key: cord-0296674-374a30ye authors: Perera, Thejanee; Schwarz, Franziska; Muzeniek, Therese; Siriwardana, Sahan; Becker-Ziaja, Beate; Perera, Inoka C.; Handunnetti, Shiroma; Weerasena, Jagathpriya; Premawansa, Gayani; Premawansa, Sunil; Nitsche, Andreas; Yapa, Wipula; Kohl, Claudia title: Molecular identification of bats from Wavulgalge cave, Wellawaya, Sri Lanka date: 2021-12-19 journal: bioRxiv DOI: 10.1101/2021.12.19.473364 sha: 5bdd5cfd2cb25cfe66bee374916901b4e5fe3f3b doc_id: 296674 cord_uid: 374a30ye This is the first report on the molecular identification and phylogeny of Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva, Miniopterus fuliginosus bat species in Sri Lanka, inferred from mitochondrially encoded cytochrome b gene sequences. Wellawaya Wavulgalge cave in Sri Lanka is one of the largest sympatric colonies found on the island, occupied by five species of bats. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even in the same species, acoustic properties of echolocation calls and morphological features such as fur colour could vary in different populations. Therefore, we have used molecular techniques for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. Bats were caught using a hand net and saliva samples were collected non-invasively from each bat using a sterile oral swab. Nucleic acids were extracted from oral swab samples and mitochondrial DNA was amplified using primers targeting the mitochondrially encoded cytochrome b gene. This study identified the bat species recorded in the Wellawaya cave as Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva and Miniopterus fuliginosus. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats. microclimate and protection from predators and extreme weather conditions (7). Sample collection was carried out during March and July of 2018 and in January 2019. Adequate personal protective equipment such as safety gloves, safety glasses and FFP3 masks 88 were used during animal capturing, handling and sample collection to reduce the potential risk 89 of zoonotic or anthropozoonotic pathogen transmission. Bats were captured using hand nets while they were emerging from the roost in the evenings. Captured live bats were placed into cotton bags and were kept in a cool dry place until further 92 processing. Bat species were macroscopically identified using external morphological features 93 (Fig 1.) . Morphometric parameters and locational data were recorded in the data sheets (Fig 2. ). Bats were released immediately after recording the morphometric data and the collection of A 250 bp length of partial sequence of MT-CYB gene was amplified using FM up and FM do 108 primers as described previously (9). Further, the full MT-CYB gene was amplified using RrFP and 109 RrRP primers as previously described (10). PCR products were purified using MSB Spin Phylogenetic trees are constructed using molecular data and used to make inferences to 142 understand the evolutionary relationship among taxa. Phylogenetic reconstruction allocates the 143 sequences of the five Sri Lankan bat species to the corresponding bat families within the species 144 tree (Fig 3.) . Reconstruction was calculated using MrBayes MCMC method (Parameters were as follows: 148 Substitution model, GTR, rate variation, equal; chain length 10 million; burn-in, 30%; Therefore, accurate species identification using molecular techniques will help to predict the 228 pathogen-host shifts and interspecies pathogen transmission in bats in the Wavulgalge cave in 229 Sri Lanka in future studies (23,26). Our study addressed the research gap of the molecular taxonomy of Sri Lankan bats. Our results are consistent with the species identification by morphological identification of the bat species in 233 the Wavulgalge cave. Accurate identification of bats plays a critical role in conservation. Therefore, our findings will also contribute to future conservation and systematic studies of bats 235 in Sri Lanka. Further data collected from our study will provide the basis for a genetic database 236 of Sri Lankan bats. Acknowledgments: We thank Angelina Targosz, Nicole Kromarek, Marica Grossegesse for 239 technical and field assistance, RKI sequencing lab for providing us Sanger sequencing results. We convey our gratitude to the Department of Wildlife Conservation, Sri Lanka and the 241 Institute of Biology, Sri Lanka for granting us the necessary permits. Competing Interests: The authors declare no financial and non-financial competing interests. Financial Declaration: This study was partly supported by the Federal Ministry of Health, Germany (Bundesministerium für Gesundheit, BMG) under the IDEA (IDentification of (https://www.ncbi.nlm.nih.gov/genbank/). 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London: Dulau and Company The national red list 2012 of Sri Lanka Breeding associated migration of Miniopterus schreibersii between two natural caves in 292 Proceedings of the 19h Annual Sessions of the Institute of Biology, Sri 293 Lanka New phonic type of the rufous 295 horseshoe bat Rhinolophus rouxii from southern India Foraging 297 behaviour and echolocation in the rufous horseshoe bat Phylogeny 300 of African fruit bats (Chiroptera, Pteropodidae) based on complete mitochondrial Page 27 of 27 301 genomes A comparison 303 of bats and rodents as reservoirs of zoonotic viruses: are bats special? Emerging diseases in Chiroptera: why bats? 306 Bat origin of human coronaviruses Betacoronaviruses in Miniopterus fuliginosus and Rousettus leschenaultii, two species of 310 Bats: important hosts of emerging viruses Bats in ecosystems and their Wide spectrum of viral infectious 315 potential threats: SARS-CoV-2 and other emerging viruses The Evolution and Genetics of 318 Virus Host Shifts The relationships observed by phylogenetic reconstructions are further supported by the heat-160 map of distances, based on the percentage of genetic identity for all five bat species (Fig 4-7) . Bat species in Sri Lanka were first described by Kelaart in 1852 (13 Prevalence of medically important pathogens such as SARS CoV-2 in bats may be linked with the 226 bat species, their geographical location, roosting nature and other behavioural aspects (24,25).