozoans, or slow-moving species without armor, such as worms, nudibranchs, frogs, toads, and salamanders, produce active allelochemicals ( , , , ) , but not so those with weapons, armor, or the possibility for an immediate flight. plants merely developed a similar strategy as these "unprotected" animal species. in this context it seems amazing that hardly anybody has doubted the defensive role of alkaloids in animals, whereas people did, and still do, where alkaloids in plants are concerned. evidence is presented in this overview that alkaloids are not waste products or functionless molecules as formerly assumed ( , ), but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were obviously developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells (i.e. they are specific inhibitors or modulators), which can clearly be seen in molecules that mimic endogenous neurotransmitters (table iv; section ii,a, ,a). on the other hand, microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. in addition, many herbivores have potent mechanisms to detoxify xenobiotics, which allows the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids obviously function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. the overall picture of alkaloids and their function in plants and animals seems to be clear, but we need substantially more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. defense in animals introduction to ecological biochemistry defense mechanisms in plants herbivores: their interaction with secondary okologische biochemie allelochemicals: role in agriculture and forestry cell culture and somatic cell genetics of plants" (f. constabel and . m. wink gifttiere und ihre waften perspectives in chemoreception behavior biochemie und physiologie der sekundaren pflanzenstoffe plant metabolites biosynthese der alkaloide biochemistry of alkaloids the alkaloids: the fundamental chemistry antiseptika baerheim svendsen lloydia . n. m. rojas hernandez vallejos and . a. roveri the merck index proc. rd inf. lupin conf insect biology in the future micromolecular evolution, systematics and ecology phytochernical ecology: allelochernicals, mycotoxins and insect pheromones phytochem biogene gifte rozniki nauk rolnikzych die gift-und arzneipflanzen in mitteleuropa antibiotics: mechanism of action of antimicrobial and antitumour agents antimicrob. agents chemo pharmazeutische biologie , biogene arzneistoffe pharmazeutische biologie drug use in pregnancy the alkaloids handbook of enzyme inhibitions cold spring harbor conf primary and secondary metabolism of plant cell cultures chemical defenses of arthropods der einfluss einer pflanze auf die andere-allelopathie the science of allelopathy allelopathy lectures on insect olfaction focus on insect-plant interactions alkaloid biology and metabolism in plants molecular aspects of insect plant associations methods of plant biochemistry secondary products in plant tissue culture proc. natl the alkaloids the work of the author was supported by the deutsche forschungsgemeinschaft. i thank dr. th. twardowski for reading an earlier draft of the manuscript. key: cord- -soaz b authors: trivellini, guido; polidori, carlo; pasquaretta, cristian; orsenigo, simone; bogliani, giuseppe title: nestedness of habitat specialists within habitat generalists in a butterfly assemblage date: - - journal: insect conserv divers doi: . /icad. sha: doc_id: cord_uid: soaz b . the habitat requirements of a species are the resources, conditions and space required for survival and reproduction. the habitat requirements of butterflies have been well studied, but the extent to which individuals within a species and between species utilise and share the habitat is poorly known. . in a butterfly assemblage in northern italy, we found that adults from species avoid deciduous high‐density forests and their ecotones, and they were positively related to open areas and their ecotones. besides these common features, five groups of species can be discriminated in relation to a gradient from open area to forest, and species within groups were not equally specialised, as observed from a bipartite network analysis. in particular, some species appeared to be specialised and others appeared to be generalist, suggesting a nested pattern of resource use, rather than a clustered pattern in which each species uses a different subset of habitat types. . the degree of variation in specialisation among species varied with the number of species falling in each group. thus, an increased number of species, and thus possibly competition, is more likely to promote the co‐occurrence of generalist and specialised species (nested patterns) rather than an increased niche segregation among species. . ascertaining how species overlap their habitat use at a local scale can be relevant for conservation purposes, because specialised populations are potentially more susceptible to network distortions. butterflies (lepidoptera) are a well-studied insect group both taxonomically and ecologically (e.g. gilbert, ; kocher & williams, ; ghazoul, ; stefanescu et al., stefanescu et al., , , particularly because they interact with plants both as larval herbivores and adult pollinators (borges et al., ; hardy et al., ) and have different habitat requirements even at each life stage (new et al., ; samways & lu, ; dennis, ) . in turn, because these interactions are important mechanisms of biological diversification (leather, ; basset, a,b; slansky, ) , the abundance and distribution of butterflies within a particular area can give insights into the environment's quality (kocher & williams, ) . correspondence : giuseppe bogliani, department of earth and environmental sciences, university of pavia, via adolfo ferrata , pavia, italy. e-mail: giuseppe.bogliani@unipv.it these largely specialised butterfly-habitat associations are influenced by many physical and biological factors (leps & spitzer, ; spitzer et al., ) , including temperature, humidity, light levels, rainfall patterns, local microclimate conditions, grassland type and host-plant species richness and distribution (hill et al., ; meyer & sisk, ; collinge et al., ; men endez et al., ) . all these variables define the resource-based habitat or habitat requirements of each butterfly species, i.e., the habitat characterised by resources and conditions required by organisms and their access to them (dennis, ) . furthermore, competition among species, presence of invasive species and avoidance strategies to escape from natural enemies (enemy-free space) also affect species occurrence in a given area (murphy, ; dennis, ; wagner & van driesche, ; hanula & horn, ) . while habitat requirements have been assessed for many butterfly species in detail, the degree to which species within an assemblage segregate their habitat use (i.e. how they are specialised in a subset of habitat types) has been less well studied. the concepts of habitat requirements and habitat specialisation are strongly linked to the classical differences between the pure ecological niche based on the physiological optimum (austin, (austin, , (niche requirements) and the true and realised ecological niche (hutchinson, ; pulliam, ) (niche specialisation). thus, while habitat requirements can be seen as a property of a species, habitat specialisation is a variable property that can vary with many interacting factors such as the presence of inter-specific competitors, the population density and thus the intra-specific competition and the body size of the individuals, among others (chase & leibold, ; dennis et al., dennis et al., , rosenfeld, ) . consider, for example, two butterfly species occurring in the same area that use similar habitat types, e.g., open grass areas and ecotones, and thus require the same habitat conditions. the two species may either use both habitat types equally (generalism) or segregate the use of the two habitat types (specialisation). species may in fact overlap to different degrees the relative use of each type of habitat (julliard et al., ; poisot et al., ) , a pattern that may occur either because of an evolutionary trade-off between the ability of species to exploit a range of resources and their capability of using each one (futuyma & moreno, ) , or because of factors such as inter-specific competition (schroder & rosenzweig, ) . it is thus important to analyse the two concepts (habitat requirements and habitat specialisation) simultaneously in a given species assemblage. here, we analysed the relationship between habitat requirements and habitat specialisation in an assemblage of butterflies in a riverine natural park in northern italy. the aim was to test whether butterfly species with similar habitat requirements overlap their habitat use or, alternatively, they segregate their habitat use despite having similar habitat requirements. to do this, we first defined the requirements, i.e., which habitat types positively or negatively correlate with species abundances, and then used a network-based approach (bl€ uthgen et al., ) to analyse the degree of habitat use overlap within groups of species with similar habitat requirements. the study was conducted in the ticino regional park (lombardy, northern italy, approximately from ° ″ n to ° ″n and from ° ″e to ° ″e, with an elevation of - m a.s.l.) in summer - . the study area is characterised by a continental climate with cold winters and hot summers and covers about hectares. the park represents one of the most important green hot spots of the po river plain, which is otherwise very anthropised. within the park, four sites were chosen for the sampling programme. the two southern sites (coded as and ) ( fig. ) were generally characterised by a thick riparian oak forest with some agricultural areas (poplar plantations, transects and ). site in particular showed a high degree of habitat diversity, including a flooded forest with alnus glutinosa, salix alba and populus alba. the two northern sites (coded as and ) ( fig. ) include areas that have previously been intensively used as military areas, and they can be considered partially anthropised areas. although some natural parts still persist, the presence of alien and invasive plant species is relevant. these two sites were mainly characterised by open areas with shrubs (calluna vulgaris, crataegus monogyna, rubus caesius, cytisus scoparius) and by a low density of trees of exotic species, such as robinia pseudoacacia, ailanthus altissima and prunus serotina. sampling was carried out from the end of june to the first days of september for weeks/year. sites and were sampled in , while sites and were sampled in . surveys were conducted between : and : h. every site contained three m line transects (n = plots), in which we followed established guidelines for butterfly monitoring to minimise observer bias (pollard & yates, ) . observations were carried out during systematic walking surveys ( min per plot visitstandardised search time) only in favourable temperature conditions (temperature > °c) and in calm to light winds. every transect walk was repeated twice a week, for a total of transects and walked kilometres. in contrast to previous research on butterflies, this study was based on a precise georeference of every sampled individual leading to a database of butterfly positions characterised by a large sample (n = ) and a good level of accuracy (breed & severns, ) . all the points were recorded by a gps (garmin csx) whose standard error is considered to be between and m in good weather conditions. we considered the average gps error to be two to seven times lower than the minimum detectable linear dimension of the available land cover, which is more accurate (minimum cartographic unit: . ha, scale : ) than land cover data used in other studies of butterfly species distributions (minimum cartographic unit: . ha, scale : , guti errez et al., ) . all the recorded points were imported and overlaid under a gis environment (esri arc view . a) with the database of land cover classes (hereafter: habitat classes) describing the whole lombardy region, thereby including the plots (dusaf, destinazione uso dei suoli agricoli e forestali, ersaf ). in the sampled areas, eight habitats were recognised together with a further seven ecotones between these habitats for a total of habitat classes (see table ). the detail level is equal to an information scale of : . for each land use feature with a polygonal configuration and a measurable surface, the minimum dimensional threshold of representability corresponds to . ha, and the smallest detectable linear dimension of the polygon is m (http://www.ersaf.lombardia.it/upload/ersaf/gestionedocumentale/web_libro_-suolo_en_ _ .pdf). all twelve m transects were first divided into sectors (n = in each transect), each characterised by a length of m and a width of m, in order to consider the influence of habitat classes contiguous to the point of the supposed selection operated by every individual (fig. ) . a double gis spatial join application associated the field-sampled points with the sectors previously associated with habitat classes (fig. ) . when more than one habitat class fell into the same sector, we considered it as a separate habitat class composed and identified by the two source-habitat classes. we then counted the abundance of sampled individuals of each species in each sector and clustered the sectors according to the habitat class they showed, thereby calculating abundances for each habitat type. data were prepared for analysis with the r package 'vegan' (version . - , the r development core team ) to associate the different habitat preferences with each species and assess habitat requirements. the matrix had species as rows and line transects as columns. we calculated morisita dissimilarity indices among species using the 'vegdist' function. morisita is an index of patchiness, and it is used to calculate overlap among samples (morisita, ) . the dissimilarity matrix obtained was clustered specifying the 'complete linkage' method in the 'hclust' function in the 'vegan' package. we applied the 'cascadekm' function to the standardised square root indices and applied the calinski-harabasz criterion (milligan & cooper, ) to select the best partition inside the dendrogram obtained by the cluster, thereby dividing the whole species group into subgroups with a similar use of habitat types. in order to understand whether environmental characteristics influenced the abundance of individuals, we applied a generalised linear mixed effects model (glmm) for count data (atkins et al., ) for each of the previously selected groups. we ran zero-inflated glmms due to the huge amount of zeros in our data set. glmms were run by applying the ad model builder procedure using the 'glmmadmb' package in r (skaug et al., ) , specifying the poisson distribution inside the model (bolker et al., ) . before starting the analysis, we applied a conservative method to calculate significance for each factor included in the model based on the evaluation of the standard deviation calculated for each estimate multiplied by . consequently, estimates were significant only if the interval enclosed by two times their standard deviations did not cross . according to breslow ( ) , generalised linear mixed models may yield biased estimates of the variance component when the mean number of counts per treatment combination is less than five, so we removed from the analysis all the species whose number of counted individuals was below this value. land cover class classification and the year of the sample were inserted in the model as fixed effects and the plot as a random effect. even though network analysis has been previously applied to feeding interactions (bosch et al., ; poulin, ; junker et al., ; polidori et al., ) , it is also adequate for species-habitat webs within given areas when habitats are well characterised and thus represent discrete nodes of the webs. ecologists continuously produce detailed classifications of habitat types based on sophisticated analyses of their components (e.g. soil, vegetation structure, climate), so that these classifications can be used as a basis for species-habitat webs. network indices based on information theory (shannon entropy) were used to characterise our species-habitat webs in each area ( and ) and site (two in and two in ) (dormann et al., ) . the nodes of each network are composed of butterfly species versus their habitat types, and link weights are the number of individuals collected in each habitat type. because we were interested in the degree of habitat segregation between species, we considered the degree of complementarity (or exclusiveness) of interactions, defined as the standardised index h (bl€ uthgen et al., ) . this index characterises the specialisation and habitat partitioning (niche overlap) in the butterfly assemblage. we first calculated the two-dimensional shannon index as: where i is a butterfly species, i is the total number of individuals collected, j is one of j total habitat types, a ij represents the number of records of the habitat type j frequented by the butterfly species i and m is the total number of habitat records associated with all butterfly species in a network. we then standardised h against its minimum (h min ) and maximum (h max ) possible value for the given habitat type frequencies (i.e. marginal totals in the i j matrix fixed), as follows: consequently, h ranges from for the most generalised to for the most specialised case. each value of h was tested against a null model of random associations (h ran ). a number of random permutations of the matrix were performed using patefield's r c randomisation algorithm (bl€ uthgen et al., ) for which probability is derived (fonseca & ganade, ; manly, ) . as a species-level measure of partner diversity, we used the kullback-leibler distance (bl€ uthgen et al., ) , which not only considers the diversity of partners but also their respective availability, thereby comparing the distribution of the interactions with each butterfly species (p j) to the overall habitat type availability (qj); the index is defined as: and then normalised as: the mean values of d i between groups of species were compared using anova. linear relationships between indices and other parameters were tested with spearman's rank correlation test. overall, species of butterflies were collected. the calinski-harabasz partition applied to the dissimilarity matrix ( fig. s ) suggested five subgroups of species based on their habitat requirements (table , fig. ). following the glmm results (fig. ) , in the first group, we found a positive influence on the abundance of open areas ( , ) and a negative influence of natural dense deciduous forests ( ) and poplar plantations ( ) on the abundance. ecotones between open and forest areas were also positively associated with abundance ( _ , _ ). similarly, in the second group, we found a positive influence on the abundance of open heath land ( ) and a negative influence on the abundance of poplar plantations and dense deciduous forests ( ). many types of ecotones ( _ , _ , _ ) and riparian vegetation ( ) were also positively associated with the abundances of these species. negative relationships between the abundance and open heath land ( ) and its ecotone and natural dense deciduous forests ( _ ) were found in the third group, while the abundances of the species belonging to the fourth group were negatively associated with open anthropised heath areas ( ) and positively associated with the ecotone between open green areas and sparse deciduous forests ( _ ). in the fifth group, positive influences on the abundance of sparse deciduous forests ( ), open green areas ( ) and poplar plantations ( ) were found. in addition, species of this group were positively associated with some ecotones, particularly those including riparian vegetation ( _ , _ ) . the year of sampling also affected the abundance of the species of groups , and (fig. ) . overall, the relationships between species and habitats were not random in the studied butterfly assemblage (h = . , p < . ). among species, habitat specialisation (d i ) varied from . (denoting a generalist species) to . (denoting a highly specialised species) with an average of . ae . , showing important variability. the mean d i calculated for each of the five groups identified by the calinski criterion differed marginally (f = . , df = , p = . ), and was highest in group (species strongly positively associated with open habitats) and more similar among the other four groups (fig. ) . the weakness of this difference was clearly due to the large variance within groups, i.e., species with similar habitat requirements were not all equally specialised (table , fig. ). in particular, within all groups but one (group ), it seemed that one or two species were much more specialised than the others. in group , this subdivision was more difficult but the difference between the highest d i (species and ) and the lowest d i (species and ) was still large. the within-group variability in d i , when expressed with its coefficient of variation (cv), increased with the number of species included in the group (spearman's correlation test, q = . , n = , p = . ). we showed for the first time that high precision of handheld gps georeferenced data can give detailed insights into how butterflies occupy possible suitable habitats based on their ecological needs, as previously shown for other animal groups (lindeque & lindeque, ; kissling et al., ; breed & severns, ) . we found a coherent picture and a general rule by which butterfly species seemed to occupy suitable habitats based on their species-specific ecological needs. in particular, species grouping seemed to be based on a continuum index of lightness, ranging from group generally being influenced by open areas to group being strongly related to closed-canopy forests. if we look at the estimates associated with forest habitats and open habitats in our model ( fig. ) , we can see that they changed sign while proceeding from group to group . in particular, forests are negatively related to the abundance of the sampled individuals in the first group, but they increase their positive influence while moving to group ; the opposite occurs for the above-mentioned general open dry areas. furthermore, all the species, regardless of the group identity, seemed to avoid natural dense deciduous forests and associated ecotones. these are closed and shaded habitats, and luminosity was shown to be very important for butterflies, determining minimum and maximum flight conditions (douwes, ) as well as flight duration (shreeve, ) . for example, in a restoration experiment of ponderosa pine forests in north america, butterfly species richness and abundance were up to three times greater in restoration treatment units where host plant and nectar plant species richness were similar to control units, but insolation (light intensity) was significantly greater (waltz & covington, ) . this differential importance of habitat light intensity in structuring our studied butterfly assemblage is confirmed by a comparison of our results with previous information on the habitat characteristics for the studied species, which revealed a general congruence. all the three species of the first group are reported to inhabit bushy places, and dry and hot areas, and are usually associated with forest ecotones and clearing margins (table s ). species of the second group showed a negative relationship with the presence of forest areas, and a positive association with open natural heath areas. furthermore, species of group seemed to be more influenced by ecotones than species of group , with grassland representing the key habitat type. we also found a positive influence of riparian vegetation on individual abundance, probably because most of the individuals belonging to group were sampled in a site characterised by a single line of riparian vegetation and large open areas, thereby representing once again a sort of structural ecotone inside an open area. according to literature, many species of group are defined as ecotone species (table s ) . groups and , on the other hand, which were negatively associated with open heath land and its ecotones, confirmed the decreasing importance of open areas while moving from group to group . indeed, species of group were clearly associated with poplar plantations and sparse deciduous forests. many species in group were previously reported as being somehow linked to a forest environment (table s ) , but some species are reported to be associated with very different habitats (from open grassland to forests) (table s ). additionally, the significant effect of the year in some groups showed a temporal shift in species abundance, suggesting population fluctuations depending on change in abiotic or biotic conditions. the degree of specialisation was not similar among species within groups in our studied assemblage. for example, hipparchia statilinus was much more specialised than the other species of group , while minois dryas frequented many more coppice deciduous forests and their ecotones than the other species of group . two species of group (everes argiades and aglais io), which in literature are reported as generalist species in many habitats including flowery bushy places, grassy banks, forest clearings, flowering meadows, forest edges and sheltered rocky gullies (table s ), were very specialised in our study. even the two closely related species (both from the genus pieris) comprising group differed greatly in specialisation. because the index of specialisation d i increases with the level of specialisation, the positive correlation between the variance of d and the number of species across groups means that an increased number of species are more likely to promote the co-occurrence of generalist and specialised species (nested patterns) rather than an increased niche segregation among species (a clustered pattern). thus, possible increased competition (i.e. a higher number of species sharing the same habitat requirements) would not increase habitat segregation, while a few highly specialised species would use a subset of habitats exploited by more generalist species. this consideration is consistent with previous network-based studies, which showed nestedness common in both mutualistic (e.g. plant-pollinator) networks (bascompte et al., ) and agonistic (e.g. hostparasite) networks (joppa et al., ) (although compartmentalisation is also observed in agonistic webs, e.g. prado & lewinsohn, ) . bastolla et al. ( ) showed that the process by which a new species enters a community will likely lead to a nested pattern of interactions, because the new species tries to minimise its competitive table for species names). note that for a few species (e.g. eight), the sample size was too small to perform the network analysis (see text for details). load, an action that forces it to interact with the most generalist species. this could also be applied to habitats: when a new species arrives in a given area, it will first select sites with certain characteristics (requirements) and then reduce the overlap with competing species, largely using only a subset of the habitats used by generalist species. our results stress the importance of considering both the habitat requirements of species and the specialisation in habitat use by each species when analysing butterfly assemblages. indeed, many species studied here, previously known as generalists, were found to be locally specialised in our analysis. this is particularly important in the light of butterfly conservation, because the more specialised species may also be those with higher susceptibility to network distortions and thus with higher fragility to co-extinction (bl€ uthgen, ) . this would provide elements for deciding protection priorities for the species at local scales. fig. . estimated parameters derived from glmm. significant influences of the parameters on the species abundance are shown by grey highlighting. the estimates (round black points) and their standard deviations multiplied by (bold black lines enclosed by vertical segments) are provided. standard deviation segments were cut when their values crossed À or + in each graph. continuous bold lines without vertical segments are used when the model was not able to estimate reliable parameters. significant influences of the parameters on the species abundance are shown by grey highlighting. digits refer to habitat and ecotone codes as in table . although none of the sampled species are listed as threatened in the european red list of lepidoptera (with the exception of hipparchia statilinus, which is listed as near threatened, van swaay et al., ) , five species (coenonympha pamphilus, lycaena phlaeas, maniola jurtina, ochlodes sylvanus and polyommatus icarus) are reported as being declined in europe since (van swaay et al., ) . these species may become locally vulnerable through persistent increased habitat specialisation associated with habitat disturbance; therefore, their ecology should be finely monitored in order to adopt adequate conservation plans. this work could not have been carried out without the help of stefano basso and ester manzini, students who helped greatly with the fieldwork. thanks are also due to marina trentin for the support she provided during and after the fieldwork. this work was made possible by funding provided by ticino regional park. cp was funded by a postdoctoral contract funded by the universidad de castilla-la mancha and the european social fund (esf). kelsey horvath kindly revised the english editing. additional supporting information may be found in the online version of this article under the doi reference: doi: . / icad. : fig. s . calinski criterion applied to the dissimilarity matrix. the number of possible group partitions is shown on the y-axis and the values obtained by the calinski-harabasz algorithm are shown on the x-axis. table s . previous published information on the habitat requirements of the studied butterfly species. tutorial on count regression and zero-altered count models for longitudinal substance use data searching for a model for use in vegetation analysis. vegetation continuum concept, ordination methods and niche theory the nested assembly of plant-animal mutualistic networks the seasonality of arboreal arthropods foraging within an australian rainforest tree leaf production of an over storey rainforest tree and its effects on the temporal distribution of 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publications office of the european union threats posed to rare or endangered insects by invasion of nonnative species ecological restoration treatments increase butterfly richness and abundance: mechanisms of response key: cord- -t qxscbp authors: losvik, mary h. title: plant species diversity in an old, traditionally managed hay meadow compared to abandoned hay meadows in southwest norway date: - - journal: nord doi: . /j. - . .tb .x sha: doc_id: cord_uid: t qxscbp a chronosequence, representing a successional series, was used for the comparison of a hay meadow site managed in an old traditional way for at least a hundred years, and hay meadow sites abandoned for about , and years, respectively. old traditional management included grazing early and late in the growing season, mowing in august and light or no fertilizing. the tree cover was the most important factor deciding the composition of vegetation. time since abandonment was not completely correlated to tree cover, as some plots had a dense canopy and others were situated in the open. the total species number decreased with number of years since abandonment in plots > . m( )and < m( ). the highest species number in m( ) plots was recorded in the managed site, with species of phanerogams. fourtyeight % of the indicators of traditional management present in the managed site was recorded in the site which had been abandoned for years. frequency — log area curves made it possible to group species according to persistence in the sward. as a result, a group of functional indicators of rare hay meadows in the region was distinguished. at small scales, temperate grasslands are the most species-rich plant communities in the world shmida & ellner ) . grasslands were once widespread in western europe, but are now greatly reduced in extent as a result from changes in agricultural land-use practices (keymer & leach ; willems ) . the grasslands may have high species numbers, up to well above per m (willems (willems , kull & zobel ) . old, traditionally managed hay meadows are rare in western norway at present ( vstedal ; lundekvam & gauslaa ; losvik a losvik , b losvik , a rosef ) . earlier they were common elements of the agricultural landscape, but most of them are now either cultivated in a modern way or abandoned (losvik a ). the few old, traditionally managed hay meadows which are left, are mostly situated on phyllittic soils and have high species diversity. no investigation in the region has so far studied what happen to the species diversity and species composition when such meadows are abandoned. in this study the species have been grouped into indicators of traditional management, common (hay meadow) species and additional species, the latter includes forest species. the species group indicators of traditional management was preliminary defined in losvik ( b) , and later (losvik b ) was shown to comprise species occurring in less than yo of plots ( - m ) of hay meadow vegetation analysed in western norway by different authors - . these species are often low-statured, creeping, slender andor with rosettes, and mostly stresstolerators (grime (grime , ) and hemicryptophytes, annuals or biennials (raunkiaer ) . the number of the indicators is usually low in abandoned sites (see data in losvik ; persson ; austad & skogen ; borgegaard & persson ) . modern management, including medium or heavy fertilizing, also reduces species number in general (see e. g. during & willems ; willems et al. ) , and especially the indicators of traditional management (losvik b (losvik , a (losvik , b . as a consequence, indicators of traditional management are becoming increasingly more rare in the region. common species are defined as being more frequent than indicators of traditional management in the agricultural landscape, tolerating medium quantities of fertilizer. these groups of species is considered to be more useful in choosing areas for hay meadow conservation, than the plain use of total number of species, as it reveals direct information on the number of target species, namely the indicators of traditional management in the sites. the changes in plant species richness during succession have been studied e. g. by nicholson & monk ( ) , bazzaz ( ) , prach ( ) and symonides ( ) . both increase and decrease in total plant species diversity have been found in these studies, and the situation may be further complicated by environmental gradients (peet ; prach ) . as the species diversity depends on the spatial scale considered (e. g. kwiatkowska & symonides ; van der maarel a) it may be better described by the species-area relationship than by single species numbers. the aim of the study was to compare the vegetation and ecology of sites which formed a chronosequence, representing a successional series from a species-rich, old, traditionally managed hay-meadow to sites which had been abandoned for about , and years, respectively. themes of special interest were: how important are the ecological factors years since abandonment and cover of tree canopy in deciding the composition of the vegetation, what differences in species diversity, as measured by species-area curves, are there between the sites, for how long are species which are characteristic for the agricultural landscape able to persist during overgrowing in such sites, and finally, what forest species and additional species are most important in the sites of this series of abandoned haymeadows. the study sites are located in a hill-side at gjuvsland, varaldsey in the hardanger fjord, western norway, generally sloping " towards south-east, from about m.a.s. . down to the shore. the hill-side was formerly common land, used as out-lying hay meadows. they were mown in august and grazed in spring and in autumn by sheep and cattle from farms at gjuvsland. in the hill-side was divided between the farms into parts which each was fenced. four of these parts constituted the study sites. the bedrock is volcanic supercrustal rocks covered by scree material containing phyllite (foslie ; see also kolderup ; askvik ). the soil is probably enriched with ca through seepage from limestone bedrock occurring above the hay meadows. the mean precipitation at the nearest meteorological station rosendal is mm year' (ferland ) . the mean temperature in january is . " c, in july . " c, and mean yearly temperature is . " c (at the nearest meteorological station measuring temperature, omastrand, aune ) . traditional management includes grazing by sheep for about days in mayijune and in september/october each year. until about years ago, mixed grazing by cattle and sheep was common, but later sheep alone were used. stocking rates are about sheepgrazingdaysha and year (including lambs), less in dry years. after the spring grazing period, dried leftovers of manure and twigs of trees are removed by raking. the grass is mown in august, using a small reaper and different forms of scythes. august may be rainy (semme ) , but the farmers usually awaits a period of dry weather to allow drying of the grass on the ground. the grass is turned on the spot twice a day till it is dry, allowing seeds to be spread evenly thoughout the grassland. usually no commercial fertilizers or wintermanure are applied, but some nutrients are recycled through urine and faeces during grazing, particularly when mixed grazing, involving higher grazing intensity than at present, was practised (brelin ) . the main features of these management practices were common in western norwegian out-lying hay meadows till about (byrkjeland ) . as long as the whole hillside was managed, there was a scattered treelayer of pollards (mostly fraxinus excelsior) or coppiced trees (corylus avellana and alnus incana). these trees and characteristic forest herbs and grasses often grew near outcrops or heaps of rocks, cleared away from the meadows, and functioned as centres of natural afforestation as management ceased. at the study site this has resulted in a mosaic-like pattern of patches with ( ) a high, dense sward of grasses and herbs, lacking a tree layer, ( ) a scattered field layer and a more or less closed canopy of trees, and ( ) a low dense field layer in areas in the open which are situated close to small outcrops, narrow paths, ant-heaps and springs. site a is managed in the old, traditional way (a. a. gjuvsland, pers. comm.) . but since about the number of sheep has been reduced to about on third. this reduction has, at least partly, been compensated for by longer grazing periods. the low-lying part (about %) of site a was lightly fertilized (about kg fertilized ha.year, type varied) in june from about till (in the ties at most kg fertilizer/ha.year). the soil is rich in minerals (base saturation: - %), and poor in organic matter (c: . - . %), nitrogen ( . - . %) and phosporous ( . - . mg/ g dry matter) with ph . - . (losvik a) . the site has a rather high species diversity (losvik , - species of phanerogams/l m ), compared to other grasslands in south-west norway ( vstedal ; lundekvam & gauslaa ; losvik a losvik , b losvik , losvik , a hundt & vevle ). species such as cynocurus cristatus, briza media, trifolium dubium and linum catharticum, which are rare in western norwegian grasslands at present (losvik b) , are abundant in the site. an assembly of uncommon grassland species of which usually only a few are recorded from each hay meadow site in western norway, such as leucanthemum vulgare. euphrasia stricta. rhinanthus minor, polygala vulgaris, centaurea jacea, pimpinella saxifraga, carum carvi, danthonia decumbens, lotus corniculatus and platanthera chlorantha, are here found together in an area of about ha. the traditional management has lowered the content of n and p in the soil and resulted in high species diversity, as have also been recorded in other studies (e. g. kowalsky ; van der maarel ; silvertown ; zoller & bischof ; collins & barber ; de leeuw & bakker ). at the time of the investigation, the managed site had a scattered tree layer of fraxinus excelsior, alnus incana, taxus baccata and old individuals of planted prunus domesticus. sites b, c and d were abandoned , and . site c was grazed by sheep the whole summer for some years after the mowing ceased. in site d light grazing might have occurred for some years after abandonment, as sheep from neighbouring parts went trough broken fences. site b was lightly fertilized in the ties. it is assumed that the vegetation was much the same over the whole hillside at the time when the management was the same, especially as the management probably had a tradition of a hundred years at least (a. a. gjuvsland, pers. comm.) . ecological factors such as as-pect and bedrock were mainly equal over the whole hillside. site c is generally steeper than the other sites and slopes about ". according to intervjues the tree structure of the sites seem to have been quite similar, with less than % tree cover of scattered trees. the nomenclature follows lid . the size of sites a-d was , . , . and . ha respectively. the whole area of each site was divided into equally-sized subsites, in order to assure that the data set should represent well the vegetation of the site. steep, stony or otherwise inaccessible areas of the sites were excluded. within each subsite the southwestern comer of one square plot of cmz was chosen at random. four subplots of x cm, placed in the comers of this plot were analysed in addition to a x cm plot, a x m plot, a x m plot and a x m plot, each made by extending the former plot towards south-east and north-east. thus the plots within each subsite were nested, while all the ten m plots were distinctly separated, each in its subsite. presence of all rooted phanerogamic species of the field layer were recorded in or . shade was estimated as the cover of the tree layer in each m plot. the number of years since abandonment, in addition to records of former management, was obtained by interviewing each farmer. main ecological gradients in the data set and length of the gradients were assessed by detrended correspondence analysis, dca (hill ; hill & gauch ) applied to presence-absence data for species (species occurring in < plots were omitted) in quadrates of m each, using canoco version . (ter braak a (ter braak , . canonical correspondence analysis, cca (ter braak (ter braak , b ) was used to evaluate the importance of the environmental factors: years since abandonment and cover of trees. standard options were used. monte carlo test (ter braak ) with permutations was used to assess the significance of first canonical axis and overall significance. specieslog area curves, frequencieslog area curves, and the significance of differences in species richness (t-test) were constructed or calculated using excel sistence in the sites and to frequency in the different plot sizes of the frequencylog area curves. dca indicated a gradient of shade along the first axis (eigenvalue . , . sd) and of nutrients along the second axis (eigenvalue . , . sd). the indicators of traditional management and the common species as expected were situated in the light part of the first axis gradient, while the additional species, comprising forest species and species common along edges and roadsides, were mainly in the opposite direction (fig. a) . when the plots were classified according to tree cover, the gradient of shade along the first axis was clearly demonstrated (fig. lb) . grouping of the plots according to sites showed small variations along the gradients in sites a, b and c compared to site d (fig. lc) . the plots within each site were more similar to each other than to any plot outside the site along axis and . cca demonstrated that tree cover (cover) as expected was positively correlated with the first axis (fig. , eigenvalue . ). years since abandonment (abandon) was positively correlated to both the first and the second axis. axis accounted for . % of the species data, axis accounted for only %. significance (p) of the first axis was . , and an overall test also gave significant results. of the species recorded in sites a-d, % were absent in the plots of site d, % were absent in site a (appendix , ) . seven % of the species was only recorded in a, % only in d. the speciesarea curves of site a for the total number of species, indicators of traditional management and common species were nearly linear, but the b-d curves increased exponentially with log area. construction of log species log area curves made the total species number curve of site d close to linear, but the other curves then turned into power function curves. none of the curves showed any tendency to flatten out at the investigated size ranges. the mean total species number for plot sizes > . m and < m was higher in the traditionally managed site a than in the abandoned sites (fig. a , p < . ). sites b and c had intermediate mean species numbers, while site d had a lower number of species than sites a-c in these plot sizes (p < . ). the highest species number in m plots was recorded in site a with species (mean , sd ) and in the m plots in site c with species (mean , sd ), followed by site a with species (mean , sd ). when all species in the plots of each site were added ('plot' size . ha) however, site b had the highest species number (appendix , ). the number of indicators of traditional . site a had the highest and site d the lowest mean number of common species in plot sizes from . m to m (fig. c) . the differences in mean number of additional species between sites were negligible for plot sizes up to m (fig. d) , but for larger plot sizes the mean number was lower in site a than in sites c-d (p < . ) and for the largest plots it was also lower in site a than in site b (p < . ). the species were grouped according to presenceifrequency of each species in all plot sizes in sites a-d (appendix , ). group mostly comprised indicators of traditional management and common species, species which are more or less dependent on high light availability. the frequency of species of group la tended to be much lower in sites b-d for all plot sizes than in site a, if they were present there at all (fig. a) . species of group lb (fig. b) persisted rather well as long as there were openings in the tree canopy. other agricultural landscape species were rather shade tolerant (group lc, fig. c) , and some species even increased in frequency after abandonment (group id, fig. d ). group a comprised border species which benefit from the lack of management in addition to the high light availability in the early successional phases (fig. a) , and group b comprised forest species (fig. sb) . the rest of the species occurred too scattered in the sites to be grouped. the ordination results confirmed that low tree cover was important for the indicators of traditional management and the common species, while the additional species may tolerate well both abandonment and a high tree cover, even if the species may be present also in the traditionally managed hay meadows. with the assumption that the vegetation in sites b-d was about the same as in site a when they were managed, dca indicated that open abandoned areas gradually became poorer in nutrients with time while areas with a closed tree canopy became richer (see figs l b and lc, plots b , d , d - ). austad & skogen ( ) recorded both rather poor betula pendula forest and rich forest types with e. g. ulmus glabra in abandoned meadows in a hillside in sogn. in losvik ( ) it is concluded that successional trends in abandoned hay meadows take different courses according to nutrient content in the soil. mown, soil analysis will be necessary to prove that the differences in vegetation resulted from differences in soil nutrients. a theory would be that litter of trees, here mainly ash (fraxinus excelsior), decomposes more rapidly than the grass sward of the open areas, releasing more nutrients to the soil in the studied time span of abandonment. bearing these statements in mind, an interesting interpretation of the cca result may be that tree cover, according to the distribution of species in the diagram, seemed to be positively correlated to nutrients, while abandonment in general seemed to be negatively correlated to nutrients. in an abandoned area, tree cover, and with it the area which is in shade will increase with time. but the increase is not evenly distributed in space, as some areas are open for a considerable time span, and as such may be poorer in nutrients than areas with a tree canopy. this implies that the composition of the vegetation in an abandoned area is dependent not only on time since abandonment, but also on the local extent of the tree cover. the occurrence of seedlings of ash (fraxinus excelsior) in at least out of plots of . m in sites b -d (fig. b) indicate that the open areas of these sites is really in a transitional phase, during which the content of nutrients in the soil may be lower than in areas with a closed canopy. the rate of increase in species number with increase in plot size is an appropriate measure of richness, instead of using number of species in one plot size (kilburn ; van der maarel a; leps & stursa ) . but this rate ought to be constant along the curve, and as the species-area curves differed too much in shape in this study, only differences in species numbers between sites at the different plot sizes were compared. singh et al. ( ) curves were close to linear, while in sites b-d, with a large turnover of species, the curves were exponential. the form of the curves is dependent on density of individuals and plant unit areas. in the traditionally managed hay meadow the species were mostly small with individuals of the species quite evenly distributed. the much smaller variance in plots of site a compared to site d (fig. lc) demonstrated that in site a the vegetation was more homogenous (van der maarel & sykes ) . wherever the analysis would have started, comparatively many species would have been added by increasing the area (fig. a) . in the abandoned meadows, the plant unit area was probably larger and there was a tendency for a few species to dominate in the plots. both invaders and persistent agricultural landscape species had a contemporary scattered presence in the plots. thus the linear log area curves may indicate homogenous stands, while exponential specieslog area curves may indicate succession or disturbance. continuos increase of the curve at and even above the investigated plot sizes is recorded e. g. by hopkins ( ) and barkman ( ) . both tree cover and management was important in deciding the species richness in the investigated chronosequence. in a wooded meadow in estonia kull & zobel ( ) also found the highest species richness where tree canopy cover was lowest (see also pausas ) , and highest species richness in sites with the most regular long-term mowing as compared to cases of cessation of mowing. for semi-natural grasslands van der maarel ( a) argued that grazing animals and mowing imply disturbance and to some extent stress, which enable more species to coexist than on a similar area without grazing or mowing. the reasons for the high mean species richness in site a compared to the other sites may be complex (see e. g. giller ), but the management itself undoubtedly plays a major role in providing light and gaps for all the species at the right time and place throughout the growing season. in the abandoned sites less light penetrated into the lowest part of the field layer and this resulted in exclusion of low-growing species (kull & zobel ) . the species may either disappear after a gradual reduction in population size, or there may be a collapse in the occurrence of the species. some of the species in this study (appendix , group la) may have experienced such rapid (in less than years) disappearance. the species diversity decline in sites b-d in - years was % - % in plots of . m - m (table ) . however, when the whole investigated area in each site was considered, the total number of species increased in all the abandoned sites (appendix , ) . this clearly demonstrated that use of total species number is very dependent on plot size and that it may be rather useless in choosing hay meadows for conservation. high species turn-over rates during the first - years of a sere was reported e. g. by houssard et al. ( ) . much more rapid decline in species diversity, % in - years, was reported from chalk grassland by willems ( ) . in time sites b-d will turn into deciduous woodland with a closed canopy and the indicators of traditional management and common grassland species will even- . ) ( . ) o( . ) ( . ) tually be lost. the fact that the mean total number of species in plots of site a was higher for areas > . m and < m than in the other sites, and at the same time total species number of the whole investigated area in site a was smaller than in the other sites, showed that in site a most species were represented by a large number of individuals which were quite evenly distributed over the whole area of the site. this can easily be seen from appendix , as it appears that in site a species are present in all plots of m each, while in sites b-d the numbers are , and , respectively. on the other hand, the number of indicators of traditional management was highest in site a, both when total number in investigated plots and when mean number at all investigated plot sizes were considered (appendix and , fig. b ). the usefulness of the group indicators of traditional management as functional indicators in choosing hay meadows in the region for conservation is thus demonstrated (see also mcintyre & lavorel ). common grassland species are quite frequent in the studied abandoned sites. they thrive when biomass is no longer removed, resulting in increased nitrogen levels in the soil. the additional species occur very scattered in the whole chronosequence, and are thus generally infrequent in small plots. in larger plots the difference, mainly between site a and site d become visible. in site d more forest species have established than in the other sites. as site c had been abandoned for a longer time than site b, more forest species had established there, and at the same time more light demanding species persisted (appendix , ) probably a result from the period with grazing after cessation of mowing, and thus species richness was higher there than in site b. similar species overlap, occurring when competitive exclusion has not yet had time to drive subordinate species to extinction is advocated also by palmer ( ) . higher species richness in grazed areas as compared to abandoned grassland was reported by regncll ( ) . the groups of species used in the present study differs from guilds sensu pianka ( ) in that no species can be a member of different groups, moreover it is no rea-son to believe that species within one group interact more intensively, mainly by competition, than species from different groups (van der maarel b). the species of group differed in their ability to persist in the abandoned hay meadows. the groups la and lb comprised some of the most rare hay meadow species in western norway at present. in abandoned hay meadows they are often recorded close to or at refuges like outcrops or heaps of stones on dry or shallow ground in the open, where the field layer is low and scattered. some of them are annuals which are dependent on gaps in the sward for germination of their seeds. for example pimpinella saxifraga is known to be very sensitive to increased density of the sward (grubb ) . these species will presumably not be able to survive the closing up of the canopy where they grow at present, and thus they face extinction at their sites in the near future. at sites in western norway where these species have been recorded during the last years, they probably will experience a collapse extinction as indicated by the differences in the frequencyarea curves. hay meadows which have been abandoned less than years ago are getting increasingly rarer, and so are the rare species within these groups. other species in these groups are as vulnerable to abandonment as the rare species, but are generally more common because they tolerate the low or medium quantities of fertilizers used in the westem norwegian small scale and part time farming system. thus a smaller group of species, tolerating neither fertilizing nor abandonment may be delimited and used as indicators in choosing hay meadows for conservation (appendix , : *). these species occur in less than % of analysed hay meadow plots in western norway published (losvik b , while indicators of traditional management occurred in less than % of the plots. several rare hay meadow species, not recorded in the plots of this study, such as botrychiurn lunaria, dianthus deltoides, galiurn verurn. gymnadenia conopsea and lychnis viscaria, will have to be added to complete this list of indicators of rare hay meadows. generally the species which gradually decreased in frequency with time since abandonment or increased in early successional stages (groups ic and id) were plants of some height and thus good light competitors in this situation. the groups comprised indicators of traditional management which are more common than species of groups la and lb, as a result from their tolerance of shade and/or light fertilization. unusual long stalks, bringing them up towards the top of the field layer were observed in individuals of potentilla erecta and lotus corniculatus in the studied abandoned sites. many border and forest species do in fact occur even in the traditionally managed hay meadow site. these species are usually growing close to stone heaps and below trees. they constitute a species pool and are able to expand as soon as the hay meadow is abandoned. the existence of a forest and border line species pool in managed hay meadows must be an old feature, as the whole of these landscapes were used very intensively under the old traditional management regimes, and thus the present forest species must have had refuges somewhere in the managed landscape. the investigation shows that in order to preserve the hay meadow diversity, the continuity in management is crucial. even with short periods of abandonment there is a risk of loosing species. the longer the 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the south of sweden evolutionary ecology succession of vegetation in abandoned fields in finland the life forms of plants and statistical plant geography a numerical study of successions in an abandoned, damp calcareous meadow in s sweden apen eng og beitemark i drift, voss kommune, hordaland -vegetasjon og skologi. -cand. scient. thesis coexistence of plant species with similar niches the dynamics of a grassland ecosystem: botanical equilibrium in the park grass experiment plant species richness and species-area relations in a shortgrass steppe in colorado floristic richness, diversity, dominance and species evenness in old-field successional ecosystems geography of norwegian agriculture. -publ. norw.univ. school of econ. & business adm. geographical series a canoco -a fortran program for canonical community ordination by (partial) (detrended) (canonical) correspondence analysis, principal components analysis and redundancy analysis (version . i). -agricultural mathematics group, wageningen. - b. the analysis of vegetation-environment relationships by canonical correspondence analysis. -vegetatio - . update notes: canoco version . . -agricultural mathematics group, wageningen. van der maarel, e. . plant species diversity in relation to management small-scale plant species turnover in a limestone grassland: the carousel model and some comments on the niche consept composition and species diversity of pine-wiregrass savannas of the green swamp, north carolina observations on north-west europaean limestone grassland communities. . phytosociological and ecological notes on chalk grasslands of southern england changes in chalk-grassland structure and species richness resulting from selective nutrient additions stufen der kulturintensitat und ihr einfluss auf artenzahl und artengegge der vegetation the vegetation of lindas and austrheim, western norway acknowledgements -the project was supported by . kishorekumar for the assistance with the input of data, and to three unknown referees for comments on the manuscript. key: cord- -qytl vqt authors: da silva, josivan soares; do nascimento, andré luiz borba; alves, rômulo romeu nóbrega; albuquerque, ulysses paulino title: use of game fauna by fulni-ô people in northeastern brazil: implications for conservation date: - - journal: j ethnobiol ethnomed doi: . /s - - - sha: doc_id: cord_uid: qytl vqt background: due to the influence of several factors on the hunting of game meat, we investigated how the seasonality of the environment, the abundance, and the biomass of wild animals, as well as the proximity to these resources, can affect the hunting. methods: the research was developed with the fulni-ô people in the municipality of Águas belas, agreste of pernambuco, northeast of brazil. in order to do this, we applied snowball sampling to select the participants. data from potentially useful game species were obtained from lists and semi-structured interviews to register their particular kind of uses, capture periods (daytime, night, or both), preferences, and perceived abundance. the hunters who allowed their game meat captured to be weighed and identified were followed for year. results: our records pointed to a vast repertoire of potentially hunting animals. however, we did not verify relationships between the abundance, seasonality, and biomass of the animals that were hunted by the fulni-ô. we observed a total of , (kg) of game meat hunted in the studied group, belonging to species, distributed in three taxonomic groups, the birds being the most representative group with % of total reported. conclusion: such consumption by the group is well below in terms of biomass when compared to other ethnic or local groups in other regions of brazil, or in caatinga areas, characterizing an activity much more of cultural character than subsistence. also, the use of game meat among the fulni-ô seems to be actively directed to the preferred species, suggesting that in the case of an urbanized indigenous community, where other sources of income are available, the demand for game meat is lower when compared to other ethnic groups. the intensive use of animals for the supply of nutritional demands by local and traditional communities worldwide has attracted the attention of several researchers due to their effects on faunal stocks caused by overexploitation [ ] . the consumption of game meat is considered the primary reason for game hunting, often occurring in developing countries with great wildlife richness, mainly in the african, asian, and south american continents [ ] . in brazil, despite its legal restriction, hunting persists in different socio-cultural contexts [ ] [ ] [ ] , being allowed in subsistence conditions [ ] . however, hunting of wild animals persists to a greater or lesser extent in all brazilian biomes, being strongly associated with cultural aspects and the nutritional needs of the human groups involved [ ] . concerning indigenous peoples, this relationship becomes even stronger, contributing to the subsistence of these peoples, as well as to the maintenance of their cultural identity [ ] [ ] [ ] . it is secured to them by the brazilian constitution their rights to maintain their lands, way of life, and traditions [ ] . for these peoples, scientific research in the amazon has pointed out a large number of species with hunting importance that varies with their size, taxonomic group, and utilitarian purposes [ , , ] . however, considering the semi-arid region, there is still little research on the dynamics of wildlife consumption by traditional communities in the northeast region of brazil [ , ] . the hunting of wild animals involves a series of factors that may influence the choice of individual species to the detriment of others [ , , , [ ] [ ] [ ] . the biological characteristics of species, such as abundance and biomass, for example, may direct the preference for specific prey among the hunters providing a higher post-activity yield [ ] . peres [ ] observed that, in several places in the amazon region, the rural population consumes between . and . , birds and mammals, representing total estimated biomass of . to . tons and a yield of . to . tons of wild meat appropriate for consumption. large animals tend to be more hunted and consequently more impacted than medium and small animals [ , ] . likewise, it is justified that taxa such as mammals and birds may be more in demand due to their biomass and abundance, respectively [ , ] . in addition to the factors cited, climatic seasonality can affect the frequency of visitation to hunting areas [ ] and the availability of these resources in the environment. these authors observed that the rainy season is considered the most favorable to hunt, due to the greater availability of food resources for the animals, easy identification of the traces left, and reduced risks of hunters being perceived. conversely, in environments with strong climatic variations, such as the caatinga, this activity may be more frequent in periods of seasonal drought, when the use of other resources becomes scarce [ ] . hunting in the caatinga plays a strong socioeconomic role. thus, the use of several vertebrate groups is an important source of protein for rural and urban communities [ ] . for indigenous people living in these regions, hunting sometimes becomes a more frequent activity in relation to other activities involving animals, such as fishing, due to the drought regime [ ] . in these areas, even cattle breeding and pasture areas, as well as plant crops, are strongly affected by droughts, which may make hunting a subsistence alternative [ , ] . another factor mentioned in the literature for the selection of game species is the proximity of human settlements to hunting areas. studies indicate that this variable influences not only the species choices but also the amount of biomass acquired [ , ] . santos et al. [ ] , evaluating the effect of migration and incorporation on the medicine repertoire of the truká indigenous people in the semi-arid region, that the proximity of other available resources, may favor the incorporation of new useful species into these communities. this distancing from the urban centers is related to a decrease in the availability of domesticated meat and may increase the demand for game meat. similarly, the authors argue that the proximity of useful resources decreases the costs of the search and investments of obtaining strategies [ ] . however, peres and nascimento [ ] point out that these meetings in the vicinity of the settlements may favor the capture of small animals that present a higher reproductive rate in relation to large animals. for the fulni-ô people, seasonality is an important factor that marks most of the activities, which are performed by the opportunities that change between the seasons [ ] . thus, this research focused on the hunting of game meat among the fulni-ô people living in the semi-arid region, being the first study carried out in an ethnic group strongly influenced by urbanization in the brazilian northeast. thus, our objective was to verify the possible influences of environmental variables and biological characteristics of the species (as perceived by the people) on hunting behavior. for this, we tested if species that are perceived as more abundant by these hunters are also more hunted. we expect that the amount of annual biomass caught (kg) of species considered more abundant will be significantly higher than that of species considered less abundant. also, we verify if the proximity to the faunal resource makes it possible to obtain it. therefore, we tested if the amount of biomass caught in the months of religious ceremony, in which the members of the group move to a settlement near the ouricuri forest, is significantly higher than in other months. we also tested whether seasonality influences the hunting of game meat in the region, and we expect that the amount of biomass (kg) is significantly higher in the dry season than in the rainy season. finally, we tested if there is a relation between the preference of game species by the hunters and the hunting of game meat. we expect that the most preferred game meat biomass will be higher than the least preferred ones. the study was carried out in the fulni-ô indigenous community, located in the municipality of Águas belas ( ° ′ ′′ s and ° ′ ′′ w) in the state of pernambuco. the fulni-ô territory occupies , ha of the municipality of Águas belas [ ] , presenting a caatinga biome, with a hot and humid semi-arid climate with an annual average temperature of . °c. the vertebrate fauna in the caatinga is composed of about reptiles and amphibians, birds, and mammals. the village presents tree divisions: the main village, the xixiaclá village, and the sacred village (ouricuriused in a specific period of the year). according to information obtained from the health clinic of the village, about indigenous people live in the main village, and about live in the xixiaxlá village located near the ouricuri forest. their dialect characterizes the members of the fulni-ô group, "yatê," belonging to the macro-jê language group, besides the portuguese language, and by their annual religious ceremony called "ouricuri." during the ceremony, the people migrate from september to december to a region distant from the main village, surrounded by caatinga vegetation, which forms one of the fewest native forest fragments in the region [ , , ] . this religious period is a time of ethnic seclusion in another village (ouricuri village) in which the fulni-ô suspend all external activities and dedicate themselves exclusively to religious demands. the main village is located a few meters from the town of Águas belas and also has urban buildings. among economic activities, campos [ ] emphasizes subsistence and commercial agriculture, handicrafts, rental of land, artistic presentations, and works in institutions in the municipality and other cities. also, in their rites or cultural presentations, they use body paintings extracted from local plants and, unlike other ethnic groups, they use hats made from the straw of the ouricuri palm (syagrus coronata (becc.) becc) instead of headdresses. regarding the faunistic use among them, campos [ ] pointed out that fishing in the year is the predominant activity performed in the village. however, given the climatic seasonality in the region, observations during the study period indicate that fishing activity is almost incipient, since areas that allow such activity are practically nonexistent, information that is supported locally. thus, with regard to fauna, hunting among the fulni-ô people is the most frequent activity today. it usually occurs in the vicinity of the village and involves only men [ ] . however, due to the proximity to the urban center of the municipality, hunting of wild animals can be replaced by the acquisition of non-wild animal protein acquired in conventional fairs and markets. the "warriors," as the hunters are called, see in this activity an important source of obtaining resources for food and religious purposes, in addition to their by-products being used in local medicine and often used for making handicrafts. the latter contains mainly byproducts of birds such as feathers that are used to make the headdresses, arrows, and decorative articles that often refer to the white man's vision of what is "indigenous" but which are not part of aldeia's traditional repertoire [ ] . also, other materials of animal origin, such as teeth, skins, and claws, have been observed in situ and are frequently used in the making of these craft artifacts. exchanges can also acquire them in cultural encounters with other ethnicities. the production of handicrafts is one of the essential activities of income generation for the fulni-ô, and many of the materials used are of plant origin, mainly the ouricuri palm (s. coronata) with which they make mats, baskets, hats, among others. the environmental conditions of the region favor the choice of areas for hunting and gathering of vegetal resources. nevertheless, many of these areas have suffered successive disturbances such as burning, deforestation, and inadequate use of the soil, which leads them to develop mechanisms that guarantee their physical and cultural existence [ ] . the fulni-ô hunting may occur in groups or individually, usually depending on the chosen taxon. we observed in situ that the foraging strategies among the fulni-ô people are facilitated by utilizing motorcycles or bicycles to the selected areas. collective hunts are usually planned hours or days earlier among the hunters involved. for animals like birds, collective hunting ends up being more frequent, usually occurring at night time with the aid of lanterns to immobilize the animals and firearms, or associating the use of the firearms and the assistance of dogs in daytime hunting as well. it is also common to use techniques like fall traps that are generally employed for medium and small mammals. the first contact of our research group occurred in the year for the recognition of the study area and the presentation of proposals. this contact and the selection of the study area occurred due to a relationship already established by members of our team who researched the village in previous years [ , , ] . this relationship facilitated the access and the establishment of a relationship of confidence to obtain the information from the hunters. subsequently, the project adjusted to the fulniô was presented to the leaders of the village, in the person of the shaman, representative of the cacique at the time, when the proposal and stages of the research were exposed. after the explanation of the objectives of the study, we were granted the prior consent term with the approval of the leadership and authorization of the local funai. the proposal was also approved by the human research ethics committee of the university of pernambuco (caae: . . . ), and the project is registered in the national system for genetic heritage management and associated traditional knowledge -sisgen (no. aa dc) in compliance with the provisions of law , of and its regulations. the group of the focus of the present research was people locally recognized as hunters with a minimum age of years. the identification of the participants was achieved through the application of the "snowball" technique (see description in [ ] ). data from potentially useful game species were obtained from free lists and semi-structured interviews [ ] , and their particular kind of uses, capture periods (daytime, night, or both), preferences, and perceived abundance were recorded. we recorded at the time of the interviews which of the animals cited by each hunter were preferred. the perceived abundance was recorded individually using the "punctuation exercise" technique [ ] , in which respondents attributed values between zero and ten, "zero" being equivalent to the disappearance of the species in the region and "ten" for the cases of very abundant animals. the monitoring of the hunting of game species was carried out monthly between june and july . for this purpose, five hunters were selected from the indication of a key informant, due to their experience or frequency in hunting activities, being trained to register the hunted animals in the absence of researchers. these informants resided in distinct areas of the village in order to facilitate the registration of hunting by as many hunters as possible. thus, the common name of the species, the number of animals, the period of the hunting, and their respective biomass (kg) were recorded. of the hunters approached in the first stage of the survey of useful species in the region, aged to years old, allowed the registration and measurement of their games during the period of the research. the captured animals were weighed using a portable scale (up to kg). to record information about local seasonality, we use the region's rainfall record as a proxy on a -year time scale. for this, we consulted the online database of the website of the pernambuco and water and climate agency (apac). the identification of the animals was performed with the assistance of a specialist from the federal university of paraíba through photographs taken by the hunters or the researchers at the time of the capture of the animals. additionally, for animals that were not possible to identify by photographic record, a checklist [ ] (see supplementary file ) containing images of the possible species cited by the interviewees was created. images of animals not occurring in the region were also added to avoid possible influences on hunters' indications. this technique is commonly used mainly in cases where there are difficulties in obtaining parts of the animals after their capture and use by the hunters. scientific records on the fauna occurring in the caatinga environments were used for the elaboration of this checklist [ , , , ] , which were later presented to the people to confirm their occurrence in the locality. the classification of species in relation to their conservation status was obtained by consulting the international union for conservation of nature (iucn) (iucnredlist.org) and the red book of brazilian endangered fauna (brazilian red list). in order to analyze if the hunting of game meat was influenced by seasonality, a simple linear regression was performed between the number of animals hunted monthly and the monthly cumulative precipitation data [ ] , as well as between the total weight of the hunted animals (kg) monthly and the cumulative monthly precipitation. also, to assess whether there were dissimilarities among the number of individuals hunted per species seasonally, a similarity matrix was built using the bray-curtis index. then the permanova analysis was used to check for dissimilarities between the rainy months and dry months (considering the apac classification) [ ] . finally, in order to test whether there were species that could be indicative of the dry period or the rainy season according to the number of individuals hunted, the analysis of indicator species was performed. in order to analyze if there were differences in the hunting of game meat between the period of ouricuri and the rest of the year, a t test was performed between the number of individuals hunted during the ritual and non-ritual months, as well as between the weight of individuals hunted during the ritual and those hunted in other periods. in addition, to evaluate whether there were dissimilarities between the number of individuals hunted per species between the ouricuri period and the rest of the year, a similarity matrix was built using the bray-curtis index. then the permanova analysis was applied to verify if there were dissimilarities between the months of the ritual and the other months of the year. finally, in order to determine if there were species that could be indicative of the period of ouricuri according to the number of individuals hunted, the analysis of indicator species (isa) was performed. in order to evaluate if species-perceived abundance influenced hunting, a generalized linear model (glm) was applied using the "quasipoisson" family between the number of individuals hunted during the year by species and the perceived abundance average. the "quasipoisson" family was used on the species' perceived abundance data in order to ensure greater control of the degrees of freedom. the same analysis (glm) was performed considering the total weight of the game obtained during the year by species and the average abundance perceived. finally, to check if the species cited as preferred were more hunted than the other species, the mann-whitney test was performed for independent samples between the annual number of hunted individuals and the total annual weight of preferred and non-preferred hunted individuals. all analyses were performed using the software r version . . [ ] ., in which all results whose p < . were considered significant were considered. the normality of the data was verified through the shapiro-wilk test. for the analysis of permanova, the vegan package was used [ ] , and the "indicspecies" package was used for the analysis of indicator species [ ] . among the potentially useful animals indicated from the semi-structured interviews (supplementary file ), species were frequently hunted within the period of the present study. a total of animals were hunted between the years and by the fulni-ô, representing , (kg) of game meat hunted. among the animals, % were hunted during the day, and birds were the taxonomic group that presented % of the game, with the highest rate for zenaida auriculata (des murs., ) ( , kg). mammals followed birds with %, among which the most hunted was galea spixii (wagler, ) ( , kg). reptiles came subsequently with %, and the species salvator merianae (duméril & bibron, ) was the most hunted within this group ( , kg) ( table ) . the perceived abundance of species did not explain the number of individuals hunted per month (estimated = . , standard error = . , t = . , p > . ) or hunting biomass (estimated = . , standard error = . , t = . , p > . ). these results show that perceived abundance is not a factor that influences the hunting of game meat. similarly, we did not observe a significant difference (t = . , p > . ) between the number of individuals hunted in the ouricuri rituals (x = , dp = . ) and the other months of the year (x = . , dp = . ), and there was no difference (t = − . , p > . ) between the biomass hunted during the ouricuri ritual (x = . , dp = . ), and after the ritual (x = . , dp = . ). furthermore, we did not find any variation between the periods mentioned above in relation to the number of hunted species (r = . , f = . , p > . ). finally, the analysis of indicator species found that only z. auriculata is significantly associated with the dry period in the region that corresponds to the same period of the ouricuri ritual (stat = . , p < . ), being it predominantly hunted in this period. these results show us that the migratory event related to the ouricuri ritual is not a factor that generates variation in the amount of game meat hunted. the rainfall variation during the year of observation neither explained the number of individuals hunted per month (r = . , f = . , p > . ) nor the biomass (r = . , f = . ; p > . ). in addition, there was no seasonal variation (dry and rainy) in the number of individuals hunted per species (r = . , f = . , p > . ). finally, the analysis of indicator species found that only patagioenas picazuro (temminck, ) is significantly associated with the rainy season (stat = . , p < . ), and it is possibly the most hunted in this period. we found significant differences (w = , p < . ) between the number of hunted individuals belonging to species cited as preferred by the informants and the other hunted species (fig. ) . also, there was a difference (w = , p < . ) between the biomass belonging to the species cited as preferred by the informants and the other hunted species (fig. ) . these results allow us to infer that the species cited as preferred by the informants are also the most hunted in the community. although the low number of volunteers monitored hunters in the second stage of hunting monitoring (n = ), we believe that these data represent the current scenario of hunting among the most active hunters that allowed the collection of such information. the results on the number of animals and taxa hunted diverge from other studies regarding the annual rate of extraction of these resources in places with high faunistic diversity, such as the amazon region [ , , ] , as well as in other tropical regions such as many african countries [ ] [ ] [ ] [ ] . these differences in relation to the amazon, for example, may be directly related to the faunistic composition and its size and the nutritional demands of the region [ , ] . even when compared to studies of wild animal consumption in the caatinga region [ , ] , the number of animals hunted is higher than that reported for the fulni-ô, which may be indicative of a lower demand for game in relation to other communities in the northeast region [ , , , ] . these differences in the hunting of game meat observed for the funi-ô in relation to other ethnic groups lead us to infer that hunting activity is much more focused on local cultural maintenance than a subsistence strategy. that low wild meat demand may suggest that hunters target their strategies at species whose attributes for choice include cultural aspects, such as the taste of meat or taboos. campos [ ] supports our idea by suggesting that hunting for this group is a food supplement for a few residential units. the author also argues that hunting is practiced for recreational purposes, domestic breeding, and the use of non-edible animals, such as some species of birds. this recent increase in wildlife utilization by the fulni-ô caused by strong drought regimes, which consequently affects fishing activity in the region, can also be interpreted as a temporal reflection that may or may not change over time, depending on the availability of other natural resources. this characteristic change in the ways of obtaining natural resources was pointed out by francesconi et al. [ ] , who argued based on their findings that hunting intensity may reflect a specific context of these communities in space and time or a cultural fusion of subsistence activities. similarly, mcnamana et al. [ ] reinforce the idea that preference for game meat may not be fixed, varying according to the circumstances experienced by such hunters. the cultural attributes, such as beliefs, attitudes, and social norms, are present and were equally important together with economic motivations for the hunting of wild animals in primarily urban areas [ ] . besides that, other factors that may reinforce the low dependency of the fulni-ô on game meat is the involvement of hunters in other external subsistence activities and the ease of obtaining non-wild animal protein, due to the proximity to the urban centers. fita et al. [ ] point out that the decrease in hunting activity may be a result of the increase of alternative productions of consumption and income generation. this possibility of external alternatives is similarly highlighted in the study by francesconi et al. [ ] , mainly among younger hunters who migrate more frequently to urban areas. this migration or the urbanization processes that local/traditional communities are subjected to, despite contributing to the well-being and providing opportunities for education and access to health, is pointed out by the authors as a factor that favors habitat fragmentation in their territories, which diminishes traditional sources of subsistence [ ] . the latter author also points out that such urbanization processes can also contribute to the exclusion, invisibility, and violence of these peoples in addition to influencing the detachment from their traditional practices. these factors also imply less involvement of hunters in this activity [ ] [ ] [ ] , which leads the indigenous people to develop these practices in their free time. these factors are suggested in the literature as potential modelers of hunting practices due to the lower cost of obtaining the animal protein. likewise, most studies report a higher frequency of hunting on mammalian species, followed by birds and reptiles [ , ] , that we did not observe in our findings. the preference for mammals is not only due to their size or availability but also to the taste of the protein in relation to the other taxa [ , , , ] . the most common mammal species observed in our research are mainly small animals, such as g. spixii. the high frequency of use of these animals can be considered an adjustment of the hunters to the reduction of the most sought species [ , ] . moreover, records in the literature point out that these small animals are less susceptible to hunting because of their high reproduction rate [ ] and are commonly abundant in more anthropic areas [ ] . on the other hand, it is important to point out that this trend towards g. spixii may also indicate a transfer of possible pressures of use in relation to other taxa of medium and large sizes [ , ] . this requires the hunter to increase the effort to capture more of these animals to meet their demands. in addition, the defaunation that has been occurring in the semi-arid region may also be a factor for this transfer for the use of other species [ , ] . this targeting of small species may indeed be a reflection of the decline of preferred species in the vicinity of human settlements, inducing hunters to search for animals other than their primary choices [ , ] . the high rate of bird reported in our study is supported by other records in the literature that indicate the taxon as one of the most sought after by local communities in the caatinga [ , , ] . however, the high frequency of avifauna caught among the fulni-ô should not only be a consequence of their high abundance but also of a greater efficiency in their attainment, since much of the incursions are carried out in groups and by applying a combination of strategies that optimize the success of the activity. this higher occurrence in the capture of birds, especially z. auriculata, by members of this ethnicity, if interpreted in terms of the benefits generated after capture, reinforces the local importance of the possibility of multiple uses of these animals to the detriment of other less versatile species. this versatility of use may justify the fact that many of these birds are caught for non-food purposes, especially between march and april, the period of highest intensity in the artisanal confection in the village. these are obtained exclusively for the extraction of their by-products with the purpose of making handcrafted artifacts [ ] , such as headdresses and arrows. silva et al. [ ] , when analyzing the artisanal production of the fulni-ô natives, also identified several artifacts that are made aiming at the external market demand, which are more related to the white man's perception of indigenous peoples. however, the need of the market and the contact with other ethnic groups can cause the indigenous people to adjust their strategies of sale. regarding the reptiles hunted in the studied region, the high rate of capture of s. merianae among the others reported may be mainly related to the return relative to biomass. in addition, like the birds, it may be related to the better use of its byproducts for local medicine, for example. this trend has been recorded in other researches that show that this lizard species is one of the most consumed in several regions of latin america, and its lard is often used in the treatment of inflammation [ ] [ ] [ ] [ ] . regarding seasonality, the migratory event related to the ritual of ouricuri (september to december), which increases the contact of fulni-ô members with natural resources, did not show a significant variation in the composition of the fauna hunted in the region. this contradicts our expectations and other studies that observed that the proximity of foraging areas is an important factor for the increase in the use of natural resources [ , ] . this intensification of faunal use was verified by van vliet and nasi [ ] in northeast of gabon, africa, where they observed a temporal and spatial variation in the period of drought in the region during the same period of the local religious ceremony. conversely, the ceremony of the fulni-ô occurs, as already mentioned, in a longer period of drought, which is a certain way that limits the encounter with animals of other taxa and favors the encounter of species of birds, mainly migratory ones like z. auriculata. this species was pointed out by mendonça et al. [ ] as one of the most hunted, which justifies the abovementioned species targeting due to their high abundance during periods of scarcity from other hunting sources. however, we cannot disregard the limitation on access to information during the reclusion period for the performance of the ouricuri religious ceremony, in which this access is allowed only to members of the ethnic group. during this period, information about hunted animals was collected only by hunters who assisted us in the research. another relevant issue among the fulni-ô is that most species were hunted during the daytime. this may also be a reflection of the very legality of the hunting activity for indigenous groups. therefore, since hunting is an integral part of these cultural reaffirmation actions, it is expected that it will be performed more frequently in shifts that pose fewer risks to hunters. this safety of daytime hunting in relation to nocturnal hunting, due to better visibility of the foraging area and the greater presence of fauna preferred by the natives, may be a determining factor in the hunting practices in the village. night hunting is reported in the literature as a more costly practice, with less prey availability [ ] , which requires different strategies that are usually applied in combination and facilitate visibility reducing risks [ ] . our results support previous studies carried out in the caatinga region in which was observed a higher avifauna catch rate, although the low rate of vertebrate hunting observed here can be interpreted as a strong contrast between the demand of game meat from other communities and fulni-ô ethnicity. likewise, the variables abundance, biomass, and the proximity of the resource did not present strong influences in relation to the species hunted in the region. this allows us to infer that the use of game meat in the village has a strong cultural aspect of maintenance and reaffirmation of the indigenous identity and not only of subsistence. this confrontation of the fulni-ô cultural practice also can be a response to the strong influence of urbanization and the external income opportunities that allow the use of non-traditional faunal resources in the markets near the village. this lower dependence of the resource as subsistence can be reinforced by the directing of the hunt to the species pointed out as the ones that are preferred by the hunters. thus, the data presented here help to understand the dynamics of how this cultural practice occurs in the face of environmental processes such as scarcity of resources, urbanization, and aspects that are inherent to hunters, such as preferences and strategies adopted to guarantee a satisfactory return of the hunting practice. on the other hand, it is also important to note that the cultural resistance of the fulni-ô members is also supported by the preservation of their native language, their religious ceremony, and their different relationships with natural resources. thus, ethnicity presents important contexts to be investigated by future research in relation to possible cultural adjustments due to socioenvironmental factors. therefore, we emphasize the importance of integrating not only environmental or biological variables of the target species, but also cultural and social aspects about the hunting practices of traditional communities. such understanding is also fundamental concerning to the food security of these peoples, especially in the current global scenario in which we live in a pandemic caused by the covid- virus, whose origin and spread may have been influenced by the cultural habit of consuming wild species. from the perspective of conservation, the information presented here helps to evaluate the natural stocks of the local fauna, emphasizing the importance of integrating these communities into the management processes through strategies that stimulate such involvement, and supports the knowledge of the practice to be transmitted to the following generations. (ppgetno -ufrpe). we also acknowledge the support of the laboratory of ecology and evolution of socioecological systems from the federal university of pernambuco (lea -ufpe), all the friends who collaborate with this material, the fulni-ô indigenous people for everything, and the funding institutions that supported the present study: capes, cnpq, and facepe. authors' contributions jss have collected the data and wrote the first draft of the paper. jss and albn performed the statistical analysis. jss, rrna, and upa conceived the study and performed data analysis. the authors read and approved the final manuscript. funding institutions that supported the present study: capes, cnpq, and facepe. the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. not applicable. the authors declare that they have no competing interests. received: january accepted: april bushmeat hunting and extinction risk to the world's mammals effects of subsistence structure in hunting on vertebrate forests community hunting strategies used in the semi-arid region of northeastern brazil the uncovered volumes of bushmeat commercialized in the amazonian trifrontier between colombia bushmeat consumption and its implications for wildlife conservation in the semi-arid region of brazil how does cultural change affect indigenous peoples' hunting activity? an empirical study among the tsimane' in the bolivian amazon sabedorias, cosmologias e estratégias de caçadores numa unidade de conservação da amazônia hunting and monitoring: communitybased research in xerente indigenous land conselho nacional de segurança alimentar e nutricional (consea) impact of game hunting by the kayapo of southeastern amazonia: implications for wildlife conservation in tropical forest indigenous reserves ethnozoology in brazil: current status and perspectives hunting, use and conservation of birds in northeast brazil hunting and wildlife use in an atlantic forest remnant of northeastern brazil cultural attitudes are stronger predictors of bushmeat consumption and preference than economic factors among urban amazonians from brazil and colombia assessment of the hunting of mammals using local ecological knowledge: an example from the brazilian semiarid region caça em assentamento rural na amazônia matogrossense game mammals of the caatinga biome os fulni-ô e suas estratégias de sobrevivência e permanência no território indígena. dissertation, programa de pós-graduação em antropologia assessing the effects of indigenous migration on zootherapeutic practices in the semiarid region of brazil landscape correlates of bushmeat consumption and hunting in a postfrontier amazonian region cultura, identidade e território no nordeste indígena: os fulni-ô. série antropologia e etnicidade memória e cultura: os fulni-ô afirmando identidade étnica. 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journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: z vbldd background: bats receive increasing attention in infectious disease studies, because of their well recognized status as reservoir species for various infectious agents. this is even more important, as bats with their capability of long distance dispersal and complex social structures are unique in the way microbes could be spread by these mammalian species. nevertheless, infection studies in bats are predominantly limited to the identification of specific pathogens presenting a potential health threat to humans. but the impact of infectious agents on the individual host and their importance on bat mortality is largely unknown and has been neglected in most studies published to date. methodology/principal findings: between and , deceased bats of european species (family vespertilionidae) were collected in different geographic regions in germany. most animals represented individual cases that have been incidentally found close to roosting sites or near human habitation in urban and urban-like environments. the bat carcasses were subjected to a post-mortem examination and investigated histo-pathologically, bacteriologically and virologically. trauma and disease represented the most important causes of death in these bats. comparative analysis of pathological findings and microbiological results show that microbial agents indeed have an impact on bats succumbing to infectious diseases, with fatal bacterial, viral and parasitic infections found in at least % of the bats investigated. conclusions/significance: our data demonstrate the importance of diseases and infectious agents as cause of death in european bat species. the clear seasonal and individual variations in disease prevalence and infection rates indicate that maternity colonies are more susceptible to infectious agents, underlining the possible important role of host physiology, immunity and roosting behavior as risk factors for infection of bats. bats are among the most successful and diverse mammals on earth. approximately chiropteran species are found on every continent except antarctica and inhabit a multitude of diverse ecological niches [ ] . bats play essential roles in maintaining healthy ecosystems, as they act as plant pollinators, seed dispersers, and predators of populations of insects including harmful forest and agricultural pests [ ] . most bat species are listed in the iucn red list of endangered species and almost half of these are considered threatened or near-threatened [ ] . to estimate and prevent further population declines, research has been primarily focused on bat biology, ecology and behavior, while disease aspects were largely neglected [ ] . in the last two decades, the importance of chiropteran species as potential vectors of significant viral diseases especially in regard to zoonoses has received growing attention. besides bat rabies that has been studied for more than half a century, extensive research efforts identified a large number of microbial agents [ ] including important emerging zoonotic viruses detected in bats across the world [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, most studies are limited to the identification of microorganisms detected and investigations regarding infectious diseases and causes of death in bats are sparse [ ] [ ] [ ] [ ] . in europe, research is predominantly focused on european bat lyssaviruses [ , ] and coronaviruses [ , ] , but first indications of bat-pathogenic bacteria [ , , [ ] [ ] [ ] and novel viruses [ , ] isolated from deceased bats in germany and great britain were found. in this study, we provide new data on infectious diseases in european bat species, considering factors likely to affect the susceptibility of bats to infectious agents including effects of seasonality, individual and species-specific heterogeneities, and possible intra-and inter-species transmission dynamics. all bat species in europe are strictly protected under the flora-fauna-habitat guidelines of the european union (http://ec.europa. eu/environment/nature/legislation/habitatsdirective/index_en.htm) ( / /eec) and the agreement on the conservation of populations of european bats (www.eurobats.org) that prohibit invasive sampling of bats for research purposes. for the animals investigated in this study, carcasses of deceased bats found in germany were kindly provided by bat researchers and bat rehabilitation centers of different federal states. between and , a total of deceased bats of european vespertilionid species (i.e., family vespertilionidae) were investigated (fig. a , [ ] ). the bat carcasses originated from different geographic regions in germany, i.e. berlin greater metropolitan area (n = ), bavaria (n = ), brandenburg (n = ), lower saxony (n = ), thuringia (n = ), and baden-wuerttemberg (n = ), and were collected by bat researchers and bat rehabilitation centers. most animals represented individual cases that were found dead, injured or moribund near human habitation. thus, the species composition in this study predominately reflected the urban and suburban bat fauna, which is characterized by a disproportionate abundance of a few bat species (fig. a , [ , ] ). two groups of and adult noctules (nyctalus noctula), respectively, were collected from tree hibernacula destroyed during wood logging. a further group of deceased adult n. noctula originated from a colony that was trapped in a rain pipe in december. nine dead juvenile pipistrellus pipistrellus were collected from a nursery roost. if bats died in care or had to be euthanized for animal welfare reasons, the carcasses were immediately stored at uc and were shipped to the leibniz institute for zoo and wildlife research, berlin, germany, for diagnostic investigations. of all carcasses examined histo-pathologically, about % were suitable for bacteriological investigation. a lesser extend ( %) was also examined for selected viral agents at the robert koch institute, berlin, germany. in addition, a brain sample of each animal was submitted to the friedrich-loeffler-institute, wusterhausen, germany, for rabies diagnosis. a full necropsy was performed on each bat and all macroscopic findings including ectoparasite infestation were recorded. for histo-pathological examination, small slices of multiple organ tissues (i.e., lung, liver, heart, kidney, adrenal gland, spleen, intestine, pancreas, brain, tongue, larynx, salivary gland and pectoral muscle) and tissues conspicuous for pathological changes were fixed in buffered % formalin, processed using standard methods and embedded in liquid paraffin. sections were cut at - mm and routinely stained with hematoxylin-eosin (he). in addition, special histological staining methods were used depending on microscopic findings, i.e. for the detection of bacteria (gram or giemsa staining), fungi (periodic acid schiff or grocott's gomori methenamine silver nitrate staining), iron (prussian blue stain), mineralization (von kossa staining), connective and collagen tissue (trichrome staining). details on pathological results are published elsewhere [ ] . the causes of mortality were rigorously standardized with the primary cause of death identified for each bat as the most serious injury, disease or event subsequently fatal to the animal. to ensure independence of primary and contributing causes of death, the categorization was based on the severity of pathological findings. samples of lung, liver, heart and kidney, and tissues conspicuous for pathological changes (e.g. enlarged spleen) of bats were plated onto columbia ( % sheep blood), chocolate, gassner, and macconkey agar (oxoid, germany) and were incubated at uc (chocolate agar % co ) for - h. specific culture media and conditions for the isolation of yersinia, salmonella and anaerobic bacteria were used if appropriate. primary identification of bacterial strains was based on colony morphology, hemolysis, gram-staining, indol production, catalase and oxidase reaction. bacterial species identification was carried out using the relevant commercial api test system (biomérieux, germany). additional conventional biochemical tests [ , ] were applied to confirm api test results where necessary. in case of ambiguous biochemical test results, s rdna gene analysis was performed for final identification [ ] . salmonella isolates were characterized at the national reference laboratory for the analysis and testing of zoonoses (salmonella) at the federal institute for risk assessment, berlin, germany. identification and characterization of yersinia and pasteurella species have been reported earlier [ , ] . homogenized organ tissue of lung, liver, heart, kidney, spleen, brain and salivary gland of bats were pooled for each individual and used for rna/dna extraction and further molecular analysis by generic pcr assays detecting flavi- [ ] , hanta- [ ] , corona- [ ] , and influenza a-viruses [ ] . also, pcr assays specific for previously described herpesviruses [ ] from european vespertilionid bats were used. for this purpose, rna/ dna was isolated using the nucleospinh rna ii kit (macherey-nagel, germany) and the nucleospinh tissue kit (macherey-nagel), respectively, according to the manufacturer's instructions. because of limitations in sample volume, for out of the bats pcr assays could only be applied for different bat herpesviruses. internal controls were used for all pcr assays to test for inhibition. for confirmation, all retrieved fragments of bat herpesvirus-specific pcr assays were checked for sequence identity to previously published isolates [ ] . for detection of lyssavirus antigen in brain tissue the fluorescent antibody test (fat) using a polyclonal antirabies conjugate (sifin, germany) was used [ ] . fat-positive brain tissues were subject of virus isolation in murine neuroblastoma cell culture (na / ) using the rabies tissue culture infection test (rtcit) as described elsewhere [ ] . lyssaviruses isolated in cell culture were characterized using both a panel of anti-nucleocapsid monoclonal antibodies (mab) [ ] and partial sequencing of a fragment of the nucleoprotein gene after rna extraction using trizol (invitrogen, germany) essentially as described [ ] . genomic dna was extracted from organ homogenates using the nucleospinh tissue kit (macherey-nagel) according to manufacturer's recommendations. genetic identification of the bat species was performed by amplification and sequencing of a bp fragment of the cytochrome b (cytb) gene [ ] using primers fm up ( -ccc chc chc aya tya arc cmg art gat a - ) and fm down ( -tcr acd ggn tgy cct ccd att cat gtt a - ). in addition, for differentiation of the distinct pipistrellus species, p. pipistrellus and p. pygmaeus, a rapid multiplex pcr assay was performed as described by kaňuch et al. the bat data were categorized in regard to different explanatory numeric and factor variables, e.g. bat species, sex and age class. the variable 'age class' ranked between and with increasing age (i.e. neonates, juveniles, subadults, and adults) and was used as numeric variable. for endoparasitic analysis, we defined a level variable 'bat size' according to the body size of a certain bat species to reduce the degrees of freedom of the full model, i.e. large species (n. noctula, eptesicus serotinus, and vespertilio murinus), medium-sized species (e. nilssonii, plecotus auritus, myotis daubentonii, m. nattereri, and p. nathusii) and small species (p. pipistrellus, and m. mystacinus). to detect effects of seasonality, different activity periods were specified according to the date of sampling, i.e. hibernation period (november to march), post-hibernation period (april/may), maternity period (june to august), and swarming period (september/october). as dependent binary variable for the respective models we either classified the mortality cause being disease or not (i.e. trauma), or the presence-absence of bacterial, ecto-and endoparasitic infections. we formulated different hypotheses to test for individual and species-specific differences in disease susceptibility and infection rates: (a) disease-related mortality in bats is influenced by sex, age and species-specific differences, and degree of endoparasitic infection. (b) bacterial infection in bats is influenced by sex, age and species-specific differences, occurrence of traumatic injuries and cat predation. (c) ecto-or (d) endoparasitic infection in bats is affected by age, sex and species-specific differences. seasonal effects were not analyzed because of too many missing data points. because the long-term dataset was highly biased towards sampling procedure, preservation of bat carcasses and following diagnostic investigations, we split and filtered the full data into several subsets reflecting the different analyses (table ) . all statistical analyses were performed using the r software v. . . (r development core team , vienna, austria). we used the chi-square test for given probabilities to evaluate significant differences in the sex ratio among bats of different species. for hypotheses a and b, we used a generalized linear mixed modeling approach (binomial glmm using function lmer in library lme ) with bat species included as random effect. this variable had not been significant as fixed effect (results not shown), but from other studies we can assume that there are speciesspecific differences in susceptibility of bats to certain infectious agents and therefore included it as random effect. we further used generalized linear models (glm with logit link and binomial error structure; for datasets with bat species . individuals) to test for individual and species-specific differences in parasite infection rates (hypotheses c and d). we created a full model for each hypothesis (a-d) to examine multiple and interaction effects of the specified variables. to select the final model variables, we used a stepwise backward algorithm (function stepaic in library mass) based on akaike's information criterion (aic) [ ] . the daic of the final model was calculated relative to a random intercept model to demonstrate the effect size of the selected variables. results of the diagnostic analyses follow the full data splitting into several subsets (see section 'statistical analysis' in material and methods; table ). all sampled bats belonged to different genera (i.e. pipistrellus, nyctalus, myotis, eptesicus, plecotus, vespertilio, and barbastella) and european vespertilionid species (fig. a) . three bat species, the common pipistrelle (p. pipistrellus, n = ), the noctule bat (n. noctula, n = ), and the serotine bat (e. serotinus, n = ) constituted about % of all bat carcasses investigated in this study, whereas p. pygmaeus, nyctalus leisleri, myotis brandtii, m. bechsteinii, m. dasycneme, plecotus austriacus and barbastella barbastellus were represented in small numbers of to animals. the overall sex ratio was . males to female with significant species-specific differences (fig. b) . animals in their first year of life (neonates, juveniles, and subadults) represented one third ( . %, n = ) of bat samples (fig. c) . overall, we were able to assign a cause of death to % (n = ) of bats investigated in this study. two thirds of mortality were due to trauma (n = ) or disease (n = ), while almost % of bats had died of other non-infectious causes like pulmonary edema, dehydration and hypoglycemia (table ). in % (n = ) no significant pathological findings could be found. among the traumatized bats, additional mild (n = ), moderate (n = ) and severe (n = ) inflammatory organ changes were noted in one half ( . %) of individuals, and % of the bats revealed bacterial (n = ) and/or parasitic infections (n = ) ( table ) . of the bats considered as dying of disease, fatal bacterial (n = ), viral (n = ) and parasitic infections (n = ) were observed in %. besides, amniotic fluid aspiration was noted in a neonate noctule bat (n. noctula), and a juvenile common pipistrelle (p. pipistrellus) was euthanized because of severe forearm bone deformation. the remaining bats ( . %) revealed moderate to severe pathological changes of unknown etiology or unconfirmed bacterial or viral cause ( table ). based on the glmm analysis, significant age-and sexdependent differences (daic = . ) were detected between the general causes of mortality, disease and trauma ( table ). the disease presence in bat samples decreased continuously with increasing age. neonates and juveniles of both sexes were significantly more affected by disease than older age classes (table ; fig. a) . we also found a significant trend in diseaseassociated mortality between the sexes, with adult females displaying higher disease prevalence ( . %) than males ( . %) ( table ). no significant association was observed between a certain cause of mortality (i.e. disease or trauma) and severity of endoparasitic infection (daic = . , result not shown). the seasonal distribution of disease-related mortality cases (fig. b ) described a trimodal pattern, with peaks in spring (april), summer (june to august) and winter (december). the proportion of traumatized individuals also increased obviously during the summer months up to and including the swarming period, but was low during the rest of the year. about % (n = ) of bat samples were examined bacteriologically. among these, different bacterial genera with more than bacterial species were identified (table s ). predominant bacteria isolated were enterococcus faecalis ( . %, n = ), hafnia alvei ( . %, n = ), serratia liquefaciens ( %, n = ), and pasteurella multocida ( . %, n = ). in % (n = ) of bats no bacterial growth was observed at all. comparative bacteriologic and histo-pathologic analysis identified different bacterial species that were clearly associated with pathological lesions and/or systemic infection, found in % (n = ) of bats investigated bacteriologically ( table ) . members of the families pasteurellaceae (above all p. multocida) ( . %, n = ), enterobacteriaceae (various bacterial species) ( . %, n = ), and streptococcaceae (above all enterococcus spp.) ( . %, n = ) were predominant bacteria associated with disease. more than half ( . %, n = ) of bacterial infections were observed in bats with traumatic injuries. the glmm analysis revealed low sex-and age-dependent differences in bacterial infection (daic = . , result not shown). female bats ( . %) and adults ( . %) showed marginally higher prevalence of bacterial disease compared to males ( . %) and to other age classes ( . %), respectively. however, we found a strong influence of cat predation (daic = ) associated with bacterial infection in bats (table ). testing for human-pathogenic zoonotic viruses, no examined bat sample ( / ) was positive for influenza a virus, corona-, hanta-and flaviviruses, respectively. no inhibition of the pcr assays was notified. out of bats tested for rabies virus infection, serotine bats (e. serotinus) were positive for lyssavirus by fat and rtcit. the viruses were identified as european bat lyssavirus type (eblv- ) sublineage a, both using mabs and sequencing. applying bat herpesvirus-specific pcr assays, out of bats proved to be infected with of the previously described bat herpesviruses ( table ). the highest prevalence of % ( / ) was observed for bat gammaherpesvirus (batghv ) in common pipistrelle bats (p. pipistrellus), followed by bat gammaherpesvirus (batghv , . %) in nathusius' pipistrelle bats (p. nathusii) and bat gammaherpesvirus (batghv , . %) in noctule bats (n. noctula). co-infection with different bat herpesviruses were recognized in n. noctula ( . %) infected with batghv and batghv , and in one n. noctula ( . %) infected with batghv and batghv . batghv was not only detected in its initially specific host p. nathusii, but also in other bat species, i.e. n. noctula, myotis myotis and m. mystacinus. although the prevalence of batghv ( . %) and batghv ( . %) differed significantly within its migrating host n. noctula, no difference was observed between the sexes. two juvenile n. noctula were found to be infected with batghv . interestingly, for the sedentary bat species p. pipistrellus being infected with batghv , a considerably higher prevalence was observed in juvenile bats ( . %) resulting in an overall prevalence of % also without difference between adult male and female bats. ectoparasites (mites, fleas, and ticks) were noted in % (n = ) of bats, but a potential bias in ectoparasite numbers collected from dead animals in comparison to ectoparasite abundance on live animals has to be taken in account. female bats ( . %) were slightly more infested by ectoparasites than males ( . %), whereas in different age classes ectoparasite prevalence was almost balanced. the glm analysis revealed significant species-specific differences in ectoparasite infestation (daic = . , table ). most bat species revealed low ectoparasite prevalence (range . - . %), while almost % (n = ) of n. noctula were infested with mites and/or fleas (fig. a) . microscopic examination of organ tissues revealed endoparasitic infection in % (n = ) of investigated bats, involving different protozoan (families eimeriidae and sarcocystidae) and helminth parasites (trematodes, cestodes, and nematodes). helminthes were predominantly found in the gastro-intestinal tract of the bats, while in some animals, granulomatous organ lesions were associated with larval migration of nematode species. based on the glm analysis, clear age-and species-specific differences (daic = . ) were observed between infected and non-infected bats ( table ). the prevalence of endoparasitic infection in bat samples increased significantly with increasing age, whereas the increase in prevalence was more rapid between juveniles and subadults ( . %) compared to the older age classes ( . %). marginal differences were also observed between the sexes, with female bats showing slightly higher ( . %) endoparasite prevalence than males ( . %). regarding species-specific differences, large bats like n. noctula, e. serotinus and v. murinus revealed higher endoparasite prevalence compared to individuals of medium-sized or small vespertilionid species (table ; fig. b ). this study was based on a passive surveillance sampling strategy that inherently influences the composition of bats sampled for diagnostic investigations [ ] and might also effect the data presented on causes of death by ecological and anthropogenic impacts of urban landscapes [ ] . trauma and disease represented the most important causes of mortality in deceased bats from germany, which differ from results of previous studies [ ] [ ] [ ] where disease-related mortality often played a subordinate role. young bats and adult females were significantly more affected by disease, indicating that sex-and age-related disease prevalence in table . pathological findings and bacterial, viral and parasitic infections specified for the general causes of mortality, trauma and disease. bats are strongly correlated with the maternal season. this assumption is further supported by the distinct increase of diseaserelated mortality from june to august, which corresponds to the maternity period of central european bat species. similar seasonal prevalence patterns in bats have also been described for parasitic (e.g. [ ] [ ] [ ] [ ] ) and viral infections (e.g. [ , , ] ). in contrast, the increase of trauma-associated mortality cases from july to october resembles major behavioral activity patterns of european bat species (i.e. weaning, mating, pre-hibernal fat storage, and migration) [ ] and could therefore predispose bats to trauma. however, both seasonal peaks also coincide with time and locations where sick, injured or dead bats are more likely to be discovered as well as with the seasonal roosting behavior of bats adapted on urban habitats [ ] . the additional seasonal peaks of disease-associated mortality corresponded to the post-hibernal and the early hibernal period of temperate zone bats. currently, there is a lack of knowledge of bat immunology. it is known for other mammalian species that hibernation reduces the innate and adaptive immune response; likewise an increasing risk of infection could be assumed for hibernating bats [ ] . with the start of the hibernation season, large aggregations of bats originating from various colonies might enhance the risk of spreading infectious agents similar to maternity colonies. equally, the post-hibernal increase of disease-related mortality is suggestive for reduced immunity in association with prolonged fasting during hibernation. bacterial diseases have rarely been documented in bats. pasteurella spp., here identified in % of bats, were the predominant bacterial pathogens reported in european bats and infection appears to be strongly correlated with cat predation [ , , , ] . in our study, bacterial infections were confirmed in % of bats investigated bacteriologically. most of these bacterial isolates represented opportunistic pathogens that usually do not harm the host unless the immune system is weakened [ ] or preceding injury to natural host barriers (e.g. skin abrasion). primary bacterial pathogens like samonella enterica serovar typhimurium, s. enteritidis and yersinia pseudotuberculosis [ ] were identified in almost % of affected bats. some of the bacterial species (e.g. burkholderia sp., cedecea davisae and clostridium sordellii) are newly described in bats. nevertheless, bacteriologic analyses can markedly be influenced by post-mortem bacterial invaders, freezing and storage of bat carcasses and the inability to detect certain bacteria by routine culture methods, resulting in some bacterial species that might have escaped detection. we found a strong association between cat predation and bacterial infection in bats as almost one half of bats ( %) caught by cats were affected by bacterial disease. various bacteria can be transmitted via cat bites [ ] ; hence bats attacked by cats are likely to succumb to bacterial infection even if non-fatal injuries were present. this relation has been proven for p. multocida infections in european bat species [ , , , ] . on the other hand, bats already debilitated by disease might easier fall prey to predators like cats. consequently, bats may also act as vectors for zoonotic pathogens, as domestic cats could pass these infectious agents on to humans. such cross-species transmission events from bats to domestic animals are well documented [ , ] . for all tested human-pathogenic zoonotic viruses no infected bat could be detected in this study except lyssaviruses. bat rabies is the only bat transmitted zoonosis in europe that is known to have resulted in human cases [ ] . unlike in other mammals table . result of the final model variables corresponding to different analyses: (a) disease-vs. trauma-related mortality, and presence-absence of (b) bacterial, (c) ecto-and (d) endoparasitic infection. where lyssaviruses ultimately cause lethal rabies, in bats nonlethal lyssavirus infections may also lead to the development of immunity [ ] . with the detection of eblv- we confirm that this lyssavirus circulates among e. serotinus as previous studies showed [ ] . in germany, bat rabies is also caused by eblv- and bokeloh bat lyssavirus (bblv) [ , ] , but while the latter was recently isolated from m. nattereri, eblv- is associated with m. daubentonii and m. dasycneme [ ] . the apparent absence of eblv- and bblv in the sampled bats is likely due to the fact that lyssavirus infections have a very low incidence in bat populations [ ] and that the sample size was too limited, especially concerning the relevant species. there is a high prevalence for herpesviruses in different insectivorous bat species in germany (this study, [ ] ). most of the previously described bat herpesviruses have been detected in low numbers in more than one bat species [ ] . here, we observed a high species-specific prevalence among herpesvirusinfected bats, indicating that a certain type of european bat herpesvirus is primarily associated with a single bat species. this is supported by batghv and batghv that were again only identified in their initial hosts p. pipistrellus and p. auritus (both sedentary), respectively, underlining the typical strong speciesspecificity of mammalian herpesviruses. however, species' range overlap and close inter-species contacts in bat roosts may result in cross-species transmission and could explain the observed overcoming of the species barrier (this study batghv , [ ] ). interspecies transmission have also been discussed for other mammalian herpesviruses, i.e. bovine and equine herpesviruses (e.g. [ , ] ). furthermore, for rna viruses (i.e. rabies virus) phylogenetic distance between different host species and overlap in geographic range have recently been demonstrated as strong predictors of host shifts and cross-species transmission in bats [ ] . some of the bat species (i.e. n. noctula, p. pipistrellus, and p. nathusii) in this study appear to be more susceptible to herpesvirus infection. in n. noctula, different gammaherpesviruses (batghv , , ) with significant prevalence differences were recognized. such type-specific differences in prevalence between the phylogenetically distant viruses batghv ( . %) and batghv ( . %) within one bat species indicates co-evolutionary virus-regulated mechanisms. parasite infestation in wildlife often occurs without clinical effects, but severe infection can reduce host fitness either in terms of survival or reproductive success [ ] . most data on infection dynamics in bats came from parasite studies focusing on individual and seasonal variations in ectoparasite prevalence (e.g. [ ] [ ] [ ] ] ). host density, roost preference and movement pattern seem to be important factors explaining individual and speciesspecific parasite infestation rates in bats [ ] [ ] [ ] . in european vespertilionid species, female-biased parasite loads are most likely associated with host physiology and differences in roosting behavior [ , ] . we also found species-specific seasonal variations in ectoparasitic infestation, with n. noctula and m. daubentonii showing higher ectoparasite prevalence in spring and autumn compared to the breeding season (data not shown), which is in accordance with zahn and rupp [ ] . additional findings of our parasite analyses are distinct variations in ecto-and endoparasite prevalence in relation to bat species. bats primarily roosting in trees or nest boxes were more frequently infested with ectoparasites like n. noctula ( %) and m. daubentonii ( %) compared to other species (range - %) investigated in this study. high ectoparasite loads have generally been described in bats preferring enclosed roosts like burrows and cavities [ , ] , suggesting that structural characteristics and the microclimate of roosting habitats influence ectoparasite survival and re-infection of bat hosts. this assumption is in accordance with results of pearce and o'shea [ ] who found differences in ectoparasite prevalence and intensity in eptesicus fuscus in relation to environmental factors (i.e. temperature and humidity) of different roost sites. in contrast to these results, the endoparasite prevalence in european vespertilionid bats seems to be correlated with the body size of the bat species [ ] . species-specific variations in diet and prey selection could possibly effect endoparasite prevalence in insectivorous bats [ ] , as larger bats feed on insects of a wider size range including hard-bodied prey [ , ] . this assumption is supported by the clear prevalence increase in subadult and adult bats compared to low endoparasite infection rates in juveniles primarily feeding on milk. a multitude of publications is restricted to pathogen presence or absence in different chiropteran species; here we demonstrate the impact of diseases and infectious agents on bats themselves. alongside to trauma-associated mortality and undefined mortality cases, disease aspects represented one third of mortality causes in investigated bats of european vespertilionid species. by comparing pathology and bacteriology results, we were able to detect different bacterial species that were clearly associated with disease in bats. at least % of all bats had died due to bacterial, viral and parasitic infections. finally, we found clear seasonal and individual variations in disease prevalence and infection rates, indicating an increased susceptibility to infectious agents in female bats and juveniles during the maternity season. our data emphasize and provide the basis for disease related studies in bat species on population level to elucidate the complexity of the ecology of infectious agents and host species likewise. table s bacteria isolated from bats found in germany. (doc) the status of the world's land and marine mammals: diversity, threat and knowledge ecosystem services provided by bats a review of the global conservation status of bats emerging diseases in chiroptera: why bats? ecological and behavioral methods for the study of bats. nd edition the isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of nipah virus from malaysian island flying-foxes fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses bats as a continuing source of emerging infections in humans isolation of genetically diverse marburg viruses from egyptian fruit bats bats, emerging infectious diseases, and the rabies paradigm revisited veterinary advances in the investigation of wildlife diseases in britain causes of morbidity and mortality in british bat species and prevalence of selected zoonotic pathogens. thesis for msc in wild animal health infectious and emerging diseases of bats, and health status of bats in new zealand veterinary treatment of evening bats (vespertilionidae) in the czech republic european bat lyssaviruses: distribution, prevalence and implications for conservation epidemiology of bat rabies in germany detection and prevalence patterns of group i coronaviruses in bats identification of sars-like coronavirus in horseshoe bats (rhinolophus hipposideros) in slovenia fatal borreliosis in bat caused by relapsing fever spirochete, united kingdom yersinia species isolated from bats genetic diversity of pasteurella species isolated from european vespertilionid bats discovery of herpesviruses in bats new adenovirus in bats diseases in free-ranging bats from germany habitat preference and flight activity of bats in a city bat ecology and public health surveillance for rabies in an urbanizing region of colorado. urban ecosystem colour atlas for the diagnosis of bacterial pathogens in animals bergey's manual of systematic bacteriology. nd edition generic rt-nested-pcr for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification hantavirus in african wood mouse generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes laboratory techniques in rabies. th edition world health organization laboratory techniques in rabies. th edition world health organization application of monoclonal antibodies for epidemiological investigations and oral vaccination studies species determination: the role and use of the cytochrome b gene a rapid pcr-based test for species identification of two cryptic bats pipistrellus pipistrellus and p. pygmaeus and its application on museum and dropping samples information theory as an extension of the maximum likelihood principle urbanization and the ecology of wildlife diseases variation of intensity of a parasitic mite (spinturnix myoti) in relation to the reproductive cycle and immunocompetence of its bat host (myotis myotis) ectoparasite load in european vespertilionid bats relationships between the parasitic mite spinturnix andegavinus (acari: spinturnicidae) and its bat host, myotis daubentonii (chiroptera: vespertilionidae): seasonal, sex-and age-related variation in infestation and possible impact of the parasite on the host condition and roosting behavior host sex and ectoparasites choice: preference for, and higher survival on female host reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes (pteropus scapulatus) host and viral ecology determine bat rabies seasonality and maintenance seasonal activity patterns of seven vespertilionid bat species in polish lowlands hibernation: the immune system at rest? bacterial pathogenesis bacteriologic analysis of infected dog and cat bites european bat lyssavirus transmission among cats human rabies due to lyssavirus infection of bat origin first isolation of eblv- in germany novel lyssavirus in natterer's bat bat rabies serological survey of herpesvirus infections in wild ruminants of france and belgium new hosts for equine herpesvirus host phylogeny constrains cross-species emergence of rabies virus in bats behavioral adaptations to parasites: an ethological approach relationships between roost preferences, ectoparasite density, and grooming behaviour of neotropical bats roosting habits of bats affect their parasitism by bat flies (diptera: streblidae) ectoparasites in an urban population of big brown bats (eptesicus fuscus) in colorado when parasites become prey : ecological and epidemiological significance of eating parasites the implications of food hardness for diet in bats prey consumed by eight species of insectivorous bats from southern illinois the authors would like to thank berliner artenschutz team-bat-e.v., f. key: cord- -dhkz co authors: chamorro, melina fernanda; ladio, ana title: native and exotic plants with edible fleshy fruits utilized in patagonia and their role as sources of local functional foods date: - - journal: bmc complement med ther doi: . /s - - - sha: doc_id: cord_uid: dhkz co background: traditionally part of the human diet, plants with edible fleshy fruits (peff) contain bioactive components that may exert physiological effects beyond nutrition, promoting human health and well-being. focusing on their food-medicine functionality, different ways of using peff were studied in a cross-sectional way using two approaches: a bibliographical survey and an ethnobotanical case study in a rural community of patagonia, argentina. methods: a total of studies were selected for the bibliographical review. the case study was carried out with % of the families inhabiting the rural community of cuyín manzano, using free listing, interviews, and participant observation. in both cases we analyzed species richness and use patterns through the edible consensus and functional consensus indices. local foods, ailments, medicines and drug plants were also registered. results: the review identified peff, the majority of which ( %) were native species, some with the highest use consensus. peff were used in different local foods, but mainly as fresh fruit. of the total, % were used in a functional way, in different medicines. the principal functional native species identified in the review were aristotelia chilensis and berberis microphylla. in the case study peff were in current use ( % were native), and consensus values were similar for native and exotic species. these were used in different local foods, mainly as fresh fruit. only % were recognized for their functional value by inhabitants (mainly as gastrointestinal and respiratory treatments). the species with the highest functional consensus were the exotic sambucus nigra and rosa rubiginosa, followed by the native a. chilensis, ribes magellanicum and b. microphylla. infusions also constituted important local functional foods. conclusions: this survey highlights the importance of studying the different local functional foods to depict the biocultural diversity of a human society. the preparation of different beverages and herbal medicines was relevant, and would be a promising subject to investigate in the future. the living heritage of peff appears to have undergone hybridization processes, such that exotic species play an increasingly significant role. a poor diet, with little consumption of fruit and vegetables, among other characteristics, is known to be strongly associated with mortality through noncommunicable diseases (such as cardiovascular diseases and cancer) [ ] . extra-nutritional substances present in plants have the capacity to reduce cellular oxidative stress, thus lessening the probability of developing these illnesses [ ] [ ] [ ] . the current challenge is to generate policies centered not just on nutrients, but on healthy foods [ ] . species with edible fruits deserve special attention not only for their value per se, but also as local foods. the importance in this study of the variety of ways of ingesting plant-based foods is highlighted by heinrich et al. [ ] . they developed the concept of "local foods" through the study of the mediterranean diet, distinguishing those recipes that are shared in a territory or culture, and which form a fundamental part of local food knowledge [ ] . this represents an interesting field of study in the subject of functional foods, a concept that originated in japan in the s as foods for specified health use (foshu). at the present time, "functional food science in europe" (fufose) proposes that functional food is that which is "satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and wellbeing and/or reduction of risk of disease" [ ] . from an ethnobotanical perspective, other concepts arise such as "folk functional foods" [ ] , or what we will call here "local functional foods", based on the multifunctional character of food use by local inhabitants [ ] . the study of plant-based local functional foods currently offers an opportunity for ethnopharmacological research, as a wide range of biocompounds is revealed, which is made available to humans in the different recipes used, capable of transforming human gastronomic habits. the study of native and exotic edible plant use in rural and indigenous communities offers an opportunity to appreciate how humans have experimented with the diversity of their plant surroundings in search of wellbeing. as established by etkin and ross [ ] , plant studies in these communities should be approached with understanding of the integral nature of food and health concepts. in their search for this functionality, humans have intentionally selected dual-purpose resources (ediblemedicinal) and have also tended towards diversification of food types and new forms of consumption. consequently, we are interested in showing the multidimensionality and multifunctionality of the peff, considering all their plant parts in an integral way. the study of the potential of patagonian native fruit species is relatively recent. progress has been made from the perspectives of agronomy [ , ] , phytochemistry [ ] [ ] [ ] and nutrition [ , ] . several studies have demonstrated the antioxidant properties of some patagonian fruits and their usefulness in regulating glucose metabolism, thus indicating their nutraceutical value [ ] [ ] [ ] . nevertheless, the diversity of existing species and their local foods has been little described up to now [ ] . one aspect highlighted in the ethnobotanical studies carried out in patagonia is the high proportion of exotic edible plant use in some local communities [ ] [ ] [ ] . for example, in rural areas, the gathering of edible plants seems to be linked to environmental changes, with the incorporation of more exotic fruit-bearing species that grow wild, including invasive plants like sweet briar, elmleaf blackberry and apple (rosa rubiginosa l., rubus ulmifolius schott, malus domestica borkh) [ ] . due to processes of colonization and imposition, since the arrival of europeans the region has tended towards the cultivation of exotic fruits [ ] . in market gardens and fruit farms % of the fruit grown is exotic in origin, such as plums and apples [ ] . this pattern, called the hybridization process [ ] , is prevalent and of great interest when considering local foods. in this study we focus on native and exotic plants with edible fleshy fruits (peff) that grow in patagonia; that is, species that may be wild, cultivated or in an intermediate state of domestication, which bear fruit that is distinguished by its flavor, preferably sweet, and its use principally as a food resource. in general, they have a high proportion of water and sugars that are easily digested and absorbed, and they have long contributed fiber, minerals and vitamins to the diet of local populations [ ] . very little information is available concerning the total number of species involved in this group and how people have used them. in addition, far fewer studies exist that deal with the safety aspect of consumption of peff and their different preparations. understanding the use patterns of peff as part of the local biocultural heritage of patagonia is crucial [ ] . in recent times, as in the rest of the world, rural and urban societies of this region have experienced sociocultural and environmental changes. these changes have led to a reduction in traditional plant use [ ] , and some elements of the flora seem to be in danger [ ] . these alterations will have serious long-term repercussions for peff use, as motivation for the generational transmission processes (oral traditions) is weakened, and consequently, the patrimony for future generations is reduced. in this study we propose a cross-sectional approach which enables exotic and native species richness, local foods, medicines and their use patterns to be evaluated, and which will also help us understand in greater depth, from an ethnobotanical perspective, that diet and health are linked concepts. we will carry out a bibliographical review and a case study of a current rural community in order to synthesize existing information on the functionality of native and exotic peff, and analyze their use patterns and potential. our main questions were: which native and exotic plants from patagonia constitute peff? which species are currently in use? what proportion is used as functional food? how many local foods are involved? this analysis of mixed information also enables us to show the continuity or change in use of functional and nonfunctional peff, shedding light on the existing biological and cultural diversity, and its potential. the bibliographical review involved quali-quantitative analysis of ethnohistorical and ethnobotanical texts published since which mention the use of native and exotic fruits in the patagonian region [ ] . the review was conducted using scopus, science direct and scielo, and google scholar search engine, as well as the library and database of the grupo de etnobiología. the key words and phrases used in the search were: native and exotic edible fruits and patagonia; edible plants and patagonia; medicinal plants and patagonia; patagonian flora and uses; ethnobotany of patagonia; medicinal and alimentary uses and patagonia, and native fruits of patagonia. the search was carried out in spanish and english, and a total of documents were examined. the works selected were mainly primary field studies, but compilations were also included given that each review used independent protocols for species evaluation, according to the author of the work. location of database publications: the articles selected were from studies carried out in argentine rural and urban communities with non-indigenous people, creoles, and people of mapuche-tehuelche-selk'nam ancestry, distributed over an area extending approximately between o and o lat. s. of the articles studied, % dealt with organized indigenous communities, while the remainder were rural and/or urban communities which were pluricultural in character. the phytogeographical heterogeneity of the bibliographical approach led to the inclusion of a high diversity of plants, life forms and botanical families that were used by the majority of the indigenous populations in the region [ , ] . fieldwork location: the fieldwork was carried out in the rural community of cuyín manzano, which is located in nahuel huapi national park, ( ° s and ° w), km from bariloche city. this location lies within the andino norpatagonica biosphere reserve (unesco). the climate in the region is temperate-cold and humid, with a mediterranean precipitation regime, such that rain and snow fall principally in winter (mat . °c). the rural community is located in an ecotonal environment between steppe and forests of austrocedrus chilensis and nothofagus spp. the population is of mestizo origin; some individuals are direct descendants of the mapuche people, while others have mixed ancestry, known locally as criollos (creoles). at present only families live here, due to strong processes of emigration. the economic activity of inhabitants has become more varied with time; they work with livestock, tourism, handicrafts and also as employees in the state school hostel and a private ranch. this community has a longstanding history of settlement in the area, according to archeological records [ ] . our previous studies in this location [ ] [ ] [ ] have shown that this population is very representative of a typical patagonian rural community. considering its low population density at present, the native and mixed ancestry of the people, its immigration and migration process, its contact with the city and the maintenance of its traditions, cuyin manzano reflects shared sociocultural patterns with other rural settlements. in addition, the fieldwork was greatly facilitated due to the close ties of trust we have built up with the people over several years. the information obtained from each source was systematized in a database according to the following criteria: ) the species should be identified taxonomically in the original study; ) it should grow in argentine patagonia; ) native and exotic species were included. native species were defined here as plants whose origin is central or southern argentina, below °southern latitude, and exotic species were those which did not fulfill this condition [ ] ; ) wild, semi-domesticated and cultivated species were included; ) the species were categorized as exclusively edible, and , functional, when the plant had at least one functional citation (edible, with also one or more medicinal uses) in the bibliography. following these criteria, studies and citations were included. each citation corresponds to a species referred to in a publication, with its corresponding ethnobotanical information. the following information was selected: species, common and indigenous names, richness of local foods, medicinal uses, herbal medicines made with peff and parts of the plant used (named plant drugs). the local foods were classified as: fresh fruit (f), jams and sweets (s), non-alcoholic drinks (d), fermented drink called "chicha" (ch), other (ot). the classification of ailments was adapted from molares and ladio [ ] : respiratory (rs), gastrointestinal (gi), urinary (u), pain and inflammation (pi), dermatologic (de), fever (f), obstetric (obs), gynecological (gyn), blood (bl), circulatory and heart (ch), nervous system (ns), cultural syndromes (cs), osteo-articular and muscular (oa), allergies (al), dentistry (den), ophthalmological (op), refreshment (re), others (ot). the different herbal medicines were classified according to method of administration: fruit ingestion (f), infusion (in), decoction (de), bathing, washing and rubbing (ba), gargling (ga), poultices and compresses (ca), creams and ointments (oi), other (ot). the plant drugs were categorized according to the part of the plant used: fruit (f), flowers (fl), leaves (l), branches (b), bark or stalk (s), root (r), the whole plant (pl). fieldwork was carried out according to the code of ethics of the international society of ethnobiology (ise) and the consensus statement on ethnopharmacological field studies [ ] . free listing, semi-structured interviews, in-depth interviews and participant observation were performed with participants [ , ] , prior oral informal consent. if the participant consented to being recorded during the interview, oral consent was also taperecorded. this procedure is supported by national research regulatory agencies and international agreements (argentinian national law n.° , ). a total of % of the population ( households) took part in the study, including several members of each family. the ages of the adult participants ranged from to years. inhabitants were consulted on their knowledge of the peff that grew in the area, and their alimentary and functional uses. the information gathered was reorganized following the categories used for the bibliographical review. the database included citations with information on peff. each citation corresponds to a species referred by each interviewee. the same quali-quantitative analysis [ ] was performed for both approaches. due to the categorical nature of the data, the analyses were principally non-parametric, using the spss package for windows. species richness (s) was calculated in total, according to biogeographical origin, and by botanical family. native and exotic species richness were compared using the binomial test (p < . ) [ ] . whether a native or exotic species was used as edible or functional was evaluated by consensus indices, mainly utilized for use pattern analysis of ethnobotanical data [ ] [ ] [ ] [ ] . these indices enable us to consider the agreement in a community or between diverse authors (and their publications) on the use of a certain species or plant family. the cultural importance index of edible plants (cie) was calculated for the species and for the families: cie = fc/n × , where fc = the frequency of citation of the species or family and n = the number of total publications/informants. we also calculated the cif index for functional species and families, as cif = fc/n × , where fc = the frequency of citation of the functional species or family. the mann whitney test was performed to compare these indexes for native and exotic species (p < . ). the percentage of each category was calculated in relation to the total reported methods of use of all the peff, that is, the total foods. %sc a = s sca / s sca..i × . where %sc a = the percentage of the subcategory a (for instance, drinks); s sca = the richness of species of the subcategory a; and s sca..i = the total of local foods. the use value is an index that indicates the versatility of a species, that is, the diversity of ailments that can be treated by a given species [ , , ] . it was obtained as follows: uvs = uvis/n [ ] , where uvis = the number of different medicinal uses registered by publication/informant for species s. similarly to local foods, the percentage of each category was calculated in relation to the sum of the plant richness for each subcategory. that is, in relation to the total medicines or plant drugs (medicinal parts of the plants). in the field study all native and exotic plant species were mentioned by their common names, and were later identified taxonomically by the authors. the collection of wild and cultivated species with fleshy fruits was performed with the assistance of local dwellers. in addition, field herbaria and photographs were utilized in the interviews to confirm the taxonomic identity of plants. plant identification followed correa [ ] [ ] [ ] plant specimens were placed in the ecotono-ethnobiology group-inibioma-university of comahue herbarium, and will be deposited in the herbarium of the centro regional universitario bariloche (bcru). voucher specimens of all reported species are shown in table . it should be noted that the berberidaceae family was treated according to landrum [ ] . all scientific names were updated using world flora online (www.worldfloraonline.org). a total richness (s) of species was found through the bibliographical review (table , fig. a ). the average richness per study was . species (min: , max: ). richness of native species ( %, species) was notably greater than that of exotic plants ( %, species) (p < . , binomial test). coincidentally, the cie of native peff was higher than for exotic plants (p < . , mann whitney test) (fig. b) . the native species with highest cie were berberis microphylla, ribes magellanicum, fragaria chiloensis, ephedra ochreata, aristotelia chilensis, gaultheria mucronata, berberis darwinii and berberis empetrifolia (fig. a) . the exotic species with highest cie were sambucus nigra, rubus idaeus, prunus cerasus, rosa rubiginosa, ribes aureum and ribes uva-crispa. the peff belonged to botanical families, the main ones being rosaceae ( species), grossulariaceae ( species), anarcardiaceae and berberidaceae ( species each). the families with the highest cie values were berberidaceae, rosaceae and grossulariaceae. in the bibliography, local foods were identified. most publications reported the use of fresh fruits ( % of the total local foods). a wide variety of preparations were found that generally included the addition of sugar, such as jams and jellies ( %). the most significant species used to make jams were b. microphylla, b. darwinii, r. magellanicum, r. cucullatum and f. chiloensis. another important way of taking advantage of peff was the preparation of drinks, mainly as soft drinks. these included non-alcoholic beverages ( %) made from the fruit of different species, such as ephedra triandra, aristotelia chilensis and berberis microphylla. the next most common drink was the fermented "chicha" ( %), which used different peff, amongst which stood out schinus polygama, geoffroea decorticans, r. magellanicum and amomyrtus luma. the production of "chicha" from a. luma mixed with fruits of other species is also mentioned. fifteen percent of local foods includes preparations with cooked, boiled or roasted fruits, as in the case of maihueniopsis darwinii. wines are also prepared, such as those made from a. chiloensis fruit, or liqueurs from b. microphylla or gaultheria poeppigi fruits. similarly, this category includes flours such as those made with condalia microphylla and geoffroea decorticans fruits. of the total number of species registered, the proportion of functional peff was % ( species, table ), similar to the non-functional species ( %, species) (p > . , binomial test). the richness of functional species was composed mainly of native species ( %, p < . , binomial test). it was also found that the cif of native species was significantly higher than that of exotic plants (p < . , mann whitney test). the principal functional species according to the cif values were the native aristotelia chilensis, ribes magellanicum, ephedra ochreata, berberis microphylla, fragaria chiloensis, luma apiculata and amomyrtus luma, and the exotic sambucus nigra, rosa rubiginosa and prunus cerasus ( table ) . the functional peff found belonged to botanical families. among these were anacardiaceae ( species); berberidaceae, ephedraceae and rosaceae with species each, and myrtaceae with species. at botanical family level, elaeocarpaceae, ephedraceae, grossulariaceae, myrtaceae, anacardiaceae, rosaceae, adoxaceae and berberidaceae were families with high cif values. the functional peff registered in the bibliography were used for a wide range of ailments (table ) , mainly gastrointestinal, respiratory, dermatological, gynecological, obstetric, related to the nervous system, heart and circulatory system, fever, pain and inflammation. the most versatile species according to their uv value were aristotelia chilensis, used to treat more than ten ailments, followed by berberis microphylla, luma apiculata, sambucus nigra, fragaria chiloensis and fuchsia magellanica ( table ) . the peff used as herbal medicines totaled (table ) , which mainly included forms that can be ingested, but also some of external use. the most important were two forms of drink: decoctions ( %) and infusions ( %). for example, infusions made of leaves or bark of r. magellanicum were used to "componer la sangre" (depurative) and for digestive ailments. a decoction of b. microphylla bark is also used to bring down a fever, or its fruit is used to combat diarrhea. the direct ingestion of a. chilensis fruit was also used to treat fever and diarrhea. among the methods of external use that stand out were: bathing ( %), for example, using luma apiculata leaves to bathe infected wounds; gargling ( %), with the same species but in this case to treat lesions of the gums. the application of exudates and poultices ( % each) has also been documented, for which the aerial parts of the plant were generally used, such as the exudate of schinus johnstonii leaves used to treat toothache. the exudate of l. apiculata as an anti-inflammatory is also frequently cited in the literature [ , , ] . poultices such as those prepared from dried a. chilensis leaves were used to clean wounds. creams and fresh fruit made up the remaining %. the parts of the plant most frequently used medicinally were the leaves ( %), fruits ( %), bark or stalk ( %) and roots ( %). a total richness of peff was found (table , fig. a) , an average of species being cited per informant (min: , max: ). this richness was divided equally between native ( species) and exotic ( species) plants (p > . , binomial test). in contrast to the findings from the review, in cuyín manzano the cie was the same for native and exotic species (p > . , mann whitney test) (fig. b) . the native species with highest cie were berberis microphylla, fragaria chiloensis and aristotelia chilensis (fig. a) . these were followed by other berberidaceae species, such as the "michay de la costa" (b. empetrifolia) and "michay de cordillera" (b. serratodentata). according to the cie values, some of the most important exotic species were rosa rubiginosa, prunus cerasus, sambucus nigra, prunus avium, prunus domestica, and finally rubus idaeus, which is known throughout the world (fig. a) . the peff in cuyín manzano belonged to eight botanical families. rosaceae contributed most to the list ( the asterisks show the species listed in the iucn red list of threatened species * = least concern, ** = near threatened, *** = vulnerable, **** = endangered, na native, ex exotic, f fruit, s sweets, d drinks, ch chicha, o others, nm not mentioned chamorro and ladio bmc complementary medicine and therapies ( ) : page of species), followed by grosulariaceae ( species) and berberidaceae ( species). however, the families with the highest cie were berberidaceae, rosaceae and elaeocarpaceae. in cuyín manzano local foods with peff were registered. of these, % were consumed as fresh fruits. in general, this method of use implied consumption at the time of gathering, with no storage involved. locals reported consuming these fruits when they were out in the countryside and felt hungry, the main reasons being that they like them a lot (such as the berberis fruits), and they are "good for them". to a lesser extent, the peff were lightly processed, through the addition of sugar or cream. also frequently consumed were sweets ( %), including jams, jellies and syrups made from mature fruit. among other preparations ( %), were the alcoholic beverages, which in this case played a role as social drinks. the most frequently cited of these was the "guindado" made with the fruit of the exotic prunus cerasus, which was served to visitors in the home. non-alcoholic drinks represented % of this category, mainly in the form of infusions, such as those prepared with the fruit of rosa rubiginosa and sambucus nigra, but also in the form of juices. other forms of consumption were also mentioned, such as the addition of fruit to baking products in the home. furthermore, the fruit tended to be used in the form of desserts, as in the case of stewed fruit, where fresh fruit such as plums were boiled in water and sugar. consumption of dried fruit called "orejones" was also registered. it is worthy of note that the preparation of "chicha" from peff was not mentioned here as it was in the review. a total of species ( %) were known to locals for their functional value (table ) ; however, the proportion of edible and/or functional species did not differ statistically (p > . , binomial test). the functional species included native and exotic species. in contrast to the review, in cuyín manzano the cif values were similar for native and exotic species (p > . , mann whitney test). as shown in table , the functional peff with highest cif were the exotic rosa rubiginosa and sambucus nigra, followed by the native aristotelia chilensis, ribes magellanicum, berberis microphylla and ribes cucullatum. the peff in cuyín manzano belonged to botanical families: grossularaciaceae ( species), and rosaceae, berberidaceae, elaeocarpaceae and adoxaceae ( species each). in terms of cif, the families that stood out were: adoxaceae ( %), rosaceae ( %), and to a lesser extent, elaeocarpaceae ( %). the peff in cuyín manzano were used principally to treat respiratory, gastrointestinal and dermatological ailments (table ) . sambucus nigra was one of the most versatile species, followed by rosa rubiginosa, aristotelia chilensis and ribes magellanicum, and finally, berberis microphylla. the inhabitants of cuyín manzano used medicines prepared from peff to treat specific local ailments (table ). these species were mainly used in the form of infusions ( %), utilized generally to treat or prevent respiratory disease. one of the most frequently used infusions was sauco (elderberry) tea, which was drunk at night to "warm up the body", and probably played a preventative role. this infusion was prepared by putting a spoonful of sambucus nigra syrup in a cup, then adding boiling water. the syrup was also consumed alone, without dilution, mainly by children but also by adults suffering cold symptoms ( %). mosqueta (rosehip) tea was used similarly as a recreational drink, to warm up the body and treat illness. fresh fruits were always consumed ( %) in the context of relieving gastrointestinal problems, and decoctions were also prepared ( %). the only external method of application was using creams ( %) made from rosa rubiginosa fruits by women from a nearby community. the medicinal parts of functional peff are mainly the fruits ( %) ( table ) , and to a lesser extent, leaves ( %), the whole plant ( %), followed by flowers, seeds, bark and roots ( % each). finally, five of the medicines were also considered as food by locals: elderberry and/or rosehip infusions, the fresh fruits of maqui and michay, and elderberry syrup, which in total represent % of local foods. the bibliographical analysis revealed a high number of native and exotic peff ( species), providing an overall picture (spanning years and an ample geographical range) that shows the study potential of all these patagonian species. the nutritional role of this plant diversity and the safety of their use is still unknown, but they presumably represent food security for locals. food security is founded on three elements: the availability, access, and utilization of food, elements that were elucidated indirectly in both the bibliographical texts and the testimonies of cuyin manzano inhabitants. it is also important to consider that the conservation status of some of the native species ( species, table ) is challenging, and must be solved by cultivation and/or sustainable management, given that their availability and access are threatened. the need for an integrated study of these aspects must be taken into account in future investigations. moreover, the field study showed that peff on this list continue to be used. although this rural community is probably experiencing a process of dietary transition from smallholder farming-based to industrialized food systems, as well as integration into the global food trade, peff continue to be part of their local heritage. lifestyle changes probably affect food production, plant gathering and consumption, with consequences for the healthfulness of diets, but these species still have an appreciated role in home-produced foods. further field research should be carried out in other patagonian communities, using this tool to survey food security in order to investigate whether local people keep peff use alive in their food traditions. this aspect is of great importance given the processes of abandonment and loss of knowledge associated with plant use observed in cuyín [ ] and numerous other rural communities throughout the world [ ] . our cross-sectional approach coincides in the cultural importance of the three native species with highest consensus: berberis microphylla, fragaria chiloensis and aristotelia chilensis. the michay plant has attractive blue-violet berries with a flavor which is sweet, but sour, and is widely distributed in forest and the patagonian steppe, therefore present also in the most arid zones. the native strawberry has strongly scented fruits, and has been used to obtain fragaria x annanasa, the strawberry which is commercially cultivated throughout the world [ ] . the berries of the maqui plant are acid to the taste, and have been designated a superfood due to their high content of anthocyanins [ , ] . at present these species are being intensively studied as target species [ ] and are attracting much attention for future innovation and development projects [ , ] . the remainder of the list of peff may also be considered by policy makers and researchers in the planning of agricultural and phytochemical research action that will support sustainable diets and local food systems. all this biodiversity is introduced into local gastronomy through a large variety of local foods. remarkably, in both studies the use of fresh fruit is high ( % in the review and % in cuyín manzano). traditional culinary habits could promote better use of nutrients, since a lower level of processing generally leads to greater availability of nutrients such as vitamin c [ ] . in second place, the preparation of preserves and beverages from the fruit indicates strategies that favor preservation over time, storage, and the use of excess fruit, as found in other parts of the world [ , ] . preserves include some forms, such as syrups, which are of great interest due to the compounds that contribute phenolic acids, fiber, and soluble solids [ ] [ ] [ ] . these plant preparations could be also highlighted, as local people could be skillfully managing the plant chemicals during food processing. patagonian communities have experimented extensively with the diversity of peff fruits in the preparation of fermented beverages, mainly in the form of "chicha". this drink is made with fruit and seeds, which in many cases were chewed by the people before being stored for fermentation. this preparation holds great cultural and spiritual value, as it is drunk in traditional mapuche festivities [ ] . nevertheless, in cuyín manzano preparation of these beverages with the peff berries was not identified. although the medicinal use of these preparations was not documented, some studies associate moderate consumption of fermented beverages with prevention of cardiovascular diseases and cancer, due to their polyphenol and alcohol content [ ] . this opens up a wide range of possibilities for the exploration of fermented beverages, taking into account ancient food traditions. these customs are considered key elements for development projects related to food sovereignty [ ] . the peff heritage possesses a great richness of species whose functional value has been well taken advantage of, and which can therefore constitute alternative healthy or phytotherapeutic food. the species taken from the bibliography and listed here are mainly native to patagonia, and represent opportunities for study, for which the consensus values can be a useful guide. in this sense, our findings are in accordance with jennings et al. [ ] as to functional species use. to understand the meaning of their functionality, contextualization of the food-plant spectrum based on both local beliefs and wider structural factors is needed. in our field study we found that dwellers consider that the fruit did them good. from the mapuche worldview, their consumption of wild fruits implies nourishing the earth's energy (called in native mapuzungum "afutum"), energy that cannot be transferred in any other way [ ] . in other words, the classification of a species as functional is inseparably supported by both the local worldview and its chemical and biological attributes. a whole universe of chemical compounds may be related to the health effects of the peff. the medicinal value of the species used as food has been explained in detail by authors such as johns [ , ] , based on the presence of a wide range of phytoconstituents. today we know that food plants have diverse constituents such as polyphenols, in addition to nutritional compounds. these molecules are recognized for their antioxidant properties; they are important radical scavengers through inhibition of prooxidant enzymes and restoration of antioxidant enzymes. however, new mechanisms of action have been proposed to explain their bioactivity, such as interaction at the level of the plasmatic membrane with proteins and phospholipids, and the regulation of signal transduction pathways [ , ] . of the peff, those which have berry-type fruits stand out as functional species, and are known worldwide as healthy foods. berries contain vitamins, minerals and phenolic compounds, and are credited with antioxidant, antiaging, chemoprotective and chemotherapeutic, anti-inflammatory and neuroprotective properties [ , ] . with a strong consensus between our two studies, the key species are: a. chilensis, ribes magellanicum, ephedra ochreata and berberis microphylla. a. chilensis is the species with the highest number of described pharmacological properties, including the ability to attenuate pain and inflammation [ ] . this activity matches the local ailment treatments described in our work. little is known about ribes magellanicum, but the antioxidant potential in vitro of the fruits found in argentine-chilean patagonia has been demonstrated [ ] . unfortunately, ephedra ochreata is one of the leaststudied species in the region. concerns about it eventual toxicity, should be carefully considered, since other ephedra spp. have been demonstrated to exhibit toxicity (https://www.health.harvard.edu/staying-healthy/thedangers-of-the-herb-ephedra). it must be used with great care, given that toxic effects have been reported for other species of ephedra, such as cardiac and psychiatric disorder [ ] [ ] [ ] . future research should focus on e. ochreata in order to test its food safety. the study of other species, such as b. microphylla, have begun only recently, revealing promising antioxidant content and antidiabetogenic activity according to chamorro in vitro and in vivo studies [ ] [ ] [ ] . again, more studies and further analysis is required to relate these chemical and biological findings to the health claims presented here. similar to the peff heritage, the use of functional species seems to be undergoing hybridization processes. this was evident in the rural community we worked in, where the cif values were similar in native and exotic plants. these results show how locals have been using a combination of native and foreign species in edible and functional terms. although this has been happening for a long time in the region, it seems to be more prevalent at the present time [ ] , probably due to changes in food procurement. the use of exotic functional peff in our region may be interpreted as a reflection of the long, strong historical influence of european immigration on the use patterns of this area. various studies carried out in south america have shown that the introduction of eurasian plants led to diversification in the use of medicinal plants [ ] , and many edible fruits form part of the pharmacopeias brought from europe [ ] . shikov et al. [ ] and totelin [ ] suggest that the use of edible fruits as medicine is due to the great influence of the food-medicine conceptions of ancient greece. however, the food-medicine interface had also been described previously, without the use of exotic plants, in patagonian indigenous communities [ ] . we suggest, therefore, that this food-medicine continuum may have been generated independently, and that the knowledge of indigenous and european communities converged at a later date. some of the exotic berries registered in this work have been extensively studied with regard to the biological activity that supports their local functional use. in the case of sambucus nigra, there is in vitro evidence to support the effectiveness of its antibacterial and antiviral properties [ , ] . hawkins et al. [ ] recently found that elderberry fruit can improve respiratory complaints associated with influenza, and so mccarty and dinicolantonio [ ] propose its use (in a dose of - mg) for the treatment of covid- infection. rosa canina, a species closely related to rosa rubiginosa, has been studied with regard to its use in treating skin ailments [ ] . the dermatological use of r. rubiginosa and use of the other functional exotic species were also registered in our field work. this pattern shows how the berry repertoire grows with input from highly reputed exotic plants. with respect to ailments, the review shows that more than ailments have been treated with peff. in cuyín manzano this number is much lower (only five ailments) and is focused on primary health care issues. the broad scope of the review shows the potential of these species, even though the main ailments treated with peff are those most frequently treated by traditional medicine, such as digestive and respiratory complaints. several studies have proposed that this redundancy gives flexibility to the regional herbal medicine, the use of a. chilensis and s. nigra fruits standing out as being the most versatile elements the population has within its reach [ ] . the peff are also important sources of herbal medicines ( in the review and in cuyín manzano) since, as previously shown, different plant parts of the peff are used in an integral way. infusions (and decoctions according to the bibliography) constitute the main method of medicinal use in the region, and this is also, within local foods, the main form of beverage in cuyín manzano. recent research has revealed how infusions of exotic rosa rubiginosa fruit can contribute compounds with antioxidant activity [ ] . the same occurs with the infusion of exotic sambucus nigra fruit, which was shown to contain considerable amounts of polyphenols and anthocyanins, comparable to an ethanolic extract [ ] . infusions are therefore, in general, good vehicles for phenolic compounds, and this is possibly the basis for their functional value in patagonian cultures. according to our analysis of the information gathered from the review, complemented by the fieldwork, we can reinterpret the comprehensiveness of the food-medicine interface in patagonia, outlined in fig. . in the review work we found a notable superposition of use (edible and medicinal) for single native or exotic species (given by the sum of citations of different authors and different study sites). however, in the analysis of each one of the studies included in the review and the fieldwork, we observed that the patagonian communities have not only experimented with native and exotic plants and discovered their medicinal properties, but they have also developed diverse forms of preparation. these make up a wide range of methods of use, but also form part of local food tradition, which considers plants from the perspective of functional, complementary logic, for their general health in their daily lives, local foods and medicines. within this group there are forms of preparation that prove effective as food and medicine (superposition of use forms depending on context), known in the literature as food medicines or healthy foods [ ] . within this group we find the subgroup of the local functional foods. as shown in our field study, this is the case of rosa rubiginosa and sambucus nigra, two exotic berries that are used in the form of infusions and indistinguishable as food or medicine. they are used as a preventative food, which in nutrition is known as a functional food [ ] . these infusions seem to be important local functional foods, given that these water solutions are good vehicles for active compounds found in plants, such as polyphenols [ , ] . finally, the functionality attributed by a culture to its native and exotic species includes superposition at different levels, and may be described in theoretical terms as different categories. however, most important is that they are principally based on the use of the entire plant. in the case of patagonian native and exotic plants with edible fruit, the people seek food, healing, and also prevention ("it warms up the body", "it does you good") through the use of infusions and the ingestion of fresh fruit. previous studies have shown the potential of some native peff, due to their polyphenol content, but little has been studied on the teas prepared with them. more integrative research is required into this use pattern of native and exotic species, their local foods and their potential as local functional foods. argentine patagonia holds great richness of native and exotic peff and the cultures who have lived on these lands have experimented with them as a source of local foods and medicines, generating a food-medicine continuum. the living heritage of patagonian peff appears to have undergone hybridization processes, such that exotic cosmopolitan species play an increasingly significant 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grateful to the families from cuyín manzano for their willingness to participate, and for sharing their knowledge. special thanks to mr. antonio bergant, who was not only a great person, but also the best field assistant. the authors acknowledge with appreciation the constructive comments made by the referees andrea pieroni and timothy johns on the earlier version of this paper. we would also like to thank the cultiva (cyted) network. authors' contributions m.f.c.: conducted the review process, the fieldwork, analyzed the data and contributed to the manuscript. a.l.: conceived and designed the research, conducted the review process, analyzed the data and contributed to the manuscript. the author(s) read and approved the final manuscript. this investigation was supported by the consejo nacional de investigaciones científicas y técnicas (conicet) of argentina (pip ), and by centro de investigación y extensión forestal andino patagónico (ciefap). the datasets used and/or analyzed during the current study are available from the corresponding author on request. this study was conducted according to the ethics guidelines of ise code of ethics (http://ethnobiology.net/code-of-ethics/) and nagoya protocol (argentinian national law n.° ). all informants orally confirmed free and informed consent prior to data collection. no other specific additional procedure is mandatory for this kind of study in our country. not applicable. the authors declare that they have no competing interests.received: august accepted: may key: cord- -m dkkbq authors: pulliam, juliet r. c. title: viral host jumps: moving toward a predictive framework date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: m dkkbq in order to predict pathogen emergence, we must distinguish between emergence phenomena that occur via different processes. focusing on the appearance of viral pathogens in new host species, i outline a framework that uses specific molecular characteristics to rank virus families by their expected a priori ability to complete each of three steps in the emergence process (encounter, infection, and propagation). i then discuss the degree to which the patterns expected, based solely on molecular-level structural characteristics, agree with observations regarding the ability of animal viruses to infect humans. this approach yields predictions consistent with empirical observations regarding the ability of specific viral families to infect novel host species but highlights the need for consideration of other factors, such as the ecology of host interactions and the determinants of cellular susceptibility and permissivity to specific virus groups, when trying to predict the frequency with which a virus will encounter a novel host species or the probability of propagation within a novel host species once infection has occurred. emerging infectious diseases present challenges to the scientist and layperson alike (morse, ; burke, ; daszak et al., ; cleaveland et al., ; dobson and foufopoulos, ; taylor et al., ) . these infections of wildlife, domestic animals, and humans seem to be increasing and more problematic every year. the cause of the recent increase in scientific and public interest in emerging infections is probably twofold. on the one hand, technological advances in detection methods and in global communications allow us to be increasingly aware of emergence events as they occur (garnett and holmes, ) . many pathogens, including west nile virus, monkeypox virus, and h n highly path-ogenic avian influenza (hpai) virus have attracted widespread media attention. online news sources provide gripping, up-to-the-minute accounts of outbreaks occurring in locations that their primary audience may not even be able to identify on a map, such as the multimedia presentations produced by bbc news in of a marburg virus outbreak in angola (phillips, ) . on the other hand, anthropogenic activities have increased the actual frequency of pathogen emergence. habitat modification brings previously separated populations into contact and disrupts natural mechanisms regulating population dynamics. for example, increases in rodent populations due to clear-cutting of forested areas for the planting of food crops (particularly corn) is thought to have been responsible for outbreaks of several hemorrhagic fever viruses in south america, including junín and machupo viruses (buchmeier et al., ) . barriers limiting pathogen transmission are altered as we change the landscape and increase global transportation, providing unprecedented opportunities for transmission to new populations and species (schrag and weiner, ; smith et al., ) . these changes have implications not only for human health but for wildlife conservation and domestic animal health. long-distance animal transport, both intentional (for agriculture or the pet trade) and unintentional, facilitates pathogen introduction (daszak et al., ) . for example, illegal bird transport is the suspected source of the recent introduction of h n hpai into a poultry farm in the uk (promed-mail, abc) , and accidental transportation of mosquitoes aboard aircraft is thought to pose the highest risk for the introduction of west nile virus into hawaii and the galapagos islands (kilpatrick et al., (kilpatrick et al., , . considering the broad public and scientific interest in infectious disease emergence, surprisingly little work has been done to quantitatively describe broad-scale patterns of emergence in light of pathogen characteristics. much work on emerging infections has focused on specific emergence events, trying to determine when, where, and how a particular pathogen entered a new host population. the emergence of human immunodeficiency virus (hiv), for example, has been well-studied on both ecological and molecular levels. strong evidence suggests that the bushmeat trade in west africa has facilitated repeated retrovirus transmission events from wild primates into humans (wolfe et al., ) , and independent introductions of hiv- subgroups (m, n, o) from chimpanzees (pan troglodytes) and hiv- subtypes (a, b) from sooty mangabeys (cercocebus atys) are believed to be responsible for the ongoing hiv/aids pandemic (sharp et al., ) . in order to predict (rather than reconstruct) pathogen emergence, however, it is necessary to extend the domain of inquiry beyond the focus on a single disease. specific ecological processes and pathogen characteristics must affect the probability of emergence, and until these factors are recognized we will have little predictive power. burke has suggested that the high ''evolvability'' of rna viruses facilitates host jumps (burke, ) . such evolvability is derived from a combination of high replication error rates and the ability to reassort and recombine. although he makes no attempt to quantitatively determine the relative frequency of emergence for different types of pathogens, burke claims that recent pandemics in humans and wildlife have mostly been caused by rna viruses, citing multiple examples (influenza a, hiv- , enteroviruses and , human t-cell lymphoma virus, three paramyxoviruses, porcine respiratory coronavirus, and a calicivirus that causes hemorrhagic disease in rabbits). he goes on to predict that future global pandemics will be caused by groups of viruses that have caused major epidemics in humans or animals and that have high intrinsic evolvability; however, this approach does not allow for identification of viral groups with high emergence potential that have not produced large epidemics to date. in particular, if undiscovered virus families exist that have a high potential for emergence-whether in humans or in animals-a priori identification of this potential may facilitate preparedness for and rapid response to emergence events. a more quantitative approach was taken by three studies published in a special issue of philosophical transactions of the royal society of london in . the first two of these studies (cleaveland et al., ; taylor et al., ) used a broad-scale literature search to construct a database of known infectious diseases of humans and domestic animals and to classify these diseases as emerging or not. the authors were then able to describe general patterns related to emergence status. they found that viruses are significantly more likely to be classified as emerging than are bacteria, fungi, helminths, or protozoa and that the ability to infect multiple host species is a significant risk factor for emergence in humans and domestic livestock. a recent update of the work on human pathogens has confirmed these findings with a larger dataset (woolhouse and gowtage-sequeria, ) . a similar approach was taken by dobson and foufopoulos ( ) , who surveyed emerging infectious diseases of wildlife based on promed reports over a -year period. emerging pathogens of wildlife were also found to be primarily viral. the results of these studies suggest that further examination of the molecular characteristics that determine viral emergence will go a long way toward being able to predict pathogen emergence. to develop a predictive framework, however, we must distinguish between the different phenomena that the term ''emergence'' commonly encompasses. these include the appearance of a pathogen in a new host species (woolhouse, ; antia et al., ; fenton and pedersen, ; , the appearance of a pathogen in a new population of an established host species (or new geographic region) (lederberg et al., ; cleaveland et al., ; taylor et al., ; smolinski et al., ; smith et al., ) , the appearance of an antimicrobial-resistant variant of a pathogen within an established host population (lederberg et al., ; smolinski et al., ) , and a change in the immunological interaction between a pathogen and an established host population (smolinski et al., ) . different mechanisms drive each of these phenomena; therefore, the ability to predict each type of emergence will require different approaches. i focus on the appearance of viral pathogens in novel host species. i first describe the process by which this type of emergence occurs and then ask the question of whether specific molecular characteristics can be expected a priori to affect the crucial steps in this process. this approach allows for the generation of testable hypotheses regarding the emergence potential of specific viral groups. i outline these hypotheses and then determine whether expectations based on the molecular characteristics discussed correspond to observed broad-scale patterns. agreement with observed patterns may suggest that the molecular characteristics explored will yield substantial predictive power and a useful framework for ranking virus groups in terms of emergence potential. inconsistencies between the patterns expected based on molecular characteristics alone and observed patterns highlight specific parts of the emergence process as highly dependent on host-host or host-virus interactions and less dependent on molecular characteristics of the viruses themselves. a glossary is provided to clarify the use of terms that may be unfamiliar to the reader or that are used inconsistently within the current emerging infectious disease literature. a virus is an obligate intracellular parasite that relies on the molecular machinery of its host for reproduction. all viruses comprise genetic material (rna or dna) and a protein coat. virus particles (virions) of many species also contain host-derived lipids and carbohydrates. to reproduce, a virus must encounter a host organism and make its way to the site of replication within a host cell that is both susceptible and permissive. the virus then makes new copies of itself, which requires transcription of the genome to produce mrna, protein synthesis, genome replication, and packaging. new virions must exit the cell to infect new host cells and, potentially, new host organisms. to persist, emerging viruses must be able to perform all of these steps within their new host species (webby et al., ) . host jumps, or cross-species transmission events that result in the successful infection of a potential host species, may encounter barriers at any step in this life-cycle. molecular characteristics of the virus itself are expected to play a large role in determining both whether a host jump occurs and the probability of transmission between individuals of a new host species. host jumps will be considered to occur as a three-step process . encounter between a virion and a potential host species, . infection, or replication within an individual of a novel host species, and . propagation, or transmission of infection from one individual of the new host species to another. the probability that, and frequency with which, a potential new host will encounter a virus circulating in another species may be determined by the nature of interaction between current and potential hosts, by the frequency, duration, and distribution of viral shedding in the current host population, and by the virion's subsequent stability in the environment. ecological interactions between current and potential host species can be divided into four categories: . those involving contact with bodily fluids, . those involving direct contact between individuals but not contact with bodily fluid, . those involving contact with a shared resource, and . those involving spatial overlap. interactions involving between-species contact with bodily fluid, such as predation, seem most likely to facilitate host jumps. slaughter and consumption of bush-meat, for example, is responsible for the transmission of multiple retroviruses from wild primates to humans (wolfe et al., ) , lending evidence to the idea that host jumps are likely to occur as the result of trophic interactions. shared vectors are also known to mediate transmission of viral infections between species, as occurs when japanese encephalitis virus is transmitted between waterfowl (particularly herons), pigs, and people (endy and nisalak, ) . because predators have direct contact with their prey, they are likely to be exposed to any infections that their prey harbor, regardless of the site of excretion or transmission route, whereas a shared vector will only facilitate cross-species transmission of pathogens with high viremia. frequent direct contact, such as commensal relationships between humans and domestic carnivores, is the second most likely type of interaction to lead to the encounter of virions shed by other host species. encounter will be high for infections transmitted via respiratory secretions, urine, feces, or skin lesions (such as an infectious rash). depending on the nature of the commensal relationship, viral shedding in saliva may also lead to frequent encounter, either directly (as a result of licking or biting) or via a fomite (such as a pet's toy). virions found exclusively in the semen, milk, and blood, on the other hand, will have a low probability of transmission between commensal species. encounter may also occur between species competing for a shared resource. while competition rarely involves direct contact between individuals of different species, dissemination of virus particles through respiratory secretions, urine, or feces may be concentrated around a shared resource. under these circumstances-as with fomite transmission between commensal species-the stability of the virion in the environment is likely to be important in determining the probability of encounter between an infectious virus particle and a potential host species. similarly, transmission between species that overlap only in their spatial distribution may occur when virus particles are resistant to degradation but may be less frequent than in the case where current and potential host species compete for a particular resource. figure summarizes the expected interaction between the ecological relationships of current and potential host species and the frequency of encounter.because direct contact with infectious fluids implies encounter, ecological considerations will dominate the encounter process for the majority of source and recipient host pairs. molecular considerations will be most important when there is no direct contact or shared vector between current and potential hosts. in such cases, the frequency with which a potential new host encounters infectious virus particles will depend on virion stability in the environment. two main properties of the virion are likely to influence the rate of inactivation: whether the virus particle requires an envelope for entry into the host cell and the arrangement of structural proteins within the virion. though the relative rates of inactivation of different virus species depend on specific environmental conditions (such as temperature and relative humidity), requiring an envelope for successful cellular infection may generally decrease the ability of a virus to survive outside a host, since the phospholipid membrane will be sensitive to desiccation, ultraviolet radiation, changes in ph, and ozone toxicity (cox, ) . thus, the ability to enter host cells without an envelope is expected to increase emergence potential. similarly, isometric and complex structural arrangements contain many contacts between proteins relative to their helical counterparts. these arrangements are therefore more stable against physical damage, reducing the rate of inactivation and increasing expected frequency of encounter. once a virion encounters an individual of a potential host species, the probability it will be able to replicate depends on the specificity of infection and the ability to generate genetic diversity. viruses preadapted for emergence are likely to enter cells via highly conserved receptors (woolhouse, ) , to have generalized immune evasion strategies that modulate the host's innate immune response (webby et al., ) , and to have the potential for rapid evolution (burke, (burke, , . since the receptors that mediate host-cell entry are not generally known, grouping viruses by the level of taxonomic conservation of the receptors required for cell entry remains an important goal for the future. similarly, while interference with host immunity has been demonstrated for several emerging viruses (webby et al., ) , the ability of other viruses to modulate the innate immune response has been largely unexplored. the site of replication within a cell may also influence infection specificity. entry into the nucleus provides an additional barrier to successful replication not present for viruses that replicate in the cytoplasm. replication in the nucleus may therefore increase specificity and decrease potential for infection of novel host species. finally, the generation of genetic diversity increases the probability of infecting a potential host species by increasing the genetic state-space covered by the virus population and therefore the chance that some virus particles will successfully infect a novel host and reproduce (burke, ; webby et al., ) . although successful invaders may make up a tiny fraction of the population within the source host species, natural selection will increase the abundance of these variants within an infected individual of the new host species. the ability to generate genetic diversity depends primarily on the replication error rate and the potential for exchange of genomic material between genetically distinct virus particles during cellular coinfection (burke, ) . while many authors (burke, (burke, , cleaveland et al., ; suggest that rna viruses are more likely to emerge than dna viruses because of their high replication error rates, it may be more useful to focus on the primary reason for the difference in error rates: the availability of proofreading mechanisms during genome replication. although host proofreading mechanisms are used when replication relies on a host-derived polymerase, most viruses that encode their own polymerase (except large dna viruses; king et al., ; huang et al., ; willer et al., ) lack error correction and therefore have relatively high replication error rates. additionally, the exchange of genetic material between closely related viruses is facilitated by two processes during cellular coinfection: . recombination (exchange of genetic information during replication, usually via template switching), and . reassortment (copackaging of genome segments from genetically distinct parent virions). packaging multiple genome copies facilitates the recombination process (lederberg et al., ; burke, ) ; however, additional mechanisms that may promote viral recombination are poorly understood. reassortment requires only that the viral genome be divided into distinct segments (analogous to eukaryotic chromosomes) and that cellular coinfection occurs in nature. the probability of transmission between individuals of a new host species is of interest in addition to the probability of infection of a novel species (may et al., ) . evolutionary potential to increase spread between individuals of the new host, however, should rely on the virus's ability to produce genetic diversity, similar to the ability to infect and reproduce in a new host species (schrag and weiner, ) . as demonstrated by antia and colleagues, even a small increase in the basic reproductive number of a pathogen within a new host species can substantially increase the probability that the pathogen will eventually evolve the ability to propagate at a sufficient level to produce an epidemic within the new host species (antia et al., ) . substantial evidence suggests that rapid adaptation of plant and animal populations to a novel environment is more likely to occur through limited gene flow between related (but significantly diverged) populations than through point mutation (lewontin and birch, ; reiseberg et al., ) . if this holds for virus populations, recombination and reassortment may be the primary evolutionary factors permitting adaptation to propagation within a new host species; however, several virus species well known for their ability to propagate within a novel host species demonstrate that figure . the expected interaction between the ecological relationships of current and potential host species and the frequency of viral encounter. the darkest boxes represent interactions where the frequency of encounter is likely to be high, the lightest boxes represent interactions where encounter is expected to occur at very low frequency, and medium boxes represent interactions where the frequency of encounter will be highly dependent on the stability of the infectious virion in the environment, as described in the text. for all ecological relationships between species, the actual frequency of encounter via different transmission routes will depend on the exact nature of the relationship. substantial genetic change is not necessary for adaptation to propagation in all cases. sars coronavirus, for example, appears to have adapted to transmission between humans through a series of point mutations in multiple genes (holmes, ) . several mutations in the receptor-binding domain of the sars coronavirus glycoprotein that increased binding affinity to human ace (the receptor for entry into the cell) are well documented and appear to have been especially important (the chinese sars molecular epidemiology consortium, ; li et al., ) . table groups mammalian virus families according to each of the molecular traits discussed above for which data are available. mammalian viruses are used for the purposes of illustration because they are particularly well studied and because mammals represent many of the source and recipient host species of primary interest (e.g., humans, domestic livestock, and domestic carnivores). a rough assessment of expected emergence potential is made by assigning scores for encounter, infection, and propagation potential, which are given and described further in table . the score indicates how many traits a virus group possesses that are expected to increase success in that step, based on two standard reference texts (van regenmortel et al., ; tidona and darai, ). thus, virus families can be ranked based on molecular characteristics alone in order of the a priori expectation that they will be able to complete a particular emergence step. complex or isometric protein arrangement and the absence of an envelope are expected to increase encounter when there is no direct contact between current and potential host species. virus families with both of these characteristics provide some examples of host-jumping viruses; however, the absence of these characteristics clearly does not prohibit cross-species virus transmission. the orthomyxoviridae, coronaviridae, filoviridae, paramyxoviridae, and rhabdoviridae all have encounter scores of / but provide a plethora of examples of host-jumping virus species: the influenzaviruses, sars coronavirus, ebola and marburg viruses, hendra and nipah viruses, and rabies virus, respectively. assuming the traits of interest are good indicators of a virion's susceptibility to environmental stresses, stability in the environment does not seem to be a necessary prerequisite for cross-species transmission, probably because many hosts interact in a way that permit direct viral transmission, as noted earlier. in order to assess how well expected patterns of cross-species infection match observed patterns, it is necessary to consider infection within a context where encounter is expected to occur. when we consider the probability that a virus population will be able to reproduce within a potential host that has close contact with a current host, the characteristics in the left-hand columns of table dominate emergence potential. reassortment or recombination, low replication fidelity (i.e., lack of proofreading), and the ability to complete replication within the cytoplasm are all expected to increase cross-species infection potential. using these three criteria to rank mammalian virus families in terms of expected infection potential indicates that three families present the highest risk: arenaviridae, which comprises multiple viral species transmitted from rodents to commensal humans, including those responsible for lassa fever and four south american hemorrhagic fevers; bunyaviridae, which includes the hantaviruses, similarly transmitted from rodents to humans; and reoviridae, of which of the species that circulate among rodents and livestock are known to infect humans (tidona and darai, ) . in addition, nearly all high-profile viral zoonoses have at least two of the three characteristics expected to promote infection of a novel host species, including hiv-like viruses (retroviridae), influenza viruses (orthomyxoviridae), rabies virus (rhabdoviridae), ebola and marburg viruses (filoviridae), hendra and nipah viruses (paramyxoviridae), and sars coronavirus (coronaviridae) (tidona and darai, ) . one exception is monkeypox virus (poxviridae), which replicates in the cytoplasm but has high replication fidelity and, according to the simple genome-packaging division used here, little recombination/reassortment potential. it is worth noting that recombination of poxviridae species has been demonstrated in laboratory experiments (see, for example, yao and evans, ) . thus, rather than implying that recombination/reassortment potential is not an important contributor to infection of novel host species, this exception may indicate that the simple molecular traits considered here are insufficient to capture the full range of recombination mechanisms available to host-jumping viruses. of the mammal viruses lacking all three traits associated with infection of a novel host (genome reassortment a recombination potential is determined by the number of copies of the genome that are packaged in a virus particle (although other, unknown factors also facilitate recombination); reassortment potential is determined by the number of genome segments (single versus multiple). high recombination or reassortment potential is expected to increase potential for both infection and propagation. b replication fidelity is determined by the origin of the polymerase (host or viral) and the associated proofreading activities. virus groups that rely on a host polymerase for replication are subject to host proofreading mechanisms and have high replication fidelity. most virus groups that encode their own polymerase have no proofreading and therefore low replication fidelity; however, polymerases encoded by members of the poxviridae, adenoviridae, and herpesviridae are known to have ' to ' exonuclease activity (king et al., ; huang et al., ; willer et al., ) , which is the primary type of error correction expected to affect viral replication fidelity. viruses in these groups are therefore expected to have high replication fidelity. low replication fidelity is expected to promote the generation of genetic diversity and therefore increase potential for infection and propagation. c the site of replication is considered to be the cytoplasm if all steps in the replication cycle take place within the cytoplasm of the infected cell; the site of replication is denoted as the nucleus if any step in the replication cycle (e.g., genome replication or transcription into mrna) requires nuclear entry. the ability to complete replication within the cytoplasm is expected to increase relative infection potential, as nuclear entry provides an additional barrier to replication. d the presence (+) or absence (-) of a lipid envelope will affect the stability of a virion in the environment. e the arrangement of structural proteins within the virion is denoted as isometric, helical, or complex. isometric and complex arrangements are more structurally stable than helical ones due to the larger number of protein contacts. f virions of some genera of the poxviridae are enveloped upon exiting the cell; however, an intact envelope is not required for the virus particle to remain infectious (tidona and darai, ) . g the lone member of the family asfarviridae, african swine fever virus, encodes a viral polymerase responsible for genome replication (fauquet et al., ) . no studies were found in web of science searches that examined whether the viral polymerase has ' to ' exonuclease activity; however, the other families of large dna viruses do encode polymerases with known proofreading activity (king et al., ; huang et al., ; willer et al., ) and it is assumed here that the polymerase encoded by african swine fever virus has undocumented exonuclease activity. or recombination, error-prone replication, and ability to replicate in the cytoplasm), none is known to infect humans, regardless of close association between humans and the animal host. though rough, these three criteria appear to yield good predictive power regarding which viruses will be able to infect and replicate within novel host species. finally, the probability that a viral epidemic will occur via transmission within the new host species is of interest. table accounts only for the role of generation of genetic diversity in propagation. drawing from examples of mammal viruses that jump to humans, it becomes clear that other factors must be important determinants of propagation. while hiv (retroviridae) and influenza (orthomyxoviridae) propagate well in human populations, as predicted, members of the arenaviridae and bunyaviridae are rarely transmitted from person to person, despite having both characteristics expected to be associated with high chances of propagation. as with encounter, molecular characteristics of the viruses themselves do not appear to provide an a priori indication of a viral group's ability to propagate within a table . encounter, infection, and propagation scores for mammalian virus families, determined from table virus family a encounter b infection c propagation d reoviridae / / / retroviridae / / / astroviridae / / / caliciviridae / / / picornaviridae / / / poxviridae / / / adenoviridae / / / circoviridae / / / papillomaviridae / / / parvoviridae / / / polyomaviridae / / / arenaviridae / / / bunyaviridae / / / arteriviridae / / / flaviviridae / / / togaviridae / / / bornaviridae / / / hepadnaviridae / / / asfarviridae / / / herpesviridae / / / orthomyxoviridae / / / coronaviridae / / / filoviridae / / / paramyxoviridae / / / rhabdoviridae / / / a families are sorted in order of decreasing encounter score, then decreasing infection score, and finally decreasing propagation score. b the encounter score denotes the number of characteristics a group has that are expected to increase exposure frequency when the current and potential hosts have little or no interaction. characteristics that increase encounter will be those that increase the stability of a virion in the environment: being infectious without a lipid envelope and having an isometric or complex virion. c the infection score denotes the number of characteristics a group has that are expected to increase the ability to infect a novel host species: high recombination/reassortment potential, low replication fidelity, and an ability to complete replication within the cytoplasm. d the propagation score denotes the number of characteristics a group has that are expected to increase propagation: high recombination/reassortment potential and low replication fidelity. novel host species. host ecology and host-virus interactions rather than general molecular characteristics appear to determine propagation potential. the approach taken so far has shown that the ability to infect a novel host species depends heavily on molecular traits of the virus, independent of host ecology and details of the host-virus interaction; however, the approach has also highlighted the need to understand the ecology of interactions between species when considering expectations of encounter and the ecology of within-species interactions when considering expectations of propagation. host-virus interactions occurring primarily at the molecular level may also to play a large role in determining encounter and propagation, and the next steps in developing a predictive framework will examine these issues more closely. stability in the environment does not appear to limit the step of pathogen encounter for most host-jumping viruses. this is not surprising in light of the expected roles of host-host and host-virus interactions in determining encounter, which are outlined above and depicted in figure . determining whether the proposed interactions work as predicted will be extremely difficult. in fact, observation of encounter independent of infection may be impossible, whether in the field, in the laboratory, or using a synthetic database approach. one useful approach may be to look within a particular category of ecological relationships and at potential source-potential recipient host pairs. the relative frequency of different sites of excretion of viruses infecting the source host could then be compared to the relative frequency of different sites of excretion of viruses transmitted from the source host to the recipient host. databases used for previous analyses of emerging infections in livestock, domestic carnivores, and humans (cleaveland et al., ; taylor et al., ; woolhouse and gowtage-sequeria, ) could be combined with data on the site of excretion as a first step in quantifying the interactions between ecological relationships, site of viral excretion, and risk of encounter. while this approach will measure infection rather than addressing encounter directly, infection implies that encounter and factors that affect infection directly, such as the molecular traits discussed here, can be controlled for in the analysis. consistent patterns of shedding in the source host across host pairs with a particular type of ecological relationship will indicate that viruses shed through particular sites of excretion are more likely to be encountered than others. as ecological relationships between species change, knowledge of these patterns will provide an indication of the risk of encounter with viral species harbored by a newly contacted host species. in the pet trade, for example, where species that are isolated in nature live in artificial, commensal-like conditions, sometimes at high densities, the expectations laid out in figure suggest that viruses shed via respiratory secretions, urine, feces, or skin lesions-or those shed in the saliva if animals are kept in close enough quarters for biting to occur-are likely to be transmitted between species. this type of change in ecological relationships was responsible for the emergence of monkeypox virus in the us in , as african rodents were brought into contact with prairie dogs, which then transmitted the virus to humans (guarner et al., ) . practical application of this type of analysis is likely to be case-specific: the approach may be useful when evaluating threats in a specific situation, such as when interested in risks to a particular endangered wildlife species (specific recipient host) or when interested in risks associated with introduction of a particular species of animal into the wildlife pet trade or wild animal markets (specific source host). the ability of some pathogens to propagate within novel host species has highlighted both our economic vulnerability to previously unknown pathogens and weaknesses in our current medical and veterinary infrastructure (and in communication between these entities; see kahn, , for a recent discussion). much epidemiological theory has been devoted to understanding the effect of within-species host interactions on the invasion and persistence of infectious diseases in novel host populations (anderson and may, ) . understanding molecular-level determinants of propagation potential is also an important goal, however difficult it may be. factors determining a virus's ability to propagate appear to depend heavily the distribution of susceptible and permissive cells within the host. malaysian nipah virus, for example, infects lung tissue of domestic pigs, causing severe respiratory symptoms that easily disseminate virus particles (hooper et al., ) . humans living and working near infected pigs can also become infected; however, very few virions are able to replicate in human lung tissue. instead, virions replicate in cerebral tissue, producing an encephalitis (hooper et al., ) . replication in the brain does not promote propagation, and transmission between humans does not occur. a variant of nipah virus found in bangladesh, on the other hand, replicates in human lung tissue, causes respiratory disease, and can propagate within the human population (hsu et al., ) . even differences in tissue tropism within a given organ system may affect propagation potential. one hypothesis as to why h n hpai has so far failed to produce large chains of humanto-human transmission is that its viral attachment is concentrated in the lower respiratory tract, whereas access to the upper respiratory tract is insufficient to produce large amounts of aerosolized virus (van riel et al., ) . some ability to generate genetic diversity may be necessary for significant transmission within a novel host species (antia et al., ) , as was seen with sars (holmes, ) ; however, a more accurate assessment of propagation potential requires a detailed understanding of the determinants of cellular susceptibility and permissivity. large-scale characterization of receptors used for cellular entry and of the distribution of these receptors within humans, domestic animals, and wildlife could provide an invaluable tool in predicting specific interactions between viruses and potential hosts. while this task may seem daunting, host-jumping viruses often gain cellular entry via membrane proteins conserved between source and recipient host groups (woolhouse, ) . thus, characterization and distribution studies should target potential receptor proteins that are conserved among and between vertebrate lineages to maximize the practical benefit of this type of basic research. knowledge of the factors that increase a pathogen's ability to jump to, and propagate in, a new host will be invaluable in the effort to prevent, prepare for, and predict viral threats to human and animal health. we currently have a good general understanding of the three steps required for a host jump to occur: encounter is heavily dependent on ecological relationships between species and often requires close contact between a recipient host and the site of viral excretion in the source host; infection depends on a viral species' evolutionary potential and the barriers to replication that it must overcome in a new host cell, which in turn are tied to specific, conserved viral molecular characteristics; and propagation may be facilitated by a viral species' evolutionary potential but also relies on conservation of underlying mechanisms of cellular entry and other determinants of tissue tropism. the next step toward being able to predict viral host jumps between species of interest is to quantify our understanding of the determinants of encounter and infection and thereby develop indicators of risk from a given viral group. virus groups for which the risk of encounter and infection is substantial, particularly those with inherently high evolvability, should be prioritized for receptor characterization, and those found to rely on broadly-conserved receptors should be considered at high risk for emergence. researchers interested in characterizing the risk to a particular target species, such as humans, domestic animals, or endangered wildlife, may then be advised to determine the distribution of receptors-and any other determinants of susceptibility and permissivity that can be characterized-within the species of interest. glossary current/established host. a species supporting active viral infection. dissemination. the distribution of infectious virus particles in the environment. emergence. the appearance (or reappearance) of an infection in a new host population or species, or a change in interaction between host and pathogen due to pathogen evolution or changes in host immunity. host jumps (see below) are the primary type of emergence discussed here. emergence potential. the probability that a given virus population will be able to infect and reproduce in a potential host species. encounter. contact between an infectious virus particle and a species other than its source host species. envelope. the host-derived lipoprotein membrane that surrounds the protein core of some virus particles. most enveloped virions require an intact membrane for receptor binding and entry into the host cell. host jump. a cross-species transmission event that results in the successful infection of the potential host species. infection. the entry and replication of a virus particle in an individual, referring here to infection of an individual of a new host species. new/novel host. a species that has acquired a viral infection as the result of a host jump. permissive cell. a cell in which a virus particle can replicate. potential host. a species that does not currently support active viral infection. propagation. transmission of an infection between individuals of a single host species, usually referring to transmission within the new host species. reassortment. the packaging of genome segments from genetically distinct parent virions in a coinfected cell. receptor. the site on the outer membrane of a cell to which a virion binds for cellular entry. recipient host. a host species, or individual of a host species, that has acquired an infection as the result of a host jump. recombination. the exchange of genetic information between closely-related virus particles during replication in a coinfected cell, usually via template switching. source host. a current host species, or individual of a current host species, that produces virus particles actually or potentially able to infect a potential host species. susceptible cell. a cell that permits viral entry. tissue tropism. the affinity of a virus particle for cells of susceptible and permissive tissues. zoonosis. an animal infection that can be transmitted to and replicate in humans. the role of evolution in the emergence of infectious diseases arenaviridae: the viruses and their replication recombination in hiv: an important evolutionary strategy evolvability of emerging viruses diseases of humans and their domestic mammals: pathogen characteristics, host range, and the risk of emergence airborne bacteria and viruses emerging infectious diseases of wildlife-threats to biodiversity and human health anthropogenic environmental change and the emergence of infectious diseases in wildlife emerging infectious pathogens of wildlife japanese encephalitis virus: ecology and epidemiology community epidemiology framework for classifying disease threats the ecology of emergent infectious disease monkeypox transmission and pathogenesis in prairie dogs adaptation of sars coronavirus to humans comparative pathology of the diseases caused by hendra and nipah viruses nipah virus encephalitis reemergence effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus dna polymerase on the fidelity of dna replication in vivo confronting zoonoses, linking human and veterinary medicine predicting pathogen introduction: west nile virus spread to galapagos quantitative risk assessment of the pathways by which west nile virus could reach hawaii processive proofreading by the adenovirus dna polymerase-association with the priming protein reduces exonucleolytic degradation hybridization as a source of variation for adaptation to new environments structure of sars coronavirus spike receptor-binding domain complexed with receptor infectious disease dynamics: what characterizes a successful invader? examining the origins of emerging viruses video: angola hit by deadly marburg virus avian influenza ( ): uk-hungary virus sequence avian influenza, poultry vs migratory birds major ecological transitions in wild sunflowers facilitated by hybridization emerging infectious disease: what are the relative roles of ecology and evolution? the origins of acquired immune deficiency syndrome viruses: where and when? globalization of human infectious disease microbial threats to health: emergence, detection, and response. washington, dc: the national academies press taylor lh molecular evolution of the sars coronavirus during the course of the sars epidemic in china virus taxonomy: classification and nomenclature of viruses molecular constraints to interspecies transmission of viral pathogens in vitro concatemer formation catalyzed by vaccinia virus dna polymerase naturally acquired simian retrovirus infections in central african hunters population biology of emerging and reemerging pathogens host range and emerging and reemerging infectious diseases emerging pathogens: the epidemiology and evolution of species jumps effects of dna structure and homology length on vaccinia virus recombination the author gratefully acknowledges l. enquist for early discussion of these ideas and insightful comments on the manuscript; p. daszak key: cord- - apj qy authors: melillo, alessandro title: applications of serum protein electrophoresis in exotic pet medicine date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: apj qy serum protein electrophoresis (spe) is a useful diagnostic and prognostic tool in human and companion animals medicine: several experiences show that it can be useful in exotic practice as well. the fundamentals of spe interpretation as well as some normal and pathological patterns for the species most commonly seen in practice are provided. allowed to clot in a test tube for to minutes, the time needed for fibrinogen to be used in the coagulation cascade. in several exotic species, especially of small size, the need to work with small volumes of blood requires the clinician to collect the blood with an anticoagulant (frequently natrium heparin or lithium heparin, sometimes heparinizing the syringe to avoid the coagulation of the sample during collection); therefore, centrifugation is achieved in plasma. the main difference between the products is the absence in the serum of fribrinogen, the protein involved in the processes of coagulation; the concentration of total solids of the plasma is thus slightly higher than that of serum (about %) and the electrophoretic pattern from it will result in a higher incidence of b-globulin fraction where the fibrinogen normally migrates. in clinical practice, the normal plasma protein concentration is measured very simply by using a refractometer: a few drops of blood are collected in a heparinized capillary whose centrifugation gives the value of the microhematocrit (percentage of blood occupied by cells compared with plasma) and a small amount of plasma. refractometry of that plasma allows easy estimation of the concentration of total solids. this method is simple and intuitive; however, it is not reliable if the sample quality is poor. among the factors that interfere with the measurement of total solids by refractometer include hemolysis, lipemia, hyperbilirubinemia, azotemia, severe hyperglycemia, hypernatremia, hyperchloremia, and administration of colloids, including synthetic hemoglobin-based oxygen carriers. in the absence of these factors, the amount of nonprotein solids present in plasma is relatively constant, therefore by subtracting the nonprotein component ( . g/dl) the value of total plasma proteins is obtained. an increase in the concentration of total solids (hyperproteinemia) must always be evaluated in correlation with the microhematocrit value and reflects mainly dehydration or increased synthesis of globulins for various pathologic conditions. the decrease in total solids (hypoproteinemia), vice versa, may reflect overhydration, decreased synthesis of albumin or immunoglobulin, or protein loss associated with hemorrhage, vasculitis, nephropathy, or enteropathy. with the exception of bleeding, which causes a loss of balanced albumins and globulins, in all pathologies involving protein loss albumin concentration falls to a greater extent than that of the globulins because of lower dimensions of the molecule, which allow easier migration through vascular endothelia. the measurement of total protein is certainly very useful, but for the purposes of a diagnosis that is as accurate as possible, it becomes essential to know the alterations of each protein fraction. for this purpose is used the characteristic of plasma proteins to be separated based on their charge and the consequent mobility in an electric field specially created. this method defines electrophoresis of proteins. the distribution of protein migrants on strips of cellulose acetate or agarose gel is represented graphically by a curve in which the extension and the height of each peak is corresponding to the breadth and the density of each protein fraction on the substrate migration. normally, protein fractions are identified in the mammalian plasma, ordered according to the electric charge: albumin (high negative charge and low molecular weight, migrates to the anode and conventionally to the left of the track), a -globulin, a -globulin, and b-globulins and g-globulins that present the greatest molecular weight and the lowest negative charge and then migrate to the right end of the curve. a different protein fraction is present in most birds and reptiles, with an even more negative charge than albumin that is positioned at the left in the same electrophoretic pattern and is therefore referred to as prealbumin. conventionally, you find a point halfway between the beginning and end of the curve as the limit between the a -globulin and b-globulins, and a point halfway to the start of b-globulins and the end of the track as the start of g-globulin. with the exception of albumin, each peak represents a large number of different proteins that can be further separated by more complex examinations. prealbumin is the protein fraction that migrates more rapidly and is positioned on the track before albumin. in species that have it, this is calculated with albumin in the ratio a/g. it is not normally detectable in mammals, except occasionally in cats, and it is normally found in human patients where it has the function of direct transport of thyroid hormones and indirectly of vitamin a by acting as a carrier protein that binds retinol. in the various avian species, it is present in variable amounts. for example, in the budgerigar (melopsittacus undulatus) or in the cockatiel (nymphicus hollandicus) it can constitute % of the total prealbumin albumin, whereas in african gray parrots (psittacus erythacus), it represents no more than % of total albumin and can also be completely absent. in reptiles, the study of plasma proteins is still sporadic and fragmentary data exist, but migrating prealbumin has been reported in some species of chelonians: caretta caretta, geochelone radiata, chelonia mydas, and sauria (gallotia spp), as well as crocodiles (caiman spp), but only with subsequent migration on polyacrylamide gels apparently have similar transport functions, as in other orders. albumin constitutes the main fraction of the plasma proteins (in general between % and % of total protein and up to % to % in primates, including humans) and represents the main and more homogeneous peak in the electropherograms of all species. the main functions of albumin are to carry many molecules and maintain the oncotic pressure of the blood. the group of globulins include the acute phase proteins (apps; a-globulins and b-globulins) and immunoglobulins (g-globulins). apps are the body's response to trauma, inflammation, or infection. the local activation of granulocytes and macrophages causes the release of cytokines (mainly interleukin- ) that stimulate the liver to produce a series of glycoproteins. alpha- antitrypsin, a- -acid glycoprotein, a -macroglobulin, c-reactive protein, haptoglobin, and fibrinogen are positive apps, because their concentration increases in response to inflammation; albumin is considered a negative app, as its synthesis decreases during inflammation, diverting liver protein synthesis to the proteins mentioned previously. the electrophoretic pattern of the apps migrates to the peak of the a (a- -acid glycoprotein, a -macroglobulin, haptoglobin, protein c) and b (c-reactive protein, fibrinogen, complement, ferritin)-globulins, but with huge specific differences. the group of g-globulins is mainly composed of immunoglobulins, although some apps, such as degradation products of the complement, can migrate into this area as well, especially in avian species. immunoglobulins are quite impressive in size, consisting of amino acid chains, so-called "heavy" and "light." depending on the composition of the heavy chains, immunoglobulins are divided into classes named by letters of the greek alphabet: gamma (igg), mu (igm), alpha (iga), and epsilon (ige). the igg antibodies are produced in response to various bacterial, viral, or toxic stimuli; ige (not present in all species) is mainly involved in allergic reactions; iga is primarily responsible for the defense processes located at the mucosal level; and igm has the characteristic of being active in pentameric form (ie, joining in groups of ), thereby forming a protein complex of imposing size. igm is secreted by b-lymphocytes and early plasma cells and its main function is to activate complement, whereas igg is produced later and then plays a predominant action of opsonization, binding to applications of serum protein electrophoresis the pathogen and allowing phagocytosis by macrophages. it is normally not possible to differentiate igm from igg through normal electrophoresis, but distinguishing the ones by the others is very important because it allows us to date the pathologic process in acute (igm only) or chronic (igg with or without igm) and to differentiate an active process (igm) by a seropositivity alone (igg). electrophoresis serum is used as a "screening" of interpretation of the humoral response and during not specific processes. recent studies in rabbits have tried to bring to the surface the true diagnostic value of this report, in particular we tried to find a link between hypergammaglobulinemia and one of the diseases most commonly encountered in clinical practice veterinary medicine in lagomorphs: encephalitozoon cuniculi. the blood for sampling is collected in plain tubes (the use of tubes with accelerant is not possible because of the reduced volume of collectable blood in this species) and left to rest for about minutes. the serum obtained is isolated and frozen at a temperature of - c to be examined in the shortest possible time. laboratory data have shown that a single cycle of freezing does not alter the electrophoretic pattern, whereas prolonged freezing determines a variation in the albumin/globulin ratio in favor of the latter. the serum of the rabbit contains, as in other mammals, various protein fractions: albumin, a-globulins, b-globulins, and g-globulins. the a-globulins and b-globulins are generally divided into subunits of a and a , and b and b (fig. ) . albumin constitutes the main fraction responsible for the oncotic pressure of body fluids, whereas the a -globulin, a -globulin, b-globulins, and g-globulins are a smaller fraction responsible for the humoral response of the organism. unlike the guinea pig, however, the area occupied by b-globulins is wider than that of the a-globulins. in rabbits, the normal serum protein electrophoresis (spe) pattern lacks a clear distinction between b -globulins and b -globulins, as present in dogs and cats, but when gammopathies in the b region occur, usually an extension of the electrophoretic band is seen, with consequent demarcation of the peaks. the a -globulins, together with the a -globulins and b-globulins, increase in case of acute inflammation, expressing a more rapid response compared with that cell mediated. the g fraction includes circulating immunoglobulins, complement, the degradation products of complement, and to a lesser extent some proteins that characterize the acute phase. fig. . normal pattern for a rabbit. c-reactive protein is one of the first proteins produced by the body following an insult to any tissue. despite being defined as an app, it is also found in the course of chronic processes. placed inside a magnetic field (principle of electrophoresis of proteins), because of its shape, weight, and molecular electrostatic charge, such protein migrates to the right (toward the anode or negative electrode) in the area of b-globulins and g-globulins to influence the path. other apps, such as aptoglobulina, a acid glycoprotein, and ceruloplasmin are normally found in the a and b fractions. in course of encephalitozoonosis we have seen a decrease of albumin, primarily because of the increased synthesis by hepatocytes of numerous immunoproteins (especially ig) with consequent widening of the electrophoretic band in the region of b and g. the b-globulin and g-globulin can be found in course of chronic inflammatory processes (like bacterial, viral, or fungal infections), parasitism (e cuniculi), cancer, or immunemediated diseases. in the specific case of protozoal infection by e cuniculi, healthy animals and seropositive animal spe patterns only differ significantly in the g fraction. results are very similar to those obtained by didier in , in a study of dogs showing clinical signs of encephalitozoon infection. the hypergammaglobulinemia during encephalitozoonosis is usually mild to moderate; this value may be altered when rabbits with impaired immune response come in contact with the parasite, which may react with a decrease of circulating igg and a possible increase in igm. the titration of igm and igg allows us to clarify the response against the parasite carried by the body at the time of collection. igm increases usually in the early stages when the parasite exerts its action, decreasing up to be absent on the th day, whereas igg increases more slowly in the course of infection but remains detectable for years. the changes of g-globulin have been shown to not be significantly different in animals with igm titratable compared with animals with igg increased. this shows that the hypergammaglobulinemia is an artifact resulting in a chronic inflammatory process. in support of this theory, it is believed that the organism remains latent in the body until it occurs in a suppression of the immune system, a phase in which the body eliminates spores in the urine. contrary to what occurs in avian species, and in accordance with what occurs in dogs, hemolysis increases the b fraction, not the g fraction. in the otherwise rich literature on diseases of the ferret, electrophoresis is quite neglected, mainly because the main source of information on ferret diseases is us research, where electrophoresis is relatively unknown: the only application is described as an aid in diagnosis of aleutian disease. actually, it is a useful diagnostic tool in ferret species and its interpretation is not very different from that of the dog and cat. being that the ferret is an animal of sufficient size, allowing access to the veins of good gauge as the cranial cava, it is recommended to draw blood without anticoagulant (heparin needle) to obtain serum instead of plasma and thus avoid the deposition of fibrinogen, which will inevitably alter the peak of b-globulins. in a healthy ferret, as in dogs, the proteins migrate in the electric field in dividing fractions: albumin, a -globulin, a -globulin, b -globulin, b -globulin, and g-globulin. albumins represent approximately half of circulating proteins, for which the a/g ratio is about . iga migrates in the field of a and b globulins, igm in the field of b globulin or "fast" g globulin, whereas the igg in the field of slow g-globulin (the far end of the track) (fig. ) . in ferrets, dehydration progresses very quickly in case of insufficient intake or increased losses, so hyperproteinemia is a frequently encountered clinical sign (for example in diarrhea, intestinal obstruction, acute gastritis, or renal failure) but in the case of noninfectious diseases, proteins raise in their entirety for hemoconcentration, so the pattern will result basically normal; in the case of inflammatory diseases, globulins increase, altering the electrophoretic curve. the most characteristic alteration is described in patients suffering from aleutian disease. this disease, fortunately is infrequent (but certainly underdiagnosed) in italy, is caused by a parvovirus (adv) that affects the mustelidae family, including mink and ferret. it is transmitted by feces and all body secretions and is particularly insidious because it evolves as a chronic disease, debilitating the ferret to the invariably fatal outcome. aleutian disease causes lymphoplasmacytic infiltration and precipitation of immune complexes in various tissues from which a variety of symptoms is shown by sick animals. blood tests are often vague, showing nonregenerative anemia, abnormal liver enzymes, and uremia; however, electrophoresis is revealing. patients with adv in fact have a marked hyperproteinemia with values up to to g/dl or more, but at the same time an important hypoalbuminemia: g-globulins rise instead to constitute % to % of total protein, drawing a characteristic "horned" path and seriously altering the a/g ratio. other diseases can cause hypergammaglobulinemia, although not as marked as adv: other viruses such as influenza or distemper, pyoderma, kidney diseases with no protein loss, certain tumors, and systemic mycoses (blastomycosis, coccidioides spp). another, more frequent, viral disease is catarrhal enteritis (ece) caused by a coronavirus: kits are often asymptomatic carriers for adults who become instead gravely ill. in case of ece, electrophoresis is characterized at the beginning by moderate hypoalbuminemia, which becomes more and more severe during the following weeks. following the evolution of the disease, an increase in a or b may be noticed, meaning the production of iga in the acute phase, then a peak of gammaglobulines even if significantly below that of adv. after the second or third week, there is often hypogammaglobulinemia, evidence of the failure of the immune response. it is also interesting to note the changes in the electrophoretic pattern in the case of liver disease. in the case of hepatic neoplasia, often the protein curve does not differ much from that of a healthy animal, except for a modest elevation of a -globulins. more marked is the alteration in case of hepatic lipidosis and acute cholangitischolangiohepatitis, when the increase of a -globulins is greater and reflects an intense inflammation. chronic hepatitis and cholestasis phenomena, intrahepatic or extrahepatic, are reflected instead in a b-globulin peak, where transferrin, c-reactive protein, and generally the igm migrate. the decrease in the percentage of globulin is uncommon and is usually associated with renal failure or lymphoma. the hypoproteinemia usually indicates a defect in protein synthesis and has a negative prognostic value. the electrophoretic pattern of the guinea pig (cavia porcellus) has been thoroughly studied in the experimental for which we know either paths in different normal farming conditions and adjustment of the normal bacterial flora (specific pathogen free subjects and the like) and the reaction to various pathologies experimentally induced; however, very little is described in the field of clinical practice about the response of animals living in uncontrolled conditions that get sick spontaneously. clinically healthy guinea pig serum proteins are separated into sections: albumin, a -globulin, a -globulin, b -globulin, b -globulin (often divided into peaks), and g-globulins, and in some subjects prealbumins have been occasionally signaled (fig. ) . the area occupied by b-globulins and g-globulins in the complex is restricted in comparison with the well-developed area of the a-globulins and a/g ratio is high. by immunoelectrophoresis, up to different protein migrants in those areas can be individuated, although not all were necessarily present in the same sample. an interesting study describes the changes in the electrophoretic pattern during the evolution of experimentally induced tuberculosis infection in a group of guinea pigs. at the end of the first week, the electrophoretic pattern was still virtually unchanged, whereas at the fulfillment of the second, a large majority of individuals showed a marked increase in a -globulins and the appearance of a new peak in the same region, characteristic of sick individuals and never found in healthy ones, named a -t. at the third week, the a started to decrease whereas a increased further with increasing importance of a -t peak, which persisted until the death of the subjects. more detailed studies have identified this protein as a glycoprotein, not macroglobulin. it is interesting to note that the response to tubercular infection occurs in the region of a in several species, including humans. guinea pig lipoproteins tend instead to migrate in the b-globulin peak. the same study also analyzed the serum of guinea pigs inoculated with killed mycobacteria: the electrophoresis of these subjects showed no increase of a -globulins nor the appearance of a -t, which can then be considered the sign of an organic reaction to the pathogen, but all the samples at the third week after inoculation showed a marked hypergammaglobulinemia. in another study, reaction to infection by entamoeba histolytica was investigated, which consisted of hypoalbuminemia and increase of a-globulins and b-globulins. the importance of electrophoresis in the diagnosis of spontaneous disease in the guinea pig and chinchilla is currently studied: patterns from clinically healthy individuals are shown as a starting point for further observations (fig. ) . the first studies of serum proteins in birds were performed on domestic chickens, showing many similarities with the layout of mammals (eg, the production of apps , ), but also several differences: the widespread presence of prealbumin, for example, the lowest concentration of g-globulin, and conversely the more marked response to inflammatory stimuli in the b-globulin field. unfortunately for the needs of clinical practice, however, not only may the electrophoretic patterns of parrots differ from those of chickens, but significant differences are found also among the different species of psittaciformes (figs. - ) . this variability has led some investigators to doubt the possibility of obtaining reliable results by electrophoresis in avian species, especially with regard to the differentiation between the various globulin fractions. the answer to this question is probably a better standardization of the techniques of electrophoresis and graphical representation, as well as a better understanding of the normal values of the different species. normally, electrophoresis of birds is run on plasma samples obtained from heparinized blood: the percentages are derived from densitometric analysis of each protein fraction deposited on the gel and absolute values (g/dl) calculated by multiplying the percentage of each fraction for the value of total protein obtained from the refractometer. the pattern of the normal chicken shows a marked peak of albumin, and globulins are divided between the b-globulin (igm, iga) and g-globulin (igg): transferrin, app, migrates mainly in the b-globulins. in birds, the albumin protein fraction is undoubtedly preeminent; an interspecific important difference is noted in the group of prealbumin. whereas in some species, such as the african gray, prealbumin is nearly or totally absent, similar to chickens, other species, such as the budgerigar or the monk parakeet (myopsitta monachus), may have even more prealbumin than albumin. conventionally, the a/g ratio is calculated by dividing the sum of prealbumin albumin for the sum of the globulins. according to some investigators, however, it may be more correct to subtract the prealbumin, because it is not properly albumin and even a globulin. the opinion of the author is that this precaution not only is of little practical value but also potentially counterproductive, being that prealbumin, as albumin, has a negative app and therefore its quantification, when present, has validity in the diagnosis of acute inflammation. the b-globulins are the predominant group of globulins in all psittaciformes: some species, such as the african gray, appear regularly divided into subfractions, but the clinical value of this feature is still unclear. the b-globulins increase from % to more than % in the psittaciformes with chlamydiosis, aspergillosis, or sarcocystosis. , an increase of b-globulins has also been described in chickens experimentally inoculated with turpentine and other irritants, , demonstrating that most apps (transferrin, fibrinogen, b lipoprotein, complement) in avian species migrate in this sector. the transferrin appears to be the major app in avian species (in contrast to what happens in mammals) and, although it is not specific to a particular disease, has an important prognostic value: its short half-life ( - hours) makes the monitoring of b-globulins an excellent indicator of the effectiveness of a therapy. the a-globulins instead represent the group less defined and the whose measurement determines the highest margin of error. altogether they represent only % to % of total protein, and already this modest quantity (in comparison with that of mammals and also of other avian orders, such as falconiformes) makes it difficult to precisely separate it from the contiguous predominant peaks, albumins and b-globulins. they are divided into fractions: the area of a -globulins hosts mainly the a antitrypsin and in normal paths is not detectable as a well-defined peak; even in sick parrots an increase of this fraction has almost never been described, unlike what was observed in mammals. in some cases has been noted a selective increase dell'alfa antitrypsin, with or without moderate hypoalbuminemia, in psittaciformes with heavy parasitism. the main protein in the a area is instead a macroglobulin. fig. . bubo bubo. the pattern from this specimen looks normal, but the bird is hypoproteinemic, possibly as a consequence of a too severe food restriction for training. the characteristic of this protein is its large size (molecular weight > , ) for which it is still retained by the filter even in the presence of serious renal damage. one of the few cases when significant elevations of a are described is precisely acute nephritis, in which most of the proteins are lost through the kidneys and the percentage of macroglobulin increases accordingly: in response to other phlogistic stimuli, instead, increments much more moderate and inconstant are noticed. modest alterations of a-globulins have also been described in relation to the activation of the ovary, similarly as described in the human species. the increase of transferrin that occurs during this stage, however, also induces a concomitant peak of b-globulins. the g-globulins are a minor fraction in the electrophoretic pattern of the psittaciformes, on average % to % of the total protein. in the area of g-globulin migrate mainly igg so we see increments in this area only at a later stage of the pathology. diseases in which we expect massive increases in g-globulin are chronic diseases such as chlamydiosis or mycobacteriosis. aspergillosis also belongs to the group of chronic diseases but, because of its immunosuppressing activity, often patients suffering from chronic fungal forms have apparently normal electrophoresis, as the humoral response is inhibited. approximately % of patients suffering from aspergillosis show cyclical peaks in the b-globulins area, and only a small percentage show hypergammaglobulinemia. the normal response to a chronic inflammatory process is the production of a wide variety of antibodies that are expressed in a series of g-globulin peaks or, more commonly, in a single peak with a large base: this phenomenon is named polyclonal gammopathy. in some cases, however, a single or very few immunoglobulins are massively produced, a phenomenon that is expressed in a peak-to-narrow base similar to that of albumin and is defined oligoclonal or monoclonal gammopathy. normally behind this phenomenon there is a neoplastic proliferation of one or a few lines of b lymphocytes, even if in some cases, monoclonal gammopathies may occur in response to non-neoplastic stimuli as well, but anyway very serious. it is worth considering that the low reliability reported by some investigators about the avian electrophoresis is to be traced back to improper collection, retention, and selection of the sample. cray and colleagues showed that serum refrigeration causes a discrete change in the albumin/globulin ratio, causing a progressive difficulty in defining and quantifying the a-globulins. conversely, a freezing of the sample did not show adverse effects. hemolysis is a phenomenon unfortunately present when sampling blood from small patients and difficult to control. in the dog, the products of hemolysis tend to migrate in the b-globulin region; conversely, those of birds seem to migrate primarily in the region of g-globulin. this may be attributable to a structural difference of the protein or by a different technique analysis. lipemia is another frequent alteration especially in the serum of some south american species (amazona spp, miopsitta, pionus spp): the electropherogram of a lipemic sample shows an artificial elevation in the region of b-globulins. in contrast, the b-globulins would be below the normal values related in the literature, in which serum would be used in place of plasma because the fibrinogen (absent in serum) migrates essentially in the b region. this is, however, the only significant difference between the tracks from the different spin cycles. electrophoresis of birds of prey basically follows the same general considerations of psittaciformes: common use of plasma instead of serum, need to standardize techniques, and to study the parameters of normality among the different species (figs. and ) . compared with psittaciformes, most falconiformes do not present prealbumin or present it in a minimal amount: vice versa, apparently healthy falconiformes have levels of a -globulins and a -globulins, markedly higher than the parrots. major differences regarding the b-globulins and g-globulins are not described. electrophoresis is an important tool for the diagnosis of chlamydiosis and aspergillosis in falconiformes. a text antibody positive for chlamydia spp acquires a very different meaning in the presence of electrophoretic curve indicative of acute or chronic inflammation than in absence of any alteration; a marked alteration of electropherogram with prevailing in the region of the b-globulins can support a presumptive diagnosis of aspergillosis also in case of a negative antibody test. the electrophoresis has proved useful as a diagnostic and prognostic tool in falconiformes even in the case of diseases such as mycobacteriosis, abscesses, and osteomyelitis. the use of electrophoresis in reptiles is still sporadic and most of the available data are anecdotal; nevertheless, they are interesting as a first element for future study. serum proteins of reptiles migrate in a manner quite similar to those of birds and are divided on the track in the fractions of albumin, a -globulin, a -globulin, b-globulins, and g-globulins. prealbumin appears in some species (eg, geochelone radiata and some turtles), in which it presumably has the same function of transport as in other animals. albumins are also the preponderant portion of the curve in reptiles, whereas the globulins as a complex, like in birds, are in minimal concentration in healthy subjects. in the fraction of a-globulins and b-globulins migrate mainly apps and complement, whereas the g-globulins are circulating antibodies; both b-globulin and g-globulin can configure a single peak or two. even in the absence of standardization, it can be seen how the electrophoretic pattern changes in subjects markedly ill compared with healthy subjects in reptiles as well. in an iguana (iguana iguana) with evidence of osteometabolic disease, for example, is frequently observed the decrease of albumins and the marked increase in b globulins (apps) that indicates suffering hepatic and renal function as a cause or consequence of the metabolic problem. the alteration of the ratio a/g is one of the most interesting parameters to observe and evaluate, as it can reveal a dysproteinemia even in the presence of a normal value of total protein, thus inducing the clinician to further investigation. an important obstacle to the use of electrophoresis, as well as the biochemistry as an assessment of the health status of a reptile, is the marked variability linked not only to the species but also to temperature, ambient humidity, photoperiod, and in general to the season, that for heterothermic animals has much more influence on homeostasis than in homeothermic animals. only the collection of an adequate volume of data will 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protein electrophoresis of psittacine plasma serologic and plasma protein eletrophoretic findings in psittacine birds with aspergillosis serologic diagnosis of sarcocystosis in psittacine birds: cases plasma protein electrophoresis: an update total protein determination in pigeon plasma and serum: comparison of refractometric methods with the biuret method protein electrophoresis as a diagnostic and prognostic tool in raptor medicine protein electrophoresis: a tool for the reptilian and amphibian practitioner key: cord- - g lio l authors: keesing, felicia; belden, lisa k.; daszak, peter; dobson, andrew; harvell, c. drew; holt, robert d.; hudson, peter; jolles, anna; jones, kate e.; mitchell, charles e.; myers, samuel s.; bogich, tiffany; ostfeld, richard s. title: impacts of biodiversity on the emergence and transmission of infectious diseases date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: g lio l current unprecedented declines in biodiversity reduce the ability of ecological communities to provide many fundamental ecosystem services. here we evaluate evidence that reduced biodiversity affects the transmission of infectious diseases of humans, other animals and plants. in principle, loss of biodiversity could either increase or decrease disease transmission. however, mounting evidence indicates that biodiversity loss frequently increases disease transmission. in contrast, areas of naturally high biodiversity may serve as a source pool for new pathogens. overall, despite many remaining questions, current evidence indicates that preserving intact ecosystems and their endemic biodiversity should generally reduce the prevalence of infectious diseases. supplementary information: the online version of this article (doi: . /nature ) contains supplementary material, which is available to authorized users. i n june , a new organization, the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes)patterned after the intergovernmental panel on climate change (ipcc)-was established to assess changes to the diversity of life on the earth and how these changes will affect human well-being . human well-being would be adversely affected by biodiversity losses if ecosystems with reduced biodiversity are less able to provide the ecosystem services-such as carbon sequestration, nutrient cycling and resistance to drought-on which humans rely. in recent years, a consensus has emerged that ecosystem functions decline as biodiversity is lost . here we examine how biodiversity affects the transmission and emergence of infectious diseases and evaluate the evidence that reduced disease transmission is an important ecosystem service provided by high biodiversity. biodiversity encompasses the diversity of genes, species and ecosystems. increases in human populations have resulted in an unprecedented and precipitous loss of biodiversity . current extinction rates are estimated to be at least - , times background extinction rates and future extinction rates (over the next years) are estimated to be to times present extinction rates . a large proportion of species in all assessed taxa are currently threatened with extinction ( % of birds, % of mammals, % of amphibians; % of gymnosperms; % of corals ) and the best estimate of population trends of birds, mammals, amphibians, reptiles and fish indicates that since global population sizes have declined by almost % (ref. ) . global and local extinction rates of some taxa, particularly microbes, have not been well characterized. for the many organisms that are symbionts of other organisms, extinction of their hosts can cause their extinction too . collectively, these declines and extinctions are caused by changing the earth's ecosystems to meet growing demands for food, fresh water, fibre, timber and fuel, and by climate change. changes in biodiversity have the potential to affect the risk of infectious disease exposure in plants and animals-including humansbecause infectious diseases by definition involve interactions among species. at a minimum, these species include a host and a pathogen; often many more species are involved, including additional hosts, vectors and other organisms with which these species interact. intriguingly, biodiversity may play a dual role in the emergence and transmission of infectious diseases. on the one hand, high biodiversity may provide a larger potential source of novel pathogens, but on the other hand, biodiversity can reduce further pathogen transmission for both longestablished and newly emerging diseases. we first review the effects of biodiversity on the transmission of established diseases and then turn to disease emergence. transmission of pathogens between species biodiversity loss might affect disease transmission through several mechanisms (box ). if the effect of each species on pathogen transmission were entirely idiosyncratic, one would expect that diversity declines would be equally likely to cause a decrease or an increase in disease transmission in the remaining species. however, in recent years, a consistent picture has emerged-biodiversity loss tends to increase pathogen transmission and disease incidence. this pattern occurs across ecological systems that vary in type of pathogen, host, ecosystem and transmission mode ( table ) . as an example, west nile virus is a mosquito-transmitted virus for which several species of passerine birds act as hosts. three recent studies detected strong correlations between low bird diversity and increased human risk or incidence of west nile encephalitis in the united states - . communities with low avian diversity tend to be dominated by species that amplify the virus, inducing high infection prevalence in mosquitoes and people, while communities with high avian diversity contain many species that are less competent hosts. for hantavirus pulmonary syndrome, a directly transmitted zoonotic disease, correlational and experimental studies have shown that a lower diversity of small mammals increases the prevalence of hantaviruses in their hosts, thereby increasing risk to humans (box ). diversity has a similar effect for plant diseases, with species losses increasing the transmission of two fungal rust pathogens that infect perennial rye grass and other plant species . recent attention has focused on assessing the mechanisms by which reduced biodiversity increases pathogen transmission (box ). biodiversity loss can clearly increase transmission if it reduces predation and competition on reservoir hosts, thereby increasing their density. however, controversy has centred around whether the loss of species can increase transmission in other ways . this is because field studies like those on west nile virus, hantaviruses and rye grass have typically not controlled for changes in host density that can result from changes in 'species richness' (the number of species present in a community, which is a measure of taxonomic diversity). as a consequence, it has been difficult to separate the effects of higher density from those of reduced diversity. recent experiments confirm that increases in disease transmission can occur when species richness declines even if host density stays constant. one of the best examples comes from a study of schistosoma mansoni, a trematode that causes schistosomiasis in humans. the parasite alternately infects snails and humans via free-living infectious stages. host snails were placed in tanks at a constant density either alone or with one or two other species of non-host snails and then exposed to the parasite . in single-species treatments, host snails were % more likely to be infected because parasites in multi-species treatments often ended up in dead-end hosts. increased parasite-host encounter rates caused by reduced diversity are sufficient to increase disease transmission for schistosoma. the loss of species can increase encounter rates between pathogens and hosts, as in the schistosoma example, when the lost species are not hosts for the pathogen. but if the lost species are indeed hosts capable of transmission, this declining diversity could also reduce the total number of hosts, thereby decreasing transmission if all else remains equal , . certainly reductions in the number of hosts can reduce the number of vectors and also their infection prevalence , , but empirical examples are relatively rare, in part because the issue has been neglected, and also because all else rarely remains equal. for example, the loss of hosts can cause compensatory increases in the abundances of other hosts, such that total host abundance changes little relative to total host abundance in more diverse communities. even when total host abundance does decline in less diverse systems, differences in host quality among species can alter simple correlations between host abundance and infection risk . pathogen transmission is not always a function of host density. for example, the number of infectious bites delivered by highly mobile vectors like mosquitoes can be independent of the density of the host population . transmission of directly transmitted pathogens like hantaviruses can also be independent of host density if transmission involves behavioural encounters, for example, aggressive interactions between rodents, and if the frequency of these encounters does not vary much with host density , . in systems like these, the loss of host species can actually increase transmission if the lost hosts are suboptimal for parasite development and reproduction; this is because these suboptimal hosts absorb pathogens but are poor at transmitting them. in sum, reducing biodiversity can increase disease transmission when the lost species are either not hosts for the pathogen or are suboptimal ones. for pathogens for which transmission is a function of host density, loss of diversity is most likely to increase transmission if the loss causes an increase in the density of competent hosts. the number and diversity of examples of pathogens for which species loss leads to increases in total transmission suggests that these conditions are frequently met (table ) . additional studies in other disease systems would better establish the generality of these relationships. the loss of particular species in a community clearly has the potential to increase disease transmission. but does reducing diversity itself increase transmission, or is increased transmission the consequence of the removal of particular species? the answer depends on how species composition changes as richness changes , . for example, if those host species most responsible for amplifying the pathogen tend to persist or even thrive as biodiversity is lost, then disease risk will consistently increase as biodiversity declines. on the other hand, if amplifying species tend to disappear as biodiversity declines, then biodiversity loss will tend to reduce disease risk. these hypothetical possibilities indicate the importance of understanding both the non-random sequences by which species are lost from communities, and whether the species that tend to occur only in more species-rich communities tend to amplify or buffer pathogen transmission. in several case studies, the species most likely to be lost from ecological communities as diversity declines are those most likely to reduce pathogen transmission. in the lyme disease system of eastern north america, for example, the white-footed mouse is simultaneously the most abundant host species, the most competent host for the lyme bacterium, and the highest-quality host for immature tick vectors the loss of biodiversity can affect the transmission of infectious diseases by changing: ( ) the abundance of the host or vector. for plants, seeding experimental fields with plant species that are not hosts for fungal pathogens decreased threefold the pathogen load of species that are hosts, apparently by reducing host density through competition . on the other hand, a greater diversity of host species can sometimes increase pathogen transmission by increasing the abundance of vectors . ( ) the behaviour of the host, vector or parasite. in a more diverse community, one of the parasitic worms that causes schistosomiasis (which infects million people worldwide) is more likely to end up in an unsuitable intermediate host. this can reduce the probability of subsequent infection of humans by - % (ref. ) . for hantavirus in utah, usa, rodent hosts on more diverse plots are more likely to come in contact with heterospecific mammals and less likely to come in contact with conspecifics, reducing the probability of transmission of the virus . in principle, higher diversity could influence behaviours with a resulting increase in disease transmission or could alter the evolutionary dynamics of virulence and transmission pathways. ( ) the condition of the host or vector. in experimental rice fields in china, rice plants in genetically diverse mixtures had drier leaves because the mixture changed microclimatic conditions . as a consequence, infection with rice blast fungus was less prevalent in diverse fields. genetically diverse plantings can also lead to induced resistance in host plants because they are exposed to similar pathogens that are specialists on the other cultivars . for some disease systems (for example, lyme disease), multiple mechanisms operate in concert, leading to a compounding effect of biodiversity loss on increased disease transmission (table ) . . ticks that attempt to feed on virginia opossums are likely to be groomed off and killed. green-andyellow circles show the mean number of ticks per hectare fed by mice or opossums; yellow shading shows the proportion of ticks infected after feeding. blue circles show the mean number of ticks per hectare groomed off and killed. ticks that feed on mice are highly likely to become infected with the bacterium that causes lyme disease, whereas those that feed on opossums are not. case study of hantavirus pulmonary syndrome hantaviruses are a group of negative-stranded rna viruses associated with murid rodents. they can cause severe morbidity and mortality in humans, with case-fatality rates near % (ref. ) . infected rodents shed hantavirus in saliva, urine and faeces; transmission to humans occurs through inhalation of aerosolized excreta as well as through rodent bites . the risk of human exposure increases as the density and infection prevalence of rodent reservoirs increase . in a field study in oregon, usa, the only variable significantly linked to infection prevalence in deer mouse host populations was mammalian species diversity, with the prevalence of the hantavirus sin nombre virus rising from % to % as diversity declined. deer mouse population density was not statistically associated with sin nombre virus infection prevalence, suggesting that high diversity reduced intraspecific encounters rather than host abundance . a study in utah, usa , also found a negative correlation between small-mammal diversity and sin nombre virus infection prevalence in deer mice. as in oregon, high diversity reduced infection prevalence apparently by reducing intraspecific encounters rather than by reducing host density, a result supported by experiments . the conclusions of these studies were supported by an experimental study of hantaviruses in small mammal communities of panamá . in replicated plots, small-mammal diversity was reduced by trapping and removing species that are not hosts for the virus; infection prevalence in hosts was compared on manipulated and unmanipulated plots (box figure) . experimentally reduced small-mammal diversity caused an increase in the density of host species and also in seroconversion rates and seroprevalence within hosts (box figure) . review research (fig. ) . as a consequence, this host species infects a high proportion of the ticks within forest communities. the white-footed mouse is also an ecologically resilient species, present in both species-rich and speciespoor communities . in contrast, virginia opossums are poor hosts for the pathogen, kill the vast majority of ticks that attempt to feed on them, and are absent from many low-diversity forest fragments and degraded forests where mice are abundant , . therefore, as biodiversity is lost, the host with a strong buffering effect-the opossum-disappears, while the host with a strong amplifying effect-the mouse-remains. the primary hosts for the pathogens that cause west nile encephalitis, hantavirus pulmonary syndrome, and bartonellosis also appear to be resilient species that increase in abundance as biodiversity is lost , , . whether an organism's host competence and its resilience to factors that reduce biodiversity are causally related is an unresolved but critical issue. traits that make a host resilient to biodiversity loss may also make them susceptible to pathogen infection and transmission. such a relationship would explain the frequency with which the link between diversity loss and disease transmission has been observed in nature ( table ). for plants, species that are fast-growing and nutrient-rich with relatively high metabolic rates-characteristics of 'weedy' speciescan be more competent hosts for arthropod vectors and plant pathogens than those with less weedy traits . plants with these weedy traits are also more likely to become more abundant when plant diversity declines . consequently, the very species that have traits permitting persistence in degraded and species-poor ecosystems are also more likely to carry high pathogen and vector burdens. a similar pattern may occur in vertebrates-resilience in the face of disturbances that cause biodiversity loss, such as habitat destruction and fragmentation, is facilitated by lifehistory features such as high reproductive output and intrinsic rates of increase . vertebrates with these features tend to invest minimally in some aspects of adaptive immunity [ ] [ ] [ ] ; we hypothesize that this may make them more competent hosts for pathogens and vectors. understanding the interrelationships among pathogen transmission, biodiversity loss and interspecific differences in immune function is an important area for future research. such studies would illuminate how frequently resilient species are also those that increase pathogen transmission, and might provide general rules about the impact of biodiversity loss on disease transmission. could changes in biodiversity within the bodies of organisms also alter pathogen transmission? recent improvements in the ability of researchers to detect unculturable microbial species have allowed documentation of the tremendous diversity of microbes upon and within plants and animals. in human bodies, for example, % of all cells are microbial . a number of studies have begun to show links between diseases and the diversity of an organism's 'microbiome'. changes in the composition of microbiomes are frequently associated with infection and disease. for example, corals suffering from white plague disease have microbial communities distinctly different from those in healthy corals . in humans, bacterial vaginosis results from changes in the composition of the vaginal microbial community , and this in turn increases the risk of hiv infection . although changes in microbial species composition associated with infection are welldocumented, few studies have investigated the effects of changes in diversity itself. in a recent investigation, patients with recurrent episodes of infection caused by the bacterium clostridium difficile had significantly lower diversity of intestinal microbes than did control patients . correlational studies such as these, though intriguing, make it difficult to determine whether changes in microbial communities are the cause or the consequence of infections. but some experimental studies clearly demonstrate that increasing microbial biodiversity can protect against infection. for example, children with a history of ear infections given a mixture of five strains of streptococcus were less likely to develop subsequent infections compared to a control group . similarly, reducing microbial diversity within a host can increase transmission. when mice with persistent infections of c. difficile were treated with antibiotics that reduced the diversity of intestinal microbes, they began shedding c. difficile spores at high rates . in some of these examples, a rich microbial community appears to regulate the abundance of endemic microbial species that can become pathogenic when overly abundant . in other cases, high microbial species diversity can prevent colonization by invasive pathogenic species. for example, the more diverse the microbiome surrounding the roots of wheat plants, the more protected the plants were against invasion by the pathogenic bacterium pseudomonas aeruginosa . similarly, piglets raised in natural environments supporting a high diversity of microbes were more resistant to invasion by pathogenic gut microbes than those raised in more sterile environments . the effects of microbial diversity within and upon host bodies show intriguing similarities to the effects of macroscopic species diversity on disease transmission in aquatic and terrestrial ecosystems. further exploration of these similarities, and particularly the specific mechanisms operating within hosts, is a critical research frontier because changes in microbial diversity might accompany biodiversity loss in their hosts. for pathogens already established within ecological communities, we have shown that biodiversity loss frequently increases the rate of transmission. but what role, if any, does biodiversity have in the processes by which new pathogens emerge? between and , over emerging disease events were identified in humans around the world . concomitantly, other emerging infectious diseases also appeared in wildlife, domesticated animals, and crop and wild plants. emerging infectious diseases include those in which the pathogen has evolved into a new strain within the same host species, for example, through the evolution of drug resistance (methicillin-resistant staphylococcus aureus or mrsa) or switched to new host species (for example, human immunodeficiency virus or hiv, severe acute respiratory syndrome or sars). in some cases, the switch to new host species is accompanied by a change in geographic range (for example, west nile virus in the americas). for pathogens that establish in new species, the emergence process involves multiple steps, including the initial invasion into the new host ('spillover'), the production of transmission stages within the new host, and the establishment of the pathogen in the host population as a whole , . the effect of biodiversity may vary for each of these steps. for the initial invasion, biodiversity may act as a source pool. this hypothesis is supported by surveys of emerging diseases of humans: most are zoonotic-jumping to humans from other vertebrate animals . in one recent analysis, the probability of emergence of pathogens from wildlife to humans was positively correlated with mammalian wildlife species richness when data were corrected for reporting bias . other environmental and socioeconomic factors that bring humans into closer contact with potentially new pathogens (for example, forest clearing for agriculture, wildlife hunting) may also contribute to this pattern. indeed, almost half of the zoonotic diseases that have emerged in humans since resulted from changes in land use, from changes in agricultural or other food production practices, or from wildlife hunting (fig. ) . these human activities increase rates of contact between humans and animals, which may be a critical factor underlying spillover. once spillover of the pathogen into a new host has occurred, high densities of that host species may facilitate pathogen establishment and transmission within the new host . for example, nipah virus spilled over from wild fruit bats to domestic pigs in malaysia; high densities of pigs in local farms appear to have facilitated establishment of pig-to-pig transmission, and the pathogen then spilled over from pigs to humans . such high densities of domesticated species are almost always associated with low biodiversity. in contrast to emergence through host-switching, % of emergence events between and arose through the evolution of drug resistance . for these cases, biodiversity of microbial communities within hosts may have a protective effect; human use of antibiotics is research review thought to select for resistant microbes by eliminating the great diversity of non-resistant microbial strains and species that suppress resistant strains in the absence of antibiotics. investigations using recent advances in microbial detection support this idea , . thus, reduced microbial diversity may be an important underlying cause of the emergence of drug-resistant pathogens; this too requires further investigation. the addition of particular species-for example, natural enemies or competitors-can reduce the impacts of established pathogens. for example, experimental addition of a naturally occurring bacterium, janthinobacterium lividum, to the skin of the endangered frog rana mucosa eliminated frog mortality from experimental infection with chytridiomycosis, which is devastating amphibian populations worldwide . for corals, application of phages isolated from natural communities can control the spread of bacterial infections . the growing interest in 'probiotics' for humans and harvested species provides another example of this approach . more broadly, biodiversity itself seems to protect organisms, including humans, from transmission of infectious diseases in many cases (table ) . preserving biodiversity in these cases, and perhaps generally, may reduce the incidence of established pathogens. to preserve high diversity in nature, conservation scientists have developed robust methods that reflect the key principle that larger areas sustain larger numbers of species . methods of conserving microbial diversity within and upon bodies or in the environment are less well developed, but avoiding the overuse of antimicrobial compounds is essential. critically, future research on the relationship between biodiversity and disease must avoid conflating the effects of biogeographic patterns of biodiversity (for example, higher diversity in lower latitudes) with those of anthropogenic reductions in extant biodiversity, because policy and management options can far more readily affect the latter than the former. for emerging diseases, the observation that a more diverse microbiome within a host suppresses strains that are resistant to antimicrobial compounds suggests that avoiding the over-use of these compounds in medicine and agriculture can prevent the emergence of resistant strains. for pathogens that emerge by switching host species, three management approaches are warranted. first, potential emergence 'hotspots' could be predictable on the basis of land-use change and underlying biodiversity patterns; these areas should be targeted for surveillance of endemic wildlife pathogens that have the potential to jump host species , . second, preserving and protecting intact habitats in these hotspots provides a simple, direct way of reducing human-animal contact and reduces the likelihood of emergence of new pathogens, although methods for achieving reduced contact are not always straightforward . and third, to reduce the probability that pathogens become established and transmissible within a new host population once spillover occurs, the husbandry of high-density monocultures of domestic animals, particularly in areas at high risk of spillover, should be subject both to more intensive surveillance and to measures that reduce contact between wildlife and livestock. managing potential emergence hotspots by attempting to eliminate them is likely to backfire because the species most resilient to habitat destruction and degradation may be those that amplify pathogen transmission. despite many recent advances in our understanding of biodiversity and disease, much remains to be learned. first, we must increase the number of disease systems for which we understand the effects of biodiversity loss on disease transmission across a range of spatial and temporal scales. we must also focus on how to implement specific policies informed by this science. future research, for example, should monitor changes in epidemiology in regions in which conservation measures are imposed compared to reference sites. a major challenge will be to untangle the complex ways in which other global anthropogenic trends-such as climate change, biotic exchange, nutrient pollution, armed conflict and economic collapse-interact with biodiversity loss to influence disease dynamics, and which of these trends have the greatest impacts on human well-being. despite remaining questions, connections between biodiversity and disease are now sufficiently clear to increase the urgency of local, regional, and global efforts to preserve natural ecosystems and the biodiversity they contain. globally, almost half of these diseases resulted from changes in land use, changes in agricultural and other food production practices, or through wildlife hunting, which suggests that contact rates between humans and other animals are an important underlying cause of zoonotic disease emergence. 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article at www.nature.com/nature. correspondence and requests for materials should be addressed to f.k. (keesing@bard.edu). supplementary information is linked to the online version of the paper at www.nature.com/nature. key: cord- -n le ix authors: saxena, abhishek; tiwari, archana; kaushik, rinku; iqbal, hafiz m.n.; parra-saldívar, roberto title: diatoms recovery from wastewater: overview from an ecological and economic perspective date: - - journal: nan doi: . /j.jwpe. . sha: doc_id: cord_uid: n le ix alarming water pollution is toxic to the aquatic ecosystem leading to a sharp decline in species diversity. diatoms have great potency to survive in contaminated water bodies, hence they can be compelling bioindicators to monitor the change in the environmental matrices effectively. around the globe, researchers are intended to evaluate the impact of pollution on the diatoms recovery and techniques used for the assessment. the diatoms are precious for futuristic need viz. value-added products, energy generation, pharmaceuticals, and aquaculture feedstocks. all these applications led to a significant rise in diatoms research among the scientific community. this review presents different isolation practices, cultivation, and other challenges associated with the diatoms. a precise focus is given to diatoms isolation techniques from highly polluted water bodies with the main thrust towards obtaining an axenic culture to elucidate the significance of pure diatom cultures. recovery of “jewels of the sea” from polluted water signifies the prospective ecological and economic aspects. diatoms are the most successful contemporary group of photosynthetic eukaryotic microbes that inhabit almost every kind of aquatic ecosystem. they can occur as endosymbionts in dinoflagellates [ , ] . diatoms have arisen because of an endosymbiotic event between red algae and heterotrophic flagellate approximately million years ago [ ] . although there are several types of microalgae present in water, diatoms are the most important and unique as they are the only species with the presence of silica in their cell [ , ] . diatoms are surrounded by a silica cell wall that allowing them to have varied shapes and sizes. their silica cell wall is popularly known as frustules that offer protection from photoinhibition; supply appropriate nutrients uptake; manage sinking rate and playing a significant role as a mechanical barrier against grazers [ ] . their silica content varies from one species to another depending upon their size, growth pattern, and environmental variables such as light, temperature, salinity, nutrients, and growth phase [ ] . there are more than , extant species of diatoms distributed to a different ecosystem [ ] . they are widely present in all aquatic habitat constituting lentic, lotic habitat and sand. it is estimated that % of species occur in freshwater, and % are benthic [ ] . aquatic ecosystems such as rivers, oceans, lakes, etc. support the lives of a large variety of organisms but excessive urbanization and industrialization transform them into the heavily polluted zone [ , ] . the rising pollution level deteriorates the water quality index leading to a huge decline in species diversity due to untreated discharge of metals, pesticides, aromatic polycyclic hydrocarbons, etc., which slows down the survival of an aquatic species, thus posing a big threat [ ] . several organisms have been used as bioindicators such as protozoa, bacteria, fishes, zooplankton, algae, etc. however, diatom-based monitoring for the assessment of ecosystem remedy is in great demand [ ] . because diatoms produce organic matter to a large extent that permits natural inbuilt capacity to withstand toxicity levels in water bodies, extended survival rate, short regeneration time than microalgae, fishes, and other micro invertebrates thus making them one of the best candidate for water quality monitoring, and excellent bioindicators of aquatic biological integrity [ , ] . water quality analysis is essential as it allowed to detect the habitat linked information of the related organisms [ ] . as diatoms also produce eicosapentaenoic acid (epa), docosahexaenoic acid (dha), and other nutritional compounds, they can be used as a food source to every aquatic organism such as zooplankton and fish. they constitute % of oxygen in water bodies, fix excessive chemical compounds and inorganic nutrients in the water, such as carbon, phosphorous, silica etc., [ ] . as fossil fuels are diminishing quickly, therefore, the demand for biofuel production has increased many folds since the last decades. the crop and food-producing plants will never be the top priority for biofuel production. in the future, diatoms will remain as the best candidate for biofuel production. some strain of diatoms produces lipids as % to % of the dry weight that can be used in the production of biofuel thereby focusing on the role of diatoms as an ideal substitute for fossil fuels [ ] . despite having all these important characteristics, they are the least explored aquatic species because isolation and maintenance of axenic culture is a tedious job [ ] . in this review, an effort has been put forward on the details related to the isolation of diatoms from polluted water bodies, the significance of diatoms, and their future potential. rapid industrialization and urbanization releasing a toxic substance into the aquatic environment. thousands of chemicals without any proper risk assessment and legislation are being introduced, which are endangered to aquatic species [ ] . algae, bacteria, protozoa, fishes, micro invertebrates, and zooplankton have been extensively used in biomonitoring purposes, but recently, diatoms have gained much more attention because they are primary producers and plays a crucial role in biogeochemical cycles of the aquatic food web [ , , ] . diatoms survive well in a variety of habitat, such as oceans, lakes, estuaries, wetlands, etc. the use of diatoms in ecotoxicological studies is advantageous to examine any possible effects of toxic elements. this makes diatom a potential tool in bioassay than any other aquatic species because of its easy sampling and frustules identification [ , ] . diatoms are highly sensitive to metal pollution than macroinvertebrates, which could very well explain gradation in metal composition, thus allows early identification of the presence of any heavy metal pollution in an aquatic ecosystem [ ] . diatom responds quickly to any organic matter or nutrient contamination, for example, metolachlor, atrazine, simazine, phenols, nitrate, phosphate, heavy metals, and pahs [ , ] . even though diatoms are ecologically diverse, their potential in biomonitoring is underutilized in evaluating any risk assessment because of traditionally available metrices such as cell density, biovolume, and relative abundance. new and advanced metrices are more advantageous in recognizing the dynamics of diatoms communities by demonstrating the sub-lethal effect that is not evident in diatom counts [ , ] . nuclear abnormalities, cell membrane, cytoplasmic content, and photosynthetic apparatus alteration change in lipid content and diatoms classification using ecological status are the main parameters that were infrequently reported [ ] [ ] [ ] . this behavior demonstrates sensitivity and efficiency in quantifying the toxic elements, stress detection after ultraviolet (uv) exposure, which remains unnoticed by the conventional method [ ] . as reported, calculating live diatom species in the biofilms seems to be a lucrative bioassessment tool. similarly, for assessing weak cytoplasmic content of freshwater species, the ecological association of diatoms is examined for evaluating the environmental health of water bodies because these newer metrices are easy to acquire, rapid detection, low labor-intensive, good reproducibility, standardized and worldwide acceptance [ , ] . diatoms as bioindicators check and observe the integrity of the precious environment by enumerating the change in water quality over large geological areas, provide variability, and any possible backdrop precondition [ ] . investigator worldwide have examined various water bodies using pollution tolerant diatom species to measure the pollution level by employing algal indices such as palmer's algal index; chlorophycean index; nygaard's phytoplankton indices to better realize the water quality. for example, some investigator utilized nygaard's algal index to evaluate the trophic status of hatia dam revealing the oligotrophic condition of water bodies [ ] . mishra and co-workers studied the bhoj wetland, employing nygaard's algal index to illustrate the wetland's eutrophic nature [ ] . palmer index was utilized in determining the seasonal variation in phytoplankton diversity of pichhola lake of udaipur, rajasthan [ ] . sharan and rekha used nygaard's algal index in their evaluation of the trophic status of hatia dam, finding the waterbody to exhibit an oligotrophic status [ ] . but still, diatoms potential is hardly exploited as bioindicators in determining the economic conditions as compared to other groups of microalgae [ ] . the environmental conditions of the target species must be taken into account as understanding their habitat and mimicking them in the laboratory play a crucial role in obtaining pure strains. each aquatic ecosystem has different attributes. river ecosystems may have less alkalinity than a marine ecosystem [ ] . these attributes directly or indirectly affect the occurrence of diatoms in a diverse aquatic ecosystem. thus, several factors, such as seasonal variation, ph, temperature, salinity, etc. should be considered as important factors [ ] . the representatives of the genus gomphonemoid can be examined from both marine and freshwater. gomphonemoid is widely present often in freshwater epiphytic and epilithic communities [ ] . gomphonema, one of the most common diatom strains, its valve and girdle view, the heteropolar cuneate shape can be recognized easily. gomphonema is attached to the substratum by a mucilage stalk, which assists in recognize strain from the rest of the other diatoms found in the freshwater ecosystem. the valves are wide at the head than foot pole with both uniseriate and biseriate nearby amid individual areolae cover-up externally by crescent-shaped volae [ ] [ ] [ ] . the raphe is straight or sinuous with terminal fissures expanding both apices to the valve margin, and inner central endings turn aside to one or the same side, respectively. the raphe is always shorter from the central nodule to the head pole, thus extending towards the foot pole. consequently, a perforated distinct pole field of different structures is also present at the foot pole. in many species, isolated punctatum near the central nodule occurs. the cingulum is arranged into many open bands; every second band is shorter than the next one to form lingual at the foot pole. each band is punctured by more than one row of pores. many other diatom species with the same cuneate shape, growing in the marine ecosystem can be compared to gomphonema by various workers [ ] . benthic diatoms are the significant controller in freshwater owing to their excellent adsorption, absorption, oxidation, decomposition, precipitation capability to store nutrients. they are very well utilized in biomonitoring purposes since the last decade because these diatoms provide crucial linkage in the aquatic ecosystem stipulating the flow of energy, cycling of materials, and reveal the water environment [ ] . diatom based indices were first produced by kolkwitz & marsson [ ] . since more than indices have been created from to such as humic degree, trophic level indicators, eutrophication, biological diatom index to name a few. however, the impact of indices has become less focal owing to the differences between floral geographical area and environment fluctuations guiding species to adapt according to the water quality criteria of the particular region in a particular instance of time [ ] . isolation and identification of benthic diatoms are problematic in comparison with planktonic species due to difficulties in sample treatment, sampling, and microscopic observation though benthic diatoms play the main role as bioindicators in the aquatic ecosystem because they attached to the substratum with secreted mucilage from their cell wall [ , ] . the morphological features help in the classification of particular species. morphological analysis by light microscope (lm) and electron microscope, for instance, transmission electron microscope (tem), scanning electron microscopy (sem) provides the better-detailed result of diatom biodiversity [ ] . the detection of diatom communities on various substrata can be observed as the slimy layer of biofilm appears brown or yellow-brown. although there are several types of substrata for isolating diatoms, the most preferred is from cobbles (epilithon). epiphytes also have diatoms attached to them [ ] . the taxonomy of the epiphyte should be recorded as specific strains attach to specific macrophytes. a typical substratum can be introduced to the water manually if the preferred substratum is absent. the isolated biofilm can be preserved or can be used for the preparation of axenic culture. a well-concentrated sample collected from a highly polluted habitat may have a more significant number of microalgae rather than diatoms. sometimes an association between diatoms and other microalgae can be seen. it may also contain empty or broken frustules. highly polluted water may have a high concentration of biogenic compounds, which may provide less nutrition or over nutrition. this may profit in the growth of those strain, which requires fewer nutrients or high nutrients, respectively. salinity, ph, light, etc. factors also play a role in the growth of such diatoms [ ] . the productivity of diatoms is higher than the other class of microalgae. they have undergone - cell divisions per day. they can survive in adverse climatic conditions since their silica cell wall is a nanoporous structure which allows them to act as a potential buffer system by improving carbonic anhydrase activity and helps in the transformation of bicarbonate to carbon-di-oxide (co ). they actively participate in wastewater remediation and quenching of heavy metal, leading to a degradation of environmental pollution [ ] . though bacteria, fungus, yeast, and algal biomass are commonly used in the treatment of heavy metal, diatoms are most preferred due to their large surface area, mucilage, nutritional requirement, and faster growth rate. bioremediation of heavy metal involves biosorption to metal-binding ligands on the cell surface, followed by bioaccumulation consists of inorganic molecules and related enzymes. in biosorption, the uptake of heavy metals is either carried out by the physiochemical pathway devoid of atp or during metabolism engaging atp energy. this mode of heavy metal remediation is a cost-effective approach through filtration. it is an extracellular passive route of adsorption of heavy metal, which is an energy-independent process occurring at the cell surface in equilibrium [ ] . biosorption is dependent upon temperature, ionic strength, ph, contact time, biomass and metal concentration, the composition of the cell wall, and metallic ions complexation. the principle behind biosorption is that diatoms are equipped with polymers, their outer wall has a negative surface charge due to functional groups like carboxyl, phosphoryl, and amine. the heavy metal with a positive surface charge attracts a negative charge through electrostatic interaction, which finally supports biosorption. bioaccumulation is an active intracellular process driven by energy. the heavy metals grow along with nutrients that assist in bioremediation. when heavy metals are accumulated, the microalgae produce reactive oxygen species (ros) to control cellular metabolism. there is an underlying passive uptake followed by an active process that is brought about by the detoxification of heavy metal at the cell surface by dehalogenation and denitrification process. this triggers the exclusion of heavy metals to maintain equilibrium conditions, thus eliminate toxic effects. despite this, a lot of work needs to be done in the treatment of wastewater by testing more species and study their capability of heavy metals removal. the biological degradation of heavy metal is quite challenging but not impossible [ , ] . previously, diatoms have been differentiating according to shape, size, pattern, and ultrastructure of their exoskeleton, which consists of silica, known as frustules. microscopic identification is based on the shape of their frustules, but still, they are challenging to identify, and improper examination reflects misleading observation upon identification beyond the genus level [ ] [ ] [ ] . to combat such a drawback, deoxyribonucleic acid (dna) sequence analysis has become a more potent approach in diatom research, thus opens a new leaf into their systematic and evolution. the main aim of the taxonomic examination is to make a hierarchical classification accurately reflecting evolutionary relationship, which then converted into a hierarchy of names related to different levels in a hierarchy of taxonomic ranks. recently, diatoms phylogenetic analysis at the generic and infrageneric levels require internal transcribed spacer (its) region of the s- . s- s nuclear ribosomal dna and among all, s ribosomal ribonucleic acid (rrna) sequencing resulting in the largest reference sequence dataset among the different markers used for diatoms [ ] [ ] [ ] . microarray technology enables parallel analysis of many genes in a single reaction. here, ordered matrices of dna sequence marked on the glass slide used for hybridization experiment with fluorescently labeled target genes and have been extensively used in both basic and applied aspects of the biological sciences [ ] . though several papers related to taxonomy and most of them provide line drawings or light microscopic images that are unable to give enough ultrastructure details of the diatom cell [ ] . a complete taxonomic survey can give the entire morphology of lesser-known microalgae or diatoms. as there can be a minor difference in the morphological structure of two or more organisms, such organisms are difficult to identify even under the lm. organisms like cyclotella brebisson may comprise more than complex species, which includes small specimens with a diameter of ≤ μm [ ] . based on frustules morphology, observation of these small specimens under a sem provides notable interspecific similarities that help in taxonomic studies. as some species may have similar morphological characteristics which may be ecologically distinct, their identification should be fundamentally accurate as it can provide quite an information about biodiversity which can be represented as an essential tool for different kind of studies such as ecological and applied studies. proschkinia is a rare diatom of the family proschkiniaceae within the naviculales. based on light microscopy, it was classified as a relative of nitzschia. the phylogenetic position of proschkinia was resolved with sequencing the complete mitochondrial genome of proschkinia complanatoides and compared the data set to other published diatom mitochondrial genes [ ] . to differentiate between diatoms strains that have similar morphology, it is vital to know the taxonomy of the target species. the nutritional requirements of various species also vary. some species require less nutrition composition, light temperature variables, while some require more. the taxonomy also helps in understanding the basic requirements of the target species [ ] . once the culture has been collected and identified, they will be preserved in the world federation of culture collection centers, which then made available to researchers and industry for intending applications and further experiments. table shows the list of available algae, microalgae, and diatom culture collection centers worldwide. the field procedures play a crucial role in the isolation of diatoms, microalgae, or other species. in a riverine environment, small boulders (rocks) and cobbles are the most favored substratum for diatoms screening. almost all diatom indexed to the eplithion community found here where epilithion`s ecology is recognized better than any other group [ ] . bricks, concrete, bridge supports, canal walls, etc. may be used as an alternative substratum, whereas simulated substrate can be set up into the stream if pebbles, cobbles, boulders, or macrophytes do not exist from the site though sampling should be done if they have been plunged for several weeks [ ] . sampling is an important step towards the collection of diatoms as the whole isolation process depends on it. the collected water sample may contain dead cells or damaged cells, which is not considered good, so it becomes very crucial to ideally collect the sample and isolate the most viable cells at the earliest. keeping the water sample for longer times is not recommended because environmental conditions change very frequently, which leads to cell death. some species multiply quickly and suddenly die, in that case, isolation should be done rapidly. sometimes, a planktonic net can be used to concentrate the sample as it removes other unwanted algal species, microorganisms, or debris but should be avoided as more concentrated sample leads to more damaged cells [ , ] . the collected sample should be kept according to the target species` environmental conditions. for example, several marine species are susceptible to sample concentration. for a sampling of these strains of species, special water bottles can be used. as several factors like water depth, pressure, temperature, or light influence the sample conditions, so instead of using a concentrated sample, a normal water sample taken in sterile containers can provide a better result. the collected sample should always be kept in sterile containers at a stable temperature [ ] . often sample collected from different water bodies contains zooplankton or other species which feed on algae or specifically on diatoms. so, it is essential to remove the unwanted organisms gently by filtering the sample at the sample site. however, sometimes tiny organisms may pass through the filter, which may pose threats to target species, in that case, dilution methods can be used to isolate the target species. the health of species in nature also plays a crucial role as an isolated cell will have successful growth only if they were in a good state at the sample collection time. it is advised to collect the sample carefully. the use of sterile equipment is mandatory, as dirty equipment may lead to contamination. all the isolation techniques should be performed under sterile conditions [ ] . a general idea about field procedure, laboratory procedure, culture preparation, and finally aim towards obtaining axenic culture is presented in fig. . the identification of a suitable substrate is a pivotal step to recognize the diatom communities in the natural environment. diatom communities form a slimy layer or thin golden-brown layer on the substratum. it can be more noticeable by feel or touch and can be identified at a specific time depending on species. the substratum can be differentiated into a preferred substrate and an alternative substrate. diatom attaches to different substrata or facilitates locomotion by releasing mucilage from various structures of the cell wall [ ] . diatom community composition is mainly influenced by chemicals present in water, water turbulence, temperature and light present in water, being eaten by large microorganisms, etc. the most preferred substratum is the solid substratum. it mainly includes cobbles/pebbles and small rocks (epilithon). fig. depicts various substrates intended for diatoms sampling. they are widely available in almost every aquatic habitat and throughout the year. in the absence of a solid substrate, submerged or emerging macrophytic plants (epiphyte) should be sampled as it also provides a suitable habitat for diatom communities. it is ideal to note the microhabitat of diatoms species [ ] . the most effective method to eliminate contaminants is achieved by various techniques such as filtration, sedimentation, and centrifugation as gravity separation. the most successful technique is filtration as it removes all unwanted microorganisms or other zooplankton, which feed on diatoms from the sample. in this, a planktonic net of a specific size is used, which does not allow any other organism to pass through except the target species. in gravity separation, diatoms settle down as sediments while leaving other microalgae in the suspension, which can be discarded further [ ] . field operators should always wear thigh waders for the protection of feet. they should wear life jackets while sampling. they should never go in-depth for sampling. they should wear globes when sampling heavily polluted water bodies. they should be very careful in habitats dominated by dangerous animals posing a threat to people and blood- fig. . flow-chart depicting isolation of diatoms from sample collection procedure to obtaining axenic culture of target species. sucking worms like leeches [ ] . anthropogenic pressure from the urbanized area causes changes in the water quality of a river and leads to the eutrophication of reservoirs and the proliferation of algae. the informal settlements without sanitary infrastructure aggravate the deterioration of water quality in urban water sources [ ] . the water quality index, conducted by using the planktonic diatom index (pdi) for monitoring the lake erie's nearshore pelagic zone, provides a robust assessment of water quality [ ] . the collected water sample should be adequately analyzed as it gives full information about the diatom habitat and its environment. several factors should be measured. hydrological characteristics of the stream, which mainly examined with stream velocity and channel depth and channel breadth. physical variables of water mainly involve water temperature and turbidity. physico-chemical variables are examined with ph, conductivity/total dissolved solids. water contains nutrients such as orthophosphate-phosphorus (po [ ] . for monitoring the pollution of water bodies, several indices have been developed, such as the index of saprobity-eutrophication (idse/ ), which utilizes the diatom population in terms of organic pollution [ ] . water can be divided into two types such as groundwater and surface water and both are at the risk of pollutants ranges from heavy metals, pesticides, fertilizers, hazardous chemicals, etc. an accepted water quality criterion according to american public health association (apha), the world health organization (who), indian standard institution (isi), indian council of medical research (icmr), and central pollution control board (cpcb) consisting of various parameters are presented in table [ ]. examination of the collected sample from different substrata should be done as quickly as possible to make sure that diatom assemblage contains live cells and not dead ones. sample with more dead cells should be discarded, and sampling should be performed again. the environmental conditions of samples collected from various aquatic habitats should be mimicked in a laboratory. it should be noted that all equipment like glass-slide, coverslip, pipette, forceps, inoculating loop, centrifuge tube, glass test tube, watch glass, etc. must be sterilized before use. uncleaned equipment may lead to contaminations that further result in the death of cells. after examination of diatoms in the collected sample, standard isolation methods should be performed [ ] . as the collected sample contains various microorganisms and other microalgae, these standard isolation methods play a crucial role in the isolation of target species of diatom. the isolator should focus on finding the target species in the collected sample. viable cells should be cultured as soon as possible while avoiding contaminants. all the following methods should be performed carefully [ ] . the simplest method of single-cell isolation is performed with the help of micropipette, first attempted by zumstein ( ) later improved by pringshiem ( ) [ , , ] . a capillary is attached to the pasteur pipette to make it ideal for single-cell isolation. this method requires a lot of practice. as diatoms have a minimal size, one single cell can also be picked separately with the help of a micropipette. single-cell isolation with micropipette is to pick a single cell of target species without getting it damage and avoiding contaminants and deposit it into a sterile tube or watch glass and then transfer it to a culture tube. the whole process is done with the help of a microscope, especially an inverted microscope, as it allows the isolator to work conveniently [ , ] . this method is very useful while isolating cells from very contaminated samples with a tiny microorganism, which is harder to separate from the sample. the aim of this method to separate one single cell of target species and deposit it in a culture test tube. usually, dilution is repeated serially up to : [ ]. it is probably the oldest and standard method available to isolate microorganisms. most of the diatom species usually grow well in agar. isolation using agar can be used with both "streaking" and "spreading" methods. later single cells or colonies can be picked from the culture plate and observed under the microscope. to study the colony/diatom community characteristics, agar plate method is advantageous. the agar concentration should be between . % to . % and . %. while observing glass slide under a microscope, if it contains a single diatom cell, it can be replicated on the agar plate for culture. agar pour plate method can also be used to isolate cells that do not grow on the surface of the agar plate [ ] . this is a physical technique for the pre-separation of single cells of diatoms from polluted water. the centrifugal technique applies gravity settling to isolate more prominent species from the microalgae. low dense cells, for example, microscopic organisms and others present in the supernatant, are emptied while diatoms species stay at the base as a pellet. the speed and time of centrifugation vary depending upon the target microalgal species. even though reasonably successful, however, it might harm delicate cells through sheer pressure [ ] . this method involves the use of enrichment media. enrichment media is rich in one constitute which provides nutrition to only one/two types of cells (species) while inhibiting the growth of other cells (microorganisms). for example, it has been noted that the presence of silica in a culture media boost the growth of diatoms but inhibit the growth of other microalgae. sometimes even a trace element makes an enormous difference in the growth of species. so, it is necessary to know the nutritional requirement of the target species, and it should be reached as it will improve the growth of the target species [ , ] . this is a pre-isolation step towards the separation of diatoms. samples can break into two portions depend upon particle size difference. sometimes enrichment media can also be utilized for better outcomes while separating fungal cells from diatoms. bigger species can be held at the membrane however, diatom cells pass through the filter quickly. this strategy is helpful, advantageous, and very adaptable [ ] . another technique where alginate beads were utilized to isolate the algal species from the mixed algal culture [ ] . in this method, the contaminated water is placed in nutrient media and incubate for one week or more to affirm the development of different species. a known concentration of sodium alginate solution containing a mixed culture of diatoms is added drop by drop to a calcium chloride solution to form alginate beads and kept for - h to make beads stronger, stiff, and firm. the beads transferred to a well enzyme-linked immunosorbent assay (elisa) microplate (one bead per well), smashed partially containing standard culture media and incubated under controlled conditions in the presence of light for one week to confirm the growth of isolated diatom species. the trapped diatom species in the beads viewed microscopically for the confirmation of isolated species. this methodology is simple, easy, cost-effective compared with existing methods and can be easily applied for the mass cultivation of specific species [ ] . there are other different strategies for the isolation of diatom species like anti-microbials, uv radiation. anti-microbial treatment hinders growth & development by eliminating microorganisms, thus help with getting the confined unadulterated culture of diatoms. uv radiation act as a disinfectant that prevents bacteria and other contaminants since diatoms are better resistant to uv radiation over bacterial cells [ ] . photoinhibition is an immediate strategy that harms biomolecules by engrossing uv light, which prompts the loss of natural capacity of microscopic organisms [ ] . diatoms are valuable for the aquatic food chain and give significant bioactive compounds to human prosperity. segregating unadulterated species or getting axenic culture from a polluted water source is a complicated and tedious procedure with the current strategies. hence, it becomes necessary to search for other advanced isolation techniques. micromanipulation is one such method permitting refined, confining single species in a more cleansed manner. prior fine capillary tubes were used focusing on target cells under microscopic observation, which is a laborious task and threat to contamination [ ] . with the arrival of a modern, sophisticated micromanipulator exploiting micromanipulator and stereomicroscope, a high level of precision can be achieved for screening and isolation of species of interest [ ] . a cell of interest can be captured utilizing a focused laser and transferred to a sterile media of interest. this system is still in its early stages because of the high state of expertise and time required [ ] . the requirement for cutting edge new methods push researcher to redesign the flow cytometer coupling facs (fluorescence-activated cell sorting). the premise of this procedure is light scattering and fluorescence [ ] . cells absorb the laser beam transmit fluorescence and give information on cell size, pigments, and reliability of species, which is identified with the morphology and other characteristics of the species accordingly, thus allowing characterization of up to , cells in a fraction of second [ ] . this technique can be employed to build up an axenic culture and get rid of any bacterial contamination. in some cases, mixed culture or aggregation of cells may make issues in the precise identification of cells, which can be overcome by cell disruption through appropriate sonication under controlled conditions. this technique is generally excellent for quick screening of organisms overproduces metabolites of interest in conjunction with fluorescent dyes. for example, bodipy or nile red facilitates the selection of desired mutants from a mixed population [ ] . an overview of the main techniques used to establish axenic diatom cultures is presented in table . diatoms, the most productive phytoplankton found all over the planet from antarctic glaciers to brick walls, have drawn a tremendous awareness in the research field, i.e., for the quantitative reconstructions of ocean surface conditions to establish palaeoceanographic records [ ] . they are one of the most promising candidates for various applications such as pharmaceuticals, bioenergy, industrial chemicals, nutraceuticals, and aquaculture [ , ] . to make most out of it, an axenic culture of diatom must be a prerequisite bearing in mind that a pure culture of diatoms is undoubtedly required in genome sequencing [ ] , to identify the producer of any novel bioactive compound for large scale manufacturing of nutraceutical [ ] , building a consortium for bioremediation [ ] and to elucidate the relationship between other microalgae using omics tool [ ] . maintaining an axenic culture for a longer duration is very difficult because bacteria are vulnerable, which frequently attacks the diatom. the primary focus is to isolate pure species of diatoms and their maintenance [ ] . nevertheless, an axenic strategy is dependent upon contamination and desirable organisms disclosing the possible relationship between them leading to the understanding, selecting, and developing an axenic culture, which is a critical step, thus alleviate the cultivation method. mimicking the natural environment for optimum growth under laboratory conditions facilitates the development of an axenic culture, but this step requires the correct isolation plan and approach [ ] . the fundamental question is, are axenic culture genuine.? what is the acceptability of their purity level.? with the direct symbiotic association between diatoms and other aquatic organisms, the development of a new advanced technique to assess the purity level cannot be ruled out. therefore, emphasis should be given towards improvement and innovation in isolation methods, to standardize and establish the correct isolation practices to solve future energy crises, nutritional requirement, nutraceuticals, pharmaceuticals by choosing the right technique for right diatom species for the benefit to the mankind, society and last but not least the ecological balance [ ] . therefore, constant effort to develop a new scientific method will surely pave the way towards the isolation of diatoms from mixed culture and maintaining their purity level to a greater extent. diatoms-virus interactions are rather difficult in obtaining pure culture. the virus kills diatoms, thus benefitting other algae. the limitation in silicon levels in the oceans facilitates infection by the virus. accelerating diatoms mortality due to viral infection is a major concern affecting the carbon cycle leading to global warming and ultimately changes climatic conditions drastically. also, ocean study is a difficult task. the main emphasis is given towards the marine environment rather than terrestrial causing disparity about diatoms-virus interactions. as a result, more isolations techniques and characterization is necessary for controlling the action of the virus in the regulation of host populations [ ] . several species of diatoms are found in a highly acidic environment, and they continue to grow consistently near or in the acidic conditions both in marine and freshwater ecosystems. they exhibited a significant response to the alterations in the growth conditions. they are capable of integrating numerous physiological as well as morphological adaptations, which favors their persistence. diatoms growth inhibited majorly in silica limitations than any other nutrients because cell division cannot sustain for a more extended period under silica deprived conditions [ ] . highly acidic conditions resulted in decreased si, indicating that close to the end of this century ocean acidification might persuade the c and si cycle and alter the composition of diatoms. [ ] . considerable human interference is turning aquatic bodies towards the acidic zone, and many countries have seen ocean acidification even in extreme low-temperature conditions such as north america, canada, and italy. due to the high concentration of hydrogen ions, diatoms flora like nitzschia, pinnularia, eunotia, and frustularia are exceptionally rich in habitats within the ph range of . - [ ] . in an acidic environment, a higher cell volume, chlorophyll, and productivity were observed due to a change in water chemistry because in acidic conditions number of grazing macroinvertebrates and microheterotrophs were less in number [ ] . however, it is unattainable to understand the natural influx of pre-acidification, natural fluctuations in conditions, and the level of nutrients that tend to vary from habitat to habitat. abundance and fall in diatom species can be explained based on physiological ph, availability of the essential nutrients, and biological interactions, which could be the factor for the productivity of diatoms in extreme conditions. very little is known about the influence of acidification on diatoms. hence the effect of acidic environment on diatoms changes from the open sea, near to the sea and deep-sea, and indeed not a decrease in diatoms productivity and growth [ ] . since diatoms nurture associated aquatic species to a greater extent within the marine environment so achieving utmost purity and break communication with unwanted organisms is a significant challenge. therefore, emphasis should be given to improve and channelize knowledge towards proper isolation techniques that will separate diatoms from contamination and any possible intervention to promote appropriate growth and development of diatoms [ , ] . an outline of isolation of pure diatom species getting affected by the surrounding contaminants is challenging since they get heavily occupied with different interfering organisms, which pose a significant threat in obtaining axenic culture, as presented in fig. . although there are several culture media are available, but the most recommended culture media for isolation and growth of diatoms are f/ -si, pm, and wc media. the composition of each culture media is prescribed according to the selection of diatoms for culturing for the desired application. f/ -si culture media is widely used as the most effective media, whereas pm media is utilized for culturing diatoms in an acidic environment. wc media is generally used for culturing diatoms existing in an alkaline environment [ ] . all these culture media differ in their composition of major and minor nutrients and provide proper nourishment to diatom culture. f/ -si media is required for marine species while pm and wc media supports the cultivation of freshwater species. for marine, it is advised to prepare both media with seawater and for freshwater prepare media with distilled water [ ] . the chemical composition of different culture media for diatoms cultivation are presented in table . elimination of contaminants is important to maintain the pure culture of isolated species. as stated, the above contamination can be eliminated with gravity separation, dilution techniques, etc. in the case of bacterial contamination, antibiotics should be used in a specific amount in culture. every equipment, glassware used in isolation or culture should be sterilized before performing experiments. culture should be maintained in aseptic and optimum conditions [ ] . after preparing the axenic culture, it should be subcultured after over a specific period. subculturing should be done before the decline phase of the primary culture. each subculture should be prepared in aseptic conditions. after each subculture, it should be observed for contaminations, and in case of occurrence of contamination that specific subculture should be discarded, and the process is repeated [ ] . an axenic culture contains only one target species and is free of all contaminations. after isolation of target species of diatoms, it is necessary to maintain the culture free from contaminants so that a pure culture can be established. pure isolated cultures can be obtained from a combination of techniques such as flow sorting, pasteur pipette, and agar plate methods. the polluted water collected from various habitat will be immediately examined by monitoring ph and temperature at the site and will be carried to the laboratory in - liters plastic bottles. the raw wastewater was filtered twice with a . μm pore size whatman filter paper to remove the large suspended solids particles and debris. the wastewater will be autoclaved for sterilization and further used for the cultivation of the microalgae. the physico-chemical parameters of wastewater will be characterized as per the standard procedure of apha guidelines [ , ] . initially, the water sample will be serially diluted in a microwell plate and test tube to get a pure culture and observed under the microscope to confirm the presence of microalgae species. isolation will also be carried out by spreading or streaking wastewater in a solid agar medium supplemented with silica. once confirmed, the microalgae species were transferred slowly and gradually in different volumes of erlenmeyer flasks containing artificial seawater enriched with f/ -si media at ph . and maintained in the culture room at h dark/ light diurnal cycle with a desired luminous intensity at - • c. finally, strains will be identified, and their taxonomic classification will be established. fig. shows the overview of obtaining pure culture diatoms from polluted water bodies. diatoms are very young as compared to other phytoplanktons but have evolved rapidly over some time. a decline return on investment in research and development (r&d) and slow-growing companies accelerating the demand for diatoms cultivation serving as a superfood. the term nutraceutical refers to nutrition that provides physiological benefits coupled with the protection and prevention of disease. these functional foods promote health by adding novel ingredients that are similar to conventional foods but with rich nutrition or bioactive compounds that may target the physiological mechanism of our body as characterized by the us department of agriculture, agricultural research service [ ] . on account of large content of polyunsaturated fatty acids (e.g., pufas n and n ), essential amino acids (e.g., leucine, isoleucine, and valine) and pigments (e.g., lutein and β-carotene) and vitamins (e.g., b ), the diatoms biomass have gained much popularity across the globe [ ] . nutraceutical industries manufacturing products on a large scale in collaboration with the food and pharmaceutical industry, thus benefitting consumers. diatoms are emerging as leading nutraceutical ingredients from a biotechnological point of view since they are equipped with polyunsaturated fatty acids, essential amino acids, pigments, and vitamins. according to the world health organization who, the most severe challenge of the st century is a lifestyle disease, i.e., noncommunicable disease. the current focus is to develop functional food and their products, which combat the disease development [ ] . diatoms are underutilized among the microalgae, and only a few species have been characterized thoroughly. diatom based products are an fig. . shows the main interfering agents or treats in obtaining axenic culture of diatoms. . × - . × - . × - excellent source for multifaceted use covering the health and nutraceutical sector. therefore, the cultivation of diatoms under different cultural conditions helps to understand their biochemistry to a large extent [ ] . diatoms cells, after degradation, settled down in the form of silica, which is known as diatomaceous earth. these death remains have tremendous applications for industrial and agriculture purposes. ongoing covid- impacted the world economy. the diatomite industry has also suffered significantly but successful in maintaining an optimistic growth for four years. the average annual growth rate of the diatomite industry will reach millions usd in . probably by , the market potential will see a significant expansion [ ] . diatomaceous earth is one of the major causes of their successful existence on the globe and also act as carbon dioxide sequester on the ocean floor. however, more information is needed about nutrient upwellings and the causes of their bloom degradation in their natural habitats. further, a more in-depth study on their self-defense mechanisms will reveal the unfolded truth about the interaction of diatoms and their feeders in the food web. to overcome the constraints of fossil fuels, the generation of bioenergy makes renewable energy particularly interesting. nowadays, renewable resources are contributing % of the total energy of the world. diatoms synthesize oil for human consumption, which is rich in nutrition showing a promising alternative to meet the demand of future growing populations. we need to emphasize the latest, more productive technologies to overcome the cost of their cultivation and harvesting for optimum utilization of diatoms as biofuels, valuable products, wastewater remediation, and aquaculture [ ] . the generation of bioenergy from novel sources like diatoms is incredibly significant as they contribute towards carbon dioxide mitigation generation of renewable energy concomitant with a plethora of value-added products. due to their unique evolutionary history and their adaptations to varied environmental conditions, diatoms have spread all around the world. undoubtedly, diatoms are playing a significant role in the reduction of global warming gases such as carbon dioxide and provide possibilities to change the present climatic conditions. but still more researches are needed on their biogeographic distribution and to learn more about factors which are responsible for their more successful existence. diatoms cells, after degradation, settled down fig. . protocol for adopting isolation strategies employed for diatom axenic culture. a. saxena et al. in the form of silica, which is known as diatomaceous earth. these death remains have tremendous applications for industrial and agriculture purposes. diatomaceous earth is one of the major causes of their successful existence on the globe and also act as carbon dioxide sequester on the ocean floor [ ] . however, more information is needed about nutrient upwellings and the causes of their bloom degradation in their natural habitats. further, a more in-depth study on their self-defense mechanisms will reveal the unfolded truth about the interaction of diatoms and their feeders in the food web. to overcome the constraints of fossil fuels, the generation of bioenergy makes renewable energy particularly interesting. nowadays, renewable resources are contributing % of the total energy of the world. diatoms synthesize oil for human consumption, which is rich in nutrition showing a promising alternative to meet the demand of future growing populations. we need to emphasize the latest more productive technologies to overcome the cost of their cultivation and harvesting for optimum utilization of diatoms as biofuels, valuable products, wastewater remediation, and aquaculture. they are the primary food of most of the herbivores in freshwater, as well as in marine water. furthermore, being the rich source of poly unsaturated fatty acid (pufa), they have a great scope in aquaculture. therefore, diatoms are the cost-effective approach which sequesters co , excess amount of heat, reduce the extra amount of nutrients, and remove metal contaminations from their habitats. they could be a possible solution to energy savers and to reduce the threat of global warming. conversely, they may be a viable source of food and feeder for living beings. diatoms and wastewater are an advanced integrated system that, on the one hand, utilize the potential of diatoms in purifying the wastewater thus, it helps in restoring the original quality of habitat. on the other hand, their biomass can be utilized in the production of antiobesity, antibacterial, antioxidant, biofuels, anticancerous, antiviral compounds. diatoms have received great attention because they are the significant producer of lipids and biomass for various commercial applications such as biofuels comprising of biodiesel and aviation fuel. they participate in cleaning the environment by fixing the atmospheric co and contributes to the reduction of greenhouse gas (ghg) [ ] . diatoms based biofuels are projected to be economically beneficial in the future, but still, some ambiguity exists whether they equate with fossil fuels or not is remain a question. obtaining a biofuel require several steps like harvesting, extraction, conversion of biomass to target biofuel is economically not feasible. one of the key bottlenecks is cell density, which is similar to the water and negative surface charge that put off settling owing to gravity, thus makes harvesting a most crucial and challenging step. there is an urgent need for an efficient harvesting technique to improve the economics and efficiency of the whole downstream process [ , ] . at present, the technologies of the water purification industry have been used for harvest and recovery of microalgae, but still, some technical glitch needs to address that are sole to microalgae harvest. firstly, harvesting techniques must be species-specific. secondly, a blend of different harvesting techniques must result in synergistic outcomes. thirdly, a complete cell separation from a diluted suspension to lower the cost of the downstream process. fourthly, steps such as lipid extraction and biofuel conversion should be minimized. finally, to come up with the idea of more advanced harvesting techniques in the future [ ] . the ultimate goal of economic feasibility is the conversion method by utilizing inexpensive chemicals and the release of the least toxic waste. microalgae biomass is the best option for fossil fuels for transportation, but the commercialization of microalgae-based biofuel is a significant task. new strategies should encourage innovative elements to the existing downstream process that can appreciably reduce the cost towards the realization of biofuel commercially and dispose of harmful co production. to sum up, harvest, extraction, and conversion steps must be environment friendly, while making biofuels as future transportation fuels [ , ] . all the above-mentioned factors enhancing the popularity of diatoms globally strengthen their market potential and growth in the coming years. their medicinal characteristics are likely to provide more interest in diatoms cultivation with a rising population. considering health benefits, the global diatoms market is expected to witness as one of the most emerging economies across the world. a general question raised by everyone about diatom`s attractive potential and advantages but still exploring their true value is a long journey because high rich diversity limits their studies making it obscure and inaccurate. it is high time to investigate new class and species of diatoms and understand their mechanism, research models, and the potential role for future perspectives. the past few decades have seen a tremendous rise in diatoms research, as reflected in the publication. an undercurrent excitement is moving around, but challenges are roaring high. practically, a good isolation practice is the only solution that will fill this void. smart isolation practices are prerequisites that must help quick isolations and speedy recovery of diatoms from polluted water. diatoms`axenic culture is the first step towards a complete study of an organism and maintaining their pure culture can be further used in different applications such as bioindicators, nutraceuticals, cosmetics, phycoremediation, aquaculture, etc. thus, growing 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challenges and current status of microalgae biomass applications in industrial wastewater treatment preparation, characterization and catalytic performance of mesoporous silicates derived from natural diatomite: comparative studies ocean acidification interacts with variable light to decrease growth but increase particulate organic nitrogen production in a diatom methods of downstream processing for the production of biodiesel from microalgae innovative microalgae biomass harvesting methods: technical feasibility and life cycle analysis dr. archana tiwari is thankful to the department of biotechnology (dbt), new delhi, india for providing financial support under project grant no: bt/pr/ /aaq/ / / . all listed authors are grateful to their representative universities for providing the literature access. key: cord- - sle d j authors: castillo-huitrón, nathalia m.; naranjo, eduardo j.; santos-fita, dídac; estrada-lugo, erin title: the importance of human emotions for wildlife conservation date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: sle d j animals have always been important for human life due to the ecological, cultural, and economic functions that they represent. this has allowed building several kinds of relationships that have promoted different emotions in human societies. the objective of this review was to identify the main emotions that humans show toward wildlife species and the impact of such emotions on animal population management. we reviewed academic databases to identify previous studies on this topic worldwide. an analysis of the emotions on wildlife and factors causing them is described in this study. we identified a controversy about these emotions. large predators such as wolves, coyotes, bears, big felids, and reptiles, such as snakes and geckos, promote mainly anger, fear, and disgust. this is likely due to the perceptions, beliefs, and experiences that societies have historically built around them. however, in some social groups these animals have promoted emotions such as happiness due to their values for people. likewise, sadness is an emotion expressed for the threatening situations that animals are currently facing. furthermore, we associated the conservation status of wildlife species identified in the study with human emotions to discuss their relevance for emerging conservation strategies, particularly focused on endangered species promoting ambiguous emotions in different social groups. since our origins, wildlife has always had a very important role in human life. the very diverse and continuous human-wildlife interactions can be seen from three main perspectives: ( ) utilitarian, in which wild species provide goods for human well-being, such as food, clothing, transport, tools, raw materials, and companionship, among others; ( ) affective, where human beings feel sympathy, admiration, and respect for animals because of religious, mystical, or philosophical reasons (kellert et al., ) , which has greatly contributed to cultural development worldwide (herzog and galvin, ; alves, ) ; and ( ) conflictive, because of the real or potential damage that wild species may inflict on people and their interests (e.g., attacks on humans, livestock predation, damage on crops, and infrastructure, among others; lescureux and linnell, ) . human-wildlife conflicts have motivated animal killings for centuries, which in many cases continue nowadays (woodroffe, ) . human-wildlife relationships have relied on the uses, values, and meanings that animals represent for people through time and space in different cultures (driscoll, ; . societies have developed a cultural predisposition for emotional reactions toward wild animals (kellert and wilson, ) , causing either positive or negative effects depending on the species (york and longo, ) . fear, anger, and disgust are emotions generating attitudes and behaviors against the presence of some species (fritts et al., ; jacobs, ) . in contrast, emotions, such as happiness, which comes out when cherished species are seen in a given place, or sadness before the vulnerability of others, may generate positive attitudes for their conservation (prinz, ) . this relationship between human emotions and attitudes has an effect on the presence, absence, and recovery of wildlife populations (herzog and burghardt, ) . understanding the transcendence of the emotional factors triggered by animals on human beings would improve our knowledge on the human dimensions of wildlife conservation. in this paper, we offer an overview of the influence that emotions have had on the relationships between wildlife and people through time. a substantial amount of the literature reviewed consists of studies conducted on large carnivores in europe, such as the brown bear (ursus arctos) and the wolf (canis lupus), as well as on snakes around the world. we analyze and discuss relevant aspects that could be considered in further studies on threatened and culturally relevant animal species across latin american countries. darwin ( ) recognized that emotions are manifested by all persons throughout their lifetime, but they vary in an individual between different moments of its life span. frijda and mesquita ( ) mentioned the main points characterizing the emotions in their theoretical perspective: ( ) emotions are considered individual responses to relevant events producing feelings of pleasure or pain; ( ) they help to find solutions to concerns that cannot be treated routinely; ( ) they are always about something, they are used to accept or decline the interaction with a real or imagined object, person, or wild animal in this case; ( ) they tend to control behaviors and thoughts (e.g., angry impulses, behaviors, and thoughts); and ( ) emotions are correlated with psychological, physiological, and social components establishing, changing, or maintaining a particular relationship with a specific object in a concrete situation. there is a wide array of studies analyzing human emotions, their origins, functions, and presence in human life (ekman, ; plutchik, a; nummenmaa et al., , among many others) . six basic emotions have been proposed: happiness, surprise, disgust, anger, fear, and sadness (ekman et al., ) . izard ( ) suggested classifying emotions into two groups: "positive, " representing interest and joy (happiness and surprise), and "negative, " including anger, disgust, fear, and sadness. this classification is an artifact of traditional psychology not informed about an evolutionary approach. here, we have focused on basic emotions to explain human-wildlife relationships because secondary emotions (the combination of basic emotions) are more useful for assessing social relationships among human beings (harelli and parkinson, ) . in this review, we consider two different approaches to explain the origins of basic emotions aiming to understand human-wildlife relationships through time. the first is the evolutionary approach, which suggests that emotions have evolved to solve adaptive problems in different environments (plutchik, b) , such as social communication, reproduction processes, and mechanisms for information processing leading to behavioral responses to specific events or objects (al-shawaf et al., ) . predator presence could have been one of such events contributing to the evolution of human emotions and the development of physiological, psychological, and morphological responses for survival (Öhman and mineka, ; prokop and randler, ) . in particular, fear and disgust are adaptive emotions helping to react toward something representing a risk for human life (ekman and cordaro, ) . fear and disgust, for instance, have been the most studied emotions due to their implications for human survival since the origin of our species . fear probably was a defense mechanism against dangerous animals, particularly large predators (Öhman, ; dalgleish, ) . it is believed that potential alert signals emitted by human groups facing predators, with whom they coexisted and sometimes competed for space, water, prey, and other resources, triggered physiological reactions such as heart rate increase, profuse sweating, and pupil dilation, allowing the generation of alert responses. in that way, human beings have historically developed greater awareness toward potentially perilous animals, such as snakes and spiders Öhman and mineka, ; lobue and deloache, ) . this adaptation mediated by fear has probably been genetically fixed throughout generations, provoking the innate physiological responses mentioned above when dangerous species are or could be present (Öhman, ) . the amygdala is the brain region where fear-generating stimuli are processed into a strong reaction that in some cases may affect human vision (phelps et al., ) . on the other side, disgust can help protect the individual against infections and disease (curtis et al., ) . disgust is saved in memory to avoid future exposure to the subject, in this case with potentially threatening animals (al-shawaf et al., ) . the second approach explaining the origins of basic emotions is the cultural context, where people integrate their physical environment with individual and collective experiences, perceptions, meanings, attitudes, and animal-related traditions to construct emotional diversification (prinz, ; johansson et al., ) . in this view, it can be said that human emotions associated with wildlife have evolved over time and continue to be gradually built and rooted in our societies all over the world. under the cultural context approach, emotions can be understood on two levels: ( ) the individual level, involving meanings, beliefs, attitudes, and behaviors based on personal experiences, knowledge, and perceptions, and ( ) the social level, where emotions are determined by collective factors such as experiences, meanings, beliefs, and myths typical of a certain region or culture, which are transmitted among individuals throughout generations (ekman, ; prinz, ) . physical characteristics of wildlife species and their "personalities" created by humans have generated a variety of emotions (kellert et al., ; kruuk, ; prokop and randler, ) . emotions such as fear and anger may be induced by predators that are bigger and heavier than persons, as in the case of large carnivores (e.g., bears, wolves, and big cats) (røskaft et al., ) or by those species unattractive for most people, like worms, small carnivores, bats, and reptiles, which are often perceived as harmful (knight, ; prokop and tunnicliffe, ; prokop et al., ) . in contrast, beloved animals such as colorful birds or small herbivore mammals (e.g., rabbits) may cause happiness providing they are not noxious for people or their livelihoods (prokop and kubiatko, ) . however, these animals are sometimes perceived in different ways. for some social groups (e.g., farmers), small mammals such as rabbits as rodents may represent a threat due the damage they can inflict on crops, cattle, properties, and human health (morzillo and merting, ; breed and moore, ) . actual or potential damage can promote negative attitudes motivated by emotions of anger, disgust, and fear. animal body shape is another physical feature that has been found to be important for the expression of emotions such as fear and disgust. in the case of class reptilia, two groups could be recognized by people according with their similar morphotype (with legs and legless). reptiles with legs (lizard, turtle, and crocodile) tend to cause fear in many people; crocodiles, specially generate intense fear in many people, in part because of the number of attacks occurring worldwide (crocbite, ) . in contrast, legless reptiles (e.g., amphisbaenia and larutia) that have thin bodies, smooth textures, small eyes, and dull colorations generate disgust rádlová et al., ) . specifically, snakes have long bodies, scales with contrasting patterns, bright coloration, and silent, rolling movements that immediately calls up human attention deloache, , ; rádlová et al., ) . it is likely that both fear and disgust can be simultaneously felt by a person observing a particular species . the ample diversity of snakes around the world makes it difficult to generalize emotions across cultures toward different taxa. species coloration has been an attribute to help identify dangerous animals (prokop and fančovičová, ) , allowing emotional responses in human beings (Öhman, ) . striking color ("aposematic") combinations such as bright red and black in some snakes and spiders intensify fearful reactions (Öhman and mineka, ; lobue and deloache, ; . on the other hand, it has been reported that striking coloration allowed perceiving snakes as beautiful animals in spite they are fearsome . it is noteworthy that aposematic species are simultaneously fearsome and attractive particularly for young persons between and years of age, promoting their interest in those animals (prokop and fančovičová, ) . on the other hand, animals' coloration could be attractive for humans and motivate "positive" feelings. in this sense, lišková et al. ( ) discovered that hues of blue and green in birds of the pittidae family promote human preference. psychologists have found that green is usually associated with happiness, relaxation, and comfort because it is related to nature, while blue elicit happiness, relaxation, and peacefulness, among other feelings (kaya and epps, ) . however, human affection for birds also represents a pressure for wild populations, especially for those charismatic species used as pets, promoting illegal trade (alves et al., ) . feeding habits of species may also influence emotions: large predators are usually regarded as hazardous and fearsome, while their prey provoke sadness (prokop and kubiatko, ) . large herbivores and omnivores in some places are often seen as less fearsome than strict carnivores. this is the case of the mainly vegetarian brown bear (ursus arctos) in some regions of europe (lescureux and linnell, ) . however, in other areas and cultures, large herbivores such as elephants (loxodonta africana) cause intense emotions of anger and fear because of the damage they inflict on crops and rural villages (lamarque et al., ) . although "dangerous" animals promote the attention of people (prokop and randler, ) , it is interesting to note that human emotions may vary depending on the life stage of the animal. for example, jaguar (panthera onca) cubs and lion (panthera leo) cubs are perceived as lovely and safe animals given their physical features, causing minor concern in societies, while adult jaguars and lions are generally considered less attractive and very dangerous, promoting fear (knight, ) . this trend is also reported for amphibians, for which people show more disgust toward the adult stage than for tadpoles (prokop and fančovičová, ) . venom in animal species is one of the most remarkable features triggering fear across cultural groups. as a consequence, snakes constitute an interesting case study in which most species produce fear all over the world, although particular species are in fact perceived as beneficial due to their role as controllers of agricultural pests, producing positive feelings in local farmers (ballouard et al., ) . in this regard, ballouard et al. ( ) observed different intensities of fear toward selected snake groups (cobras, vipers, and boas) depending on the nationality and cultural background of their interviewees. animal activity patterns constitute one more physical factor influencing human emotions toward wildlife. humans are not adapted for living in the darkness; they have a poor vision to act in this kind of environment, hence they may associate nocturnal species such as felines, some snakes, rodents, and bats with danger (buss, ) . in addition, these animals historically have been linked to "evil forces" damaging human beings worldwide (prokop et al., ) . contrastingly, many diurnal species (e.g., most of the birds and ungulates) are usually related to positive values such as peacefulness and wisdom that have inspired leaders and rulers to make better decisions (cano-contreras, ). physical characteristics have been useful to classify animals depending on the emotions they produce on people. in this sense, tarantulas, snakes, sharks, and mosquitoes have been categorized as perilous, generating agonistic emotions. contrastingly, large, charismatic species that have traditionally been regarded as dangerous but intelligent at the same time motivate emotions that may result in actions for their protection, as it has occurred for lions (panthera leo), tigers (panthera tigris), leopards (panthera pardus), and polar bears (ursus maritimus) (driscoll, ; landová et al., ) . these categories have emerged after the anthropomorphization of animals, a process in which cultural groups attribute human features and "personalities" to wildlife species (kruuk, ) . for instance, the panda bear (ailuropoda melanoleuca) inspires tenderness and happiness when it is observed, but those emotions are overcome by sadness after considering its high vulnerability to extinction. in this case, positive attributes facilitate particular species to become flagships for wildlife conservation (root-bernstein et al., ) . in rural communities where people frequently interact with wildlife, knowledge about the behavior of culturally relevant species develops better than in other areas. this facilitates the anthropomorphization of certain animals calling them "shy, " "noxious, " and "monstrous, " among other adjectives, which intensifies fear and rejection toward them (lescureux and linnell, ) . furthermore, if the presence of an animal implies economic losses for residents of a community, their predominant perception will be negative and will produce anger that may end in lethal management (naughton-treves, ). contrastingly, animals inspiring greatness and qualified as "kings" of the wilderness will likely motivate local people to feel happiness and pride because of their presence in the region (lescureux and linnell, ) . these examples help identifying the relevance of animal physical features in emotions, which transform throughout history according to the natural, social, and economic context of each human generation. in some cases, emotions produce attitudes against the conservation of unpopular species (knight, ) . therefore, we propose to highlight the ecological role of dangerous or disgusting species as a potential way to mitigate negative emotions toward them. emotions induced by wildlife differ among individuals according to variables such as their sex, age, cultural and natural environment, and perceived vulnerability to each species (johansson et al., ) . it has been shown that young children (under years of age) of both sexes take more time to detect a snake and react toward it than their parents (lobue and deloache, ). that behavior was explained by deloache and lobue ( ), proposing that fear and alert signals in front of this kind of animals develop later, when individuals start to explore their environment and link adult behaviors with animal species. fear and disgust have been the most studied emotions between genders. in general, women tend to express stronger negative emotions (fear and disgust) toward invertebrates, amphibians, predatory mammals like bears, wolves, lynx (lynx lynx), and wolverine (gulo gulo) and toward snakes compared to men (Öhman and mineka, ; røskaft et al., ; ballouard et al., ; bajwa et al., ; . this difference seems to be related to the female gender role taken since the start of human evolutionary history, where men developed skills for both hunting and escaping from predators (prokop and fančovičová, ) . likewise, men gradually reduced their fear of large animals, while women kept distance from those species in part because of their household activities and their care for children in safer places (røskaft et al., ; prokop et al., ) . however, differences within genders are usually present in different cultural and geographic contexts (kellert and berry, ; bjerke et al., ; de pinho et al., ) . in some societies, women, particularly adolescents, have a greater disposition to spend more time in wildlife related activities as compared to men (e.g., volunteer programs; kidd and kidd, ) . this information could be useful to direct conservation programs in spaces as zoos where experiences with uncharismatic and endanger animals could help to promote positive emotions and attitudes. age is a significant variable determining the presence and intensity of agonistic emotions toward animals, which may be related to personal experiences. childhood is the critical life stage when fear of predators starts and when attitudes and behaviors to avoid encounters with them develop (Öhman, ) . it is likely that fear of predators intensifies with learning from parents, given that as the child gets older, his/her reactions become faster when facing species such as snakes (lobue and deloache, ) . in this regard, fear of animals may either decrease (kaltenborn et al., ) or increase (røskaft et al., ) with age. besides age, the natural and cultural environments in which an individual grows determine the knowledge, perceptions, and emotions related to animals (frynta et al., ) . for a person raised in close contact with nature, an encounter with a wild animal can induce happiness, while the same species may produce fear in an individual that has always lived far away from natural spaces (kellert, ; manfredo, ; almarcha, ) . the presence or absence of different species in human territories has a role in the generation of emotions. residents of rural areas who frequently interact with wildlife are usually less fearful of animals than city dwellers. this is because closeness with native animals promotes knowledge about their ecology and behavior, allowing for building better management strategies and reactions toward them (røskaft et al., ) . likewise, recreational activities involving contact with wildlife such as hiking, bird watching, fishing, and hunting have direct influence in emotions, facilitating the overcoming of fears and phobias by promoting learning through first-hand experiences, although in some cases, these activities decrease with age (bjerke et al., ; røskaft et al., ; prokop et al., ) . in particular, emotions produced by hunting deserve further discussion. subsistence hunting as a traditional practice in many rural areas of the world usually involves local regulations to avoid overexploitation and feelings of respect by the hunters toward their prey (e.g., santos-fita et al., ) . in contrast, sport hunting is more focused on the pleasure of the hunter for finding and killing his target species, which has been a motive social dispute in different contexts, generating anger in broad sectors of society considering this an unacceptable practice (nelson et al., ) . some of these recreational activities involve parents and their children, who get used to those practices at an early age (amiot and bastian, ) . this can be an important inter-generational strategy to avoid negative attitudes toward fearsome and disgusting animals and promote positive emotions (i.e., happiness and surprise), especially in areas where humanwildlife conflicts may arise. significant differences have been found among people with different levels of study with respect to fear of wildlife species: individuals with higher levels of education are generally less fearful of wild animals than those with lower degrees of studies (røskaft et al., ) . it is likely that individuals with higher education had more opportunities to receive information on the environment and wild animals in particular, which may have reduced their negative prejudices and perceptions about non-charismatic species, maximizing their perspectives on the ecological benefits provided by those animals. the geographic space where an event occurs triggers distinct emotions, which have varied according to the lifestyles of societies (mesquita and frijda, ) . this argument could be used to understand emotions historically induced by wildlife, considering the different worldviews of each culture. for example, snakes were regarded as deities in mesoamerican cultures, including quetzalcoatl or kukulkan (the feathered serpent), which was the most important deity for the aztecs and the maya, respectively (díaz, ) . snakes were also given high rankings among the deities of the ancient greek, egyptian, hindu, and roman civilizations, where some of these reptiles were associated with values of wisdom, justice, and power (stanley, ; al-rawi, ) . these reptiles have also starred countless stories and myths around the world (ménez, ) , but for christians, muslims, and jews, snakes have traditionally represented evil and death (gonzález, ; al-rawi, ) . nowadays, myths about the damage caused by snakes are important elements to promote and intensify fear in rural communities (fita et al., ) . the social fear could be learned, inherited, and used by societies across generations, driving particular attitudes toward wild species (Öhman, ) . in this case, the relevant ecological role of snakes as predators and pest controllers has been largely neglected. another interesting example is that of wolves, which have been protagonists of many stories and myths worldwide. these carnivores have traditionally been portrayed as fearsome and dangerous animals, producing social rejection in most areas where they are present, nonetheless, in particular cases such as that of ancient rome (whose founders were suckled by a shewolf) and that of native north american cultures, for whom wolves were spiritual symbols related to power and intelligence (fritts et al., ; prokop et al., ) . beyond mythology, other elements that have facilitated the development of cultures (e.g., art, literature, symbolism, religion) have had their foundations in the relationships between humans and wildlife, involving emotions promoting respect and admiration (fritts et al., ; alves, ; almarcha, ) . these emotions frequently lead to attitudes favorable for animal care and conservation. other events that have always happened, but which have received special attention in recent decades because of the human population growth and expansion, are the attacks of large carnivores on people and livestock, and crop damage by large herbivores (inskip and zimmermann, ). these events make jaguars, tigers, lions, leopards (panthera pardus), hyenas (crocuta crocuta), african wild dogs (lycaon pictus), and african elephants (loxodonta africana), among others, be considered problems in rural communities, giving place to misunderstandings and false beliefs about their behavior (marchini and macdonald, ; dickman et al., ) . this situation has contributed to magnification of the actual damages of those species, stimulating even more fear, disgust, and rejection toward them (lescureux and linnell, ) . in this sense, the individual background and experiences of humans contribute to their emotions and behaviors. for example, the presence of large predators may produce fear and thoughts of escape in most people, while some others may feel encouraged to confront the danger (al-shawaf et al., ) . the context of the encounter with an animal may also be relevant for the emotions manifested. for a given person, the sighting of a carnivore such as a female puma with their offspring while hiking on a forest trail may produce fear and desire to escape. in contrast, the same person may feel surprised and delighted to have the same sighting from the safety of a car (narratives collected by the first author in chiapas, mexico). furthermore, local knowledge and the emotional links between people and wildlife could be useful to identify flagship species to foster interest in nature (bowen-jones and entwistle, ) . flagship species [e.g., giraffe (giraffa camelopardalis), elephants, and lions, among others] are usually charismatic and popular and may be relevant for promoting positive emotions in a public that has been distant from wild animals. differently, more complex sets of emotions (both positive and negative) are usually present where people are in constant interaction with these animal species (bowen-jones and entwistle, ; de pinho et al., ) . zoos represent spaces where emotional confrontations take place. for instance, marseille et al. ( ) observed visitors watching imposing and charismatic polar bears. the authors found that visitors felt happy in front of the bears, but at the same time they felt sad after recognizing the small size of the enclosures and the stereotyped behavior of the captive animals. interestingly, visitors' emotions transformed into fear and even greater sadness when they were told about observing polar bears in their natural habitat, which was associated with concerns about human safety and habitat vulnerability. another element that has an effect in the affection of children for wild animals is the presence of pets (bjerke et al., ) . pets can boost appreciation emotions, such as happiness, while naturalistic, ecological, humanistic, and moralistic attitudes may also be encouraged (prokop and tunnicliffe, ) . although knowledge about animals usually differs between urban and rural communities, the lack of accurate information about the species and their contribution to ecosystem services is persistent in both environments (gomes et al., ) . it promotes the intensification of emotions such as danger and disgust, especially for species that are unattractive to people. disgust has also been identified as one of the emotions inducing human rejection. it may arise when people perceive nasty odors in animals, or when unpleasant feelings emerge while touching (or thinking about) the fur of certain mammals (johansson et al., ) or the skins of amphibians such as frogs (lobue and deloache, ) . in other cases, disgust may be brought after linking animals such as spiders and rats with dirtiness, pollution, disease spreading, and potential crop damage (kellert, ; davey, ; prokop and tunnicliffe, ) . furthermore, animals that cause disgust are often perceived as ugly . contempt of human societies for amphibians and reptiles intensifies misinformation about them and favors negative attitudes toward them (manzano- garcía and martínez, ) . for example, it has been documented that non-venomous snakes are killed just because of their resemblance to poisonous species (breed and moore, ) . moreover, misinformation is an intensifier of disgust, for instance, when considering geckos (hemidactylus turcicus) as venomous animals or vectors of skin diseases (ceríaco et al., ) , or bats as a threat for fruit crops and responsible to infect people with parasites and viruses (musila et al., ) . in this sense, the case of bats and pangolins (pholidota) could be cited, which are considered the main transmitting agents of the novel coronavirus (covid- ; van staden, ). the respiratory illness has become a pandemic infecting million and killing many thousands of people around the world (nature, ). it is likely that the disease has a zoonotic origin as a result to the food and medicinal uses of animals (van staden, ). therefore, in some places there has been motivation to eliminate these animals (zhao, ) . this event might increase the negative perception and emotions of anger, disgust, and fear for this kind of animals and will encourage the eradication of populations without considering their importance in ecosystems. in this regard, it has been found that women and residents living near caves tend to believe in myths about bats more than men and people living far from caves (musila et al., ) . fearsome and disgusting species frequently induce rejection attitudes in social groups (Öhman and mineka, ), a phenomenon known as "biophobia" that is used to express the feeling of panic, fear, and disgust in front of a particular non-human living being. phobia for animals (agrizoophobia) is one of the most frequently reported biophobias in the general population (antony and mccabe, ) , but there are actually around twenty-five documented phobias to particular animal groups, such as that for snakes (ophidiophobia), spiders (arachnophobia), insects (entomophobia or insectophobia), ants (myrmecophobia), bees (apiphobia or melissophobia), and birds (ornithophobia), among others (fredrikson et al., ; antony and mccabe, ; prokop and fančovičová, ) . however, there are no specific phobias for carnivores, probably because the coevolution between humans and these animals has been too short in comparison with other groups such as snakes (prokop and randler, ) . biophobia may promote persecution and extermination attitudes (zhang et al., ) . avoiding contact with animals or killing them are the most frequent reactions without considering their long-term impacts on ecosystems (antony and mccabe, ; al-shawaf et al., ) . orr ( ) mentioned that one of the causes of biophobia is social distancing from nature. in a parallel way, biophilia has a genetic basis and consists of the interest and empathy of humans for other living beings (wilson, ) . as industrialization and urbanization increase around the world, lifestyles change in human societies, sometimes in radical ways (steffen et al., ) . these processes have contributed to the distancing of people from their natural environment even in rural communities (louv, ; lescureux and linnell, ) . however, there are still spaces such as zoos and natural parks facilitating social approach and understanding of wildlife in most of the cities and large towns all over the world. in those spaces, visitors are generally safe in front of animals that otherwise would be considered dangerous or harmful, and they may feel sadness and even culpability after recognizing the impact of the human population on those species. in this sense, vining ( ) suggested that visiting zoos and natural parks may represent opportunities for reconnecting people and wildlife to enhance social cooperation in conserving biodiversity. human emotions transcend over time. a specific emotion is saved by the individual as an experience that may be used in future behavior and decision-making (izard, ) . protection attitudes toward spiders, insects, amphibians, and reptiles are milder than those shown for other groups, such as birds and mammals due the sentiments of danger or disgust that these animal groups provoke in humans (prokop and fančovičová, ; . in addition, emotional experiences may have an effect on wildlife management techniques (larson et al., ) . this has occurred during experiences of invasive species management. one example is that of the house sparrow (passer domesticus), which competes for food and space with native birds and generates anger or disgust when managed through nest and egg removal, repellents, and traps. in contrast, bluebirds (sialia sialis) stimulate happiness in people watching them and listening to their songs, who at the same time feel sadness for these birds due to the negative impact of human activity on their populations. these feelings motivate protection attitudes favoring the persistence of the liking bird species (larson et al., ) . it is important to recognize that fear impacts human attitudes and behaviors toward keystone species, particularly those regarded as dangerous or harmful (e.g., wolves, bears, and big cats). fear may limit the involvement of local communities in managing predator populations because of the high costs implied or because the social acceptance of certain techniques, such as reintroduction, may be difficult (johansson et al., ) . examples of this include reintroducing wolves in mexico and the united states, where emotions have played fundamental roles in the acceptance of new wolf populations (straka et al., ) . mexican wolves (canis lupus baileyi) were eradicated from the mexican territory in the s because of conflicts with farmers and negative perceptions due to livestock predation (leopold, ; moctezuma et al., ) . wolf reintroduction projects have been started recently in northwestern mexico, where it has been clear that social acceptance is the primary limiting factor for their success (araiza et al., ; garcía, ; lara-díaz et al., ) . society's emotions toward wildlife may be key elements for decision-making on conservation issues. anger is one of the primary collective emotions that can lead to positive changes for natural resource management when social pressure is put on government leaders to improve and enforce environmental legislation. however, anger may have other implications and cause social fragmentation (buijs and lawrence, ) . in these cases, participation of wildlife management agencies is crucial given their social confidence. if the capacity of these agencies is not appropriate, collective distrust and fear of dangerous and disgusting animals may stimulate hostile environments for their proper management (johansson et al., ) . community confidence in environmental agencies is especially relevant where threatened species are under recovery, as is the case with wolves in different countries (swenson and andrén, ) , or where people take action by themselves, such as in the case of the killings of andean bears (tremarctos ornatus; figueroa, ) . it seems clear that some wildlife species are far more significant to humans than others (herzog and burghardt, ) , perhaps linked to their evolutionary closeness (e.g., primates, and particularly the great apes; gunnthorsdottir, ; miralles et al., ) or because of their cultural, aesthetic, or affective attributes favoring more interest and attention toward them. interest and attention favor people's attitudes for conserving these species, differently from others without a transcendental meaning for social groups. this idea highlights the relevance of designing conservation strategies fomenting interest for wildlife through generating affective links between humans and animals both in rural and urban areas. beautiful and attractive animals causing "positive" emotions (e.g., happiness and surprise) receive special attention driving in situ and ex situ conservation actions (gunnthorsdottir, ) . this could be a limitation for conservation efforts focused on species considered unattractive particularly in zoos. the preferences of human societies to watch specific animals have promoted that zoos keep attractive species more than those needing protection due to their conservation status (frynta et al., (frynta et al., , . mammals constitute the preferred group among zoo visitors around the world (moss and esson, ) . however, these spaces keep only , individuals belonging to , species (frynta et al., ) , which represent just . % of known living species (burgin et al., ) . this preference is strongly biased toward large, attractive, and active mammals belonging to the families ailuridae, felidae, phascolarctidae, ursidae, giraffidae, elephantidae, equidae, macropodidae, mephitidae, and cervidae, among others (frynta et al., ) . the same correlation between human preference and species kept in zoos was found for large, colorful, and long-tailed parrot species (frynta et al., ) . in contrast, small and unpopular species do not motivate the same appreciation, even if they are endangered. as a consequence, zoos generally keep a few of those local species (frynta et al., ) . in this sense, zoos and other places keeping wildlife need to implement exhibition strategies to promote human interest on less attractive but highly relevant animal species of threatened ecosystems (bitgood and patterson, ; frynta et al., ) . considering this distinction in preference, it is relevant to spread information about the ecological importance of animals in ecosystems, especially regarding native and endangered species (conde et al., ) , messages to promote "positive" emotions in people could be a way to support the appropriation of endangered species by societies and improve their attitudes toward them in the long term. massive media communication may be of utmost importance for these purposes, especially if the appropriate images of and messages about target species are transmitted to the general public (gunnthorsdottir, ) . following breed and moore ( ) , successful conservation projects require focusing on promoting wide social empathy for wildlife species, particularly those that generate fear and disgust (e.g., large predators, venomous species, and many amphibians) motivating their killing or removal (bishop et al., ; prokop and fančovičová, ; . individual and collective idiosyncrasies have promoted a diversity of attitudes toward wildlife species (herzog and burghardt, ) motivated in part by a diversification of emotions built with dynamic biological and cultural elements. identifying and understanding diversified emotions and their local precursors (e.g., in areas where protected areas and human presence are relevant) would allow analyzing wildlife problems and their solutions through multidisciplinary strategies. considering that knowledge is a relevant element for the expression of emotions, we propose that regional strategies to integrate information on the biology, ecology, and management of culturally important animal species (particularly those regarded as fearsome, dangerous, harmful, and disgusting) should be included in national education systems and massive media campaigns throughout the neotropics (espinosa and jacobson, ) . these strategies must be carefully designed by taking into account the impact of mass media (e.g., news, television shows, documentaries, films, and public text books, among others) may have on the public about wildlife conservation (røskaft et al., ; knight, ; ceríaco et al., ; wieczorek, ) . when an animal species is projected as aggressive, a negative emotional experience can be produced in the public. this negative experience may in turn lead the individual to believe the species is a dangerous agent or threat to human life, bringing about attitudes against its conservation (prokop and fančovičová, ) . on the contrary, if wildlife species are positively seen by children through different media outlets, where the real facts about unpopular animals are shown, it is more likely that fear and disgust decrease, while empathy may grow (prokop et al., ) . ensuring the continuity of transmitting traditional ecological knowledge about animal species will be equally important to stimulate positive emotions and a long-term interest of the new generations in wildlife conservation (jacques-coper et al., ) . another strategy that could have a positive impact on emotions toward fearsome and disgusting animals is promoting physical interactions with them (e.g., touching snails, rays, amphibians, mice; randler et al., ; ; the new knowledge about the animals and physical contact with them could reduce the anxiety of danger. recognizing that emotions are culturally influenced, we propose developing outreach strategies by retrieving traditional aspects that formerly favored empathy with animal species, including the noncharismatic or unpopular ones, even if they are threatened. this review aimed to discuss the role of emotions in the conservation of species which a have been transcendent for the human 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biophobia and conservation attitude in china covid- drives new threat to bats in china mexico's national council of science and technology (conacyt) provided a scholarship to nc-h for her doctoral studies. we thank ph.d. angela may steward for reviewing and editing the manuscript. key: cord- -io jmp o authors: roche, benjamin; guégan, jean-françois title: ecosystem dynamics, biological diversity and emerging infectious diseases date: - - journal: c r biol doi: . /j.crvi. . . sha: doc_id: cord_uid: io jmp o in this article, we summarize the major scientific developments of the last decade on the transmission of infectious agents in multi-host systems. almost sixty percent of the pathogens that have emerged in humans during the last – years are of animal origin and about sixty percent of them show an important variety of host species besides humans ( or more possible host species). in this review, we focus on zoonotic infections with vector-borne transmission and dissect the contrasting effects that a multiplicity of host reservoirs and vectors can have on their disease dynamics. we discuss the effects exerted by host and vector species richness and composition on pathogen prevalence (i.e., reduction, including the dilution effect, or amplification). we emphasize that, in multiple host systems and for vector-borne zoonotic pathogens, host reservoir species and vector species can exert contrasting effect locally. the outcome on disease dynamics (reduced pathogen prevalence in vectors when the host reservoir species is rich and increased pathogen prevalence when the vector species richness increases) may be highly heterogeneous in both space and time. we then ask briefly how a shift towards a more systemic perspective in the study of emerging infectious diseases, which are driven by a multiplicity of hosts, may stimulate further research developments. finally, we propose some research avenues that take better into account the multi-host species reality in the transmission of the most important emerging infectious diseases, and, particularly, suggest, as a possible orientation, the careful assessment of the life-history characteristics of hosts and vectors in a community ecology-based perspective. the pathogens responsible for the infectious diseases that affect human populations are seldom strictly confined to our species. among the about currently known human infectious diseases, c.a. to % have an established animal origin [ , ] . ruminants, carnivores, rodents, birds and primates constitute, in the order, the five major animal categories that have transmitted pathogens to human populations [ ] . this percentage increases to - % if only the ''emerging'' or ''reemerging'' diseases, which have appeared in the last to years, are considered [ , ] . the work by woolhouse et al. [ ] has also shown that these pathogens have an important range of host reservoir species as about % of them have or more host species (in addition to humans), thus highlighting the notion that pathogen dynamics driven by a multiplicity of hosts is a more realistic picture for emerging infectious diseases [ ] . without doubt, the origin of the most important pathogens and parasites that affect human populations is zoonotic (in this work, we will use the term ''parasite'' to in this article, we summarize the major scientific developments of the last decade on the transmission of infectious agents in multi-host systems. almost sixty percent of the pathogens that have emerged in humans during the last - years are of animal origin and about sixty percent of them show an important variety of host species besides humans ( or more possible host species). in this review, we focus on zoonotic infections with vector-borne transmission and dissect the contrasting effects that a multiplicity of host reservoirs and vectors can have on their disease dynamics. we discuss the effects exerted by host and vector species richness and composition on pathogen prevalence (i.e., reduction, including the dilution effect, or amplification). we emphasize that, in multiple host systems and for vector-borne zoonotic pathogens, host reservoir species and vector species can exert contrasting effect locally. the outcome on disease dynamics (reduced pathogen prevalence in vectors when the host reservoir species is rich and increased pathogen prevalence when the vector species richness increases) may be highly heterogeneous in both space and time. we then ask briefly how a shift towards a more systemic perspective in the study of emerging infectious diseases, which are driven by a multiplicity of hosts, may stimulate further research developments. finally, we propose some research avenues that take better into account the multi-host species reality in the transmission of the most important emerging infectious diseases, and, particularly, suggest, as a possible orientation, the careful assessment of the life-history characteristics of hosts and vectors in a community ecology-based perspective. ß acadé mie des sciences. published by elsevier masson sas. all rights reserved. refer to agents of infectious diseases). therefore, it is easy to recognize the importance of a better understanding of animal species as potential hosts and sources of novel contaminations in humans [ ] . the recent appearance and propagation of the highly pathogenic h n avian influenza (bird flu), west nile fever, lyme disease, rabies as well as of severe acute respiratory syndrome (sars), all of animal origin, have truly highlighted the role of animal species as major reservoirs for (new) human infections. understanding how they are transmitted in animal species communities and identifying the underlying ecological and evolutionary mechanisms is essential to lessen the transmission risk to humans [ ] . anthropogenic changes in landscapes, particularly habitat destruction and fragmentation [ ] and their subsequent effects on biological diversity have led to disease emergence in a number of situations [ ] . here, we discuss new insights gained from studies that explicitly acknowledge the contribution of multi-host species systems to the dynamics of emerging pathogens. throughout this review, we also argue on the necessity to better cross-fertilize the two separate research fields of epidemiology and ecology of infectious diseases if we want to anticipate future epidemics. finally, we recommend an increased attention to the role of the many epizootic host species and the weakly competent vectors in disease transmission not only among themselves, but also in driving the infection towards human communities. many definitions can be used to describe what an ''ecological community'' is. this profusion of definitions does not simplify communication within this research field. in this article, we have chosen to use the relatively neutral, but also vague, definition by putman [ ] . an ecological community is thus defined as ''a group of species that interact in a given geographical area and whose functions and dynamics are somehow interdependent''. classically, a community structure is described using a species abundance/richness curve that associates a number of species for each abundance ''rank'' (e.g., species involving between and individuals each). these relations can take different forms according to the animal communities under study. for instance, fisher et al. [ ] showed statistically that this relation follows a geometrical law ( fig. ) for butterfly communities. a few years later, preston showed that this relation takes more frequently the shape of a log-normal distribution in mammal communities [ , ] (fig. ) . these two explanations have been unified only recently following hubbell's demonstration that both relations correspond to two specific situations of another more general distribution called zero-sum multinomial [ ] . these species abundance/richness relationships are the results of all the interactions that occur within local communities (e.g., preys/predators, competition. . .) and as such they may constitute a good integrator of all these relations. since it is not sufficient to account for the observed community structure, trophic networks should not be dismissed as they might be another important contributing mechanism to the dynamics of ecological communities [ ] . however, within these networks, the importance of parasitic organisms (e.g., viruses, bacteria, protozoans, helminth worms.) has been frequently ignored due to sampling issues. parasites are surprisingly important in food webs and their role in food web stability is increasingly recognized [ ] [ ] [ ] . first, by inducing a differential mortality among the host species, parasites can positively or negatively influence the abundance and the spatial distribution of the host species they use as resources. second, and this aspect has been less studied, parasites can affect food webs and interacting host species especially by altering trophic cascades. the first ''visible'' effect exerted by parasites is the more or less important regulation that virulent infections can have on the size of host populations in wildlife [ ] . the literature in this domain is teeming with scientific evidences that describe the parasitic pressures on natural host populations. the case of the accidental introduction of bovine rinderpest in the wild ungulate communities of the serengeti national park in is here emblematic [ ] . the disease decimated different animal species. this, in turn, caused the decrease in the predation pressure on the vegetation, leading, at the same time, to a modification of the vegetation cover of the savannah that made it less favourable to fire development. although this example describes well the primordial role exercised by parasites in ecosystems, it is however necessary to distinguish the effects of generalist (nonspecific) and host-specialized parasites on host species communities. in the case of a generalist parasite that can thus infect a significant range of host species, the consequences are generally destabilizing for the host communities. indeed, if a parasite species can simultaneously attack many host species, the ones that are more tolerant to the parasite (i.e., the host species that can support the reproduction of the parasite while sustaining little damage) will engender an ''apparent'' competition with the less tolerant host species that will pay for the cost of the parasite virulence. in scotland, the parasitic nematode heterakis gallinarum infects both pheasants and partridges [ ] . this pathogen multiplies very well in pheasants without decreasing the size of their population that, as a result, shows a great tolerance towards this parasite. for partridges, the situation is the opposite because a large proportion of partridges infected by this nematode will rapidly die. the degree of abundance of pheasants in the community has thus the consequence of increasing locally the nematode incidence and this will directly affect the abundance of partridges, which are more susceptible to the parasite (fig. ) . although we cannot propose other examples as representative as this one, in nature such situations must be frequently encountered. on the other hand, the effects of host-specialized parasites on host species communities are the opposite of the ones previously described, because generally an important diversity in host species is maintained locally. indeed, by limiting the abundance of the species they infect, these host-specialized parasites also decrease the local competition pressure among host species and allow the rarest species to freely coexist in the environment [ ] . to sum up, parasites can influence the structure of species communities and the consequences of this on the ecosystems can be contrasting due to the leverage action they exert on the coexistence of host species within local communities. moreover, within natural ecosystems, parasites are undeniably key elements that contribute to the shaping of the observed variety of local species communities. in return, this diversity in species richness and composition can also influence the transmission mode of pathogens. asking about the role played by a community of hosts on the dissemination of a parasite boils down to consider the influence that a local change in the host species composition can have on its transmission. one or more host species can either disappear or appear locally, and this in turn can have important repercussions on the dynamics of parasite transmission. the consequences of greater host species richness may strongly depend on the transmission mode of the pathogen. for pathogens with ''density-dependent transmission'', in general any local addition of host species is likely to enhance disease transmission due to the increase in contact rates among individual hosts within the community [ ] . this situation is often observed in the case of directly-transmitted infectious pathogens, like rodent-borne hantaviruses or avian influenza viruses [ , ] . a second situation is that of pathogens with ''frequency-dependent transmission'' in which contacts among host individuals occur at a constant frequency and do not depend on the host population density. this type of transmission is assumed for sexually transmitted pathogens, like hiv, in which the number of sexual contacts does not increase with the size of the population. another example of frequency-dependent transmission concerns vector-borne diseases, e.g. malaria, in which the transmission is from host to host via a mosquito vector. this type of transmission is also encountered with pathogens transmitted by ticks, such as lyme disease for instance. here, fig. . example of indirect competition induced by a parasite within a local community of hosts. the introduction of individuals of a host species (here symbolized by a pheasant), which is tolerant to the parasite (here a heterakis nematode worm), leads to an increase of the parasite prevalence within the community. if this tolerant host species interacts with another host species, which is less or nontolerant (here partridges), the increase in the disease prevalence will then cause extra mortality in the non-or less tolerant host species, independently from the type of interactions between tolerant and nontolerant host species. the frequency of blood meals by the arthropod vectors generally does not increase with the abundance of host individuals [ ] . for the sake of simplicity, we consider here only the case of vector-borne transmission. during a blood meal from an infected reservoir host, the vector can be contaminated and transmit the parasite to healthy host individuals. in this context, the individuals challenged by the pathogen can be from a single host species (for a hostspecialized parasite) or can belong to different host species (for a generalist, nonspecialized parasite). in the case of a nonspecialized, multi-species system, the local conditions of species richness and composition in reservoir hosts will have important consequences on the disease transmission, as it has been shown by richard ostfeld et al. [ , ] . these authors have analysed the circulation of the bacterium borrelia burgdorferi, responsible of lyme disease in north america. this biological model, which involves numerous reservoir host species and only one vector species (the tick species ixodes scapularis), represents an interesting first step for the study of pathogen transmission in multispecies systems. each of these potential reservoir species has naturally a different competence to multiply the pathogen and to transmit it again during a blood meal by noninfected ticks. here, we will define as susceptibility the probability that a healthy host individual might become infected following a contact with an infected vector [ ] . hence, in animal species communities, each reservoir and vector species has a different susceptibility that is characteristic of that species. in reality, within a host (reservoir or vector) species, important differences in susceptibility can be observed, but it is difficult to integrate this intraspecies variability due to the simplicity of the epidemiological models that are currently used. by focusing on the interspecific variability of susceptibilities, ostfeld et al. have carried out a series of experimental and field studies that have demonstrated the importance of the local composition in reservoir host species on the transmission of the bacterium responsible of lyme disease [ ] . these authors observed a decrease of pathogen prevalence in ixodes ticks and a lower human prevalence with greater reservoir host species richness [ ] . further studies on the west nile virus, which is transmitted by different mosquito species and many bird species in the usa, have also highlighted a tendency to lower pathogen circulation in the presence of greater host bird richness [ ] . the results of the few empirical studies, which have been carried out, converge and indicate that local communities with low richness in reservoir host species tend to present higher levels of disease prevalence (fig. ) , whereas local communities that involve a larger number of reservoir hosts species see this level decreasing (fig. ) . ostfeld et al. [ ] [ ] [ ] ] have called this phenomenon the ''dilution effect'' in reference to the process of deviation to which is subjected the pathogen in communities which are rich in reservoir species and a significant proportion of which are less or not susceptible to infection. to the best of our knowledge, only two theoretical studies tried to formally explain the dilution effect. first, using an epidemiological model of the susceptible-infected-resistant (sir) type that relies on allometric laws to quantify the different parameters, dobson [ ] analysed the epidemiological consequences of host reservoir species inclusion (up to six, and progressively less susceptible) in a virtual host community. among other outputs, dobson [ ] studied the basic reproduction number (r ) that quantifies the mean number of new infections caused by a single infected host individual. he underlined that, in the case of frequency-dependent transmission, the value of r decreases when the richness in host species increases, which represents a direct application of the previously described ''dilution effect'' phenomenon. this first theoretical formalism had the merit of introducing a theoretical [ ( ) t d $ f i g ] fig. . schematic representation of the dilution effect proposed by richard ostfeld and his collaborators. for the sake of simplicity, it is considered that one vector individual can bite only twice during its lifespan. in the left panel, an infected vector transmits the pathogen to two individuals from a susceptible reservoir species. these two highly susceptible reservoir individuals will infect in turn two new vector individuals. on the right, the same initial conditions, but with the introduction of an individual from a nonsusceptible reservoir species. if the vector is generalist, there is a nonnegligible probability that the infected vector will bite this nonsusceptible reservoir individual. hence, only one reservoir individual will be infected and transmit the disease to one vector individual. overall this results in a decline of the pathogen prevalence in the vector populations. framework to explain the dilution effect observed empirically. nevertheless the low reservoir species richness involved ( species) and the fact that dobson's work considered only directly transmitted infections did not allow the generalization of these results to vector-borne diseases, especially when multiple vector species are involved. in a more recent theoretical work, begon [ ] has discussed the effects of the addition of a second, less competent host species on transmission pathways, pathogen persistence and abundance. in the case of louping ill virus, which is transmitted in red grouse by the vector tick species ixodes ricinus, begon [ ] concluded that in the presence of a second, less competent host species, a transmission event linked infectious individuals with incompetent susceptible host individuals that generated many fewer new infections than a competent host would have. the second host species therefore served only to dilute the transmission process, but in general, it is difficult to separate a dilution effect due to greater host species richness or composition. to continue the works by dobson [ ] and begon [ ] , a recent study by roche et al. [ ] has proposed a more general theoretical formalism by borrowing empirical observations derived from community ecology to mimic the relationships observed in nature between local species richness and relative abundance in species (see section , i.e., species richness-abundance curves). this study concentrated on the intensity of transmission of a pathogen in a local community of host species by studying the observed maximum prevalence of locally infected cases that represents a close estimate of the r [ ] . by taking into account that, in the case of vector-borne zoonotic diseases, two categories of animal communities interact, on one side, the reservoir species and, on the other side, the insect species, this study allowed highlighting the following findings [ ] . for two local communities with the same reservoir species richness and abundance but with different species compositions, an increase in the variation of the reservoir species susceptibility corresponded to a greater number of reservoir species with lower level of susceptibility. an immediate consequence is that an important number of vectors, all other proportions kept identical, will feed on reservoir individuals which are less able to transmit the pathogen. thus, the interspecific variation in susceptibility of the host reservoirs is an important criterion that on its own can explain the decrease in prevalence of a pathogen in vector populations. in practice, these pathogen dynamics are usually the result of a balance mainly between efficient transmission by competent hosts and abortive transmission by poorly competent hosts that can likely lead to a dilution effect (fig. ) . indeed, it is not, strictly speaking, the local richness in reservoir species that acts on the level of prevalence of the pathogen in the vectors, but the proportion of reservoir species with low susceptibility, which tends to augment in species-rich communities, that decreases the intensity of vector-borne transmission. so far, we have focused on the community of host reservoir species and its role in the transmission of pathogens in multi-host systems. nevertheless, the vector species community also plays an important part in disease transmission. the dilution effect has been most thoroughly studied in vector-borne diseases, especially lyme disease, which, in the usa, is transmitted by a single tick species, i. scapularis [ , , ] . this, therefore, might not be representative of the many indirectly transmitted zoonotic pathogens for which several vector species may be more or less competent in disease transmission. for zoonotic vector-borne infections, there are two types of contacts: (i) between infectious vectors and more or less susceptible reservoir hosts and (ii) between infectious host reservoirs and more or less susceptible vectors. indeed, the dilution effect requires that vector contacts must be wasted on poorly competent reservoirs, but also that vector species richness, by increasing their abundance, cannot compensate for these wasted contacts to overcome the dilution effect. in the case of vector-borne pathogens, the analysis of the species richness-abundance curves of the local vector communities in the different models allows the assessment of the effect of the number of vector species in a given community on the pathogen persistence and prevalence. this raises two important questions concerning: (i) the effects on local disease transmission of the accidental introduction or biological invasion by exotic vectors, even when they show a low competence to transmit the infection; and (ii) the role of low to very low in species-rich reservoir communities, generally a decrease in the prevalence of disease pathogens in the vectors is observed. this is illustrated by the case of borrelia burgdorferi that is transmitted by the ixodes scapularis ticks in many vertebrate hosts in the usa. the diversity of reservoir hosts dilutes disease transmission (dilution effect) and the pathogen dynamics drops. on the other hand, in species-rich communities of vectors, a larger proportion of vectors, even if poorly competent, can acquire the pathogen from infectious hosts and then amplify the transmission of the disease to susceptible hosts (amplification effect). in natural conditions, these two antagonistic effects can lead to very contrasting epidemiological situations depending on the characteristics of the local ecosystems and their biological diversity, as observed for the emerging disease caused by west nile virus (see text for further explanations). competent resident vector species on the general circulation of pathogens. moreover, it highlights what is perhaps a key issue in the understanding of vector-borne emerging pathogens. although medium to very high competent vector species are usually credited with the maintenance and transmission of a disease, the role of the many poorly to very poorly competent vectors should not be underestimated as recent evidences have shown for bovine catarrhal fever (also known as blue tongue disease) in northern europe [ ] , chikungunya in italy [ ] and reunion island [ ] , or malaria in corsica [ ] . it is interesting to note from the multi-host dynamic model by roche et al. [ ] that, while an increase in the number of reservoir host species generally dilutes the transmission of a pathogen, a local increase in vector species richness amplifies transmission (fig. ) . to understand why, it is necessary to consider the number of new additional bites that occur when local vector species richness increases, even though some vector species might be poorly to very poorly competent for the pathogen locally. these opposite effects of dilution and amplification of the pathogen circulation must be taken into consideration when analysing vector-borne disease transmission. indeed, depending on the local species richness and composition in host reservoirs and vectors, we may observe different disease trends ( fig. ; and see also begon [ ] ). this model clearly illustrates the mechanics and the potential role played by the biological diversity of host reservoirs and vectors in vector-borne infections. a possible example of this is represented by the transmission of west nile virus. west nile virus is a flavivirus commonly found in africa, west asia, middle east, eastern europe, northern and central america and west indies that causes encephalitis (inflammation of brain and spinal chord) in humans and horses. west nile disease is transmitted by different mosquito vectors, which bite and infect birds. many mosquito species have been tested positive for this pathogen [ ] , but in experimental studies the most susceptible one seems to be the culex species complex [ ] . infections have been identified mostly in wild birds (and even domestic ones), which are the primary reservoirs for the virus. although most wild bird species are not affected and rather act as more or less amplifying reservoirs, members of the corvid family, including crows, blue jays, magpies and ravens, are very susceptible to the effects of west nile virus. the virus can also infect many different dead-end hosts in terms of transmission, including humans and other mammals. in the case of west nile virus, the ''take home'' message from the multi-host/multi-vector models is that the local richness and composition in host reservoirs and vectors may lead to different combinatorial effects on disease transmission. in southern europe, like in the camargue area, where the bird reservoir species are poorly to moderately susceptible to infection and only two vector species, one being moderately and the other one poorly susceptible, are present, the prevalence of west nile virus is highly sporadic [ ] . conversely, in the usa, where there are a multitude of bird reservoir species (of which a large part appears to be very susceptible) and an important diversity in vector species (some vectors being highly competent), huge epidemics that have propagated to the entire country, from east to west, in only a few years have been reported [ ] . the absence of previous immunity in bird populations to this virus (west nile fever appeared in north america in ) cannot explain this pattern due to the fast demographic turnover within avian communities. in the simplified context of fig. , the combination(s) of local reservoir and vector species richness and composition that can either amplify or dilute the infection may help to explain many other (new) emerging disease spillovers [ ] . bearing in mind that, generally, pathogen dynamics are driven by the dynamics of the overall biological diversity of the community and not of one single reservoir or vector host species, then future research should concentrate on revisiting the idea of disease transmission using a broader community-scale perspective than the one generally applied. our understanding of the influence of reservoir host and vector diversity on pathogen epidemiology and disease dynamics is still rudimentary. the possibility of strong effects due to a local multiplicity of hosts and vectors is however high for a large number of emerging pathogens [ ] . for some pathogens, like for instance west nile virus, different species of reservoirs (birds) and vectors can be involved locally, and changes in both reservoir and vector diversity from place to place can result in functional changes in transmission that have strong effects on the disease dynamics [ ] . indeed, animal viruses with multiple hosts are more likely to be designated as emerging threats than the ones with single hosts, a finding that emphasizes the importance of better examining the role played by host and vector diversity [ , ] . acknowledging a multiplicity of hosts in many zoonotic diseases [ ] may also complicate the way these diseases should be studied and analyzed when compared to simple one host-one vector systems. our examination of emerging infectious diseases with a zoonotic origin indicates that an explicit assessment of the influence of multiple host reservoirs and vectors must be taken into account. undoubtedly, it is realistic to acknowledge that the local and regional heterogeneity in vector and reservoir diversity can more or less affect disease behaviour and spread in nonspecialized parasites. overall, the evidence that the local host diversity influences the outcomes of disease dynamics seems clear for both lyme disease and west nile virus. as a consequence, not considering a priori the potential role played by poorly to very poorly competent hosts -the immersed part of the iceberg -, and particularly of vectors, under the pretext of a low or very low supposed or proven participation in the transmission, can have important ecological and epidemiological consequences. in many situations, the onset of emerging infectious diseases might be due to the multiplicity of hosts and vectors and its local variability and the role of the many epizootic reservoirs and poorly competent vectors should not be underestimated, not only in driving the disease dynamics in the system, but also in increasing the risk of infection to humans. this highlights possible research key issues for understanding pathogens in multi-species systems. given the recent emergence of important infectious diseases in several complex communities [ ] , more attention should be given to generalist pathogens and to recording the number and identity of all potential vector and reservoir species locally, instead of focusing only on the more competent, well-accepted hosts and vectors. it is clear from this review that to better understand multi-species dynamics of diseases, we should rather collect qualitative information on the largest possible number of actors in disease dynamics than to obtain quantitative and substantial data but only for one or two reservoir hosts or vectors that are a priori suspected to be involved in the transmission. moreover, since we can observe both spatial and temporal variations in local species diversity, such analysis should be repeated in several different communities and for different periods of time in order to infer on disease regional dynamics and on its effects on host assemblages, and vice versa. in addition, the potential for an accidental introduction in an originally untouched geographical area of some vector specimens, even if they are not very competent in transmitting the pathogen, should not be underestimated especially because they might originate or exacerbate the onset of an epidemic. the same could be said also about resident vectors and reservoir hosts that are accepted as being putatively low to very low competent hosts, but might play a key starting role in the epidemiology of a disease. besides, it comes back also to better understand the links that exist between the relative competence of reservoir and vector species and their life-history strategies, particularly the fact that competent hosts and vectors are often ubiquitous and generalist organisms. other lifehistory characteristics of reservoir and vector species should be further explored, such as reproductive strategies, feeding habits or body features, for instance. from an evolutionary point of view, pathogens should select for the best host vessels that facilitate their own survival and dissemination. a multiplicity of hosts might thus complicate the evolutionary dynamics of pathogens, if it introduces conflicting selective pressure between being embarked into the main reservoir host(s)/vector(s) or into a more occasional host/vector [ ] . on the other hand, there is no clear evidence that the pathogen prevalence for vector-borne infections is lower in richer reservoir communities. indeed, by studying the transmission of a generalist plant virus in experimentally manipulated plant communities, power and mitchell [ ] showed that the pathogen prevalence was entirely driven by the single, most competent plant species. in this case, therefore, the link between pathogen prevalence and species richness was a reflection of the community composition rather than of the community richness. to what extent, this observation might be applied to other multi-species systems is still a matter of debate. nevertheless, this suggests again that it would be more fruitful to explore the links between biological diversity and emerging infectious diseases by focusing on community composition and species life-history strategies. the community epidemiology perspective we advocate here may present also some practical aspects. indeed, it could be envisaged to exploit the natural complexity of ecological systems by introducing locally animal baits in the form of reservoir species with low susceptibility that might thus divert vector species from their human targets. by choosing preferentially these baits, vectors will consequently reduce disease transmission to humans [ ] . this type of control is called zooprophylaxy. the development of livestock rearing in western countries, by increasing the herd of animals with low susceptibility, has been suggested to be responsible, according to bruce-chwatt [ ] , to the disappearance of malaria in europe. this method, which has been theoretically studied for malaria [ ] , is still considered for application in some regions of asia [ ] . this calls into question the importance, in practice, of the community function in the epidemiology of infections. of course, more complete theoretical and experimental studies must be carried out because the introduction of these animal baits could have other indirect and even more dramatic consequences. community epidemiology is still in its early days. limited so far by a too simplistic perspective (the triad ''one agent-one vector-one reservoir'') on the complexity of natural communities, its development in the next years should stem from a partnership between specialists of complex systems, especially those working on multi-agent systems, ecologists, evolutionary biologists and epidemiologists. we conclude this review with an idea that has been pervasive throughout this essay: different epidemiological and ecological outcomes for a same disease can occur in 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/j.tree. . . sha: doc_id: cord_uid: t g u the – megafires in australia brought a tragic loss of human life and the most dramatic loss of habitat for threatened species and devastation of ecological communities in postcolonial history. what must be done now to keep impacted species from extinction? what can be done to avoid a repeat of the impacts of such devastating bushfires? here, we describe hard-won lessons that may also be of global relevance. the - megafires in australia brought a tragic loss of human life and the most dramatic loss of habitat for threatened species and devastation of ecological communities in postcolonial history. what must be done now to keep impacted species from extinction? what can be done to avoid a repeat of the impacts of such devastating bushfires? here, we describe hard-won lessons that may also be of global relevance. despite the familiarity of australia with fire, the timing, ferocity, and extent of the - fires was shocking. by area burnt, it was the largest fire season in eastern australia since european occupation. the total area burnt in eastern australia from august to march was almost km or . million hectares, almost the area of england ( million ha) ( figure ). megafires have occurred intermittently in australia over the past years, possibly facilitated by the removal of traditional land practices of indigenous people. for example, in , fires in eastern australia burnt an area b % of the most recent fires, killing people and destroying n dwellings. however, this most recent fire season was unprecedented in geographical scale, duration, and intensity, and has had major impacts on species and ecosystems that were already under immense stress from prolonged drought. the comprehensiveness of the destruction is striking. postfire aerial reconnaissance revealed vast landscapes of grey ash extending as far as the eye can see: grey, not a hint of green, bounded only by the blue of sea and sky ( figure ). the full impacts on biodiversity will not be fully understood for years to come as extinction debts are realised. some coarse surrogates paint a stark picture: ( plants, and animals, including five invertebrates) of the~ listed threatened species in australia had a significant portion (n %) of their known distribution within the fire footprint i , of which were already critically endangered. among the significantly impacted species, have lost at least half of their habitat and have lost over %. although these numbers are still being refined, this is likely to result in significant population losses. the conservation status of many species [e.g., gang gang cockatoo (callocephalon fimbriatum) and yellow-bellied glider (petaurus australis)] previously considered secure, will now need to be reconsidered. impacts will be long-lasting, because many of the fire-affected species were dependent upon long-unburnt habitats that take decades to re-establish and many have slow reproductive output and, thus, it will take many years for populations to re-establish. thousands of less wellknown species, including invertebrates and plants, many yet to be described and many with very localised distributions, will have suffered dramatic impacts. some may even have become extinct before being discovered or named. most fires leave a scattering of unburnt patches within the fire footprint, often in small topographical features such as sheltered, wetter gullies, but sometimes also due to the vagaries of sudden windshifts that send the fire in a different direction. although a comprehensive analysis of the spatial variation in fire intensity across the entire fire-affected areas is yet to be done, it appears that, at least in some regions, the - fires burnt extraordinarily thoroughly, even burning through landscape features such as deep gullies, rainforest edges, rocky outcrops, and riparian strips, that have acted as fire refuges in past fire events (figure ). this creates a new and more serious challenge for the recovery of species. immediately following fire, animals that survived the blaze by sheltering underground, in water, or in rocks are faced with the challenge of finding food and avoiding predation in a moonscape environment. many will perish due to lack of food and shelter. animals able to find the rare patches of unburnt vegetation will likely find it to be suboptimal habitat or the territory of another animal. plants regenerating after fire are vulnerable to herbivores and desiccation. fish die in warm, deoxygenated water caused by heavy loads of sediment and ash following the first post-fire rains. a particular challenge of megafires is that, as the bushland slowly recovers, the prospects for recolonisation by dispersal-limited species is greatly diminished due to the larger distances over which species need to travel to recolonise suitable habitat. in many cases, those dispersal pathways are now interrupted by cleared land and human settlements or dams and weirs on rivers. recovery will also be stymied if, as is likely, the interval between successive fires decreases. in the midst of emergency, the response was, understandably, almost solely about human life and property. unfortunately, there was little strategic priority in fire control operations for the protection of key populations of threatened species, critical habitats, and threatened ecological communities. thus, aside from a small number of exceptional cases, biodiversity destruction was unabated by human intervention. in the immediate aftermath of fires, the wildlife response focussed primarily on animal welfare. the triage effort by veterinarians was impressively rapid, with significant government support. images of koalas (phascolarctos cinereus) in bandages being offered water by fire fighters resulted in~$au million being donated to animal welfare organisations. one of only a few pre-emptive responses aimed at species conservation was for the critically endangered wollemi pine (wollemia nobilis), a highly restricted species of great antiquity: the species was saved by an air operations crew who deposited fire retardant on the ridge-lines surrounding the few populations, and by conservation managers setting up irrigation systems around trees. this was a wonderful, but sadly exceptional, proactive nature conservation success. the absence of strategic protection targeting the protection of threatened species, ecosystems, and iconic places is disappointing, given that we know how much harder it is to restore or repair nature than it is to avoid its loss or damage. (i) (ii) (iii) (i) (ii) (iii) (a) (b) by contrast, the postfire response of agencies has been energetic, focussed, and adept. before the fires were out, federal and state governments pledged nearly $au million for conservation. expert panels were formed, impacts on species and ecosystems were analysed with support from science and nongovernment organisations. experts salvaged birds, plants and their seed, and fish species that faced postfire demise or were in imminent danger from further fire. actions included targeted control of feral herbivores and predators, erection of artificial nest boxes, and supplementary feeding of endangered macropods. for the most part, it was an impressive start to the long journey of postfire conservation. at the time of writing, the australian government and civil society organisations continue to allocate significant resources to support the recovery of fireaffected species and environments. such actions include postfire reconnaissance to quantify impacts on species and ecosystems, to manage postfire threats, and to monitor changes in species and ecosystems following fire and in response to recovery actions. some of the most widely deployed actions have included aerial culling of feral herbivores (mostly deer) that threaten the regeneration of sensitive forest and alpine ecosystems, and intensified baiting of introduced predators (mostly foxes) in fire-affected areas and fire fringes where they hunt native wildlife more intensively and successfully [ ] . compared with the limited consideration of biodiversity protection in preparatory fire management plans and during the fire emergency, the immediate postfire response was generally well organised, science based, and relatively well resourced. the increased attention given to the plight of wildlife during the fires in the media has created momentum around postfire biodiversity conservation. given that recovery efforts may need to be sustained for years, even decades, a key challenge will be to maintain support for those efforts as the collective memory of the fire fades and governments and society now grapple with another disaster, covid- . monitoring the outcomes of recovery efforts and tracking the fate of species after the fires is crucial, not only to inform where ongoing investment of conservation effort is most needed and which recovery actions are working best, but also for public engagement [ ] . numerous challenges remain; systematic prioritisation is required to determine where spending and conservation efforts, such as predator and herbivore control, are most beneficial, and to correctly time and implement the re-introduction of species that were rescued during and immediately following the fire. translocations will also be needed to 'rescue' or bolster populations, and to re-establish populations in areas from which they were extirpated. reassessment of conservation status will be required to identify and list species that now face a palpable risk of extinction. active restoration, including seed harvesting and aerial sowing of some forest ecosystems, will be needed if they are to persist following repeated recent fires [ ] . there is a significant opportunity to couple postfire forest restoration efforts with carbon sequestrationfunding initiatives to leverage biodiversity conservation. however, most of these actions must be deployed under imperfect knowledge, making rigorous monitoring of the outcomes crucial for improving understanding and maximising ongoing effectiveness in an adaptive management framework [ ] . avoidance of damaging postfire 'salvage' logging is key to the survival of many species, such as the threatened greater glider (petauroides volans) and sooty owl (tyto tenebricosa) [ ] . protecting residual oldforest habitats is one of the most crucial but politically challenging postfire actions to achieve because conserving habitat for threatened species competes with economic considerations. unfortunately, each new fire confers greater importance on the diminishing old-forest habitats that remain, making their protection ever more critical to the survival of old-forest-dependent species. careful analysis of the importance of remaining habitats for the survival of species, based on population viability analysis, provides evidence to support their protection in the face of economic pressures [ , ] . consistent with an escalating global trend of warming and drying, was the hottest and driest year on record in australia ii . national annual rainfall was % lower than the long-term average and maximum temperatures were, on average . °c above the long-run average maxima. the forest fire index in december was the highest on record for almost all of eastern australia [ ] . these conditions will increase in frequency with a changing climate, and catastrophic fire events will also become more frequent. there is little joy to be derived from reflecting on the ever-increasing size of fires and the ever-diminishing interval between them. however, it drives home the importance of learning from each experience and doing better next time, for there will be lots of next times. clear guidance on how to most effectively organise species and ecosystem conservation activities before, during, and immediately following a major fire event can be helpful (figure ). reflecting on the recent fire season, we failed nature, both in the heat of the moment and beforehand. precautionary precatastrophe actions should include: the establishment of more insurance populations (which can be used as sources after such extensive fires); translocations to better allow risks to be spread; more effective, sustained, and extensive control of other threats that can compound fire impacts; and collection of baseline monitoring and survey data to help identify places critical to protect, prioritise emergency responses, and provide clarity around what has been lost immediately following a fire event. most of the - fires were ignited by lightning strike, and little can be done to stop this. however, many fires are anthropogenic in origin [ ] . reducing anthropogenic ignitions and providing more effective suppression before fires get out of control could both be helpful. as a society, we could make the choice to invest more in early-strike fire suppression capacity. there is also a need for strategy, planning, and a greater recognition in fire control centres of the importance of protecting natural assets during fire. at present, natural assets feature only in some of the plans that form the basis for fire-fighting strategy. in most fire management and control plans, there is little spatial information on the occurrence of critical biodiversity features that must be protected, acknowledging that it is impossible to replace millions of years of evolution. it may be unrealistic to expect critical habitats of our most precarious species to compete for fire-fighting resources with houses and farms. we are far too self-interested. however, could we imagine the last remaining habitat for a brush-tailed rock-wallaby (petrogale penicillata) might feature as an asset for protection in a fire that is burning through a wilderness area? surely that needs doing. it will require prioritisation, mapping habitats of precious fire-sensitive species, and a signal to the fire controller that these maps should sit alongside critical human infrastructure maps in the fire room. inclusion of biodiversity assets in fire management plans and fire control operations also needs to be complemented by increased recognition of the risks of wildfire within conservation management and recovery planning: very few of such existing plans for threatened species anticipated such catastrophes or provided useful guidance on how to respond. we should learn from what went well this past fire season and ensure that successful responses are hardwired for next time, here in australia and in comparable biomes elsewhere in the world. the rapid production of spatial statistical summaries activities are organised according to the relevant timing (columns) and under broad families of activities (rows). crucial elements in the preparatory phase 'before the fire' that were not adequately addressed before the - australian megafires include analysis and synthesis of species sensitivity to fire, monitoring, surveying, and mapping to delineate critical habitats for protection and emergency postfire action. undertaking adequate preparation will help ensure that, during and immediately post fire, actions will be efficiently deployed to protect sensitive and critical biodiversity assets and rapidly drive their recovery. many of the medium-long-term postfire activities support preparation for the next fire event, such as policy and management changes, to reduce the likelihood and minimise the impacts of future events. identifying species that were most impacted facilitated prioritisation of immediate care, and what form that care should take; these actions probably saved species. fortunately, some of those processes have been documented and made publicly available i . there is a role for everyone in reducing risks of fire, minimising losses during fire, and recovery after fire. we have focussed here primarily on the role of land managers, wildlife rescue, policy makers, and ecologists. however, the most crucial role may reside in the media and social sciences. there is a significant risk that mega-fires will be attributed to the occurrence of natural vegetation. we need to better understand these perceptions and design messages that dampen the desire to punish the bush for what happened to rural communities and avoid an outbreak of illegal or statesanctioned land clearing in the name of fire prevention. it is vital to communicate the awe and wonder of our species and ecosystems, and the message that, while we will always live with the risk of fire in our landscapes, those risks to people and property can be managed and mitigated without the need for further sacrifice of nature. extraterritorial hunting expeditions to intense fire scars by feral cats monitoring threatened species and ecological communities aerial sowing stopped the loss of alpine ash (eucalyptus delegatensis) forests burnt by three short-interval fires in the alpine national park towards adaptive management of native vegetation in regional landscapes salvage logging in the montane ash eucalypt forests of the central highlands of victoria and its potential impacts on biodiversity ranking conservation and timber management options for leadbeaters possum in southeastern australia using population viability analysis modelling human impacts on the tasmanian wedge-tailed eagle (aquila audax fleayi) unprecedented burn area of australian mega forest fires spatial patterns of wildfire ignitions in south-eastern australia thanks to many people who contributed knowledge, ideas, and data. thanks to natasha cadenhead and casey visintin for assistance with editing and figures. thanks to the community and many agencies for their extraordinary efforts to recover biodiversity. key: cord- -vj o b authors: crous, p.w.; wingfield, m.j.; chooi, y.-h.; gilchrist, c.l.m.; lacey, e.; pitt, j.i.; roets, f.; swart, w.j.; cano-lira, j.f.; valenzuela-lopez, n.; hubka, v.; shivas, r.g.; stchigel, a.m.; holdom, d.g.; jurjević, Ž.; kachalkin, a.v.; lebel, t.; lock, c.; martín, m.p.; tan, y.p.; tomashevskaya, m.a.; vitelli, j.s.; baseia, i.g.; bhatt, v.k.; brandrud, t.e.; de souza, j.t.; dima, b.; lacey, h.j.; lombard, l.; johnston, p.r.; morte, a.; papp, v.; rodríguez, a.; rodríguez-andrade, e.; semwal, k.c.; tegart, l.; abad, z.g.; akulov, a.; alvarado, p.; alves, a.; andrade, j.p.; arenas, f.; asenjo, c.; ballarà, j.; barrett, m.d.; berná, l.m.; berraf-tebbal, a.; bianchinotti, m.v.; bransgrove, k.; burgess, t.i.; carmo, f.s.; chávez, r.; Čmoková, a.; dearnaley, j.d.w.; de a. santiago, a.l.c.m.; freitas-neto, j.f.; denman, s.; douglas, b.; dovana, f.; eichmeier, a.; esteve-raventós, f.; farid, a.; fedosova, a.g.; ferisin, g.; ferreira, r.j.; ferrer, a.; figueiredo, c.n.; figueiredo, y.f.; reinoso-fuentealba, c.g.; garrido-benavent, i.; cañete-gibas, c.f.; gil-durán, c.; glushakova, a.m.; gonçalves, m.f.m.; gonzález, m.; gorczak, m.; gorton, c.; guard, f.e.; guarnizo, a.l.; guarro, j.; gutiérrez, m.; hamal, p.; hien, l.t.; hocking, a.d.; houbraken, j.; hunter, g.c.; inácio, c.a.; jourdan, m.; kapitonov, v.i.; kelly, l.; khanh, t.n.; kisło, k.; kiss, l.; kiyashko, a.; kolařík, m.; kruse, j.; kubátová, a.; kučera, v.; kučerová, i.; kušan, i.; lee, h.b.; levicán, g.; lewis, a.; liem, n.v.; liimatainen, k.; lim, h.j.; lyons, m.n.; maciá-vicente, j.g.; magaña-dueñas, v.; mahiques, r.; malysheva, e.f.; marbach, p.a.s.; marinho, p.; matočec, n.; mctaggart, a.r.; mešić, a.; morin, l.; muñoz-mohedano, j.m.; navarro-ródenas, a.; nicolli, c.p.; oliveira, r.l.; otsing, e.; ovrebo, c.l.; pankratov, t.a.; paños, a.; paz-conde, a.; pérez-sierra, a.; phosri, c.; pintos, Á.; pošta, a.; prencipe, s.; rubio, e.; saitta, a.; sales, l.s.; sanhueza, l.; shuttleworth, l.a.; smith, j.; smith, m.e.; spadaro, d.; spetik, m.; sochor, m.; sochorová, z.; sousa, j.o.; suwannasai, n.; tedersoo, l.; thanh, h.m.; thao, l.d.; tkalčec, z.; vaghefi, n.; venzhik, a.s.; verbeken, a.; vizzini, a.; voyron, s.; wainhouse, m.; whalley, a.j.s.; wrzosek, m.; zapata, m.; zeil-rolfe, i.; groenewald, j.z. title: fungal planet description sheets: – date: - - journal: persoonia doi: . /persoonia. . . sha: doc_id: cord_uid: vj o b novel species of fungi described in this study include those from various countries as follows: antarctica, cladosporium arenosum from marine sediment sand. argentina, kosmimatamyces alatophylus (incl. kosmimatamyces gen. nov.) from soil. australia, aspergillus banksianus, aspergillus kumbius, aspergillus luteorubrus, aspergillus malvicolor and aspergillus nanangensis from soil, erysiphe medicaginis from leaves of medicago polymorpha, hymenotorrendiella communis on leaf litter of eucalyptus bicostata, lactifluus albopicri and lactifluus austropiperatus on soil, macalpinomyces collinsiae on eriachne benthamii, marasmius vagus on soil, microdochium dawsoniorum from leaves of sporobolus natalensis, neopestalotiopsis nebuloides from leaves of sporobolus elongatus, pestalotiopsis etonensis from leaves of sporobolus jacquemontii, phytophthora personensis from soil associated with dying grevillea mccutcheonii. brazil, aspergillus oxumiae from soil, calvatia baixaverdensis on soil, geastrum calycicoriaceum on leaf litter, greeneria kielmeyerae on leaf spots of kielmeyera coriacea. chile, phytophthora aysenensis on collar rot and stem of aristotelia chilensis. croatia, mollisia gibbospora on fallen branch of fagus sylvatica. czech republic, neosetophoma hnaniceana from buxus sempervirens. ecuador, exophiala frigidotolerans from soil. estonia, elaphomyces bucholtzii in soil. france, venturia paralias from leaves of euphorbia paralias. india, cortinarius balteatoindicus and cortinarius ulkhagarhiensis on leaf litter. indonesia, hymenotorrendiella indonesiana on eucalyptus urophylla leaf litter. italy, penicillium taurinense from indoor chestnut mill. malaysia, hemileucoglossum kelabitense on soil, satchmopsis pini on dead needles of pinus tecunumanii. poland, lecanicillium praecognitum on insects’ frass. portugal, neodevriesia aestuarina from saline water. republic of korea, gongronella namwonensis from freshwater. russia, candida pellucida from exomias pellucidus, heterocephalacria septentrionalis as endophyte from cladonia rangiferina, vishniacozyma phoenicis from dates fruit, volvariella paludosa from swamp. slovenia, mallocybe crassivelata on soil. south africa, beltraniella podocarpi, hamatocanthoscypha podocarpi, coleophoma podocarpi and nothoseiridium podocarpi (incl. nothoseiridium gen. nov.) from leaves of podocarpus latifolius, gyrothrix encephalarti from leaves of encephalartos sp., paraphyton cutaneum from skin of human patient, phacidiella alsophilae from leaves of alsophila capensis, and satchmopsis metrosideri on leaf litter of metrosideros excelsa. spain, cladophialophora cabanerensis from soil, cortinarius paezii on soil, cylindrium magnoliae from leaves of magnolia grandiflora, trichophoma cylindrospora (incl. trichophoma gen. nov.) from plant debris, tuber alcaracense in calcareus soil, tuber buendiae in calcareus soil. thailand, annulohypoxylon spougei on corticated wood, poaceascoma filiforme from leaves of unknown poaceae. uk, dendrostoma luteum on branch lesions of castanea sativa, ypsilina buttingtonensis from heartwood of quercus sp. ukraine, myrmecridium phragmiticola from leaves of phragmites australis. usa, absidia pararepens from air, juncomyces californiensis (incl. juncomyces gen. nov.) from leaves of juncus effusus, montagnula cylindrospora from a human skin sample, muriphila oklahomaensis (incl. muriphila gen. nov.) on outside wall of alcohol distillery, neofabraea eucalyptorum from leaves of eucalyptus macrandra, diabolocovidia claustri (incl. diabolocovidia gen. nov.) from leaves of serenoa repens, paecilomyces penicilliformis from air, pseudopezicula betulae from leaves of leaf spots of populus tremuloides. vietnam, diaporthe durionigena on branches of durio zibethinus and roridomyces pseudoirritans on rotten wood. morphological and culture characteristics are supported by dna barcodes. . bayesian posterior probabilities (pp) > . are shown at the nodes and thickened lines represent nodes with pp = . . the scale bar represents the expected changes per site. families and orders are indicated with coloured blocks to the right of the tree. genbank accession and/or fungal planet numbers are indicated behind the species names. the tree was rooted to diaporthe perjuncta (genbank ng_ . ) and the taxonomic novelties described in this study for which lsu sequence data were available are indicated in bold face. the alignment and tree were deposited in treebase (submission id s ). . bayesian posterior probabilities (pp) > . are shown at the nodes and thickened lines represent nodes with pp = . . the scale bar represents the expected changes per site. families and orders are indicated with coloured blocks to the right of the tree. genbank accession and/or fungal planet numbers are indicated behind the species names. the tree was rooted to diaporthe perjuncta (genbank ng_ . ) and the taxonomic novelties described in this study for which lsu sequence data were available are indicated in bold face. the alignment and tree were deposited in treebase (submission id s ). consensus phylogram ( % majority rule) of trees resulting from a bayesian analysis of the lsu sequence alignment ( sequences including outgroup; aligned positions; unique site patterns) using mrbayes v. . . a . bayesian posterior probabilities (pp) > . are shown at the nodes and thickened lines represent nodes with pp = . . the scale bar represents the expected changes per site. families, orders and classes are indicated with coloured blocks to the right of the tree. genbank accession or fungal planet numbers are indicated behind the species names. the tree was rooted to saccharomyces cerevisiae (genbank z . ) and the taxonomic novelties described in this study for which lsu sequence data were available are indicated in bold face. the alignment and tree were deposited in treebase (submission id s ). . bayesian posterior probabilities (pp) > . are shown at the nodes and thickened lines represent nodes with pp = . . the scale bar represents the expected changes per site. families and orders are indicated with coloured blocks to the right of the tree. genbank accession or fungal planet numbers are indicated behind the species names. the tree was rooted to xylaria hypoxylon (genbank ay . ) and the taxonomic novelties described in this study for which lsu sequence data were available are indicated in bold face. the alignment and tree were deposited in treebase (submission id s ). . bayesian posterior probabilities (pp) > . are shown at the nodes and thickened lines represent nodes with pp = . . the scale bar represents the expected changes per site. families and orders are indicated with coloured blocks to the right of the tree. genbank accession and/or fungal planet numbers are indicated behind the species names. the tree was rooted to ramularia endophylla (genbank ay . ) and the taxonomic novelties described in this study for which lsu sequence data were available are indicated in bold face. the alignment and tree were deposited in treebase (submission id s ). notes -phacidiella alsophilae is related to p. podocarpi (conidia -septate, ( -) - (- ) × ( -) . (- ) μm; crous et al. ), although they are morphologically distinct. because the type species of phacidiella, p. salicina (conidia aseptate, on twigs of salix viminalis, finland), is presently not known from culture, the phylogenetic relationships between species in the genus remains unresolved. phacidiella alsophilae and p. podocarpi are thus tentatively retained in phacidiella. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to phacidiella podocarpi (strain cbs , genbank nr_ . ; identities = / ( %), gaps ( %)), fitzroyomyces cyperi (strain cbs , genbank mg . ; identities = / ( %), gaps ( %)), and fitzroyomyces cyperacearum (voucher mflu - b, gen-bank mk . ; identities = / ( %), gaps ( %)). closest hits using the lsu sequence were phacidiella podocarpi (strain cbs , genbank ng_ . ; identities = / ( %), gaps ( %)), stictis radiata (voucher palice (ess ), genbank ay . ; identities = / ( %), no gaps), and carestiella socia (strain gg a, genbank ay . ; identities = / ( %), gaps ( %)). (crous) crous, comb. nov. mycobank mb . basionym. phacidiella eucalypti crous, fungal diversity : . . description & illustration - crous et al. ( b) . typus . south africa, western cape province, stellenbosch mountain, on eucalyptus sp., jan. , p.w. crous (holotype cbs h- , cultures ex-type cbs = cpc , cpc , ; its-lsu sequence genbank ef . ). notes -the genus hormodochis was resurrected by crous et al. ( a) to accommodate taxa with erumpent, globose pycni dial conidiomata with aseptate conidia, arranged in cylindrical chains, olivaceous brown, smooth, subcylindrical to somewhat doliiform, with truncate ends. morphologically and phylogene tically, phacidiella eucalypti is better accommodated in hormodochis than phacidiella, as the latter has hyaline conidia (sutton ) . another genus to consider with subhyaline conidia is trullula, which differs in mode of conidiogenesis and conidium morphology (see crous et al. a ). notes -poaceascoma was introduced by phookamsak et al. ( ) to accommodate a genus of saprobic ascomycetes on poaceae with setose ascomata and filiform ascospores. although p. filiforme lacks setae, its spirally twisted, filiform ascospores are a good fit for the genus. based on a megablast search of ncbis genbank nucleotide database, the its sequence had distant, partial hits to poaceascoma taiwanense (strain mflucc - , genbank mg . ; identities = / ( %), gaps ( %)), setoseptoria phragmitis (strain cbs , genbank kf . ; identities = / ( %), gaps ( %)), and setoseptoria englandensis (strain mflucc - , genbank mg . ; identities = / ( %), gaps ( %)). closest hits using the lsu sequence are poaceascoma aquaticum (strain mflucc - , genbank ng_ . ; identities = / ( %), gap ( %)), poaceascoma halophilum (strain mflucc - , genbank mf . ; identities = / ( %) , gaps ( %)), and poaceascoma taiwanense (strain mflucc - , genbank mg . ; identities = / ( %) , no gaps). closest hits using the rpb sequence had highest similarity to poaceascoma aquaticum (strain mflucc - , genbank kt . ; identities = / ( %), no gaps), poaceascoma helicoides (strain mflucc - , genbank kp . ; identities = / ( %), no gaps), and wettsteinina lacustris (strain aftol-id = cbs . , genbank dq . ; identities = / ( %), gaps ( %)). closest hits using the tef sequence had highest similarity to darksidea zeta (strain cbs , genbank kp . ; identities = / ( %), gaps ( %)), darksidea beta (strain cbs , genbank kp . ; identities = / ( %), gaps ( %)), and darksidea gamma (strain cbs , genbank kp . ; identities = / ( %), gaps ( %)). closest hits using the tub sequence had highest similarity to pleurophoma acaciae (strain cpc , genbank ky . ; identities = / ( %), gaps ( %)), crassiclypeus aquaticus (strain kh , genbank lc . ; identities = / ( %), gaps ( %)), and flabellascoma minimum (strain kt , genbank lc . ; identities = / ( %), gaps ( %)). associated with brown leaf spots. conidiomata (on podocarpus leaves and on sna), black, round, flattened, acervular, - μm diam; wall of several layers of brown textura epidermoidea, splitting open all along outer margin, appearing saucer-shaped on leaf. conidiophores reduced to conidiogenous cells, arising from basal layers of stroma, hyaline, smooth, subcylindrical to ampulliform, annellidic, - × . - μm. conidia fusoid, slightly curved, smooth-walled, guttulate, pale brown, unequally -euseptate; basal cell obconic with truncate hilum, hyaline; median cells pale brown; apical cell obtuse, hyaline. apical cell . - μm long; second cell . - μm long; third cell - μm long; fourth cell - μm long; basal cell - μm long; conidia ( -) - (- ) × ( . -) μm; apical appendage filiform, flexuous, unbranched, excentric, - μm long; basal appendage filiform, flexuous, unbranched, excentric, - μm long. culture characteristics -colonies spreading, with moderate aerial mycelium and smooth, lobate margin, covering dish after wk at °c. on mea surface smoke grey, reverse olivaceous grey. on pda surface and reverse olivaceous grey. on oa surface pale olivaceous grey. notes -seimatosporium and allied genera have recently been revised (bonthond et al. , liu et al. , with genera being accepted in sporocadaceae. nothoseiridium podocarpi is allied to seiridium ( -septate, appendaged conidia) and nonappendiculata ( -septate, non-appendaged conidia), but is distinct in having -septate, fusoid conidia with unbranched, excentric apical and basal appendages. nothoseiridium is further characterised by forming submerged acervuli nothoseiridium podocarpi crous, sp. nov. that break through the epidermis with a saucer-like appearance, being associated with prominent leaf spots. it is not possible to distinguish nothoseiridium from seiridium based on lsu sequence data. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to seimatosporium lichenicola (as discostroma fuscellum; strain gsaa- , genbank jf . ; identities = / ( %), gaps ( %)), sporocadus rosarum (as seimatosporium pseudorosarum; strain mflucc - , genbank kt . ; identities = / ( %), gaps ( %)), seimatosporium lichenicola (strain cbs . , gen-bank mh . ; identities = / ( %), gaps ( %)) and millesimomyces rhoicissi (strain cpc , genbank nr_ . ; identities = / ( %), gaps ( %)). closest hits using the lsu sequence are seiridium unicorne (strain cbs . , genbank mh . ; identities = / ( %), no gaps), seiridium pseudocardinale (strain cbs , genbank mh . ; identities = / ( %), no gaps), and seiridium phylicae (strain cpc , genbank ng_ . ; identities = / ( %), gap ( %)). closest hits using the rpb sequence had highest similarity to seiridium cardinale (strain cpc , genbank lt . ; identities = / ( %), no gaps), seiridium unicorne (strain cbs , genbank mk . ; identities = / ( %), no gaps), and seiridium aquaticum (voucher mflu - , genbank mn . ; identities = / ( %) , no gaps). closest hits using the tef sequence had highest similarity to seiridium marginatum (strain cbs , genbank lt . ; identities = / ( %), gaps ( %)), seiridium papillatum (strain cbs . , genbank lt . ; identities = / ( %), gaps ( %)), and seiridium podocarpi (strain cbs , genbank lt . ; identities = / ( %), gaps ( %)). closest hits using the tub sequence had highest similarity to seiridium cupressi (strain cbs . , genbank lt . ; identities = / ( %), gaps ( %)), seiridium papillatum (strain cbs . , genbank lt . ; identities = / ( %), gaps ( %)), and seiridium podocarpi (strain cbs , gen-bank lt . ; identities = / ( %), gaps ( %)). associated with prominent brown leaf spots. conidiomata pycnidial, grey-brown, - μm diam, with central ostiole. conidiophores lining the inner cavity, intermingled among paraphyses, - -septate, - × - μm, or reduced to conidiogenous cells, hyaline, smooth, guttulate, doliiform to ampulliform, - × - μm. paraphyses intermingled among conidiophores, hyaline, smooth, cylindrical, aseptate, - (- ) μm diam, up to μm long, with age becoming multiseptate and with intercalary conidiogenous cells. conidiogenous cells hyaline, smooth, guttulate, doliiform to ampulliform, - × - μm, phialidic, with minute periclinal thickening. conidia aseptate, hyaline, smooth, guttulate, subcylindrical to fusoid to irregular, straight to somewhat curved, apex subobtuse, base truncate, ( -) notes -coleophoma includes species that are plant pathogenic or saprobic, occurring on a wide range of plant hosts (crous et al. b (crous et al. , b . the genus was revised by , and shown to reside in the dermateaceae (leotiomycetes), with morphologically similar taxa also clustering in dothideomycetes. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to coleophoma parafusiformis (strain cbs , genbank nr_ . ; identities = / ( %), gaps ( %)), coleophoma ericicola ( ( %)). the first of two equally most parsimonious trees obtained from a phylo genetic analysis of the coleophoma its/acta/tef /tub alignment ( strains including the outgroup; characters including alignment gaps analysed: constant, variable and parsimony-uninformative and parsimonyinformative). paup v. . b (swofford ) was used to analyse the data. the novel species was added to the alignment of , where also the genbank accession numbers of the reference sequences can be found. the tree was rooted to two strains of davidhawksworthia ilicicola and the scale bar indicates the number of changes. parsimony bootstrap support values higher than % are shown at the nodes (pbs/njbs) and the novel species is highlighted in bold. type status is indicated in superscript. branches present in the strict consensus tree are thickened. tree statistics: tl = , ci = . , ri = . , rc = . . the alignment and tree were deposited in treebase (submission id s ). notes -the genus chalara as circumscribed by nag raj & kendrick ( ) is polyphyletic and awaits revision. hamatocanthoscypha podocarpi is phylogenetically allied to the type species of hamatocanthoscypha, h. laricionis (svrček ) , and placed in this genus based on dna similarity. several species of 'chalara' have been described from podocarpus, namely c. brevipes (conidia ( -) . (- ) × . - µm), c. novaezelandiae (conidia ( -) . (- ) × - . µm), c. cylindrosperma (conidia ( . -) (- ) × ( . -) . (- . ) µm), c. fusidioides (conidia ( . -) . (- ) × ( . -) . (- . ) µm), c. acuaria (conidia ( -) (- ) × ( -) . (- . ) µm) and c. bicolor (conidia -septate, ( -) - (- ) × . - µm) (nag raj & kendrick ) . of these, h. podocarpi is most similar to c. brevipes, but can be distinguished in having smaller conidiogenous cells, and conidiophores that are aggregated in clusters. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to numerous sequences wrongly labelled as 'infundichalara microchona' (e.g., strain krp - , genbank hm . ; identities = / ( %), gaps ( %)), chalara holubovae ( myrmecridium phragmiticola crous & akulov, sp. nov. etymology. name refers to the host genus phragmites from which it was isolated. classification -myrmecridiaceae, myrmecridiales, sordariomycetes. on sna: mycelium consisting of hyaline, smooth, branched, septate, - µm diam hyphae. conidiophores unbranched, erect, straight, medium brown, thick-walled, - -septate, up to µm tall, - . µm diam; basal cell - µm diam. conidiogenous cells terminal, integrated, subcylindrical, - µm long, pale brown, forming a rachis with pimple-shaped denticles less than µm long and . µm diam; slightly thickened. conidia solitary, aseptate, pale brown, thin-walled, smooth, guttulate, with or without a wing-like gelatinous sheath, ellipsoid to fusoid, ( -) - × ( . -) µm; hilum unthickened nor darkened, . µm diam. culture characteristics -colonies flat, spreading, with mo derate aerial mycelium and smooth, lobate margin, reaching mm diam after wk at °c. on mea surface isabelline, reverse hazel. on pda surface and reverse greyish sepia. on oa surface isabelline. notes - arzanlou et al. ( ) established the genus myrmecridium to accommodate taxa with hyaline mycelium, pigmented, solitary conidiophores with pimple-like denticles, and - -septate, ellipsoid conidia with a mucoid sheath. myrmecridium phragmiticola should be compared to m. phragmites (phragmites australis, netherlands), which has - -septate conidia, ( . -) - (- ) × ( . -) (- . ) μm (crous et al. etymology. name refers to the closure or lockdown experienced in many countries during the covid- pandemic. mycelium consisting of branched, septate, hyaline to pale brown, smooth to finely roughened, - µm diam hyphae. conidiophores solitary, erect, flexuous, mostly reduced to a terminal conidiogenous cell. conidiogenous cells pale brown, smooth, subcylindrical to slightly clavate, - × - µm, proliferating via single apical blastic locus, and remaining attached to acropetal chain of conidia that remain attached to one another via narrow isthmus. conidia brown, thin-walled, smooth, guttulate, granular, ellipsoid to obovoid, ( -) - (- ) × ( -) - (- ) µm; conidia remaining attached in chains of - propagules, disarticulating at maturity into single propagules or shorter chains. culture characteristics -colonies flat, spreading, with sparse to moderate aerial mycelium and feathery, lobate margin, reaching mm diam after wk at °c. on mea surface and reverse cinnamon. on pda surface and reverse hazel to brown vinaceous. on oa surface hazel. crous, sp. nov. notes -diabolocovidia is reminiscent of genera such as ampullifera (but conidiophores different and hyphopodia present) and junctospora (but conidiophores sparingly branched, subhyaline; seifert et al. ) . phylogenetically, it is allied to vamsapriya, which is characterised by having brown, synnematous conidiophores, mono-to polytretic conidiogenous cells, and dark brown, septate conidia arranged in acropetal chains (dai et al. ) . based on these differences, diabolocovidia is herewith introduced as a new genus. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to vamsapriya khunkonensis (voucher mflu - , genbank nr_ . ; identities = / ( %), gaps ( %)), didymobotryum rigidum (strain jcm , genbank lc . ; identities = / ( %), gaps ( %)), and vamsapriya bambusicola (voucher mflu - , genbank nr_ . ; identities = / ( %), gaps ( %)). closest hits using the lsu sequence are vamsapriya bambusicola (strain mflucc - , genbank ng_ . ; identities = / ( %), no gaps), fasciatispora petrakii (strain hkucc , genbank ay . ; identities = / ( %), gap ( %)), and vamsapriya indica (strain mflucc - , genbank km . ; identities = / ( %), no gaps). crous, sp. nov. notes -juncomyces is closely related to graminopassalora, which was introduced to accommodate passalora graminis, a widespread pathogen occurring on a broad range of grass (poaceae) hosts (videira et al. beltraniella podocarpi crous, sp. nov. etymology. name refers to the host genus podocarpus from which it was isolated. classification -beltraniaceae, xylariales, sordariomycetes. setae solitary to aggregated, erect, flexuous, arising from a lobate basal cell, - µm diam, dark brown, warty, chiefly unbranched, up to -septate, thick-walled with large central guttules, tapering in upper part to acute apex, - × - µm. conidiophores arranged in dense clusters around the base of setae, brown, smooth, subcylindrical, frequently branched at basal cell, - -septate, - × - µm. conidiogenous cells integrated, terminal and intercalary, - × - µm, pale brown, smooth, obclavate, tapering toward - denticulate loci, - . µm long, µm diam. separating cells clavate to fusoid-ellipsoid, pale brown, smooth, finely guttulate, tapering toward long basal stalk and short apical locus, - × - µm. conidia obovoid to narrowly turbinate, tapering toward base, apex rounded to subtruncate, aseptate, finely verruculose, guttulate, pale brown, ( -) - (- ) × ( -) µm. culture characteristics -colonies flat, spreading, with moderate aerial mycelium and feathery, lobate margin, covering dish after wk at °c. on mea surface olivaceous grey, reverse honey with olivaceous grey margin. on pda surface olivaceous grey, reverse iron-grey. on oa surface iron-grey with dirty white margin. notes -beltraniella is characterised by brown, unbranched setae, setiform conidiophores, polyblastic, denticulate conidiogenous cells, and turbinate conidia with a distinct hyaline transverse band (rajeshkumar et al. ) . beltraniella podocarpi is closely related to several species that tend to have some overlap in conidial length, but have narrower conidia (rajeshkumar et al. , crous et al. a ). based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to beltraniella portoricensis (strain bcrc , genbank gu . ; identities = / ( %), gaps ( %)), beltraniella ramosiphora (strain lcg - , genbank mg . ; identities = / ( %), gaps ( %)), and beltraniella pseudoportoricensis (strain cbs , genbank nr_ . ; identities = / ( %), gaps ( %)). neofabraea eucalyptorum crous, sp. nov. etymology. name refers to the host genus eucalyptus from which it was isolated. classification -dermateaceae, helotiales, leotiomycetes. associated with brown, amphigenous leaf spots, - mm diam. conidiomata - µm diam, acervular, erumpent, associated with dark brown, amphigenous leaf spots. conidiophores hyaline, smooth, branched, septate, subcylindrical, phialidic, up to µm long, - µm diam. conidiogenous cells hyaline, smooth, subcylindrical, terminal and intercalary with visible periclinal thickening, - × - µm. conidia subcylindrical to fusoid-ellipsoid, variously curved, hyaline, smooth, guttulate, apex subobtuse, base with flattened hilum, aseptate, but becoming up to -septate in older cultures, ( -) - (- ) × ( . -) - (- ) notes -the neofabraea generic complex was revised by chen et al. ( ) , and neofabraea eucalypti was subsequently placed in coleophoma . neofabraea eucalyptorum is thus the first confirmed species of the genus associated with leaf spots on eucalyptus (crous et al. b ). based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to neofabraea alba (strain uasws , genbank hq . classification -ploettnerulaceae, helotiales, leotiomycetes. mycelium consisting of hyaline, smooth, branched, septate, - µm diam hyphae. conidiophores integrated, subcylindrical, hyaline, smooth, septate, sparingly branched, mostly terminal on hyphal ends, - × - µm. conidiogenous cells integrated, terminal and intercalary, subcylindrical, smooth, - × - µm; proliferating sympodially. conidia solitary but aggregating in mucoid mass, y-shaped, smooth, hyaline; central cell obclavate, base with truncate hilum, µm diam, apex subobtuse, - -septate, ( -) - (- ) × ( -) - (- ) µm, with - lateral branches inserted below the median, pointing upwards, aseptate, obclavate, apex subobtuse, ( -) - (- ) × (- . ) µm. culture characteristics -colonies erumpent, spreading, with moderate aerial mycelium and folded surface (on mea), with smooth, lobate margin, reaching mm diam after wk at °c. on mea, pda and oa surface dirty white, reverse ochreous. notes -although the ecology of ypsilina remains unknown, y. graminea has been isolated from freshwater foam, roots and leaves of various plants (descals et al. ) . ypsilina buttingtonensis was isolated from an ancient pedunculate oak quercus robur in buttington, wales (longitude and latitude: . , - . ). the tree, known as the buttington oak, was an open-grown lapsed pollard. at the time when the tree fell in february , it had a trunk girth of . m at breast height and was believed to be the second oldest oak tree in wales. the tree had a . m diam hollow through centre where brown cubical rot could be seen, attributed to fistulina hepatica. the significance of the tree was realised in when it was 'discovered'. cores of wood were extracted from the tree with a . mm increment bore. wood chips were taken from the cm cores at cm intervals and placed on low ph % malt agar petri dishes and incubated at °c in the dark. ypsilina buttingtonensis was cultured from a chip cm into the heartwood. in addition to ypsilina buttingtonensis, fistulina hepatica, and eight species of ascomycete were also cultured from the wood chips including cryphonectria radicalis, a close relative of the aggressive canker pathogen cryphonectria parasitica, responsible for chestnut blight. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to helgardia anguioides ( notes -pseudopezicula accommodates two species of apothecial ascomycetes that cause angular leaf scorch on vitis vinifera. an epitype is here designated for one of these, namely p. tracheiphila. in culture they produce phialophora-like asexual morphs (korf et al. ) , that resemble the phialidic asexual morph isolated in the present study. although pseudopezicula betulae was associated with prominent leaf spots, its occurrence was inconsistent, and therefore it is unknown whether it is a primary pathogen. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to gyoerffyella entomobryoides (strain cbs . , genbank nr_ . ; identities = / ( %), gap ( %)), gyoerffyella rotula (strain jb , genbank ku . ; identities = / ( %), gap ( %)), and fontanospora eccentrica (strain umb- . , genbank kf . ; identities = / ( %), gaps ( %)). gyrothrix encephalarti crous, sp. nov. etymology. name refers to the host genus encephalartos from which it was isolated. culture sterile, morphology based on sporulation on dead leaf spots. mycelium consisting of brown, smooth, septate, branched, . - µm diam hyphae. setae erect, - µm long, - µm diam, brown, multiseptate, thick-walled, verrucose, subcylindrical with apical taper, base bulbous, - µm diam, apex spirally twisted with twisted lateral branches in apical region. conidiophores reduced to conidiogenous cells around base of setae, ampulliform to subcylindrical, pale brown, smooth, - × - µm, proliferating percurrently at apex. conidia hyaline, smooth, aseptate, fusoid, inaequilateral, inner plane flat, outer plane convex, apex subobtuse, base truncate, ( -) - (- ) × (- . ) µm. culture characteristics -colonies flat, spreading, with moderate aerial mycelium and smooth, lobate margin, reaching mm diam after wk at °c. on mea surface buff, reverse cinnamon. on pda surface buff, reverse rosy buff. on oa surface rosy buff. notes -gyrothrix encephalarti is closely related to g. eucalypti (eucalyptus sp., south africa; conidia ( -) - (- ) × ( -) . μm, setae - μm tall, - μm diam at base; crous et al. c ), but has wider conidia and shorter setae. dna sequences of g. eucalypti and g. encephalarti are related to the type sequence deposited for neoanthostomella viticola (ng_ . ), which has a completely different asexual morph. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to neoanthostomella viticola (strain mflucc - , genbank nr_ . ; identities = / ( %), gaps ( %)), gyrothrix eucalypti ( satchmopsis metrosideri crous, sp. nov. etymology. name refers to the host genus metrosideros from which it was isolated. classification -cochlearomycetaceae, leotiales, leotiomycetes. conidiomata cupulate, superficial, - µm diam at apex, - µm deep, dark brown, attached to a basal stroma of dark brown cells that occupy the stomatal chamber; wall consisting of two regions, the lower region having thick-walled dark brown cells up to layers thick; upper region on thin-walled paler cells, cylindrical, - × - µm, with even, smooth flat edge. in culture conidiomata are paler in colour and much larger, flattened, cupulate, and margins have cells that are lobate due to expanding growth (not flat as in vivo). conidiogenous cells restricted to lower part of basal wall, - × - µm, doliiform to lageniform, phialidic with periclinal thickening, hyaline with indistinct collarette. conidia hyaline, smooth, aseptate, guttulate, subcylindrical, predominantly straight with obtuse ends, ( -) - (- ) × - . µm. culture characteristics -colonies flat, spreading, with sparse aerial mycelium and feathery, lobate margin, reaching mm diam after wk at °c. on mea, pda and oa surface umber with patches of sepia, reverse umber. notes -the genus satchmopsis, based on s. brasiliensis (eucalyptus paniculata, brazil; conidia . - . × - . μm) (sutton ) was introduced for a genus of cupulate coelomycetes with aseptate conidia. satchmopsis is commonly isolated from eucalypt leaf litter in south america (crous et al. ). the present collection, from metrosideros excelsa leaf litter collected in south africa, differs from s. brasiliensis in being phylogenetically distinct, and also having longer conidia. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to satchmopsis brasiliensis (strain cpc , genbank dq . ; identities = / ( %) satchmopsis pini crous, sp. nov. etymology. name refers to the host genus pinus from which it was isolated. classification -cochlearomycetaceae, leotiales, leotiomycetes. conidiomata cupulate, superficial, - µm diam, and - µm deep, dark brown, attached centrally to a brown stroma via a dark brown stalk, up to µm tall, µm wide; conidiomatal wall of two regions, the lower region of brown cells, the upper region of cylindrical cells with flat to obtuse edge, - × - µm; terminal - cell layers are prominently thick-walled, darker brown, and can give rise to hyphal outgrowths on outside of conidiomatal margin. conidiogenous cells restricted to lower part of basal wall, - × - µm, doliiform to lageniform, phialidic with periclinal thickening, hyaline with indistinct collarette. conidia hyaline, smooth, aseptate, guttulate, subcylindrical, straight with obtuse ends, ( -) - (- ) × - . µm. culture characteristics -colonies flat, spreading, with sparse aerial mycelium and feathery, lobate margin, covering dish after wk at °c. on mea, pda and oa surface umber with patches of sepia, reverse umber. notes -satchmopsis pini is morphologically distinct from s. brasiliensis and s. metrosideri in having cupulate conidiomata with a prominently thick-walled, darker brown upper region, giving rise to hyphal outgrowths on outside of conidiomatal margin. furthermore, conidiomata are centrally attached to a brown stroma via a long, dark brown stalk, which is absent in s. brasiliensis and s. metrosideri. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to satchmopsis brasiliensis ( notes -the phylogeny and morphology of torrendiella and hymenotorrendiella was discussed in detail by johnston et al. ( ) . although the name torrendiella eucalypti has commonly been used for the species occurring on eucalyptus leaf litter (crous et al. ) , johnston et al. ( ) showed that the type of t. eucalypti occurred on fallen phyllodes of an acacia sp. (tasmania, australia), which then became the type species of the new genus hymenotorrendiella. however, this resulted in the common endophyte and saprobe occurring on eucalypt leaf litter not having a name. several collections from eucalyptus leaf litter were investigated in the present study, and two taxa were found to be present. the first, described here as h. communis, occurred in a clade with isolates from australia, colombia, spain, and south africa. morphologically, however, the south african isolates differ from others in this clade based on macromorphology. apothecia have shorter stalks, - µm high; setae vary from - per apothecium, but are much shorter, and wider that those from other collections in this clade, being - µm long, with obtuse apices, (- ) µm diam, and slightly inflated bases, - µm diam. asci are similar however, being - × - µm, as well as ascospores, ( -) - (- ) × ( . -) µm. hymenotorrendiella communis can be distinguished from the second species, h. indonesiana (ascospores - × - μm), which occurs in indonesia, by its shorter and wider ascospores. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence had highest similarity to hymenotorrendiella indonesiana (as torrendiella eucalypti; strain , genbank fr . ; identities = / ( %), gaps ( %)), hymenotorrendiella andina (as torrendiella andina; strain prj sa , genbank kj . ; identities = / ( %), no gaps), and hymenotorrendiella madsenii (as torrendiella madsenii; voucher pdd , genbank ay . ; identities = / ( %), gap ( %)). crous & p.r. johnst., sp. nov. etymology. name refers to indonesia, the country from which it was collected. micromorphology (on malt extract agar; mea): hyphae hyaline to brownish, coenocytic, smooth, finely roughened to definitely roughened near crustaceous, - µm diam. sporangiophores hyaline to brown near dark brown, simple or branched, arising solitarily, occasionally in pairs, never grouped in whorls, arising from aerial hyphae or substrate, most commonly - × - μm; smooth, finely roughened to definitely roughened near crustaceous walls, with a single septum below the sporangium and rarely with additional septum at the base. sporangia hyaline to brown to dark greyish brown, most commonly pyriform, ( -) - (- ) µm diam, smooth-walled. apophyses funnelshaped, smooth-walled. columellae globose, hemispherical, with a short collarette, occasionally with one projection, smoothwalled, ( -) - (- ) µm diam. sporangiospores of two types: sub-globose to globose, hyaline, smooth-walled, and oval, occasionally slightly irregular, brown, rough-walled (formed in the different sporangia), ( . -) . - (- ) × ( . -) . - μm. chlamydospores (terminal and intercalary) occasionally present in the aerial mycelia. zygospores not observed. culture characteristics -(in darkness, °c after d / d): colonies on mea - /> mm diam, cottony, mycelium at first white, then becoming grey to grey-brown (light mouse grey to mouse grey, r ; ridgway ( ) ), abundant sporulation, reverse colonial buff to deep colonial buff (r ), smooth and wavy zonate. colonies on potato dextrose agar (pda - /> mm diam, cottony, mycelium at first white, then becoming grey to grey-brown [r ), very good sporulation, reverse grey to grey with buckthorn brown shades (r ), radially sulcate. colonies on oa - /> mm diam, cottony, mycelium at first white, then becoming light mouse grey to mouse grey (r ), good sporulation. colony diam at °c (in mm after d): . no growth on mea, pda and oa at °°c. typus. usa, new york, jericho, bathroom, air, dec. notes -blast analyses with the its and lsu sequences of a. pararepens showed greatest similarity with a. repens extype cbs (~ % and ~ % similarity, respectively). the american isolates kas (genbank fj ), fsu (genbank ay ), cbs . = fsu (genbank ef ), cbs . = fsu (genbank ef ), nrrl (genbank af ) and a (genbank ay ) also represent a. pararepens, while european isolates cbs (genbank eu , hm ) and fsu (genbank eu ) represent a. repens s.str. however, this geographic pattern should be confirmed by analysis of additional strains. hesseltine & ellis ( ) invalidly designated a neotype for a. repens. in conflict with art. . (turland et al. ) , the authors selected a living culture, nrrl . this culture originated from a collection of a.f. blakeslee, and was probably isolated in america. however, as pointed out by hoffmann et al. ( ) and hoffmann ( ) , there are large genetic differences between european and american isolates of 'a. repens'. consequently, the neotype of a. repens should be selected from among european strains in accordance with the original description of van tieghem ( ), who collected a. repens on fruit of bertholletia excelsa lying on a layer of moist sphagnum in france. the specimen cbs originating from england, uk, was mentioned as isotype of a. repens by hoffmann et al. ( ) and hoffmann ( ) , but formal typification has never been published. to formalize the typification, we designate here a lectotype of a. repens (illustration from the original material): pl. , f. - (not paginated) in p. van tieghem, annales des sciences naturelles botanique ser. , vol. . , vol. . [ . mycobank typification no. is mbt . epitype designated here: specimen cbs (preserved in metabolically inactive state), ex-epitype culture cbs . mycobank typification no. is mbt ). absidia pararepens has on average shorter sporangiophores ( - × - μm), and larger sporangiospores (( . -) . - (- ) × ( . -) . - μm) than the closely related a. repens (( -) - (- ) × . - μm), and ( . - . (- . ) × - μm), respectively. etymology. named after the american bioinformatician john l. spouge who contributed to the discovery of this species, and for his efforts to implement tools for dna barcoding analyses within the genus annulohypoxylon. classification -hypoxylaceae, xylariales, sordariomycetes. stromata glomerate to hemispherical, effused-pulvinate, with perithecial mounds / to / exposed and not covered by the outermost stromatal layer, . - cm long × . - cm broad and - . mm thick; surface dark brown vinaceous, becoming black with reddish brown hues, finally black and shiny; black granules immediately below the surface, koh pigments green olivaceous. perithecia spherical, . - . mm diam. ostioles conical papillate, surrounded by a flattened bovei-type disc, . - . mm diam. asci - × - . µm, the spore bearing parts - µm long with stipes - (- ) µm long, with apical ring bluing in melzer's iodine reagent, discoid . µm high × . - µm broad. ascospores pale brown, unicellular, ellipsoid-inequilateral with narrowly rounded ends, - . × - . (- . ) µm with straight germ slit along the full length of the spore; perispore dehiscent in % koh, epispore smooth. culture characteristics -colonies on potato dextrose agar (pda) covering petri dish in wk, at first white, becoming hazel to dull green, azonate, with diffuse margins, with scattered black patches; reverse dull green to dark brown. conidiogenous structure nodulisporium-like, brown. conidia hyaline, smooth, ellipsoid, . - . × - µm. additional materials examined. herbarium number is indicated, as well as the its, α-actin, β-tubulin and ef -α genbank sequences between brackets, absent sequences are indicated with '-'. annulohypoxylon spougei: thai-notes -during extensive studies of the hypoxylaceae in thailand over a period of almost yr, problems were encountered in the identification of several taxa, especially a. nitens. a previous study on species of hypoxylon and annulohypoxylon using morphology and its nrdna sequences (suwannasai et al. ) indicated that this taxon was not monophyletic but could be separated into a. nitens and another species. twenty-eight fungal specimens of a. nitens and a cryptic species collected from thailand, previously named 'a. nitens' in our study (suwannasai et al. ) , were carefully re-analysed based on morphological and asexual morph characters. the comparison of morphological characters between a. nitens and a cryptic species showed unclear distinction of these species. the cryptic species, here named as a. spougei possesses spherical perithecia ( . - . mm diam), which are slightly narrower than those of a. nitens described by ju & rogers ( ) the ostiolar discs of both species groups are bovei-type and have the same dimensions of . - . mm. ascospore sizes of a. nitens and the cryptic species are . - × . - . µm and - . × - . (- . ) µm, respectively. these are similar to the species description for a. nitens (as h. nitens) ( . - (- ) × - . µm) from ju & rogers ( ) . the cultural and asexual morph characters were observed from both pda and oatmeal agar. colonies of a. spougei are white at first becoming hazel and dull green with scattered black patches. the asexual morph is nodulisporiumlike and conidial size ( . - . × - µm) is similar to a. nitens ( - × . - µm). with those similar features, it is very difficult to separate the a. spougei from a. nitens by using only morphological and asexual morph characters. however, although morphological data for all of the collections initially identified as a. nitens failed to provide clear separation of the two entities, there are clear supporting dna data for their separation. in the present study based on α-actin, β-tubulin and elongation factor -α sequences, we confirm the separation of two taxa mentioned in suwannasai et al. ( ) . colour illustrations. thailand, chaiyaphum province, phu khiao wildlife sanctuary, where the specimens were collected. from top to bottom: stromata with ostiolar discs (swuf-h ); ascospores under sem (swuf-h ); fungal culture on pda (swuf-h ); nodulisporium-like anamorph (swuf-h ); ascospores with apical apparatus (swuf-h ). scale bars = . mm (stromata), µm (ascospores sem), cm (fungal culture), µm (asexual morph), µm ( aspergillus banksianus pitt, sp. nov. etymology. named for the australian endemic tree banksia integrifolia, from the rhizosphere of which this species was isolated. culture characteristics -czapek yeast extract agar (cya), °c, d: colonies - mm diam, low and dense, plane or irregularly wrinkled, with narrow margins of white mycelium; conidiogenesis moderate to heavy, dark grey to dark grey blue (m. - d -e - ); exudate absent, soluble pigment brown; reverse deep green (m. f - ). mea, °c, d: colonies - mm diam, low and plane, with wide uncoloured margins, light to heavily sporing, coloured as on cya or slightly greener (m. d ); exudate and soluble pigment absent; reverse centrally dark green (m. f ), paler towards the margins. % glycerol nitrate agar (g n), °c, d: colonies up to mm diam, of white mycelium. °c, cya, d: colonies - mm diam, heavily sporing, dull green to grey green; reverse dark green, greyish green or black. conidiophores borne from aerial hyphae, sometimes unbranched, and then ( -) - × . - µm, sometimes bearing a short lateral stipe - µm long as well; broadening slowly to spathulate vesicles, - µm diam, fertile area characteristically hemispherical but sometimes asymmetrical to give a 'nodding' appearance. phialides short and stout, . - × . - µm, with narrow bases and very short narrow necks, sometimes almost ellipsoidal. conidia . - µm diam, smooth to finely roughened, borne in short disordered chains, separating in wet mounts. media formulations are from pitt & hocking ( ) ; (m.) colours are from kornerup & wanscher ( notes -aspergillus banksianus clusters in aspergillus subgenus fumigati, in a small clade that includes a. brevipes and a. duricaulis, with which it shares slow growth at °c, green conidial colouration and intermittent production of asymmetrical fruiting structures. colonies of a. banksianus on cya have a deep green reverse colour, in contrast with a. duricaulis, 'colorless to pinkish drab' or a. brevipes 'becoming purple-red' (raper & fennell ) . molecularly, a. banksianus is particularly close to a. quadricinctus, from which the most obvious difference is lack of the neosartorya sexual morph. aspergillus banksianus when grown on agar, liquid media or grain, displays a unique chemotaxonomic profile comprising banksialactones a-i, and banksiamarins a and b, which are not present in the closely related species a. quadricinctus and a. duricaulis (chaudhary et al. ) . aspergillus banksianus also produces known metabolites clearanol and dothideomynone a, together with the pigments endocrocin and questin previously reported from other aspergillus species. colour illustrations. a specimen tree of the endemic species banksia integrifolia, planted on a street in collaroy, nsw, from under which a soil sample included a. banksianus. colonies grown on cya (upper) and malt extract agar (mea) (lower) for d at °c; fruiting structures and conidia. scale bars = µm (fruiting structures) and µm (conidia). classification -aspergillaceae, eurotiales, eurotiomycetes. conidial heads radiate. conidiophores uniseriate. stipes smooth, frequently septate - (- ) × - (- ) μm, sometimes with subterminal branches and mycelial coils occasionally present. vesicles pyriform to subglobose, pigmented, - (- ) × . - (- ) μm (av. ± . × ± . ), phialides - (- ) × - μm (av. ± . × ± . ) covering half to upper half of vesicle. conidia globose to subglobose, - × . - μm (av. ± . × ± . ), brown to greyish brown, with coarsely roughened to echinulate surface, average width/length = ± . , n = . sclerotia observed. culture aspergillus kumbius pitt, sp. nov. etymology. named for the small town of kumbia, south burnett district, queensland, australia, near where this species was collected. conidiophores borne from aerial hyphae, stipes - (- ) × - µm, uncoloured to pale brown, smooth walled. vesicles spherical, - µm diam, fertile over the upper hemisphere or two thirds; metulae - × . - . µm; phialides acerose, - × . - . µm. conidia spherical, . - . µm diam, walls smooth to finally roughened, borne in disordered chains. culture characteristics -czapek yeast extract agar (cya), °c, d: colonies - mm diam, plane, low and relatively sparse, lightly sulcate, velutinous; margins entire, wide; mycelium white to pale yellow; abundant sclerotia borne on the agar surface, white at first, at maturity pale orange to orange grey (m. a-b ), spherical or near, - µm diam; conidial production sparse, pale yellow brown (m. notes -aspergillus kumbius belongs in aspergillus subgenus circumdati sect. circumdati. molecularly, it is very close to aspergillus bridgeri and a, subramanianii. it is distinguished by rapid growth at °c with abundant buff coloured spherical sclerotia. when grown on agar, liquid media or grain, a. kumbius displays a unique chemotaxonomic profile including kumbicins a-d, which are not present in the closely related species a. bridgeri, a. subramanianii, a. salwaensis, a. persii or a. sclerotiorum. aspergillus kumbius also produces known metabolites asterriquinol d dimethyl ether, petromurins c and d, aspochracin, jbir- , and neohydroxyaspergillic acid, compounds previously reported from other aspergillus species. colour illustrations. a scene of pasture near kumbia, queensland, similar to the one from which this species was described. colonies grown on cya (left) and mea (right) for d at °c; fruiting structures and conidia. scale bars = µm (fruiting structures) and µm (conidia). classification -aspergillaceae, eurotiales, eurotiomycetes. conidiophores borne from aerial hyphae, slender, ( -) - (- ) × - . µm, with thin smooth walls, enlarging slowly to very small spathulate vesicles, - (- ) µm diam; bearing few short phialides, - × . - µm. conidia spherical, - . µm diam, smooth-walled, borne in short disordered chains. media formulations are from pitt & hocking ( ) ; (m.) capitalised colours and notation are from kornerup & wanscher ( notes -aspergillus luteorubrus clusters in aspergillus subg. fumigati, near a. fennelliae. this heterothallic species produces cleistothecia and ascospores characteristic of the sexual genus neosartorya. as only a single strain of a. luteobrunneus is known, it is not clear whether this is an asexual species or, perhaps more likely, heterothallic. aspergillus luteorubrus differs from this and other closely related species in colony colours, conidial size, shape and ornamentation. differences also exist in molecular phylogeny and chemistry (unpubl. data). a maximum likelihood tree inferred from the combined bena, cam and actin sequences of taxa within aspergillus sect. fumigati. the combined sequence alignment was partitioned by marker; substitution models for each partition were chosen according to the corrected information criteria using modeltest-ng v. . . (darriba et al. ). the k +g was used for bena sequences, k +g for cam and tpm+i for actin. the tree was constructed using raxml-ng v. . . (kozlov et al. aspergillus nanangensis pitt, sp. nov. etymology. named for the town of nanango, south burnett district, queensland, australia, near which this species was collected. conidiophores borne from surface hyphae, - × - µm, with thick, smooth, pale yellow walls, bearing very small vesicles. vesicles - µm diam, ellipsoidal to somewhat irregular, bearing metulae and phialides over almost all of the vesicle surface, but sometimes bent to form only a hemispherical head; metulae - × . - . µm; phialides ampulliform - × . - . µm. conidia spherical, . - . µm diam, with walls varying from almost smooth to conspicuously spiny, borne in compact spherical heads, even at age. culture characteristics -czapek yeast extract agar (cya), °c, d: colonies growing slowly, - mm diam, rather sparse, lightly floccose; margins narrow and entire; mycelium white to off white; conidial production light, pale greenish grey (m. - c ); exudate and soluble pigment absent; reverse greyish orange (m. b - notes -aspergillus nanangensis clusters in aspergillus clade jani, a small clade within aspergillus subg. circumdati, but is molecularly distinct. it is close to aspergillus janus and aspergillus brevijanus, but differs from both by lack of the larger white conidial heads that characterise these species. culturally, growth rates of a. nanangensis on standard media are much slower. microscopically, a. nanangensis produces smaller vesicles, fertile over a reduced area. when grown on agar, liquid media or grain, a. nanangensis displays a unique chemotaxonomic profile comprising isonanangenine b and d, nanangelenin, nanangenic acid, nanangenines a-h and nanoxepin not present in the closely related species a. janus and a. brevijanus (lacey et al. classification -agaricaceae, agaricales, agaricomycetes. basidiomata growing solitary, epigeous, incrustations in the rooting base, subglobose and - mm wide × - mm high. exoperidium < . mm thin, fragile, slightly tomentose, evanescent, white to yellowish white ( a , a ; kornerup & wanscher ) . mesoperidium < . mm thin, fragile, membranaceous, persistent at the base, smooth with senescence, greyish brown to brown ( c , d , e , f ). endoperidium < . mm thin, fragile and brittle at the apex, resistant and persistent at the base, papyraceous, olive brown to brown ( d , e ). rhizomorphs not seen. subgleba reduced, woolly, compact, brownish beige ( e ). gleba powdery, not persistent, brownish beige, brown to dark brown ( e , e , f ), at maturity. exoperidium hyphalic, . - . µm diam, intertwined, frequent and non-regular septa, double v branching, walls ≤ . µm thin, straight for curves, hyaline, dextrinoid, low reaction and cyanophilic. mesoperidium compacted, collapsed, hyaline, not dextrinoid and cyanophilic. endoperidium apical composed of two layers of hyphae continuous, all brown, not dextrinoid and cyanophilic, hyphae . - . µm diam, frequent and non-regular true septa, double v branching, and mycosclereids globose, subglobose, pyriform, ovoid, ellipsoid, or rectangular, . - . µm high × . - . µm diam, weakly interconnected, branched, breaking in the septa, regular and thick walls ≤ . µm thin and straight for curves. endoperidium basal hyphalic, . - . µm diam, rare and non-regular true septa, v-shaped branches, single and double, and in t, walls < . µm thin, tortuous and regular, brown, dextrinoid, and cyanophilic. subgleba hyphalic, . - . µm diam, rare true septa, branching v, single and double, and t, cyanophilic nodes frequent, regular walls ≤ . µm thin, straight for curves, reddish brown, not dextrinoid and cyanophilic. paracapillitium absent. capillitium calvatia-type, . - . µm diam, hyaline to light brown, dextrinoid and cyanophilic; septa frequent and non-regular, v-branching, single and double, and in t, fragmenting in any part of the capillitium or frequent in the septa; walls ≤ . µm thin and regular, straight, with large and numerous conspicuous pits ( - µm wide). basidiospores globose to subglobose, . - . µm wide × . - . µm high (χ = . ± . × . ± . ; q m (medium coefficient) = . ; n (measurement numbers) = ), verrucose, ornamentation < µm length; pedicels present in some basidiospores ≤ . µm in length. habit & habitat -basidiomata growing solitary on moist soil. notes -calvatia baixaverdensis is morphologically related to species of sect. calvatia: c. craniiformis, c. subtomentosa, c. rugosa, c. nodulata, and c. holothurioides. calvatia craniiformis, c. rugosa and c. subtomentosa have a capillitium with large conspicuous pits ( - µm wide) similar to c. baixaverdensis. however, c. craniiformis presents subglobose to globose basidiospores with punctate ornamentation, and well-developed cellular subgleba. calvatia rugosa has exoperidium granulose, furfuraceous to subvelutinous, endoperidium smooth, membranous, very thin (< . mm), subgleba well-developed and lanose to cellular (reid ) . calvatia subtomentosa has basidiospores . - . µm diam, and capillitium . - . µm, branched, septate, rather short fragments (dissing & lange ) , but is easily distinguished from c. baixaverdensis in the ornamentation of the basidiospores, equinulate, and in the absence of pedicels, besides the absence of large pits in the capillitium and nodules in the hyphae of subgleba in c. subtomentosa. calvatia nodulata and c. holothurioides are other morphologically close species to c. baixaverdensis mainly by the basidiospores - μm diam and capillitium - μm diam; however, c. nodulata has exoperidium granulose to pilose, subgleba occupying half of the basidiomata, and capillitium with spaced nodules (alfredo et al. ) , and c. holothurioides has subgleba prominent, cellular, capillitium with pores up to μm diam (rebriev classification -debaryomycetaceae, saccharomycetales, saccharomycetes. on glucose peptone yeast extract agar (gpya) and % malt extract agar (mea), after d at °c, streak is white-cream, semi-glistening, with a smooth surface and entire margin. cells are ovoid to elongate ( - × - μm) and occur singly or in pairs, dividing by polar and multilateral budding. rare pseudohyphae are produced on potato dextrose agar (pda) and cornmeal agar (cma). ascospores and true hyphae have not been observed during wk at and °c in culture (pure cultures and in mating test) grown on gpya, mea, pda, cma and yeast nitrogen base with . % glucose (ynb) agar. fermentation of glucose, galactose (delayed weak), trehalose and maltose (delayed) are positive, but negative for sucrose, lactose and raffinose. glucose, sucrose, galactose, maltose, cellobiose, trehalose, melezitose, methyl alpha-d-glucoside, dxylose, l-arabinose, d-glucosamine, ethanol, glycerol (weak), ribitol, d-mannitol, d-glucitol, salicin (weak), dl-lactic acid (weak), succinic acid (weak), citric acid, -keto-d-gluconate, arbutin are assimilated; no growth occurs on lactose, melibiose, raffinose, soluble starch, inulin, d-arabinose, d-ribose, l-sorbose, l-rhamnose, galactitol, erythritol, myo-inositol, -keto-d-gluconate, d-glucuronate and methanol. nitrogen compounds: ammonium sulfate, potassium nitrate (weak), creatinine, creatine, l-lysine, d-glucosamine (weak) are assimilated. growth on vitamin-free medium, on mea with % nacl and on % w/w glucose / yeast extract ( . %) agar is positive. growth with . % and . % cycloheximide is weak. (zhai et al. ) , the placement of the new species is demonstrated using the combined its and lsu rdna phylogeny. candida pellucida can be differentiated from the phylogenetically most close species c. viswanathii based on its ability to grow on vitamin-free medium, good growth at the temperature °c, and negative growth on soluble starch. maximum likelihood (ml) tree obtained from the combined analysis of its and lsu sequence data. bootstrap support values above % are shown at the nodes. the alignment included bp and was performed with mafft v. (katoh et al. ) . the general time reversible model (gtr) with gamma distribution and invariant sites (g+i) was used as the best nucleotide substitution model. the phylogenetic analysis was conducted in mega v. (tamura et al. ) . saccharomyces cerevisiae (ab /jq ) was used as outgroup (hidden). © naturalis biodiversity center & westerdijk fungal biodiversity institute fungal planet - june cladophialophora cabanerensis maciá-vicente, sp. nov. etymology. named after the cabañeros national park in central spain, where the soil sample was collected. classification -herpotrichiellaceae, chaetothyriales, eurotiomycetes. mycelium consisting of hyaline, branched, septate hyphae, ( . -) . - . (- . ) µm diam, forming hyphal strands. conidiophores mostly single, sympodial, erect, subcylindrical, hyaline, smooth, bearing one phialide, often reduced to a conidiogenous cell. conidiogenous cells phialidic, hyaline, smooth, fusiform with one locus at the apex that leaves a scar, ( . -) . - . (- . ) × ( . -) . - . (- . ) µm. conidia aseptate, produced in mass, hyaline, smooth, globose with a scar, ( . -) . - . (- . ) µm diam (n = ). chlamydospores absent. sexual morph unknown. culture characteristics -colonies slow-growing, reaching - mm diam on malt extract agar (mea), - mm diam on potato-dextrose agar (pda), and - mm diam on cornmeal agar (cma) after d at °c. colonies velvety, white, becoming light earthy after - wk, with a compact and suede-like surface; reverse white-cream. notes -the three isolates examined have identical morphologies and partial its and lsu sequences. since they originate from the same soil sample, they likely represent clonal isolates. based on a megablast search of ncbis genbank nucleotide database, the its sequence has low similarity with several unidentified chaetothyriales strains (e.g., genbank kx . , identities / ( %), gaps ( %); genbank kf . , identities / ( %), gaps ( %); gen-bank kf . , identities / ( %), gaps ( %)) and with cladophialophora immunda (genbank mh . , identities / ( %), gaps ( %)). however, the low identity values result from a long insert at the ' end of the s rdna gene, similarly to what has been found in other fungi (e.g., tedersoo et al. , cross et al. ), but that is not present in most genbank records. when analysing only the partial its region (nt - ) that is homologous to other sequences in genbank, the megablast search yields highest similarity with environmental sequences originating from a single study (e.g., genbank mf . , identities / ( %), no gaps), and to two unidentified fungi (genbank mg . , identities / ( %), no gaps; genbank gq . , identities / ( %), gap ( %)) and two cladophialophora sp. isolates (genbank lc . , identities / ( %), no gaps; and genbank lc . , identities / ( %), gap ( %)). the closest hits using the lsu sequence are an unidentified fungus (genbank gu . , identities / ( %), gap ( %)), cladophialophora sp. (genbank mf . , identities / ( %), gap ( %)), unidentified chaetothyriales (genbank kf . , identities / ( %), gap ( %)), and cladophialophora carrionii (genbank af . , identities / ( %), gap ( %)). the genus cladophialophora is polyphyletic, including species that are commonly isolated from soil and living plants, but also found as causal agents of human infections. cladophialophora cabanerensis is phylogenetically placed outside the carrionii and bantiana clades defined by badali et al. ( ) that contain most species pathogenic to humans. all the closest hits in the megablast search using the insert-free its sequence originate from fungi associated with plant roots, like the type specimen of c. cabanerensis, suggesting a preference of the species toward this habitat colour illustrations. wet heathland ('trampal') located in the cabañeros national park, ciudad real, spain. seven-day-old colonies growing at °c on pda; from top to bottom, overview of mycelium bearing conidiophores under phase-contrast microscopy; conidiophores under light microscopy; loose conidia under light microscopy. scale bars = µm (mycelium) and µm (conidiophores and conidia). culture characteristics -(after wk at °c in the dark): on potato dextrose agar (pda), colonies reach - mm diam, round shape, flat, dark olive green, dusty, aerial mycelium absent, profuse sporulation, margin white and glabrous, exudates (blackish droplets) produced mainly on the outermost colony surface; reverse olive green to olive black. on malt extract agar (mea), colonies reach - mm diam, irregular flat growth, elevated centre, dusty, olive green to yellowish green, aerial mycelium absent, exudates absent, white filiform margin; reverse, irregular olive-black. on synthetic nutrient-poor agar (sna), colonies reach a - mm diam, irregular flat growth, dusty, olive-green, profuse sporulation mainly in the centre of the colony, exudates absent; reverse olive grey with white filiform margin. on oatmeal agar (oa), colonies reach - mm diam, round shape, flat, olive-green, abundant velvety aerial mycelium, absent on the outermost colony surface, profuse sporulation, exudates absent, margin grey-green, narrow and glabrous. cardinal temperature for growth -optimum °c, maximum °c, minimum °c. notes -based on the combined analysis of its, acta and tef markers, cladosporium arenosum belongs to the c. cladosporioides complex (bensch et al. ) and is phylogenetically related to cladosporium asperulatum. however, c. asperulatum exhibits asperulate surface ornamentation of its conidia, conidiophores and mycelium (bensch et al. ), characters not found in c. arenosum. in addition, c. asperulatum has longer conidiophores (( -) - (- ) × ( -) - (- ) μm) and ramoconidia ( - × - μm)) (bensch et al. ) . finally, c. arenosum produces exudates on pda, limoniform conidia, and its colonies have a characteristic yellowish green colour after wk at °c on mea, characters not found in c. asperulatum (bensch et al. classification -cortinariaceae, agaricales, agaricomycetes. pileus up to mm diam, plano-convex to applanate, slightly glutinous when young, reddish golden to greyish orange ( b - b ); margin smooth or slightly innately fibrillose, incurved. lamellae emarginate, moderately crowded, up to mm broad, greyish when young, later greyish orange ( b - b ), lamellulae present, of various lengths. stipe × mm, cylindrical, with slightly clavate base, up to mm broad, greyish orange to brownish orange ( b - b ). context dull lilac ( c ) to purplish in pileus and in stipe base. odour distinct, earth-like. taste indistinct. spore print light brown ( d ). basidiospores ( . -) . - . (- . ) × ( . -) . - . (- . ) μm, av. = . × . μm, q = ( . -) . - . (- . ), qav = . , n = , amygdaloid, verrucose. basidia -spored, - × - μm, clavate. pileipellis more or less simplex ( -layered); strongly coloured in koh. epicutis at surface of narrow ( - μm wide), loosely erect-entangled, gelatinous, distinctly brownish yellow hyphae (many more or less collapsed); below wider ( - μm wide), parallel hyphae, with yellow thick walls or distinctly zebrastriped, encrusted pigment; the basal part of epicutis of more or less cemented hyphae up to ± μm wide, with distinctly thickened, yellow walls, some filled with dark brown granulate to oleiferous pigment (most pigment in the basal, cemented part). habitat & distribution -solitary, occurring among leaf litter in temperate forests dominated mainly by quercus leuco trichophora and pinus roxburghii. typus. india, uttarakhand, pauri garhwal, teka, m asl, n ° ' " e ° ' ", sept. , k.c. semwal (holotype kcs ; its and lsu sequences genbank mt and mt , mycobank mb ). phlegmacioides based on morphological and molecular (nrdna its and lsu regions) data, belonging to the /balteatocumatilis clade. it forms a well-supported (bs = %) lineage with three sequences known from the americas: usa, tennessee (gen-bank mf ), usa, minnesota (genbank ky ) and mexico (genbank eu ). the closest sequence is the one from minnesota; they differ by nucleotide and indel positions, but only in the its region. further studies are needed to unveil whether this sequence belongs to c. balteatoindicus with such a disjunct distribution. the other north american sequence from tennessee differs by nucleotide and indel positions, so it might well represent a separate species. the phylogenetically more distant european species of this clade have more robust basidiomata too, e.g., c. balteatocumatilis, c. balteatobulbosus, c. pseudonebularis, and the recently described c. hemicaeruleus (brotzu et al. ; its sequence genbank mt ) and have slightly larger spores. the special ecology and the unique its sequence are, however, the best delimiting characters for the time being. classification -cortinariaceae, agaricales, agaricomycetes. pileus up to mm diam, plano-convex to applanate, slightly inflated at centre, surface glabrous, slimy when young, slightly bluish greyish when young, but soon becoming reddish golden to light brown ( c - d ); margin smooth, fairly undulate. lamellae emarginate, crowded, greyish when young, later greyish orange ( b ), brownish orange ( c ) when mature, lamellulae present, of various lengths. stipe - × - mm, prominently clavate at the base, bulb up to mm wide, pale brown, becoming brownish orange to reddish orange ( d , b - b ) with greyish lilac ( b - ) tinge throughout the stipe, especially at apex. context greyish to bluish lilac. odour and taste not recorded. spore print brown ( e ). basidiospores ( . -) . - . (- . ) × ( . -) . - . (- . ) μm, av. = . × . μm, q = ( . -) . - . (- . ), qav = . , n = , amygdaloid, verrucose. basidia -spored, - × - μm, clavate. pileipellis more or less simplex ( -layered); rather weakly coloured in koh. epicutis at surface of narrow, - μm diam, loosely erect-entangled, gelatinous, pale yellow hyphae; below a few layers of slightly wider, - μm diam hyphae with slightly thickened yellow walls, a few with pale, weakly encrusted wall pigment; the basal part of epicutis of hyphae up to approx. μm diam, with distinctly thickened, yellow walls, forming tightly cemented bundles which in surface view forms a zig-pattern. habitat & distribution -caespitose, occurring among leaf litter of quercus leucotrichophora, on humicolous soil, in temperate broadleaved forests dominated by mainly q. leucotrichophora, rhododendron arboreum, and myrica esculenta. typus notes -cortinarius ulkhagarhiensis belongs to sect. phlegmacioides based on both morphological and molecular (nrdna its and lsu regions) data. within the section it belongs to the /daulnoyae clade, where it forms a close sister species of the european c. caesiocolor. they differ by nucleotide and indel positions, and in morphological characters. the spores of c. ulkhagarhiensis are significantly larger than those of c. caesiocolor (av. . × . μm vs . × . μm, respectively), and they are also longer (qav = . vs . ). macromorphologically they are rather similar, with e.g., bluish context. another closely related species is the european c. daulnoyae (syn.: c. chromataphilus and c. sabuletorum) which has a strong earth-like smell, yellowing, never bluish context, and phylogenetically is more distant. morphologically, other species in sect. phlegmacioides might also resemble c. ulkhagarhiensis, but the ecology and its sequence data will be helpful in identification. classification -cortinariaceae, agaricales, agaricomycetes. basidiomata rather small. pileus - (- ) mm diam, at first hemispheric, later convex with a persistent, obtuse, rounded and low umbo; margin first very incurved and highly lobulated and later extended and slightly serrate, retaining whitish veil remnants; surface hygrophanous, smooth to fibrous, dark grey, dark grey-brown (caill. t , t ; cailleux ) to ochraceous, pale ochraceous or reddish brown (caill. m , m , m ) when dry; mature pilei with necropigments. lamellae moderately dense, uncinated, pale ochraceous to beige ochraceous (caill. m , n ); lamellae edges slightly paler, and slightly mustard brown with age; lamellulae present. stipe ( -) - (- ) mm long and - (- ) mm wide, cylindrical to clavate or subglobose at the base; surface white, later pale beige, with universal veil copious towards the base, partial veil fugacious, not forming an annular area. context generally fibrous, pale ochraceous, and brownish in the stipe cortex. taste mild and smell indistinguishable. macrochemical reactions: negative to koh, guaiac tincture, ph.a. and methol. basidiospores broadly ellipsoid in front and side view, ( -) - . - . (- ) × ( . -) - . - . (- ) µm in size, with a q (length/width ratio) = ( . -) . - . - . (- . ), and with a marked apical depression; spore surface densely ornamented with projecting warts of moderate size. basidia - × - µm, -spored; lamellar edge with basidia and some claviform cells, - × - µm. pileipellis a cutis formed by a layer of - µm wide, clamped, more or less cylindrical hyphae, with scattered pale ochraceous incrusted wall pigments; subcutis composed of short and irregularly-arranged, septate hyphae, - × - µm; hyphae of the veil remnants - µm diam. habitat & distribution -restricted to the alpine belt (> m asl) in association with dryas octopetala. so far found in the pre-pyrenees (north-eastern iberian peninsula). the existence of an its sequence in genbank (fr ) identical to the ones obtained in the present study indicates the presence of c. paezii in the hyrcanian forests of iran. notes -cortinarius paezii is a rather small telamonioid species with relatively large spores that we initially considered to conform to the morphological variability of c. casimiri due to the general size, habitat and pigmentation. however, basidiomata of the latter species are in general slenderer than those of c. paezii, and show reddish and somewhat lilaceous tinges, their smell is more or less raphanoid, and the spores are smaller, - . × - µm (brandrud et al. ) . cortinarius paezii produces hygrophanous pilei that are very dark when hydrated, without lilaceous traces, and instead shows pale ochraceous to reddish brown tinges with time. furthermore, c. casimiri distributes preferentially in altimontane-subalpine habitats, and more rarely forms mycorrhizal associations with salix spp. in the alpine belt. considering other species growing in the alpine belt, c. cavipes would share two additional characters with c. paezii: the evident change in colour of pilei after drying and the clavate stipe (favre ) . as indicated by its epithet, however, c. cavipes has a hollow stipe; additionally, it shows lilaceous traces in the stipe apex and context (as in c. casimiri), and produces smaller, less ornamented spores. two additional alpine species described by favre ( ) were c. levipileus and c. rusticellus. the former differs from c. paezii in producing smaller basidiomata, with a finely granulose pileus cuticle, with the surface dark to reddish brown, and by the less abundant veil remnants and the slightly smaller, more ovoid spores (lower q value). lamoure ( ) obtained similar values for spore size in c. levipileus and provided further evidence of its habitat on calcareous soils in the alpine belt. cortinarius rusticellus produces spores more similar in size to those of the new species but has smaller basidiomata, pilei are more umbonate and fibrous to felty, lamellae are darker, and there is an abundant and persistent veil forming an evident annulus on the stipe. the two its sequences obtained for the new species were bp (plus four indels), bp (plus eight indels), and bp (plus six indels) different from those of c. casimiri /subsertipes, c. levipileus and c. rusticellus, respectively. the phylogenetic tree revealed c. tatrensis as a close relative of c. paezii. this species was described from salix and dryas communities in the alpine belt of the belaer tatras, in northern slovakia (fellner & landa ) . apart from the similar habitat, c. paezii and c. tatrensis share the general habitat of basidiomata, the hygrophaneity of pilei and their pigmentation, and the spores, which the authors described as broadly ovoid, ( -) . - . × ( . -) - . µm. however, lilaceous to vinaceous tinges were originally noticed in the surface of the stipe base and in the stipe context of c. tatrensis while these characters are absent in c. paezii. additionally, the stipe in c. tatrensis is described as 'cylindrical, slightly narrowing towards the base', whereas in the new species it is markedly clavate. the its sequence of c. tatrensis is provided for the first time in the present work, and shows five different nucleotides from c. paezii at the its region. classification -cylindriaceae, amphisphaeriales, sordariomycetes. asexual morph: mycelium consisting of smooth hyaline hyphae, branched and septate, - µm diam. conidiomata foliicolous, - wide and - µm tall, often in scattered groups, stromatic, immersed but erumpent when moist after pushing up a flap of host tissue and revealing a whitish jelly content. peridium composed of a single subepidermal inner layer of brown cells arranged as textura angularis, presenting paler pigmentation towards the conidiogenous region. ostiole absent. setae dark brown, - × - µm (length/width), smooth, dichotomously branched at the base, with - transversal septa, tapered towards the apex. paraphyses hyaline, scattered between setae and conidiophores. conidiophores arising from lageniform or cylindrical cells with hyaline or brownish walls at the internal wall of the peridium, formed of - µm long cylindrical cells (tapered towards the apex), septate and branched. conidiogenous cells integrated, hyaline, cylindrical (tapered towards the apex), lageniform, phialidic or percurrent, - × - µm (length/width). conidia hyaline, smooth, falcate, wider in the middle, tapering towards the apex, truncate at the base, measuring - × . - . µm (length/width), completely filled with small droplets. culture characteristics -(day/light °c, after wk): colonies slow-growing, with sparse aerial mycelium, rounded margins, reaching mm in wk. on malt extract agar and potato-dextrose agar white on surface, salmon in reverse. notes -on the basis of a combined phylogeny using its and s nrdna data (available in mycobank mb ), c. magnoliae is probably related with c. aeruginosum, c. algarvense, and c. purgamentum. lombard et al. ( ) proved that c. aeruginosum is phylogenetically related with the type species c. elongatum. crous et al. ( ) created a new family, cylindriaceae to accommodate this genus, proposing c. algarvense and c. purgamentum, and combining c. syzygii. recently, the new species c. grande was added to the genus (crous et al. c) . morphologically, c. magnoliae differs from other species of cylindrium because of its stromatic conidiomata, the specialised method of dehiscence, and the presence of setae and paraphyses. cylindrium magnoliae does not produce a pigmented stipe or sympodial loci and lacks ramoconidia which are present in c. purgamentum . cylindrium grande (crous et al. a ) produces sympodial conidiogenous cells and solitary conidia, features not present in c. magnoliae. colour illustrations. conidiomata on host. section of conidioma with brown setae; conidiophore; conidiogenous cells with successive percurrent proliferations (annellations); conidiogenous cells giving rise to conidia. scale bars = µm (section of conidioma), µm (others). etymology. name refers to the colour of conidial masses produced by conidiomata in culture. classification -erythrogloeaceae, diaporthales, sordariomycetidae, sordariomycetes. conidiomata after wk / h l/d cycles on c. sativa bark strips, pycnidial, separate, globose to subglobose, ( -) (- ) × ( -) (- ) μm with central ostiole, exuding a luteous, pale luteous to hyaline conidial mass. conidiophores reduced to conidiogenous cells, ampulliform to doliiform with prominent taper towards narrow cylindrical apex, enteroblastic, ( -) (- ) × ( -) (- ) μm wide. conidia aseptate, hyaline, smooth, ellipsoid, straight to curved, apex obtuse, smooth, thin-walled, guttules of varying sizes sometimes visible, ( -) (- ) × ( -) (- ) μm. culture characteristics -after mo in the dark at °c. colours determined from rayner ( ) . ex-type culture on potato dextrose agar . × . mm diam. surface undulating, woolly to velvety in texture. centre vinaceous buff, followed by dark mouse grey, hazel, fawn with an irregular pale vinaceous margin. reverse: centre sepia, extending to a mm ring of fuscous black, extending to brick to dark brick. on malt extract agar × mm diam. surface flat, woolly. centre rosy buff, extending to buff, rosy buff, vinaceous, and rosy buff. reverse: centre chestnut extending to flesh coloured. ( ) described four new species of dendrostoma from europe, and also updated the description of d. leiphaemia. on the concatenated lsu, its, tef -a tree, d. luteum was a strongly supported species (maximum parsimony bootstrap support %), sister to d. leiphaemia. morphologically, conidiomata of d. luteum are longer and wider than d. leiphaemia. dendrostoma luteum was consistently associated with branch lesions, but pathogenicity testing on detached c. sativa branches ( cm diam, cm length, n = ) did not produce lesions significantly longer than the control. therefore d. luteum is currently considered as an endophyte of c. sativa. the concatenated phylogenetic tree was inferred using maximum parsimony. * indicates ex-type cultures, with taxonomic novelty in bold. branch supports were determined using maximum parsimony bootstrap replicates. branch support values < % were excluded. scale bar on tree indicates number of changes. etymology. name refers to the host genus durio from which it was isolated. classification -diaporthaceae, diaporthales, sordariomycetes. conidiomata pycnidial, black, globose to subglobose, solitary or aggregated, embedded in tissue, - µm diam; forming up to six well-defined necks that can arise from a single conidioma. conidiophores formed from the inner layer of the conidiomatal wall, reduced to conidiogenous cells, cylindrical, hyaline, - × . - . µm. alpha conidia rare or absent in culture, hyaline, aseptate, biguttulate, ellipsoidal, smooth, ( . -) . - . (- . ) × ( . -) . - . (- ) µm. beta conidia abundant, hyaline, aseptate, hamate, ( . -) . - . (- . ) × . - . (- . ) µm. culture characteristics -on potato dextrose agar (pda), colonies white, fast-growing, covering dish after d at °c, surface buff with patches of purplish grey (rayner ), reverse purplish grey, with patches of dirty white. on malt extract agar (mea) with moderate aerial mycelium, and even, smooth margins; surface dirty white with patches of grey olivaceous, reverse dark mouse grey with patches of greyish sepia. on oatmeal agar (oa) surface dirty white with patches of dark mouse grey. the phylogenetic tree generated by raxml from the combined sequences of three loci demonstrated that the isolates of this study, kcsr . and kcsr . , grouped separately as a novel species. diaporthe ueckerae on cucumis melo has larger alpha conidia (( -) . - . (- . ) × ( -) . - . µm), and lacks beta conidia (udayanga et al. ) . diaporthe miriciae on helianthus annuus has larger alpha conidia, - . (- ) × - . (- ) μm, and longer beta conidia ( - × . - . μm; thompson et al. ) . previously, phomopsis durionis (dna data unavailable), was reported to be the causal agent of the durian leaf spot. however, the original description of p. durionis reports smaller conidioma, - μm diam, smaller alpha conidia ( - . × - . μm), with no beta conidia being observed (sydow ) . based on the characteristically large conidiomata with multiple necks, the canker disease and dieback symptoms, the present collection is herewith introduced as a new species. colour illustrations. dieback symptoms occurring on durio zibethinus at dak lak, central highlands, vietnam. colony on pda; conidioma on a durian twig on water agar; alpha conidia; conidiophores; beta conidia; conidiophores. scale bars: twig = mm, all others = µm. notes -macroscopically elaphomyces bucholtzii closely resembles the e. muricatus group, being differentiated by the variable height of its cortex warts. moreover, a section of the peridium is marbled, forming ellipsoidal (of cerebriform aspect) patches on a purple background, unlike the e. muricatus group that has a peridium marmorised in circles on a light background (white-cream), and the spores are decorated by thick, very curved sticks that usually form loops. a recently described european species e. barrioi (paz et al. ) has a marmorised peridium in red-purple tones forming small ellipses, on a vinous background, a dark brown gleba with red tones and smaller spores than e. bucholtzii. another species of the group is e. decipiens, but its cortex presents flat warts that are slightly oxidised after manipulation, a purple vinous peridium with cream-white veins arranged radially outward from the ascoma (paz et al. ) . elaphomyces violaceoniger has a dark violet peridium and some spores decorated with canes that are joined at maturity by drawing plaits, that clearly distinguishes this species from all the others in the e. muricatus group (paz et al. mycelium on leaves, epiphytic, amphigenous, producing dense, white patches mostly on the upper leaf surfaces. hyphae hyaline, thin-walled, - µm wide; hyphal appressoria mostly simple, nipple-shaped or knob-like, and rarely slightly lobed. conidiophores erect, consisting of a foot-cell, straight or occasionally slightly curved-sinuous at the base, - × - μm, basal septum at the branching point, followed by ( -) - cells up to the same length as the foot-cell. conidia produced singly, mostly cylindrical or ellipsoid-cylindrical, and occasionally doliiform, - × - µm. germ tubes terminal or subterminal, . - (- ) times longer than conidia (longitubus pattern when × longer), terminating in simple, often swollen, or rarely lobed appressoria. sexual morph not observed. notes -erysiphe contains approximately species of powdery mildew (braun & cook ) , including many common, widespread, plurivorous taxa (takamatsu et al. ) . some are taxonomically unresolved species complexes that are difficult to distinguish morphologically. these have similar or identical its sequences, and overlapping, or little-known, host ranges. an example is e. aquilegiae (jankovics et al. , kovacs et al. , takamatsu et al. . powdery mildews with its sequences that were identical or highly similar to e. aquilegiae were recorded on diverse host plants in different parts of the world (takamatsu et al. ) , including australia (cunnington et al. , southwell et al. , and belonged to the e. aquilegiae clade as defined by takamatsu et al. ( ) . phylogenetically, e. medicaginis belongs to a well-defined lineage that is sister to the e. aquilegiae clade. morphologically, it differs from e. aquilegiae, and also from the asexual morphs of other taxa that had its sequences identical, or highly similar, to e. aquilegiae, by having mostly simple, and not lobed or multi-lobed hyphal appressoria. the closest hits using the its sequence of e. medicaginis were two powdery mildew specimens from japan, mumh and mumh , collected from fabaceous hosts, baptisia australis and sophora flavescens, respectively. their its sequences (genbank lc and lc ) were identical to the five e. medicaginis specimens. these two specimens from japan were recognised as representing a distinct lineage, sister to the e. aquilegiae clade, without being identified at the species level (takamatsu et al. ) . erysiphe medicaginis is commonly found on m. polymorpha in queensland, australia. its host plant is a common weed globally, therefore it is likely that e. medicaginis is also widespread on m. polymorpha in different parts of the world, and likely on other fabaceous hosts, as indicated by the two specimens from japan. colour illustrations. medicago polymorpha with powdery mildew-infected older leaves in a weedy area in tipton, queensland, australia. conidiophores; a non-germinating and a germinated conidium; and simple, nipple-shaped and knob-like hyphal appressoria of erysiphe medicaginis. micrographs were taken following rehydration of the powdery mildew mycelium by boiling small pieces of infected plant tissues in lactic acid. scale bars = μm (conidiophores, conidia), μm (hyphal appressoria) the majority rule consensus phylogram inferred from the internal transcribed spacer sequences of the nuclear ribosomal dna and the intervening . s nrdna region using bayesian inference. the analysis was performed using mrbayes v. . . ) based on the gtr+g nucleotide substitution model selected using paup v. . b (swofford ) and mr-modeltest v. . . (nylander mycelium composed of pale olivaceous brown, septate, branched, smooth-and thin-walled hyphae, - μm wide; older hyphae being more strongly pigmented. spirally twisted hyphae present. moniliform cells scarce, globose to ellipsoidal, in short chains (- cells) . conidiophores semi-micronematous, pale olivaceous brown, smooth-and thin-walled, mostly laterally disposed on the vegetative hyphae, sometimes terminally disposed, erect, rarely once branched near the base, cylindrical, with a rounded or pointed apex, - -septate, with a terminal conidiogenous locus, sometimes with additional conidiogenous loci, - × - μm. conidiogenous cells enteroblastic, monoor polyblastic, integrated to the conidiophores, on vegetative hyphae or well-developed, in the latter case ellipsoidal, ovoid or flask-shaped, - × - μm, conidiogenous loci cylindrical or conic-cylindrical, with small percurrent proliferations. conidia aseptate, occasionally -septate, pale olivaceous brown, smooth-and thin-walled, ellipsoidal to reniform, - × - μm, sometimes with a truncate base, solitary. budding cells scarce, ellipsoidal, ovoid or barrel-shaped, - × - μm, in chains up to elements. chlamydospores scarce, olivaceous, globose, - μm diam. culture characteristics -colonies on potato dextrose agar (pda) reaching - mm diam after wk at °c, slightly raised, velvety, margins regular, brownish grey (m. e ; kornerup & wanscher ) , sporulation absent, exudate absent; reverse brownish grey (m. e ), diffusible pigment absent. colonies on oatmeal agar (oa) reaching - mm diam after wk at °c, morphologically similar to those on pda, with sparse sporulation. colonies on malt extract agar (mea) reaching - mm diam after wk at °c, slightly raised, velvety, margins regular, olive brown (m. e ), sporulation absent, exudate absent; reverse olive brown (m. f ), diffusible pigment absent. colonies on potato carrot agar (pca) reaching - mm diam after wk at °c, slightly raised, velvety, margins regular, olive brown (m. e ), sparse sporulation, exudate absent; reverse brownish grey (m. f ), diffusible pigment absent. colonies on pda reaching - mm diam after wk at °c slightly raised velvety, margins regular, brownish grey (m. e ), sporulation absent, exudate absent; reverse brownish grey (m. e ), diffusible pigment absent. minimum, optimal and maximum temperature of growth, °c, °c, and °c, respectively. notes -exophiala frigidotolerans was recovered from a soil sample collected in guayaquil, ecuador. the genus exophiala pertains to a group of fungi known as 'black yeasts', because of the production of yeast-like colonies and budding cells with dark, melanised cell walls. the genus exophiala is characteri sed by an annellidic conidiogenesis and the production of solitary conidia grouping in slimy masses, and its phylogenetic affiliation to the ascomycete order chaetothyriales (de hoog et al. ). this genus contains numerous potential opportunists or pathogens of immunocompetent humans (sudhadham et al. , li et al. and are isolated from a broad spectrum of substrata, environments and geographic areas (de hoog et al. , ferrari et al. . as in e. psychrophila, e. frigidotolerans exhibited the ability to grow at low temperatures. however, e. frigidotolerans presents more developed conidiophores than e. psychrophila (which are reduced to a unique discrete conidiogenous cell in this latter species), and produces shorter chains of moniliform cells (scarce and of up to cells in the former species, and very abundant and of up to several hundred of cells in the latter). based on a megablast search of ncbis genbank nucleotide database, the closest hit using the its sequence is the ex-type strain of exophiala brunnea cbs . (genbank jf ; identities = / ( %), gaps ( %)); and using the lsu sequence the ex-type strain of exophiala brunnea cbs . (genbank mh ; identities = / ( %), gap ( %)). the its-lsu-bena phylogenetic tree corroborated the placement of our isolate as a new species of exophiala, being located phylogenetically close to e. brunnea. exophiala brunnea is easily distinguished from e. frigidotolerans by the production of -celled conidia (mostly -celled in e. frigidotolerans) and absence of budding cells (formed in e. frigidotolerans). maximum likelihood tree obtained from the its-lsu-bena alignment of our isolate and sequences retrieved from genbank. the tree was built by using raxml cipres (http://www.phylo.org/sub_sections/portal/) and the analysis of probability was run in mrbayes v. . . . bootstrap support values ≥ % and bayesian posterior probability values ≥ . are presented at the nodes. fully supported branches ( % bs / pp) are thickened. cyphellophora laciniata cbs . and cyphellophora pauciseptata cbs . were used as outgroup. the new species proposed in this study is indicated in bold. t represents the ex-type strains of the taxa employed in this analysis. etymology. from latin calyx (cup) and coriaceum (leather). in reference to the coriaceous surface of mycelial layer, peeling-off to form a cup under basidiomata. classification -geastraceae, geastrales, agaricomycetes. unexpanded basidiomata epigeous, golden brown ( d , kornerup & wanscher ) to brown ( e ; f ), subglobose to obpyriform, . - × - mm, surface coriaceous, with little triangular processes when young, velutinous to papery with age, slightly encrusted with debris. subiculum white ( a ). expanded basidiomata saccate, - mm high (including peristome) × - mm wide. exoperidium splitting into - triangular rays, revolute or sometimes involute, rolling up under basidiomata, non-hygroscopic. mycelial layer honey yellow ( d ) to brown ( f ), non-persistent, ephemeral, peeling-off forming a cup under basidiomata, surface coriaceous, not encrusted. fibrous layer greyish yellow ( b ) to white orange ( a ), coriaceous. pseudoparenchymatous layer reddish ( e ) when fresh, brownish orange ( c ) to dark brown ( f ) when dried, rimose, persistent or peeling-off in irregular patches, with an inconspicuous collar. endoperidial body greyish brown ( d ) to brownish orange ( c ), subglobose to pyriform, - × - mm, sessile, surface glabrous, non-pruinose. apophysis absent. peristome fimbriate, distinctly delimited by greyish brown ( e ) line, mammiform, lighter than endoperidium, < mm high. gleba greyish brown ( f ). basidiospores brownish, globose to subglobose, . - . × . - . μm (x = . ± . × . ± . µm, q m = . , n = ), ornamentation conspicuous under lm. warts cylindrical ( . - . μm high), sometimes with some confluent tips. apiculous reduced. basidia yellowish, oval, lageniform to clavate, thick walls ( . - . μm), . - . × . - . µm, - sterigmata. eucapillitium pale brown hyphae, . - . μm diam, surface encrusted, covered by warts, thin walls ( . - . μm) and lumen evident. mycelial layer composed of yellowish to hyaline, some sinuous and inflated hyphae, . - . μm diam, surface non-encrusted, some branched, thin-walled ( . - . μm) and lumen evident. fibrous layer composed of yellowish to hyaline hyphae, . - μm diam, surface non-encrusted, non-branched, thin-walled ( . - μm) and lumen evident. pseudoparenchymatous layer composed of yellowish, subglobose, oval to elongated cells, . - . × . - . μm, thick-walled ( . - . μm). rhizomorphs composed of hyaline, thin hyphae, surface covered by acicular crystals, . - . × . - . μm, in an irregular arrangement. notes -geastrum calycicoriaceum is characterised mainly by its ephemeral yellowish mycelial layer with coriaceous surface, peeling-off forming a cup under basidiomata and persistent rhizomorph with acicular crystals, also by distinct delimited peristome and basidiospores with . - . μm diam and small warts (up to . μm high). our phylogenetic analyses (concatenate its and lsu) grouped g. calycicoriaceum in the mycelisotroma section, velutina subsection. this subsection comprises, until now, the species geastrum velutinum, which has some features in common with g. calycicoriaceum: both have a yellowish mycelial layer, delimited and fimbriate peristome and presence of subiculum. however, g. velutinum has lighter colours in peridium layers (yellowish pseudoparenchymatous layer when fresh and pale brown endoperidium) than g. caly cicoriaceum; moreover, g. velutinum lacks an ephemeral, coriaceous mycelial layer (dissing & lange ) . geastrum javanicum is another species which could be grouped in subsect. velutina based on its morphological features. presently there are no molecular data from the type to support g. javanicum as morphologically similar to g. calycicoriaceum, and it is distinct based on its smaller basidiospores ( . - . μm diam), conical peristome and felted endoperidium surface (ponce de leon ). geastrum argentinum is another species morphologically close to g. calycicoriaceum. however, it has a non-delimited peristome, and larger basidiospores ( . - . μm diam) (zamora et al. colour illustrations. woncheon stream, located in namwon city, jeonnam povince, republic of korea. once branched sporangiophore with fertile and sterile sporangium (branch); branched sporangiophore with two branches in whorls of two and columellae; sympodially branched sporangiophore with columellae and sterile sporangia; unbranched sporangiophore with apophysis and columella; giant cells; unbranched sporangiophore with apophysis and columella; sporangiospores. scale bars = μm. etymology. name refers to the isolation site, namwon city, from where the strain was first isolated. classification -cunninghamellaceae, mucorales, mucoromycotina, mucoromycota, mucoromyceta. mycelium hyaline. rhizoids hyaline, coenocytic, branched; stolons hyaline, coenocytic, smooth-walled. sporangiophores mostly arising from stolons or directly from aerial hyphae and stolon-like, erect or slightly recumbent, apophysate, simple or commonly sympodially and/or monopodially branched, smooth-walled, up to mm in length and µm diam, with up to septum (majority) below the sporangium. short and long branches may be found on the same sporangiophore at short or long distances from the main sporangium, and frequently rebranching. branches in whorls of or may be found on some sporangiophores. apophyses globose ( . -) - . (- ) µm, subglobose and ellipsoid, some with a truncated base, . - . × . - µm, smooth-walled. sporangia pale yellow, globose, wall transparent, deliquescent and smooth, up to µm diam. sometimes sterile sporangia are formed. columellae hyaline, globose, subglobose, . - µm diam, hemispherical, . - . × . - . µm, nipple-like, ellipsoidal, - . × - µm. sporangiospores hyaline, reniform, ellipsoidal, some ovoid, . - . × . - . µm, rarely irregular, up to × . µm. giant cells globose, subglobose and branched. chlamydospores mostly globose and subglobose. zygosporangia not observed. culture characteristics & temperature tests -colony white, reverse cream, low to moderate growth, taking the whole petri dish ( cm diam) after d on malt extract agar (mea) at °c; odourless; at °c -lack of growth; at °c -slow growth ( . cm diam after h); at °c -slow growth ( . cm diam after h); at °c -slow growth ( . cm diam after h), better than at °c; at °c -moderate growth ( . cm diam after h); at °c -optimum growth ( cm diam after h); at °c -moderate growth ( cm diam after h); at °cslow growing ( . cm after h). gongronella namwonensis exhibited slightly better growth on mea than on potato dextrose agar (pda) at , , , and °c with similar growth at and °c. the growth was also slightly better on mea than on synthetic mucor agar (sma), except at °c, where g. namwonensis grew . cm after h in sma. typus. south korea, namwon city, jeonbuk province, n ° ' . " e ° ' . ", from freshwater samples, july , h.b. lee (holotype cnufc-ww - ; its and lsu sequences genbank mn and mn , mycobank mb ). notes -gongronella namwonensis differs from other species based on its morphological characters and the phylogenetic relationships established based on the its and lsu rdna regions. morphologically, g. namwonensis differs from the other species by producing concomitantly strongly sympodially and/ or monopodially branched sporangiophores, some showing branches in whorls of , columellae varied (some nipple-like) and apophyses (some with a truncated basis), as well as giant cells. so far, giant cells had only been visualised in g. brasiliensis. sporangiophores of g. namwonensis presents up to one septum below the sporangia and are long (up to mm in length), different from the shorter (up to μm in length) and with up to two septa sporangiophores of g. brasiliensis. the columellae of g. brasiliensis are globose, subglobose and conicalcylindrical, never hemispheric or nipple-like, as observed in g. namwonensis. additionally, as observed in g. brasiliensis, our species produces sporangiospores varied in shape, but they are never falciform or ellipsoid to fusiform like the ones of the brazilian species (tibpromma et al. ). in the its and lsu rdna trees (data not shown) g. namwonensis was placed in a well-supported clade separate from the other species. a more closely related species is g. guangdonguensis. morphologically, both species can be easily distinguished by the shape of sporangiospores, being globose in g. guangdonguensis, as well the fact that it lacks rhizoids and stolons (adamčík et al. ) , both structures which are present in g. namwonensis. phylogenetic tree of gongronella namwonensis cnufc-ww - and cnufc-ww - - based on maximum likelihood analysis of the internal transcribed spacer (its) nrdna region. the numbers at the branches are bootstrap support value (≥ %) from replications. gongronella lacrispora was used as the outgroup. ex-type strains are indicated by t . pathogenic on leaves of kielmeyera coriacea. leaf spots up to cm diam, rather irregular, sometimes confluent and covering almost the whole blade, often at the margins, amphigenous, showing small dark points of conidiomata at the upper side of the leaves. conidiomata acervular at maturity, - µm diam, with brownish wall layers of textura angularis, sub cuticular to intraepidermal, scattered. conidiophores hyaline to pale brown, - -septate, - × . - µm, branched, smooth. vegetative hyphae internal, hyaline to pale brown, smooth, intermingled with the host cells, branched, - μm diam. conidiogenous cells hyaline, phialidic with a conspicuous collarette, - × - . µm, percurrent proliferation with a serrate collarette. conidia hyaline to pale brown, variable, sometimes elongate-fusoid, fusoid to ellipsoidal, aseptate, smooth and thick-walled, attenuate and papillate at the apex, truncate at the base, guttulate, - × - µm, - µm. conidial cirrhi arising from conidiomata on the surface of infected leaves. culture characteristics -colonies on potato dextrose agar (pda), malt agar (ma %) and oatmeal agar (oa) (near-uv light, h photoperiod) with a pale brown centre surrounded by a greyish olivaceous ring containing dark spore masses, followed by a pale brown area with aerial feltose mycelium and irregular margins. a reddish to vinaceous pigment is produced in all media (either side of the plates), more intensely in pda. slow growth on all media, no growth at , , and °c on pda and ma % and at , , and °c on oa. growth / d at °c on pda was . and . mm (for isolates cml and cml , respectively) while on ma % it was . and . mm. growth at °c on pda ( and . mm), on ma % ( . and . mm), on oa ( . and . mm). growth at °c on pda was . mm for both isolates, on ma % was . and . mm and on oa . and . mm. conidia were produced at approximately , and d, respectively on oa, ma % and pda at °c. notes -greeneria kielmeyerae is related to the other species of the genus, g. uvicola (farr et al. ) and g. saprophytica (tangthirasunun et al. ) , but differs from these species in the production of a reddish to vinaceous pigment on plates, for having larger conidia ( × . in g. kielmeyerae, . × . in g. uvicola and × . in g. saprophytica) and a larger length to width ratio of the conidia ( . in g. kielmeyerae, . in g. uvicola and . in g. saprophytica). the closest phylogenetic relative of g. kielmeyerae cml t (accession mn . ) with its sequences was melanconiella spodiaea spod (genbank jq . ; . % identity), it had . % identity with the its sequence of g. uvicola fl (genbank hq . ) and . % identity with the its of g. sapro phytica ntcl - (genbank kj . ). with lsu sequences the closest relative of g. kielmeyerae cml t (genbank mn . ) was g. uvicola usvitis (genbank jn . ; . %) and it was . % identical to the lsu sequence of g. saprophytica ntcl - (genbank kj . ). with ssu sequences, the closest relative of g. kielmeyerae cml t (genbank mn . ) was coryneum heveanum mflucc - (genbank ng_ . ; . % identity), and had . % identity with the ssu sequence of g. saprophytica ntcl - (genbank kj . ). the phylogenetic relationships of the genus greeneria and closely-related genera are not well defined as observed by tangthirasunun et al. ( ) . unrooted maximum likelihood tree obtained by phylogenetic analysis of the combined its and lsu sequences from greeneria kiemeyerae and phylogenetically related species performed in the software mega v. . (tamura et al. ) employing the gtr+g model with bootstrap re-samplings. bootstrap support values > % are presented. the new species is shown in red text ( t = ex-type) and the genus greeneria is delimited in a pale red box. genbank accession numbers are given after each strain (its = blue, lsu= green). hemileucoglossum kelabitense v. kučera, fedosova & sochorová, sp. nov. etymology. name refers to the kelabit highlands where the fungus was collected. classification -geoglossaceae, geoglossales, geoglossomycetes. ascomata scattered to gregarious, capitate, stipitate, - × - . mm, dry, black throughout. ascigerous part capitate or broadly clavate, / - / of the total ascomata length, black, compressed or oval in cross section, sharply delimited from the stipe, smooth both in fresh and dry conditions. stipe terete, cylindrical, oval in cross section, - × . - mm, slender to robust, with dark brown setose hairs in tufts mainly at the upper part of the stipe, rough to squamulose. asci cylindrical-clavate, ( -) - (- ) × . - μm (all measurements of microscopic characters refer to rehydrated material in tap water), q = . - . , unitunicate, inoperculate, -spored, with euamyloid ascoapical apparatus and inamyloid wall in mlz and iki, arising from croziers. ascospores elongate, ellipsoid baculiform, sometimes slightly curved, ( -) - (- ) × - . μm, q = ( -) - , hyaline, finally becoming brown, -septate at maturity, smooth. ascoconidia ellipsoid, ovoid or dacryoid, . - . × . - . μm, q = . - . , brown, aseptate, formed already on ascospores inside the asci, smooth. paraphyses straight, sparsely septate, - μm wide at lower part, hyaline at basal part to pale brown at the apex, agglutinated by dense brown amorphous matter. apical cells of paraphyses usually inflated, clavate to capitate, sometimes constricted and proliferating, - × ( -) - (- ) μm. flesh of the fertile part formed by grey cylindrical cells, - × - μm. flesh of the stipe formed by grey cylindrical cells, - × - μm, oriented with their long axis in the same direction as the stipe. stipe surface squamulose due to tufts of septate dark brown setose hairs, ( -) - (- ) × - μm at the middle part and - μm at the apical part, crumpled, sometimes bifurcated, thick-walled ( . - μm), with rounded apex. habit, habitat & distribution -in small groups on soil among mosses. the species is known only from the type locality. notes -predominantly hyaline ascospores finally becoming brown, paraphyses agglutinated by a dense brown amorphous matter and setose hairs on the stipe (but lacking on the fertile part), as well as the genetic profile rank the new species with the genus hemileucoglossum (arauzo & iglesias ) . hemileucoglossum kelabitense is characterised by the longest ascospores and asci within the genus. morphologically, the most similar species is h. alveolatum, which is alike in number of septa (up to ) in ascospores, but its ascospores are shorter and slightly thinner ( - × - μm). moreover, h. alveolatum prefers very rotten wood and logs (durand (durand , nannfeldt , crous et al. , kučera & fedosova . colour illustrations. tropical rainforest in bario town surroundings, the kelabit highlands. macro-and microscopic structures of holotype: ascomata; ascospores (in tap water); ascospores germinating by ascoconidia inside the ascus (in tap water); amyloid reaction of the ascoapical apparatus (in iki); paraphyses (in % koh); setose hairs of the stipe surface (in tap water). rehydrated material was used for all photos of microscopic characters. scale bars = cm (ascomata), μm (microscopic structures). heterocephalacria septentrionalis kachalkin, m.a. tomashevskaya & t.a. pankratov, sp. nov. etymology. the epithet refers to the species distribution in the northern regions of russia. classification -filobasidiaceae, filobasidiales, tremellomycetes. on glucose peptone yeast extract agar (gpya) and % malt extract agar (mea), after d at °c, streak is cream-coloured, shiny and mucoid, with an entire margin, and flat profile. after a month, the colour of the streak is pinkish cinnamon. cells are globose, ovoid to ellipsoid, - × . - μm, occur singly or in pairs, dividing by polar and multilateral budding. sexual structures, pseudohyphae, true hyphae and ballistoconidia have not been observed during wk at and °c in culture (pure cultures and in mating test) grown on gpya, mea, potato dextrose agar (pda), yeast nitrogen base with . % glucose (ynb) agar and cornmeal agar. glucose is not fermented. glucose, galactose, sucrose, maltose, cellobiose, trehalose, lactose, melibiose, raffinose, melezitose, soluble starch (variable and weak), d-xylose, l-arabinose, d-arabinose, d-ribose, l-rhamnose, d-glucosamine (variable and weak), ethanol (weak), glycerol (weak), ribitol (weak), galactitol, d-mannitol, d-glucitol, methyl alpha-d-glucoside, salicin, d-gluconate, succinic acid, citric acid, -keto-d-gluconate, myo-inositol and arbutin are assimilated; no growth occurs on l-sorbose, inulin, erythritol, dl-lactic acid, methanol. nitrogen compounds: ammonium sulfate, potassium nitrate, l-lysine (variable), d-glucosamine (variable), cadaverine (variable), creatinine (variable), creatine (variable) are assimilated, and no growth occurs on ethylamine. growth on vitamin-free medium is variable. growth on mea with % nacl and on % w/w glucose / yeast extract ( . %) agar is negative. growth with . % and . % cycloheximide is variable. starch-like compounds are produced. diazonium blue b colour and urease reactions are positive. maximum growth temperature is - °c. notes -analysis of the its-d /d regions of the surveyed yeasts suggested that they were conspecific ( subst. between strains from nadym and kandalaksha) and represented a hitherto undescribed species of heterocephalacria. the genus heterocephalacria comprises three sexual mycoparasites of lichens (h. bachmannii and h. physciacearum) and mushrooms (h. solida), and several asexual species of yeasts (kachalkin et al. , kunthiphun et al. , li et al. . two new yeast strains were isolated as a minor component from cladonia lichens whose thallus did not have basidioma-like structures. although no sexual morph was discovered for new species, its mycoparasitic lifestyle cannot be excluded. based on the ncbi genbank database, the best hit using the its and lsu sequences is h. sinensis cbs t (its -genbank mg ; . % similar, subst. and gaps, lsu -gen-bank ky ; . % similar, subst. and gaps), using ssu it is h. bachmannii am (genbank jn ; . % similar, subst. and gap), using tef and rpb the number of nucleotide substitutions was considerable -without hits with heterocephalacria. in compliance with a recent phylogenetic analysis of the genus (kachalkin et al. ) , the placement of the new species is demonstrated using the combined its and lsu rdna phylogeny. heterocephalacria septentrionalis can be differentiated from h. sinensis based on its ability to assimilate galactose, maltose, trehalose, melezitose, l-rhamnose, glycerol, d-mannitol, d-glucitol, citric acid, and negative growth on l-sorbose. notes -kosmimatamyces is a new genus that groups in the capnodiaceae, a family whose members are known as sooty molds whose dark hyphae cover the surface of living leaves and twigs of many plants (hughes , abdollahzadeh et al. . hypersaline soil represents a new ecological niche, reinforcing the hypothesis of crous et al. ( ) and chomnunti et al. ( ) that plant surfaces are not the only environmental niche for this group of fungi. based on a megablast search of ncbis genbank nucleotide database, the closest hit using the its sequence was micro xiphium theae cbs . (genbank mh ; identities = / ( %), gaps ( %)), antennariella placitae as (genbank mg ; identities = / ( %), gaps ( %)), and leptoxyphium kurandae mcc (genbank kf ; identities = / ( %), gaps ( %)); using the lsu sequence the closest hit were capnodium coartatum mflucc - (genbank jn ; identities = / ( %) , no gaps), microxyphium aciculiforme cbs . (gen-bank gu ; identities = / ( %), no gaps), and conidioxyphium gardeniorum cpc (genbank gu ; identities = / ( %) , no gaps). the lsu phylogenetic tree corroborated the placement of our isolate close to the genus leptoxyphium. the species of leptoxyphium are characterised by pycnidial conidiomata with a bulbous swollen base and cylindrical neck that expands at the apex to become funnel-shaped (hughes , chomnunti et al. , whereas kosmimatamyces produces single conidio phores. etymology. from greek αλατος-, salt, and -φιλος, lover, because of the environment from which the fungus was recovered. mycelium consisting of branched, septate, thick-walled, . - . µm wide hyphae. conidiophores solitary, macronematous or semimacronematous, erect, straight to flexuous, from hyaline to dark brown, thick-and smooth-walled to verrucose along its length, branched or unbranched, branches terminal or lateral, in an angle to °, - × . - µm. conidiogenous cells determinate, integrated, terminal or intercalary, pale to dark brown, verrucose, mono-or polyblastic, - × - . µm, scars truncate of - . µm wide. ramoconidia aseptate, pale to dark brown, thick-and smooth-walled to verrucose, subcylindrical, . - × - µm. conidia - -septate, brown to dark brown, thick-walled, with a spinulose, digitate, pustulate to crater-like ornamentation, globose, limoniform to ovoid or ellipsoid, - × - µm, with one or more notorious scars, arranged in branching acropetal chains, of schizolytic secession. culture characteristics -(after wk in darkness at °c). colonies on oatmeal agar (oa) up to mm diam, flat, slightly dusty to floccose, greyish sepia (rayner ) , aerial mycelium scarce, margins entire, exudates as olivaceous brown; reverse black, diffusible pigments absent. colonies on potato dextrose agar (pda) up to mm diam, flat, velvety, radiate and sulcate, greyish sepia at centre, greyish white to the borders, margins regular, scarce droplets of olivaceous exudates; reverse olive black to greyish sepia, diffusible pigments absent. on malt extract agar (mea) up to mm diam, velvety, zonate, radially folded and somewhat elevated, pale olivaceous grey, mostly consisting of vegetative mycelium, margins irregular; reverse greenish black, diffusible pigments absent. on potato carrot agar (pca) up to mm diam, floccose, olivaceous black, radiate, margin filamentous; reverse olivaceous black, diffusible pigments absent. on sna up to mm diam, flat, radiate, olivaceous at the centre and isabelline to the margins, margin entire; reverse olivaceous black at the centre, borders olive, diffusible pigments absent. fungal planet - june kosmimatamyces bianchin., reinoso f., rodr.-andr., cano & stchigel, gen. nov. etymology. from greek κοσμήματα-, jewellery, and -μύκης, fungus, because of the microscopic look of the fungus. classification -capnodiaceae, capnodiales, dothideomycetidae, dothideomycetes. mycelium consisting of branched, septate, pale to dark brown, thick-walled hyphae, sometimes coarsely ornamented. conidiophores solitary, macronematous or semimacronematous, erect, straight to flexuous, from hyaline to dark brown, thick-and smoothto rough-walled, cylindrical, narrow, branched or not, branches terminal and lateral, in angles of to °. conidio genous cells determinate, integrated, terminal and intercalary, monoor polyblastic, pale to dark brown, verrucose, scars truncate. conidia holoblastic, - -septate, brown to dark brown, thickwalled, globose, ovoid or ellipsoid, ornamented with spines and crater-like warts, with dark scars at one or both ends, arranged in branching acropetal chains. lactifluus albopicri t. lebel & l. tegart, sp. nov. etymology. named for the colour and hot taste of the basidiomata, albo = white, picri-= hot. classification -russulaceae, agaricales, agaricomycetes. basidiomata robust, lactarioid. pileus - (- ) mm diam, convex with decurved margin, planoconvex with depressed centre when mature; dry, smooth to very finely tomentose, white becoming very pale yellowish buff to pale honey cream with cream margin and slightly honey coloured centre; context solid, white becoming very pale cream coloured, no staining. lamellae at first broadly adnate, then subdecurrent to decurrent, narrow ( . mm), close (c. - per cm) with some forking, and - rows lamellulae, concolourous with pileus or slightly paler. stipe - (- ) × - (- ) mm, cylindrical or slightly tapered to base in older material, central, smooth, dry, whitish tinged with yellowish buff or hints of pale orange to pinkish buff; context firm, white, unchanging, dry, chalky. basal mycelium white. latex copious, white, unchanging, very hot and peppery. spore deposit white. phenol: rapidly wine red/pink; feso rapidly brown. spores . - . (- . ) × . - . µm (n = , . ± . × . ± . µm), q = . - . , subglobose to broadly ellipsoid, hyaline, asymmetric; ornamentation and spore wall amyloid, up to . - µm high, composed of isolated irregular warts that join together to variable degree in very fine lines to form a very partial reticulum, plage inamyloid. basidia - × . - µm, cylindrical to subclavate, - -spored; sterigmata - µm long. pleuromacrocystidia - × - µm, moderately common to abundant, cylindric to fusiform. pleuropseudocystidia - × - µm, cylindrical, moderately abundant, apices occasionally mucronate. cheilomacrocystidia - × - µm, cylindrical to subclavate with capitate apices, scattered. hymenophoral trama predominantly composed of hyaline hyphae - µm diam, interwoven with abundant sinuous lactifers up to µm broad; subhymenium cellular, - tiers of parenchymatous cells, - × - µm. pileipellis a hyphoepithelium, -layered: subpellis up to µm thick, composed of globose to subglobose cells - (- ) µm diam; suprapellis - µm thick, composed of mostly upright thin-walled hyaline hyphae, - (- ) µm diam, and abundant dermatocystidia - × - µm, cylindrical to clavate, with granular contents. pileus context heteromerous, with abundant sinuous laticiferous hyphae, - µm diam. habit, habitat & distribution -gregarious on soil amongst eucalypt leaf litter in wet sclerophyll forest. widely distributed from cool temperate forest in southern australia (vic, tas) with eucalyptus regnans and nothofagus cunninghamii dominant in canopy, scattered acacia dealbata and a. melanoxylon, with ferny understorey of dicksonia antarctica, blechnum watsii and asplenium sp., up to subtropical eucalypt and lophostemon woodland in northern australia (nt, qld). notes -the overall diversity of lactifluus in australia is poorly known (may et al. ) , perhaps in the order of - species, with only a few sections examined (i.e., gerardii; stubbe et al. ) . the main characters used to distinguish species in lf. sect. piperati are the shape and ornamentation of the spores, the composition of the lamellar edge, the form of the cheilomacrocystidia and the hypoepithelium pileipellis that lacks thick-walled elements (heilmann-clausen et al. , de crop et al. . the majority of other species in lactifluus have pilei with thick-walled elements (verbeken & walleyn ) . two morphologically distinct species are recognised from europe and an additional or so asian species remain to be morphologically documented and described (de crop et al. ) . up until recently, no species of lf. sect piperati were known to occur in south america, africa or australasia. lactifluus albopicri is a widespread species in eastern australia, from tasmania up to subtropical queensland and into the northern territory, occurring in wetter forests in association with eucalyptus and nothofagus. lactifluus albopicri resembles many of the known species from lf. sect. piperati, in the robust, pale coloured basidiomata, hot peppery latex, and spores with fine, low ornamentation. lactifluus albopicri differs from another australian sect. piperati species, lf. austropiperatus, in the typically larger sporocarps, slightly darker yellowish to pale orange pileus and lamellae, larger and subglobose vs broadly ellipsoid spores with finer and lower ornamentation. lactifluus albopicri sits in a well-supported clade with a single sequence from thailand (genbank kf ), amongst other se asian clades. colour illustrations. cool temperate rainforest dominated with eucalyptus regnans and nothofagus cunninghamii with scattered acacia spp. and understorey of dicksonia antarctica (photo g. lay). northern habitat of subtropical lophostemon woodland; basidiomata; section through hypoepithelium pileipellis; sem of spores. scale bars: mm; µm; µm. lactifluus austropiperatus t. lebel & l. tegart, sp. nov. etymology. named for its similarity to lactifluus piperatus from the northern hemisphere and its distribution in australia. basidiomata robust, lactarioid. pileus - mm diam, convex with decurved margin, planoconvex with depressed centre when mature; pileipellis dry, glabrous, azonate, whitish variously tinged with yellowish or hints of pale orange when younger, to pale-biscuit-buff overall in older specimens. lamellae subdecurrent, close (c. - per cm) with some forking closer to margin, and - rows lamellulae, very pale orange, bruising a little darker with handling. stipe - × - mm, cylindrical, whitish tinged with yellowish or hints of pale orange; context white, unchanging, taste hot and peppery. latex copious, white, unchanging or barely yellowing slightly after - min, very hot and peppery. spore print white. spores ( . -) . - . × . - . µm (n = , . ± . × . ± . ), q = . - . ± . , barely globose to subglobose, asymmetric, hyaline; ornamentation amyloid, up to . µm high, composed of irregular warts that join together to variable degree in short thin fine lines; plage inamyloid. basidia - × - µm, cylindrical to subclavate, -spored; sterigmata short, robust. pleuromacrocystidia - × - µm, abundant, narrowly cylindrical to fusiform, with tapering apex. pleuropseudocystidia similar size and shape to pleuromacrocystidia, moderately abundant, sometimes with irregular mucronate apices. cheilomacrocystidia ( -) - (- ) × - . (- ) µm, cylindrical to filiform with acute or capitate apices, with crystalline contents, scattered, more obvious in younger specimens. hymenophoral trama up to µm wide, composed of interwoven hyaline hyphae of - µm diam and abundant sinuous lactifers up to µm thick, sphaerocytes rare; subhymenium layer up to µm thick, parenchymatous, cells - µm diam. pileipellis a hyphoepithelium, -layered: subpellis up to µm thick, composed of globose to subglobose cells - µm diam; suprapellis - µm thick, composed of mostly repent thin-walled hyphae, frequently septate, - (- ) µm broad; context broad, composed of hetero merous tissue, sphaerocytes up to µm diam interwoven with hyaline hyphae - µm diam, and scattered to abundant sinuous laticiferous hyphae of - µm diam. habit, habitat & distribution -in savanna eucalypt woodland with eucalyptus pilularis or e. delegatensis, and e. cypellocarpa near creek lines with syzygium, allocasuarina, acacia spp., with tall grass understorey, rarely in mixed nothofagus moorei forest leaf litter; solitary but common. notes -lactifluus austropiperatus morphologically closely resembles lf. subpiperatus, described from japan (hongo ) ; unfortunately, no sequence data are currently available for comparison. lactifluus dwaliensis, lf. allardii, lf. glaucescens, and lf. subpiperatus grow respectively in association with species of oak in temperate deciduous forests in india, hardwood or pine-oak forests in central to southern usa, mixed deciduous forests in europe, or deciduous oak forest in japan (das et al. , verbeken et al. . lactifluus dwaliensis is a rare all-white species with quite a long stipe, and context and tissue that slowly stains light greenish yellow, while lf. allardii is stockier, with pinkish brown colours and flesh that stains purplish pinkish then green, and white copious latex that slowly turns greenish then brownish (das et al. , de crop et al. . lactifluus glaucescens is an elegant, allwhite species with densely crowded lamellae and latex that turns slowly olive to pastel green. all five species have smallish spores with low ornamentation under . µm high, as isolated warts with scattered connecting lines, grading into a partial reticulum. lactifluus subpiperatus is morphologically most similar to lf. austropiperatus, also having white then patchily pale ochraceous, somewhat stocky basidiomata, forked lamellae, and small subsphaerical spores (hongo ) . however, lf. austropiperatus has sporocarps with more yellowish to pale orange tinges, flesh and latex that does not change colour or only very slowly and slightly, with no green tones, and pleuromacrocystidia are present (de crop et al. ) . lactifluus austropiperatus grows in association with subtropical forest of eucalyptus, and more rarely nothofagus, in northern nsw and southern qld, australia. in our analysis lf. austropiperatus is in a strongly sup ported clade with a specimen from thailand (genbank kf , h.t. le ), however, at this time we maintain the australian material as distinct until further collections from thailand can be examined and sequenced. preliminary morphological examination shows the spores of h.t. le to be slightly smaller, and the ornamentation to be slightly finer than the australian material. lactifluus austropiperatus is sister to l. dwaliensis, a specimen from honduras (lmunah ; no plant associate listed), and an environmental sample from florida, usa associated with pinus clausa. lecanicillium praecognitum gorczak & kisło, sp. nov. etymology. prae (latin: before, ahead of) + cognitum (latin: known, noted; neut. part. adj.); known before; referring to the fact that the species was noted several years before formal description. classification -cordycipitaceae, hypocreales, sordariomycetes. on sabouraud dextrose agar (sda): conidiophores erect, mostly single, sometimes in whorls up to four. unfrequently secon dary phialides arise, sometimes in whorls up to three. phialides . - . (av. = . ) µm long × . - (av. = . ) µm wide. conidia hyaline, smooth, granular, oblong to slightly fusiform, solitary or in small clusters, ( . -) - . (- . ) (av. = . ) µm long × - . (- ) (av. = . ) µm wide, usually trice as long as wide, with one to two guttules. vegetative hyphae smooth, hyaline, regularly septate, . - (av. = . ) µm wide. crystals octahedral, translucent, . - (- . ) (av. = . ) µm long in medium, less regularly in substrate mycelium. culture characteristics -(in darkness, ± °c). colonies cottony, margin even to slightly irregular, with dense and abundant aerial mycelium. on sda and potato dextrose agar (pda) averse white, reverse creamy to yellow, reaching cm in d, . cm in d. octahedral crystals produced in the medium and substrate mycelium. sometimes yellowish droplets of exudate on the surface of older cultures. growth is slow but not arrested in °c. notes -based on a megablast search of ncbis genbank nucleotide database, four similar its sequences were found ( . - . % identity). two of them belongs to strains isolated from wood: historic construction wood in chiloé, chile (genbank kf . ) and picea abies wood from sweden (genbank ay . ) and other two sequences were generated during research on mycorrhizae of ericaceae: pyrola media in scotland, uk (genbank fn . ) and epacris pulchella in south-eastern australia (genbank ay . ). this variety suggests that the species have a global distribution and much wider ecological niche than known strains. however, if l. praecognitum can thrive on frass of minute arthropods as we observed, the actual substratum might have been overlooked in previous cases. no specimens are available from a study in sweden (a. menkis pers. comm.) or study in chile (r. blanchette pers. comm.). lecanicillium longisporum is morphologically most similar to l. praecognitum, but it has longer (up to . µm) and sometimes septate spores (zare & gams ) . other species with similar spores includes akanthomyces muscarius (formerly lecanicillium), which differs in size of conidia and more often has phialides in the whorls; a. attenuatum, which differs in phialide size and has at least some conidia with attenuate base; l. flavidum and l. fungicola which produce spores in slimy heads; l. fusisporium which produces characteristic broad conidia, and l. nodulosum which has characteristic swellings of hyphae. related l. acerosum is most similar when it comes to size of phialides and conidia but it can be easily distinguished by its long, thin, acerose spores. colour illustrations. białowieża primeval forest logging site, poland. fourteen-day-old colonies of l. praecognitum on sda at °c, obverse (left) and reverse (right); solitary phialides; octahedral crystals in medium; conidia; apical hyphae with whorls of phialides and secondary phialides. scale bars = µm. notes -prior to this study, macalpinomyces contained host-specific species restricted to eriachne (poaceae) in australasia (li et al. ) . macalpinomyces collinsiae is the twelfth species, known only from the type specimen on e. benthamii in north-western australia. all species of macalpinomyces are morphologically similar and can only be reliably separated by host range and molecular phylogenetic analysis. phylogram obtained from a maximum likelihood analysis of the its region of rdna in iqtree v. . beta (nguyen et al. ) with a model test for each partition (command -m test -spp) . arlt values (≥ %) (guindon et al. ) and ultrafast bootstrap values (≥ %) (hoang et al. ) from replicates above nodes. minimum spanning network (bandelt et al. ) mallocybe crassivelata ferisin, bizio, esteve-rav., vizzini & dovana, sp. nov. etymology. from the latin crassus (thick) and velatus (with a veil), referring to the presence of a thick, abundant veil on the pileus surface. classification -inocybaceae, agaricales, agaricomycetes. basidiomata stipitate. pileus - mm diam, at first convex, then applanate to plano-convex, without umbo, with an inflexed margin when young, fibrillose-tomentose to woolly-tomentose, sometimes scaly, when moist almost smooth; initially ochraceous yellow (mu yr / ) to ochraceous brown (mu . y / ), brown with an olivaceous tinge when moist, sometimes fulvous orange (mu . yr / ) at disc; in young basidiomes with a thick, white velipellis. lamellae rather crowded to crowded (l = - ), with lamellulae (l = - ), adnexed to arcuate, sometimes subdecurrent, initially pale ochraceous with a faint olivaceous hue, then brown; edge whitish to concolourous, crenulate. stipe - × - mm, cylindrical, solid, then becoming fistulose, pale yellow to concolourous with pileus in aged basidiomes; surface fibrillose, white towards the base for the presence of a white velipellis; white cortina present in young basidiomes. context yellowish in pileus, somewhat and ochraceous brownish in stipe. smell earthy sometimes mixed with a subspermatic component. taste indistinct. basidiospores ( . -) . - . - . (- . ) × ( . -) . - - . (- . ) µm, q = ( . -) . - . - . (- . ), smooth, yellowish, very variable in shape, ellipsoid to subphaseoliform, sometimes amygdaliform in side view with obtuse or sub-ogival apex; presence of anomalous long spores (over μm, probably discharged from bisporic basidia), walls up to . μm thick. ) × ( . -) . - . (- . ) µm, clavate to cylindrical, -spored, sometimes - -spored, with inner olivaceous guttulae and brown necropigment, sterigmata up to µm long; sometimes they are rarely present on lamella edge. hymenophoral trama regular, formed by cylindrical to ellipsoidal, - µm wide elements, with a brownish wall; subhymenium consisting of up to µm long elements, - µm wide. cheilocystidia very numerous, ( . -) - . (- . ) × ( . -) . - . (- . ) µm, hyaline, usually thin-walled, very variable in shape, cylindrical, oblong to clavate, with a few septa; mixed with basidia. pleurocystidia absent. caulocystidia present at stipe apex ( / ), at least partly catenulate with terminal element as true cystidium, from ellipsoid to ovoid, up to µm long. pileipellis an undifferentiated cutis with some ascending hyphae; terminal elements cylindrical to subcylindrical, - × - µm, with ochraceous-brown parietal pigment. clamp-connections present. habitat & distribution -gregarious in deciduous (fagaceae) or coniferous (picea abies, pinus sylvestris) forests. so far known from italy, slovenia and spain. typus. slovenia, pregarje, m asl, under fagus sylvatica, june , g. ferisin (holotype mcve ; its and lsu sequences genbank mn and mn , mycobank mb notes -terminology for descriptive terms is according to kuyper ( ) and vellinga ( ) and colour codes are taken from munsell ( ) . in our phylogeny m. crassivelata belongs to a well-supported clade (bootstrap support value = %) together with m. leucoloma, m. malenconii, m. myriadophylla and three sequences of 'uncultured inocybe sp.' (genbank jx , jx , jx ) from the usa and associated with dryas integrifolia. mallocybe crassivelata shows, as major morphological features, a rather fleshy, predominately ochraceous basidioma, fibrillose-tomentose to woolly-tomentose pileus covered with a thick white velipellis, narrow subphaseoliform spores and an earthy smell (similar to that of inosperma cervicolor) often associated to a subspermatic component, though in some collections (ah ) nearly indistinct. mallocybe leucoloma differs from the new species mainly by a smaller and slender habit, different shape of cheilocystidia (often pyriform), sub-odourless context and being associated with dwarf salix or dryas (kühner ) . mallocybe malenconii can easily be distinguished by its longer spores ( - × - . μm) with mean q-value of c. . (vauras & larsson ) and an indistinct smell (heim ) . compared to m. crassivelata, m. myriadophylla has a pale grey cortina, very narrow and crowded lamellae (- mm wide), smell 'indistinct to somewhat fungoid and slightly metallic' and seems strictly associated with betula pendula (vauras & larsson ) . mallocybe hebelomoides is characterised by a smaller size, broadly elliptical to subovoid spores with q = . - . and habitat under dwarf salix species (kühner classification -marasmiaceae, agaricales, agaricomycetes. basidiomata small to medium sized, collybioid. pileus - (- ) mm, initially hemispherical, convex, becoming plane at maturity, apricot ( ; flora of british fungi chart ), sometimes paler orange ( ) margin, and darker sienna ( ) centre, dry, smooth to finely matt, margin entire, not in-rolled. pileus colours display much variation depending on weather, tending to wash out in rain, and increase in intensity in dry weather. flesh thin, white. lamellae white, margins white or concolourous with pileus, free to adnexed, close, - , - mm deep, with - series of lamellulae, very fine shallow cross-anastomoses, mostly in outer half of cap, and not always present in juveniles. stipe central, cartilaginous, - × - mm, white to cream full length of stipe, or occasionally yellowish brown lower half, smooth, hollow, cylindrical, sometimes bi-tubular; basal hyphae forming a white tuft. spore print white. basidiospores variable between collections, with holotype at lower end of range, ( . -) - . (- . ) × ( . -) - (- . ) μm (av. × . μm, q = . - . , q m = . ± . , n = , s = specimens), slightly curved ellipsoid to elongate, hyaline, inamyloid, with some granular contents. basidia - × - . μm, sterigmata short, rounded, - . μm, - -spored. basidioles - × - μm, clavate. cheilocystidia common, siccus-type broom cells, with short to very long apical divergent projections, main body - × - μm, digits - × - μm, with - (- ) digits, mostly thin-walled, with body also thin-walled except for outer / at base of projections; narrowly to broadly and irregularly cylindrical, clavate, occasionally branched; rare smooth, mucronate cheilocystidia also found, × μm. pleurocystidia absent. pileipellis consists of a hymeniderm of siccus-type broom cells, main body - (- ) × . - . μm, digits . - . × - μm, broadly clavate, cylindrical, ± branching with sparse to common digits, usually thin-walled at base, often thick-walled and refractive in upper two-thirds, and including the digits, which may be bifid; pileal hyphae . - μm. caulocystidia absent. stipitipellis of parallel hyphae, . - μm diam. clamp connections present in all tissues. melzer's reaction -pileal and lamellar trama inamyloid, stipe trama mildly dextrinoid. habit, habitat & distribution -gregarious in habit and at times caespitose, it may also fruit in rings. a terrestrial saprotroph in accumulated leaf litter, the natural habitat in undisturbed sites varies from shaded microsites in tropical savanna woodland, to grassland and margins of tropical rainforest across more than km of northern australia. for approximately yr it has also been found growing in suburban lawns and highly disturbed habitats in florida, usa. typus. australia, queensland, mt carbine, s ° ' . " e ° ' . ", in savanna grassland leaf litter, mar. , f. guard & s. mcmullan-fisher smf (holotype aq ; its and lsu sequences genbank mt and mt , mycobank mb ). notes -marasmius vagus is characterised by a small to medium, orange to apricot, smooth pileus, close gills with cross-anastomoses and an all-white or pale cartilaginous stipe. these characters, with cheilocystidia of siccus-type broom cells, in the absence of pleurocystidia and caulocystidia and a well-developed, non-collariate, non-instititious stipe place this species in sect. globulares (group sicci ) subsect. siccini ser. leonini. marasmius vagus is sister to a well-supported m. hypochroides/ m. vladimiri clade. marasmius hypochroides (berkeley & broome ) described from sri lanka, but found across southern asia, forms more robust, darker basidiomes ( - mm) with longer stipes ( - mm) that have dark reddish brown bases. marasmius vladimiri (crous et al. ) from india, is brighter in colour (orange scarlet with orange chestnut disc), has a coloured stipe with slightly shorter spores and larger basidia ( - μm). marasmius vagus also bears a superficial resemblance to the australian species marasmius elegans (grgurinovic ) that has bicoloured stipes (white above, brown below) and lacks cross-anastomoses in the lamellae. our analyses of its data show that m. elegans and m. vagus are not genetically closely related. marasmius vagus is native to northern australia where it is widely distributed amongst native vegetation in the monsoon tropics; it has been recorded there for more than yr. however, it has also been found in lawns in the tourist mecca, cairns, and several other towns in southeast queensland. in florida this species has been collected almost exclusively in suburban lawns and highly disturbed habitats, with the oldest known observation (mushroom observer, obs. ) from , suggestive of a recent introduction to florida, usa. there are no records that this species was collected by florida mycologists from previous generations, such as william murrill ( - (weber ) or james kimbrough ( - (smith & healy microdochium dawsoniorum c. lock, vitelli, holdom, y.p. tan & r.g. shivas, sp. nov. etymology. named after the dawson family from taunton, queensland, on whose property the fungus was first collected. classification -michrodochiaceae, xylariales, sordariomycetes. conidiophores abundant in a dense compact layer, occasionally branched, mostly reduced to conidiogenous cells. conidiogenous cells cylindrical to irregular, flexuous, - × - μm, narrowed towards the tip, hyaline, smooth. conidia flexuous to falcate, - -septate, sometimes with a geniculation, - × - μm, acute at the tip, narrow at the base. sexual morph not seen. culture characteristics -colonies on oatmeal agar (oa) after wk covering cm diam plates, flat, mycelium in compact irregular to concentric scattered salmon tufts, with abundant slimy apricot sporodochia up to mm arranged in irregular to concentric rings. reverse pale saffron with sporodochia apparent as darker patches. mycelium immersed or superficial, hyphae hyaline, septate, smooth. mollisia gibbospora i. kušan, matočec, pošta, tkalčec & mešić, sp. nov. etymology. named after the protuberances on living, mature ascospores. ascomata apothecial, shallowly cupulate to plate-shaped when young, becoming sub-pulvinate to pulvinate when fully mature, superficial, sessile, ± circular from the top view, * . - (- . ) mm diam, solitary or gregarious (up to few apothecia). hymenium whitish grey to pale lead-grey, not wrinkled but notably finely pruinose; margin slightly irregular, ± sharp, whitish, not concolourous with the hymenium, smooth, entire, finely wavy; excipular surface brownish from base to the upper flank, smooth. basal hyphae macroscopically indistinguishable. asexual morph not seen. hymenium * - µm thick. asci cylindrical with conical-subtruncate apex, * - × ( . -) . - . µm, pars sporifera * - µm, -spored, of which - are gibbose, in living state protruding above ordinary paraphyses up to µm, base cylindrical-truncate, arising from croziers, in lugol's solution (iki) apical ring of medium amyloidity ( bb) of calycina-type. ascospores subscutuloid, with rounded poles, majority of them having lateral or apical protuberation(s) already in *mature asci, -celled, * . - . - . (- . ) × . - . - µm, *q = . - . - . (- . ), - (- ) protuberations per spore, up to . µm high and . - . µm wide, hyaline, smooth, uninucleate, *sporoplasm with one to few non-refractive vacuoles, freshly ejected apically with sheath remnants persisting mostly around protuberations, biseriate inside *asci, lipid bodies scanty, overmatured partly with single septa; in iki unstained, nucleus slightly contrasted, vacuoles hyaline and non-refractive. paraphyses cylindric-obtuse, widest in the subapical or in the middle part, apical cell * - . × ( . -) . - . µm, some far projecting, exceeding living asci ('macroparaphyses'), * - × . - . µm, straight, simple, *containing single cylindrical strongly refractive vacuolar body (vb), wall thin and hyaline; in koh without yellow reaction; in iki vbs not stained, soon collapse. subhymenium * - . µm thick at the middle flank, hyaline, composed of densely packed epidermoid cells * . - . µm wide. medullary excipulum * . - µm thick at the middle flank, reaching µm in the central part, hyaline, composed of textura intricata, cells * . - µm wide, at the border with ectal layer somewhat swollen, reaching . µm in width, thin-walled, koh-soluble globules present, in iki not stained, . - µm wide, devoid of crystals. ectal excipulum * - µm thick at the middle flank, reaching µm in the basal part, composed of textura angularis, cells * . - . × . - . µm, upper flank and inner layers of lower flanks subhyaline and contain refractive koh-soluble and iki unstainable globules, while outer layer of lower flanks tobacco brown with cell walls * . - . µm thick, most of the terminal clavate cells in the cortical layer of upper and middle flank contain single, hyaline and highly refractive vb. marginal tissue very thin, * - . µm thick, composed of several cylindricalclavate cells, * . - . µm wide, thin-walled, each containing short cylindrical or elongated vb. subicular hyphae confined to an apothecial base only, forming plaques, smooth, greyish brown, * . - . µm wide, walls * . - . µm thick. asterisk (*) denotes living material. ascus amyloidity is termed after baral ( ) and spore shape after kušan et al. ( ) . distribution & habitat -sporogenous phases of the species are known so far from the type locality on mt velebit, croatia, and (?)new zealand (unpubl. data). croatian collection is found on a decorticated fallen branch of fagus sylvatica (originally a part of the trunk), lying in a moist litter at the edge of a natural karstic pond in a subalpine type of forest while new zealand collection originates from decorticated wood. notes -tanney & seifert ( ) performed the first multigene analysis of the mollisiaceae s.lat. a detailed polyphasic taxonomic analysis in this group is still missing. according to our analysis of its - . s-its nrdna data (see fp ), m. gibbospora is conspecific with mollisia sp. (genbank mg ) whose dna sequence is derived from germinated ascospores of specimen pdd (unpublished) as well as with ascomycota sp. (genbank kc ) isolated from wooden structures on antarctica (held & blanchette ). several other unidentified sequences from genbank share identities higher than % with m. gibbospora (kauserud et al. , klaubauf et al. ). this group of isolates form the youngest phylogenetic lineage in mollisiaceae among analysed sequences. it is evident that the mollisiaceous group comprises a number of generic representatives whose species, assigned to large genera such as mollisia and pyreno peziza are mixed together thus clearly showing polyphyly in both genera. numerous species attributed to some other genera, such as asexual acidomelania and phialocephala (cf. crous et al. a) , sexual-aquatic loramyces spp. and obtectodiscus aquaticus form phylogenetic groups along with certain members ascribed to the genus mollisia. tanney & seifert ( ) synonymised acidomelania with mollisia, while loramycetaceae falls into synonymy with mollisiaceae, which is supported in our analysis. until now, regularly present lateral and apical protuberations in fully mature, dormant and freshly ejected ascospores were not reported in the genus mollisia. even though m. gibbospora is macroscopically very much alike to a number of species in the genus (including its type m. cinerea), it is readily distinguishable according to the following microscopical differential characters: ) living inner ectal excipular and some medullary excipular cells contain freely floating, hyaline and moderately refractive globules which are not stainable by crb nor by iki, and are soluble in koh; ) living mature asci containing four to eight gibbose ascospores; ) ascospore sheath is fairly longlived after spore ejection but retained around individual spore protuberations; and ) some paraphyses are extremely long, far projecting above living mature asci, giving finely pruinose appearance of hymenial surface on living apothecia. colour illustrations. croatia, mt velebit, subalpine beech forest in javornik area -type locality. *apothecia; vertical median section of the apothecium; upper excipular flank; lower exc. flank; margin; *asci and paraphyses, asci in iki; mature ascospores in *asci; *ascospores; *'macroparaphyses'. scale bars = mm (apothecia), µm (apothecial anatomy) and µm (microscopic elements). fp maximum likelihood phylogenetic tree inferred from the dataset of its - . s-its gene sequences from mollisia gibbospora and related species. classification -didymosphaeriaceae, pleosporales, dothideomycetes. hyphae pale brown to brown, smooth-and thin-walled, septate, - µm wide. conidiomata pycnidial, brown to dark brown, solitary, superficial (on oatmeal agar, oa), globose to subglobose, × - µm, covered by brown, asperulate, septate setae of - µm long and . - µm wide at the base, pycnidial wall of textura angularis, - -layered, - µm thick, composed of brown to dark brown, flattened polygonal cells of - µm diam, neck absent, ostiolate. conidiogenous cells phialidic, ampulliform to doliiform, hyaline, smooth-walled, × . µm. conidia aseptate, hyaline, smooth-and thin-walled, cylindrical, sometimes slightly curved, - × . - µm, guttulate. culture characteristics -colonies on oa reaching mm diam after d at ± °c, flattened, white (m. a ; kornerup & wanscher ( ) notes -this fungus differs from all known species of montagnula by the in vitro formation of a coelomycetous asexual morph, and by the absence of a sexual morph (tennakoon et al. , valenzuela-lopez et al. ). based on a megablast search of ncbis genbank nucleotide database, the closest hit using the lsu sequence is montagnula cirsii strain mflucc - (genbank kx ; identities = / ( %), no gaps). closest hit using its sequence is montagnula scabiosae type strain mflucc - (genbank nr_ ; identities = / ( %), gaps). the closest hit using the tub sequence is montagnula saikhuensis strain mflucc - (genbank ku ; identities = / ( %), no gaps). the closest hit using the rpb sequence is montagnula opulenta strain aftol-id (= cbs . ) (genbank dq ; identities = / ( %), gaps). the closest hit using the tef sequence is bimuria novae-zelandiae type strain aftol-id (= cbs . ) (genbank dq ; identities = / ( %), gaps). maximum likelihood (ml) tree obtained from its of our isolate and sequences retrieved from genbank. alignment and tree building were performed by mega v. . (tamura et al. ). the ml bootstrap support values (≥ %) are provided at the nodes. tremateia arundicola mflucc - and tremateia guiyangensis gzaas were used as outgroup. the new species proposed in this study is indicated in bold. t represents ex-type strains of the species used in this analysis. jurjević, Čmoková & hubka, sp. nov. notes -blast analysis with the its sequences of m. oklahomaensis showed low similarity with members of different genera, including austroafricana parva ( . - %), pseudotaeniolina globosa ( . %) and camarosporula persooniae ( . %), other taxa had similarity lower than %. the lsu nrdna sequence showed - % similarity to a wide variety of genera in the teratosphaeriaceae with devriesia shelburniensis having the highest degree of similarity ( . %). the position of muriphila within teratosphaeriaceae is unresolved. neither lsu nor ssu phylogenetic analyses comprising teratosphaeriaceae genera (quaedvlieg et al. ) were able to resolve its position with satisfactory support. in the resulting phylogenetic trees, the genus muriphila was most commonly placed close to genera batcheloromyces and devriesia (data not shown). muriphila oklahomaensis resembles morphologically meristematic rock-inhabiting fungi that are relatively common in teratosphaeriaceae (egidi et al. mycelium on synthetic nutrient-poor agar (sna) consisting of branched, septate, olivaceous grey (rayner ) , moniliform hyphae with aerial hyphae absent. chlamydospores not observed. conidiophores arising from hyphae occasionally reduced to conidiogenous cells, thick-walled, cylindrical, straight to slightly curved, long, septate, brown with an apical conidiogenous apparatus. conidia smooth, cylindrical, sometimes in acropetal chains, apex and base truncate with one and occasionally two septa, ( . -) . (- . ) × ( . -) . (- . ) μm (n = although morphologically very similar to devriesia it is phylogenetically distinguishable. neodevriesia aestuarina is the first member of the genus isolated from saline water, but other species have been found in a marine environment associated with macroalgae. based on a megablast search of ncbis genbank nucleotide database, the closest hits using the its sequence are neodevriesia capensis (genbank mk ; identities = / ( %), gaps ( %)) and an uncultured marine ascomycete (genbank af ; identities = / ( %), gaps ( %)). closest hits using the lsu sequence had highest similarity to neodevriesia grateloupiae (genbank ku ; identities = / ( %), gaps ( %)), neodevriesia cladophorae (genbank ku ; identities = / ( %), gaps ( %)) and neodevriesia strelitziae (genbank gu ; identities = / ( %), gaps ( %)). conidia subcylindrical, fusoid or ellipsoid to fusoid, individually hyaline, olivaceous green at maturity, with transverse septum, thin-and smooth-walled, ( . -) . - . (- . ) × ( . -) . - . (- . ) µm, mean ± s.d. . ± . × . ± . µm, l/w ratio = . . culture characteristics -colonies on malt extract agar (mea) reaching - cm diam in the dark, at °c, after wk, slow growing, white to dirty white in the first week, becoming yellow-green with pale irregular margin after wk, moderate aerial mycelium, reverse iron-grey to umber, with age. notes -based on a megablast search of ncbi nucleotide database, the closest hits using the its sequence had the highest similarity to neosetophoma aseptata (genbank nr_ . ; identities = / ( %) , no gaps), neosetophoma lunariace (genbank nr_ . ; identities = / ( %) , no gaps) and neosetophoma shoemakeri (genbank nr_ . ; identities = / ( %), gap ( %)). the closest hits using the lsu sequence had the highest similarity to loratospora aestuarii (genbank gu . ; identities = / ( %) , no gaps), ophiosphaerella herpotricha (genbank dq . ; identities = / ( %), gap ( %)) and phoma cladoniicola (genbank jq . ; identities = / ( %), no gaps); closest hits using the rpb sequence are brunneomurispora lonicerae (genbank mk . ; identities = / ( %), no gaps), ophiosphaerella herpotricha (genbank dq . ; identities = / ( %), gaps ( %)) and phaeopoacea festucae (genbank ky . ; identities = / ( %), no gaps). sequences of all known neosetophoma species were retrieved from genbank and aligned with sequences of the isolate obtained in this study. alignments were done with clustalx v. . (thompson et al. ). kimura's two parameter model with gamma distribution (k +g) was used as the best nucleotide substitution model. the maximum likelihood (ml) analysis was performed using mega v. software (kumar et al. ) . the robustness of the ml tree was evaluated by bootstrap replications. maximum likelihood tree obtained from the its and lsu gene sequences of neosetophoma species of our isolates and sequences retrieved from genbank. the tree was built using mega v. . . bootstrap support values above % are shown at the nodes. the species described here is highlighted in bold. the alignment and tree are available in treebase (study s ). conidiophores terverticillate; stipes coarsely roughened, - × - . µm; synnemata up to mm long; branches - µm; metulae - , - × . - . µm; phialides ampulliform, - per metula, - . × - µm. conidia smooth, broadly ellipsoidal, - . × . - µm. culture characteristics -( °c, d) czapek yeast autolysate agar (cya): colonies slightly radially sulcate in centre, low; mycelium white; margins irregular; texture fasciculate; soluble pigments brown, moderately produced; exudate droplets small, copious, brown; sporulation moderate; conidia en masse pale grey-green; reverse brown, dark brown in centre. malt extract agar (mea): colonies plane, elevated in the centre; mycelium white; margins slightly irregular; texture fasciculate; soluble pigments absent; exudate droplets large, pale brown; sporulation strong; conidia en masse dull to grey-green; reverse brown in centre, pale brown at edge. yeast extract sucrose agar (yes): colonies slightly radially sulcate, raised; mycelium white; margins entire; texture floccose; soluble pigment present, brown, weakly produced; exudates absent; sporulation moderate to strong; conidia en masse dull to grey-green; reverse reddish brown (copper). dichloran % glycerol agar (dg ): colonies plane, raised at the centre; margins entire or slightly irregular; mycelium white; texture fasciculate; soluble pigments present, light brown, weak; exudates absent; sporulation strong; conidia en masse dull green; reverse reddish brown (copper) or reddish brown in centre, yellowish brown at edge. oatmeal agar (oa): colonies plane, low; margins regular, thin; mycelium white; texture fasciculate; soluble pigments light brown present, moderately produced; exudates brown present, small; sporulation strong; conidia en masse dark green. notes -a blast search of bena, cam and its sequences of p. taurinense against an in-house reference sequence database containing data of all accepted penicillium species, re trieved the highest similarities with penicillium glandicola, p. geumsanense and p. synnematicola, clearly indicating that the species belongs to penicillium sect. robsamsonia ser. glandicolarum (houbraken et al. unpubl. data) . phylogenetic analyses showed that p. taurinense is sister to a clade containing cbs (ex-type strain of p. synnematicola), cbs . (extype of p. granulatum) and cbs (ex-type of p. granulatum var. globosum). the latter two strains were identified as p. glandicola; however, the ex-type of p. glandicola is more distantly related. frisvad & samson ( ) treated p. granulatum as a synonym of p. glandicola based on morphology and extrolite patterns. however, our phylogenetic analysis shows that p. granulatum is an accepted species, with p. granulatum var. globosum being a synonym of that species. furthermore, p. schneggii is confirmed to be a synonym of p. glandicola. penicillium taurinense is phylogenetically distinct from p. synnematicola and p. glandicola (houbraken et al. , guevara-suarez et al. . penicillium taurinense grows faster than p. synnematicola on cya ( - vs - mm), yes ( - vs - mm) and mea ( - vs - mm) at °c. both grows at °c while p. glandicola is not able to grow at this temperature (frisvad & samson , guevara-suarez et al. . furthermore, p. taurinense produces brown exudates on cya compared to hyaline exudate droplets of p. synnematicola and clear to pale yellow ones of p. glandicola. in addition, p. taurinense has a different colony reverse colour on cya, mea, dg and yes, no acid production on crea and shorter phialides compared to p. synnematicola. a taxonomic study dealing with all accepted species in penicillium ser. glandicolarum is lacking, and could reveal more phenotypic differences. classification -peronosporaceae, peronosporidae, oomycota. hyphae hyaline, aseptate, tortuous, . - µm diam. hyphal swellings absent. sporangia produced abundantly in non-sterile soil extract, noncaducous, papillate, mainly ovoid ( %), globose ( %), limoniform ( %), distorted shapes ( %), other shapes ( %), ( -) . - (- ) × ( -) - (- . ) μm (av. . ± × . ± ), length/breadth ratio . ± . . sporangiophores sympodial. chlamydospores not observed. homothallic, abundant gametangia on % carrot agar with β-sitosterol (cas) after d. oogonia globose, smooth-walled, ( -) - (- . ) μm diam (av. . ± . ), borne laterally and terminally. antheridia amphigynous, -celled, ( . -) . - (- . ) × . - (- . ) μm (av. . ± . × . ± . ). oospores observed after wk on cas, globose, plerotic, ( -) - . (- ) μm diam (av. . ± . ), wall thickness ( . -) - (- ) μm (av. . ± . ). culture characteristics -colonies on cas showed appressed to submerged mycelium, without distinct growth pattern, reaching . ± . mm diam after d at °c in darkness. colonies on corn meal agar (cma) white, with moderate to profuse aerial mycelium, cottoned, reaching . ± . mm diam. © naturalis biodiversity center & westerdijk fungal biodiversity institute fungal planet - june phytophthora personensis z.g. abad, w. gut. & t.i. burgess, sp. nov. etymology. named after person county, north carolina, the location where the first specimen of the species was isolated. classification -peronosporaceae, peronosporidae, oomycota. sporangia produced abundantly in non-sterile soil extract; persistent and produced usually on unbranched sporangiophores, non-papillate, most commonly ovoid ( %), often ellipsoid ( %) and rarely limoniform or obpyriform; . ± . × . ± . µm (overall range . - . × . - . µm), length/ breadth ratio . ± . . sporangial proliferation in chains of internally proliferating sporangia, both nested and extended. hyphal swellings common, catenulate to globose -( . ± . )- . µm. chlamydospores common, globose . -( . ± . )- . µm. gametangia not produced in single culture or when paired with a and a tester strains of p. cinnamomi, p. tropicalis, p. cryptogea and p. cambivora. radial growth rates on v agar at optimum temperature ( - °c) and near the maximum temperature ( . °c), . ± . mm/d and . ± . mm/d, respectively. culture characteristics -submerged colonies with no pattern were produced on malt extract, carrot and v agar. cottony colonies with regular margins were produced on potato dextrose agar. © naturalis biodiversity center & westerdijk fungal biodiversity institute fungal planet - june kiyashko, sp. nov. etymology. name refers to roridomyces irritans, a species which is morphologically similar. classification -mycenaceae, agaricales, agaricomycetes. pileus at first convex, then plano-convex with depressed or subumbilicate centre, up to . mm diam (dried specimens), radially pellucid-sulcate-striate almost up to centre, margin reflexed, crenulate, membranaceous, surface dry, velvety at centre, pallid, greyish orange ( b - , kornerup & wanscher ) , turning yellowish white ( a ) to white towards margin, sometimes with blurred reddish brown ( d - , c - ) spots. lamellae arcuate decurrent, moderately distant ( - reach to the stipe), with - series of lamellulae, thin, whitish to pale greyish orange (like cap centre), sometimes also with spots, edge concolourous, slightly eroded. stipe cylindrical, slightly attenuated towards apex, up to × . mm, shiny, polished, faintly pellucid, whitish at apex, darkening to brownish orange or brownish yellow ( c - ) towards base, covered with thick, glassy glutinous sheath, without strigose hairs at base. context thin, concolourous with cap and stipe surfaces, odour not recorded. basidiospores ellipsoid to oblong, rarely subcylindrical, . - . (- . ) × - . (- . ) mm (x av = . ± . × . ± . mm, q = ( . -) . - . (- . ), q av = . ± . , n = , s = ), with small apiculus, smooth, hyaline, amyloid. clamped, subclavate, . ) × . - mm, thinwalled, inamyloid. basidiolae subclavate or subfusoid, more rarely subutriform, - . × . - mm, thin-walled, inamyloid. lamellar edge sterile. cheilocystidia short and not much exceeding basidia, - . × . - . mm (n = , s = ), mostly clavate to subcapitate, rarely clear capitate, sometimes bifid or septate, thin-walled, occasionally with slightly thickened walls or yellowish content, colourless, smooth, inamyloid. pleurocystidia absent. hyphae of subhymenium cylindrical, smooth, hyaline . - . (- . ) mm diam. pileipellis hymeniform, composed of spheropedunculate (sometimes on very long pedicel) or broadly clavate cells with or without constrictions, . - . × . - . mm, sometimes connected in short chains, smooth, with slightly thickened colourless or brownish walls, sometimes with yellowish content. stipitipellis hyphae - (- . ) mm diam, cylindrical, smooth, uncoloured, thin-walled, parallel, with not abundant caulocystidia. caulocystidia . - . (- . ) × . - . (- . ) mm, narrowly clavate to subcylindrical, rarely more or less capitate, thin-walled, sometimes with slightly thickened walls, smooth, uncoloured, inamyloid. clamp connections on all hyphae. habitat & distribution -gregarious to caespitose on rotten wood in montane mixed forest of broad-leaved trees and pinus kesiya. typus . vietnam, Đắk lắk province, lắk district, chư yang sin national park, ≈ km to the south from krông kmar town, n ° ' . " e ° ' . ", h ≈ m asl, on rotten wood, may , a.a. kiyasko, -ak- (holotype le ; its and lsu sequences genbank mt and mt , mycobank mb ). notes -roridomyces includes species, the most of which are described from the southern hemisphere. morphologically, r. irritans from new caledonia and papua new guinea is the closest to r. pseudoirritans. although they have overlapping spore dimensions, r. pseudoirritans clearly differs from r. irritans by having short cheilocystidia: - . mm vs - mm according to horak ( ) . furthermore, cheilocystidia of r. pseudoirritans are not clearly capitate and may even be bifid or septate. its caulocystidia are also short and narrowly clavate to subcylindrical. among other small-spored species r. lamprosporus and r. pruinosoviscidus both have cheilo-and caulocystidia which are irregularly clavate to bifid with diverticulate projections (horak , chew et al. , mycena yirukensis possesses cylindrical-ventricose, broadly ventricose-rostrate or strangulate cheilocystidia and cylindrical caulocystidia with one or few large branches (grgurinovic ) . roridomyces mauritianus differs in having a dark brown cap and abundant pigmented caulocystidia with flexuous, contorted excrescences (robich & hausknecht ) . roridomyces praeclarus has an orange-red pileus, lageniform cheilocystidia and coralloid caulocystia; r. palmensis and r. subglobosus both are characterised by subglobose spores (rexer , miersch & dähncke . the other species (r. albororidus, r. appendiculatus, r. austrororidus, r. fuscororidus, r. ornatororidus and r. roridus) possess larger spores with no overlapping dimensions (rexer , maas geesteranus & meijer . the majority of roridomyces species from the southern hemisphere still lack dna sequence data, and thus their phylogenetic relationships remain unknown. trichophoma cylindrospora magaña-dueñas, cano & stchigel, sp. nov. notes -based on the phylogenetic analysis of the its, lsu and tef- α combined dataset, the closest relative of t. cylindrospora is forliomyces uniseptata. forliomyces uniseptata differs from t. cylindrospora in producing shorter and broader conidia ( - × - µm vs - × - µm), which are brown-coloured when mature (hyaline in t. cylindrospora) (phukhamsakda et al. ). based on a megablast search of ncbis genbank nucleotide database, the closest hit using the lsu sequence was forliomyces uniseptata mflucc - (genbank ng_ , identities = / ( %), no gaps); the closest hits using its was forliomyces uniseptata mflucc - (genbank nr_ , identities = / ( %), gaps ( %)); and the closest hits using the tef- α sequence was forliomyces uniseptata mflucc - (genbank ku , identities = / ( %), no gaps). etymology. from greek κυλινδρικό-, cylindrical, due to the shape of the conidia. hyphae hyaline to brown, septate, branched, thin-walled, smooth to tuberculate, . - µm wide. conidiomata pycnidial, brown to blackish brown, immersed to semi-immersed, solitary, scattered, subglobose to pyriform, - × - µm, ostiolate, setose. conidioma wall - -layered, - µm thick, covered by a mass of interwoven, brown to dark brown hyphae, followed by an outer layer of textura angularis, composed of brown to dark brown, flattened polygonal cells of - µm diam, incrusted with a dark brown to carbonaceous material around the neck; neck dark brown to carbonaceous, cylindrical, - × - µm, covered by brown to dark brown, septate, erect, nodose, thick-walled setae - × - . µm, tapering towards the apex, mainly disposed around the ostiole. conidiophores absent. conidiogenous cells phialidic, determinate, hyaline, smooth-walled, ampulliform, - × - µm. conidia - -septate, hyaline, smooth-and thin-walled, long cylindrical, - × - µm, guttulate, with a narrowly flattened base and rounded at the end. culture characteristics -( d at °c). colonies on potato dextrose agar (pda) reaching mm diam, flattened, velvety, margins regular, yellowish white to reddish brown (m. a / d ; kornerup & wanscher ) ; reverse reddish brown to dark brown (m. e / f ), exopigment reddish brown to golden yellow (m. d / b ). colonies on oatmeal agar (oa) reaching mm diam, flattened, slightly floccose, margin regular, grey (m. f ); reverse grey (m. f ). colonies on malt extract agar (mea) % reaching - mm diam, flattened, velvety to slightly floccose, margins lobate, dark brown to greyish yellow (m. f / c ); reverse greyish brown to greyish orange, with yellowish brown patches (m. f / b / f ). cardinal temperatures for growing -optimum °c, maximum °c, minimum °c. classification -tuberaceae, pezizales, pezizomycetes. ascomata hypogeous, - cm, subglobose, covered with brown-black pyramidal warts, - -sided, - (- ) mm across, - mm high, often depressed at the apex. peridium - μm thick, pseudoparenchymatous, composed of subglobose, angular cells, - μm diam, pale yellow and thin-walled in the innermost layers, dark red-brown and with thicker walls in the outermost layers. gleba firm, solid, white when immature, becoming dark brown at maturity, marbled with numerous, thin, white, meandering veins that do not change colour when exposed to the air. pleasant odour. asci inamyloid, - × - μm, walls thickened, - μm, ellipsoid to subglobose, with a short stalk, - × - μm, ( -) - (- )-spored. ascospores - × - μm, q = . - . , excluding ornamentation, yellowish, ellipsoid to subglobose, ornamented with a coarse irregular reticulum, - µm high, sometimes bending at the top. meshes variable, usually - across width of spore and often with incomplete secondary crests inside. ascospores from -spored asci - × - μm, -spored asci - × - μm, -spored asci - × - μm, -spored asci - × - μm and -spored asci - × - μm. ecology & distribution -tuber alcaracense grows in mediterranean quercus ilex subsp. ballota forest, in limestone mountains of the southeast of the iberian peninsula, - m alt., from december to february. notes -tuber alcaracense is a black truffle of the aestivum clade characterised by its brown-black warty peridium, brown gleba marbled with thin white veins and reticulate-alveolate spores. it resembles tuber mesentericum, but in addition to genetic differences it differs from t. mesentericum (vittadini ) by having a pleasant odour and lacking a basal cavity. colour illustrations. spain, alcaraz mountain range (albacete), mediterranean quercus ilex subsp. ballota forest. ascocarps; mature ascospores. scale bar = μm. . gtr + g selected as model of evolution for analysis. the sequences obtained in the present study are highlighted in bold. bootstrap support values (≥ %) are indicated at the nodes. tuber macrosporum af was used as outgroup. the scale bar indicates the expected changes per site. classification -tuberaceae, pezizales, pezizomycetes. ascomata hypogeous, - cm, subglobose or irregular in form, sometimes lobed, sometimes with a basal depression, fissured in age, yellow brown to reddish brown, minutely warted with pyramidal, flattened warts. peridium - μm thick, composed of hyaline, agglutinated, interwoven hyphae (intricate texture), becoming pseudoparenchymatous towards the surface and forming pigmented, subangular, thick-walled cells, in a superficial layer - μm thick. gleba firm, solid, whitish at first, becoming light-brown, dark-brown or red-brown at maturity, marbled with numerous, branching, white and dark veins. odour pleasant. asci inamyloid, - × - μm excluding stalk, pyriform to clavate or subglobose, with a long or short stalk arising from a crozier, - μm long, walls - μm thick, - (- )-spored. ascospores - × - μm, q = . - . , excluding ornamentation, at first hyaline, yellowish brown at maturity, ellipsoid to ovoid or subglobose, ornamented with short spines, sometimes curved, - (- ) µm long, often connected by lower ridges, making the ornamentation an irregular and incomplete spiny reticulum. ascospores from -spored asci - × - μm, -spored asci - × - μm, -spored asci - × - μm, -spored asci - × - μm, -spored asci - × - μm. ecology & distribution -tuber buendiae grows in mediterranean quercus ilex subsp. ballota forest, in limestone mountains of the southeast of the iberian peninsula, - m altitude. the species occurs all year; maturing during autumn and winter. notes -tuber buendiae is a reddish brown truffle that clusters in the rufum clade, and is characterised by its minutely warted peridium, brown gleba marbled with white and dark veins and spiny-reticulate spores. healy et al. ( ) previously identified it as a hypothetical undescribed species tuber sp. . tuber buendiae resembles tuber pustulatum, but in addition to genetic differences, it differs from t. pustulatum (leonardi et al. ) , by having a pleasant odour, a gleba with numerous veins and spores with shorter spines. classification -venturiaceae, venturiales, dothideomycetes. lesions on leaves and stems, amphigenous, predominantly adaxial, circular to irregular, pale to dark brown, - mm diam, stem lesions pale to dark brown. mycelium internal, . - mm, subcuticular. stromata oblong to subcircular, ( -) - (- ) × ( -) - (- ) µm, formed by swollen thickwalled cells. conidiophores in loose to dense fascicles on stroma, unbranched, thin-walled, straight to slightly curved, pale brown and lighter towards the apex, occasionally thickened at the base, smooth, ( -) - (- ) × ( -) - (- ) µm, - -septate. conidiogenous cells integrated, terminal, one to several conidiogenous loci, slightly guttulate, proliferating sympodially, loci flat, hila convex and slightly thickened and darkened-refractive. conidia solitary or catenate, fusiform, subcylindrical, obclavate, clavate, straight or slightly curved, ( -) - (- ) × ( -) - (- ) µm, - -septate, slightly or not constricted at the septa, brown to pale brown, smooth to verruculose, thickened, apex obtuse, truncate or papillate. culture characteristics -colonies on potato dextrose agar (pda) flat to slightly raised, aerial mycelium feathery, circular to irregular with entire to undulate margin, mm diam after d at °c under h photoperiod. outer edge of colony grey olivaceous, inner part olivaceous; reverse olivaceous to olivaceous black. notes -based on a megablast search of ncbis genbank nucleotide database, the closest matches for its sequence were venturia oleaginea (genbank mn , identities = / ( %), gaps ( %)), fusicladium phillyreae (gen-bank eu , identities = / ( %), gaps ( %)) and venturia inaequalis (genbank mn , identities = / ( %), gap ( %)). closest similarities using the tef -α partial gene sequence were to venturia polygoni-vivipari (genbank kf , identities / ( %), gaps ( %)), venturia ditricha (genbank kf , identities = / ( %), gaps ( %)) and venturia chlorospora (genbank kf , identities / ( %), gaps ( %)). venturia paralias was shown in pathogenicity tests to cause disease on e. paralias and euphorbia segetalis (unpubl. data). venturia paralias is morphologically similar to fusicladium euphorbiae (schubert et al. ) , which has been recorded from e. amygdaloides, e. cy parissias, e. esula, e. exigua, e. lamprocarpa, e. villosa and e. virgata (schubert et al. ) . we were not able to obtain lectotype material of f. euphorbiae from le for comparison. for taxonomic stability we have therefore described the fungus isolated from e. paralias as v. paralias. fusicladium fasciculatum var. fasciculatum, f. fasciculatum var. didymium and f. fautreyi are also morphologically similar to v. paralias. venturia paralias produces distinctive pale to dark brown elongated stem lesions, dense fasciculate conidiophores with conidiogenous loci that are less conspicuous or prominent than those of f. fasciculatum var. fasciculatum and f. fasciculatum var. didymium (deighton , schubert et al. . venturia paralias is distinguished from f. fautreyi by its pale brown conidiophores, less conspicuous conidiogenous loci and -septate conidia (deighton vishniacozyma phoenicis kachalkin, a.s. venzhik & m.a. tomashevskaya, sp. nov. etymology. name phoenicis refers to the date palm, from which fruits the strains were isolated. dipterocarpaceae forest swuf-h (fn , kp , kp swuf-h (fn , fr , fr swuf-h (fn , kp swuf-h (fn , fr , fr dipterocarpaceae forest sut (dq sut (dq annulohypoxylon nitens: thailand, chiang rai province, dipterocarpaceae forest swuf-h (fm , fr , kp swuf-h (fm , fr , kp blast search of species of macalpinomyces, the its sequence of m. collinsiae differs from the sister species m. vankyi (genbank kx ; identities = / ( %), gaps ( %)), and from m. cookei gaps ( %)). the species of macalpinomyces are further illustrated on the smut fungi of australia lucid key eriachne benthamii tussock grassland, mulga downs station centre for crop health queensland alliance for agriculture and food innovation ecosystem science, department of biodiversity, conservation and attractions fungal planet description sheets © naturalis biodiversity center & westerdijk fungal biodiversity institute fungal planet from the latin nebula, meaning cloud, in reference to the fluffy, white, aerial mycelia. classification -pestalotiopsidaceae, xylariales globose or clavate, scattered or aggregated, semi-immersed, black, up to μm diam; exuding dark brown to black conidial masses. conidiophores reduced to conidiogenous cells. conidio genous cells discrete, ampulliform, hyaline, smooth, - × - μm. conidia fusoid, cylindrical, straight to slightly curved, -septate, - × - μm, basal cell conic, hyaline, smooth and thin-walled, - μm long; three median cells doliiform, - μm long, smooth, versicoloured, septa darker than long, hyaline, conic, thin-walled, smooth °c, margin irregular to undulating, whitish, zonate, with fichera (holotype brip , includes ex-type culture; its, tub and tef a sequences genbank mk , mk and mk notes -the multilocus phylogenetic analysis placed n. nebuloides in a clade with n. asiatica, n. chrysea and n. umbrinospora. blastn searches in genbank, restricted to ex-type strains, showed that n. nebuloides differs from n umbrinospora from unidentified plant material in china sporobolus natalensis infestation near conondale, australia. colony on pda at wk; sporulating conidiomata on pda; conidiogenous cells; conidia. scale bars = μm (conidiomata) and μm (conidiogenous cells and conidia) department of agriculture and fisheries plant pathology herbarium, department of agriculture and fisheries centre for crop health benátská , prague , czech republic and laboratory of fungal genetics and metabolism, institute of microbiology of the cas, v.v.i, vídeňská e-mail: petr.hamal@fnol.cz colour illustrations. human skin. fourteen-day-old cultures of paraphyton cutaneum grown at °c on sga, mea and pda (left to right); conidiophores bearing multi-celled macroconidia and one-celled microconidia, free macroand microconidia, macroconidia in sem. scale bars = µm department of agriculture and fisheries plant pathology herbarium, department of agriculture and fisheries dense infestation of sporobolus natalensis near collection site. conidiomata sporulating on pda analyses were done on the geneious v. . . platform (biomatters ltd.) using raxml v. . . (stamatakis & alachiotis ) and mrbayes v. . . (ronquist & huelsenbeck ), both based on the gtr substitution model with gamma-distribution rate variation. branch lengths are proportional to distance. raxml bootstrap (bs) values > % and bayesian posterior probabilities (pp) > . are given at the nodes (bs/pp). pestalotiopsis camelliae was used as outgroup m. gonzález (holotype rgm , culture extype ccct . ; its, lsu, tub, cox , nad and rps sequences genbank mn , mn , mn , mn , mn and mn , mycobank mb ). additional material examined. chile, aysén, on soil associated with rot of a. chilensis notes -phytophthora aysenensis was isolated for the first time from root and collar rot of aristotelia chilensis, a native chilean plant known as maqui or chilean wineberry. phytophthora aysenensis is a homothallic species, belonging to the waterhouse's group ii, which is characterised by amphigynous antheridia and papillate sporangia p. mengei (genbank jq ; identities = / no gaps) and p. botryosa (genbank jq aristorelia chilensis exhibiting dieback and mortality by phytophthora aysenensis in the collection site (photo credit milixsa gonzález oogonia with amphigynous antheridia; plerotic oospore; papillate sporangia. scale bars = µm (others) and µm (hyphae) unidad de fitopatología e-mail: maria.asenjo@sag.gob.cl & monica.gutierrez@sag.gob.cl maximum likelihood tree inferred from the combined its, lsu, tub, cox , nad and rsp regions for selected phytophthora species. dna sequences were aligned using mafft v. . (katoh & standley ) with automatic strategy. the ml analysis was performed in raxml-hpc black-box v. . . (stamatakis ) via the cipres science gateway v. . (miller et al. ), using a gtr+g+i model of evolution fungal planet description sheets © naturalis biodiversity center & westerdijk fungal biodiversity institute fungal planet mediterranean quercus ilex subsp. ballota forest. the collector and her truffle-hunting dog at the collection site; ascocarps gtr + g selected as model of evolution for analysis. the sequences obtained in the present study are highlighted in bold. bootstrap support values (≥ %) are indicated at the nodes. tuber spinoreticulatum (genbank fj ) was used as outgroup. the scale bar indicates the expected changes per site. species hypotheses for undescribed species (tuber sp ethanol (weak), glycerol, erythritol, ribitol, galactitol, d-mannitol, d-glucitol, myo-inositol, methyl alpha-d-glucoside (weak), salicin, dl-lactic acid (weak), citric acid, succinic acid, d-gluconate, d-glucoronate, d-glucosamine (weak), n-acetyl-d-glucosamine, -keto-d-gluconate, -keto-d-gluconate and arbutin are assimilated (holotype kbp y- preserved in a metabolically inactive state, ex-type cultures vkm y- = dsm = cbs ; ssu, its-d /d domains of lsu nrdna, tef and rpb sequences genbank mn , mn , lr and lr , mycobank mb ). additional material examined. russia, moscow, from dates fruit bought on local market leninskie gory str. / , russia and all-russian collection of microorganisms, g.k. skryabin institute of biochemistry and physiology of microorganisms ras leninskie gory str. / , russia; e-mail: rhysenn skryabin institute of biochemistry and physiology of microorganisms ras, , pushchino, pr. nauki , russia; e-mail: tomkotik@rambler.ru colour illustrations. russia, moscow, dates fruit on local market t (genbank mk ; . % similar, subst. and gaps); using lsu these are v. peneaus cbs t (genbank ng_ ; . % similar, subst.) and some strains genbank fj ); using ssu these are strain v. pseudopenaeus cgmcc . t (genbank mk ; . % similar, subst.) and v. peneaus cbs t in compliance with a recent phylogenetic analysis of the genus (tsuji et al. ), the placement of the new species is demonstrated using the combined its and lsu rdna phylogeny. vishniacozyma phoenicis differs from other species of the genus by good growth (v. taibaiensis with weak growth) on % w/w glucose media. the new species can be also differentiated from v. peneaus based on its ability to assimilate ethanol, creatinine, potassium nitrate, growth on vitamin-free medium and on mea with % nacl, and differ from v. pseudopenaeus by its ability to assimilate dl-lactic acid and soluble starch, production of starch-like compounds and its inability to growth at °c maximum likelihood (ml) tree obtained from the combined analysis of its and lsu sequence data. bootstrap support values above % are shown at the nodes. the alignment included bp and was performed with mafft v pileipellis cutis, consisting of septate and elongate, non-gelatinous, slightly thick-walled hyphae, - μm wide. cheilocystidia numerous, variable in size and shape: predominantly ventricoselageniform, broadly fusiform, more rarely narrowly to broadly clavate or utriform -) -spored, broadly clavate, thickwalled vagayskiy district, near kobjakskaja village, low (minerotrophic) swamp, n ° ' " e ° ' notes -volvariella paludosa is characterised by its rather large basidiospores compared to other known species of volvariella, medium-sized basidiocarps with light grey and hairy pilei, whitish volva and variable hymenial cystidia, and rich fen habitat. the preferred habitat, hairy pileus and spore size are the key characters for separating v. paludosa from other species with grey, fibrillose pilei, such as v. volvacea, v. murinella, v. taylorii. the phylogenetically closest species, shape (justo & castro vagayskiy district, near kobjakskaja village, rich fen, where the holotype was collected. top: mature basidiocarp; median: pileus (view from above); bottom: lamellae and volva; bottom: basidiospores and basidia; on the right: four various cheilocystidia and two various 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beltraniaceae the genus aspergillus. williams & wilkins a mycological colour chart calvatia holothurioides sp. nov., new species from vietnam. mikologiya i fitopatologiya some gasteromycetes from trinidad and tobago die gattung mycena s.l. studien zu ihrer anatomie, morphologie und systematik mycena mauritiana, a new species of sect antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants mrbayes : bayesian phylogenetic inference under mixed models mrbayes . : efficient bayesian phylogenetic inference and model choice across a large model space an evolutionary perspective on morphology and ecological characters in the mushroom family inocybaceae (agaricomycotina, fungi) polyphasic taxonomy of the heat resistant ascomycete genus byssochlamys and its paecilomyces anamorphs a monograph of fusicladium s.lat. (hyphomycetes) multilocus dna sequencing of the whiskey fungus reveals a continental -scale speciation pattern the genera of hyphomycetes online identification guides for australian smut fungi (ustilaginomycotina) and rust fungi (pucciniales) raxmlgui: a graphical front-end for raxml first conclusive report of an erysiphe sp. causing powdery mildew on moth vine (araujia sericifera) in australia and worldwide raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies time and memory efficient likelihood-based tree searches on phylogenomic alignments with missing data a fast bootstraping algorithm for the raxml web-servers the australasian species of lactarius subgenus gerardii (russulales) the neurotropic black yeast exophiala dermatitidis has a possible origin in the tropical rain forest eucalyptus microfungi: satchmopsis gen. nov., and new species of coniella, coniothyrium and harknessia the coelomycetes. fungi imperfecti with pycnidia, acervuli and stromata fungi in thailand: a case study of the efficacy of an its barcode for automatically identifying species within the annulohypoxylon and hypoxylon genera new or less known discomycetes paup phylogenetic analysis using parsimony (and other methods). version . sinauer associates novae fungorum species -xxi first comprehensive phylogenetic analysis of the genus erysiphe (erysiphales, erysiphaceae) i. the microsphaera lineage estimation of the number of nucleotide substitutions in the control of mitochondrial dna in humans and chimpanzees mega : molecular evolutionary genetics analysis version . greeneria saprophytica sp. nov. on dead leaves of syzygium cumini from chiang rai mollisiaceae: an overlooked lineage of diverse endophytes response to comment on 'global diversity and geography of soil fungi taxonomy and phylogenetic appraisal of montagnula jonesii sp. nov. (didymosphaeriaceae, pleosporales) the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools green and brown bridges between weeds and crops reveal novel diaporthe species in australia fungal diversity notes - : taxonomic and phylogenetic contribution to fungal taxa vishniacozyma ellesmerensis sp. nov., a psychrophilic yeast isolated from a retreating glacier in the canadian high arctic international code of nomenclature for algae, fungi, and plants (shenzhen code) adopted by the nineteenth international botanical congress shenzhen, china the diaporthe sojae species complex: phylogenetic re-assessment of pathogens associated with soybean, cucurbits and other field crops coelomycetous fungi in the clinical setting: morphological convergence and cryptic diversity troisième mémoire sur les mucorinées inocybe myriadophylla, a new species from finland and sweden glossary new combinations in lactifluus. . l. subgenera lactifluus and piperati monograph of lactarius in tropical africa additional records of volvariella dunensis (basidiomycota, agaricales): morphological and molecular characterization key to the species of phytophthora de bary on geastrum argentinum, a forgotten species the genera lecanicillium and simplicillium gen candida yunnanensis sp. nov. and candida parablackwelliae sp. nov., two yeast species in the candida albicans/lodderomyces clade lecanicillium cauligalbarum sp. nov. (cordycipitaceae, hypocreales notes -phylogenetically, p. personensis resides in a strongly supported terminal clade and shares a common ancestor with p. inundata (brasier et al. ), p. condilina (burgess et al. ) , p. humicola (ko & ann ) , p. balyanboodja (burgess et al. ) and p. chesapeakensis (man in 't veld et al. ). together with p. gemini (man in 't veld et al. ) these species form a species cluster within clade of the phytophthora phylogeny (burgess et al. ) . in a multigene phylogeny of the its, hsp , bt, nadh and coxi gene regions, p. personensis differs from both p. condilina and p. humicola by . %, p. inundata by . %, p. balyanboodja and p. chesapeakensis by . % and p. gemini by . %. all these species are morphologically similar; they all produce ovoid persistent, nonpapillate sporangia that are borne terminally and they all have high temperature optima and maxima for growth. phytophthora personensis appears to be sterile in culture and thus differs from p. inundata, p. humicola and p. condilina as these three species readily produce homothallic oogonia. phytophthora personensis produces chlamydospores and thus differs from the three other sterile species in the clade, p. balyanboodja, p. chesapeakensis and p. gemini. phytophthora personensis has been recovered from a variety of hosts on two continents, north america and australia, and at this point in time its origin cannot be determined. colour illustrations. grevillia sp., host of the type isolate. typical ovoid and ellipsoid sporangia; proliferation is internal and extended and chlamydospores were common; cottony colony on potato dextrose agar. scale bar = µm.bayesian inference tree based on a concatenated its, ß-tubulin, hsp , coxi and nadh sequence alignment showing the placement of p. personensis in phytophthora clade a generated in mrbayes v. . . (ronquist & huelsenbeck ) as a plugin in geneious prime® . . (biomatters ltd.) using the gtr substitution model. the posterior probability values are shown at the nodes. the tree was rooted to p. rosacearum (not shown) and the novel species is shown in bold font.colour illustrations. vietnam, southern annamite range, chư yang sin national park, montane mixed forest from broad-leaved trees and pinus kesiya at the type locality. in situ basidiomata; spores; basidiae and basidiolae; cheilocystidia; cells of pileipellis; caulocystidia. scale bars = cm (basidio mata), µm (all others).anna kiyashko, komarov botanical institute of russian academy of sciences (bin ras), saint petersburg, professora popova str., , russia; e-mail: anna.kiyashko@binran.ru classification -thermoascaceae, eurotiales, eurotiomycetes.micromorphology (on malt extract agar; mea): hyphae hyaline to pale yellow-brown, . - µm diam, conidiophores borne on the surface or from aerial hyphae, commonly - × ( . -) - μm diam; with smooth walls, bearing terminal whorls of verticillately arranged branches. phialides - per branch, cylindrical, occasionally flask shaped, - (- ) × . - . (- ) μm diam, tapering abruptly toward a long cylindrical collula, up to μm long and - μm diam, solitary phialides rarely present. conidia in long divergent chains, ellipsoidal or cylindrical with conspicuously truncated ends, rarely subglobose, - (- ) × - . (- ) μm diam. chlamydospores very rare, smooth. sexual morph was not observed even after prolonged incubation at °c.cultural characteristics -(in darkness, °c after d): colonies on mea > mm diam, colony texture, floccose, mycelium white to yellow-brown (deep colonial buff to honey yellow, r ; ridgway ( ) ), sporulation very good, conidia en masse light-buff to warm-buff (r ), exudate absent, soluble pigments absent, reverse mustard yellow to primuline yellow (r ). colonies on czapek yeast autolysate agar (cya) - mm diam, colony texture floccose, mycelium white yellow ochre (r ), sporulation good, conidia en masse light buff to warm buff (r ), exudate absent, soluble pigments absent, reverse light buff to warn buff (r ). colonies on potato dextrose agar (pda) > mm diam, colony texture floccose, mycelium deep colonial buff to honey yellow (r ), sporulation very good, conidia en masse light-buff to warm-buff (r ), exudate absent, soluble pigments absent, reverse amber yellow to primuline yellow (r ). colonies on czapek yeast agar with % sucrose, (cy s) - mm diam, colony texture floccose, mycelium white yellow ochre (r ), sporulation good, conidia en masse light buff to warm buff (r ), exudate absent, soluble pigments absent, reverse light buff to warm buff (r ). colonies on dichloran glycerol agar (dg ) - mm diam, colony texture light floccose, sporulation very good, mycelium white to cream colour (r ), reverse warm buff (r ). no growth on cya supplemented with % (w/v) nacl (cyas). colonies on oa - mm diam, colony texture floccose, mycelium white to honey yellow (r ), sporulation very good, conidia en masse light-buff to warm-buff (r ), exudate absent, soluble pigments absent. colonies on creatine sucrose agar (crea) - mm diam, poor growth, no acid production, mycelium white, colony subsurface to submerged into the agar. colony diam (in mm after d) at °c/ °c; mea > / - ; cya - / - ; pda > / - ; cy s - / - ; dg - / - ; oa > / - ; crea - /ng; no growth at °c. a best scoring maximum likelihood tree based on the its region and the β-tubulin gene sequences shows the relationships of p. penicilliformis with other paecilomyces and byssochlamys species. the dataset contained taxa and a total of characters of which were variable and parsimony-informative. partitioning scheme and substitution models for analyses were selected using partitionfinder v. (lanfear et al. ): the gtr+i+g model was proposed for the its , its and β-tubulin gene exons; jc model for the . s region; and k +i model for the β-tubulin gene introns. the tree was constructed with iq-tree v. . . (nguyen et al. ) . support values at branches were obtained from bootstrap replicates. only bootstrap support values ≥ % are shown; ex-type strains are indicated by t and the novel species in bold text. the tree is rooted with sclerocleista ornata nrrl . paecilomyces penicilliformis produces predominantly long cylindrical conidia with conspicuously truncated ends, - (- ) × - . (- ) μm compared to smaller and predominantly globose conidia with flattened base produced by the closely related b. lagunculariae, . - . × . - . μm (samson et al. ). in addition, b. lagunculariae produces a sexual morph in culture (homothallic) and grows faster on mea at °c ( - mm after d) (samson et al. ). paecilomyces penicilliformis is similar to p. dactylethromorphus by its cylindrical or ellipsoidal conidia and regularly branched conidiophores (penicilliumlike). these species can be distinguished by wider conidia, - . (- ) μm produced by p. penicilliformis compared to p. dactylethromorphus, . - . μm wide (samson et al. ). micromorphology (on malt extract agar (mea), °c, wk): mycelium consisting of branched, septate, hyaline, smooth, . - . mm diam hyphae; racquet hyphae, spiral hyphae and peridial hyphae not observed. conidiophores simple, usually poorly differentiated from vegetative hyphae; conidiogenous hyphae unbranched or sparsely laterally branched. microconidia sessile, borne laterally or terminally, clavate or pyriform, truncate, aseptate, smooth-walled, . - . × . - . μm (mean ± standard deviation: . ± . × . ± . μm), l/w . - . . macroconidia borne singly or on sparsely and irregularly branched conidiophores, fusiform or clavate with rounded apex (less frequently slightly acuminate) and truncate base, straight or slightly to strongly curved, multi-celled, thick-walled, usually with - (- ) septa (median = ), smooth-walled, hyaline to pale yellow en masse, - (- ) × - μm ( ± . × . ± . μm), l/w . - . . chlamydospores globose, subglobose to irregular, usually - μm diam. sexual morph unknown.culture characteristics -colonies on sabouraud glucose agar (sga) at °c - mm diam after wk, covering dish after wk, flat, centrally raised to umbonate, granular, pale yellow ( a ; kornerup & wanscher ) to yellowish white ( a ), margins filamentous, reverse light brown ( d ) to orange yellow ( b ). colonies on mea at °c - mm diam after wk, covering dish after wk, flat with elevated centre, granular (with or without cottony centre), pale yellow ( a ) to pinkish white ( a ), pink ( a ) sectors or concentric zone may be present in old cultures, margins filamentous, serrate to irregular, reverse greyish orange ( b ) to orange white ( a ), bright red pigment inconstantly exuded into the medium. colonies on potato dextrose agar (pda) at °c - mm diam after wk, - mm diam after wk, centrally raised to raised, downy to delicately granular, pale yellow ( a ) to yellowish white ( a ), margins filamentous, reverse light brown ( d ) to greyish yellow ( b ). colonies on mea at °c after wk - mm diam, covering dish after wk; no growth on mea °c.typus. south africa, skin scrapings from human patient, before , unknown collector (holotype prm , isotype prm , cultures ex-type uamh = ccf ; its, lsu, β-tubulin and tef α sequences genbank mt , mt , mt and mt , mycobank mb ).additional material examined. czech republic, skin scales, heel, -yr-old woman with suspected dermatophytosis, oct. , p. hamal, culture ccf ; its, lsu, β-tubulin and tef α sequences genbank mt , mt , mt and mt .notes -blast analyses with the its and β-tubulin sequences of paraphyton cutaneum showed the following similarities with currently accepted paraphyton species (de hoog et al. ) : p. cookei ( . % and . %, respectively), p. mirabile ( . % and . %, respectively) and p. cookiellum ( . % and . %, respectively); lsu and tef α sequences are not available for all accepted species.paraphyton cookei and p. cookiellum have echinulate to verrucose macroconidia and can be easily distinguished from p. cutaneum having smooth-walled macroconidia. additionally, macroconidia of p. cookiellum are oval ( - × - μm) and predominantly -celled (currah ) . paraphyton mirabile is a slow-growing species compared with p. cutaneum; its colonies attain approximately mm diam after wk on sga (choi et al. ).both isolates of p. cutaneum were isolated from patients with skin lesions suggestive of dermatophytosis but its pathogenicity is questionable because the detailed anamnestic data are not available. the strain from the czech patient was isolated from a skin lesion on the heel (direct microscopic examination not performed due to insufficient amount of material). a complete clinical healing was observed after mo treatment with topical oxiconazole. the species probably naturally occurs in soil, similarly to the remaining paraphyton species which are also occasionally isolated from clinical material (choi et al. ).classification -pestalotiopsidaceae, xylariales, sordariomycetes.conidiomata pycnidial on / potato dextrose agar (pda), globose or clavate, scattered or aggregated, immersed or semiimmersed, dark brown to black, up to μm diam; exuding dark brown to black conidial masses. conidiophores reduced to conidiogenous cells. conidiogenous cells discrete, cylindrical, hyaline, smooth, - × - μm. conidia fusoid, cylindrical, straight to slightly curved, -septate, - × - μm, basal cell conic, hyaline, smooth and thin-walled, - μm long; three median cells doliiform, - μm long, smooth, concolourous, septa darker than the rest of the cell (second cell from base - . μm long; third cell - . μm long; fourth cell . - μm long); apical cell . - . μm long, hyaline, conic, thin-walled, smooth; with three tubular apical appendages, unbranched, filiform, - μm; basal appendage tubular, centric, - . μm long. sexual morph not seen.culture characteristics -colonies on pda after d cm diam, adpressed with no aerial mycelium, margin entire, dark tan in the centre becoming lighter towards the margin, with dark radial striations in the middle part. notes -the multilocus phylogenetic analysis placed p. etonensis in a well-supported clade with p. parva. based on a blastn search, p. etonensis differs from p. parva in its (gen-bank nr_ ; identities = / ( %), gap ( %)), tub (genbank km ; identities = / ( %), gaps ( %)) and tef a (genbank km ; identities = / ( %), gap ( %)). morphologically, p. etonensis conidia size and shape is indistinguishable from p. parva (fusoid, straight to slightly curved, -septate, - × - μm; maharachchikumbura et al. ) . geographically, p. etonensis is only known from one location in australia, while the origin and distribution of p. parva is unknown (maharachchikumbura et al. ) . pestalotiopsis etonensis has only been isolated from sporobolus jacquemontii in australia, while p. parva is known from delonix regia (caesalpiniaceae) and leucothoe fontanesiana (ericaceae) (maharachchikumbura et al. ) .