key: cord-0279661-zfvjhmed authors: Stork, Sydney; Jalinsky, Joseph; Neiman, Maurine title: Asexual freshwater snails make poor mate choice decisions date: 2021-08-05 journal: bioRxiv DOI: 10.1101/2021.06.08.447504 sha: 9900b970ace2096811ef4b4dc3306ec9efb0efd4 doc_id: 279661 cord_uid: zfvjhmed Once-useful traits that no longer contribute to fitness tend to decay over time. Here, we address whether the expression of mating-related traits that increase the fitness of sexually reproducing individuals but are likely less useful or even costly to asexual counterparts seems to exhibit decay in the latter. Potamopyrgus antipodarum is a New Zealand freshwater snail characterized by repeated transitions from sexual to asexual reproduction. The frequent coexistence of sexual and asexual lineages makes P. antipodarum an excellent model for the study of mating-related trait loss. We used a mating choice assay including sexual and asexual P. antipodarum females and conspecific (presumed better choice) vs. heterospecific (presumed worse choice) males to evaluate the loss of behavioral traits related to sexual reproduction. We found that sexual females engaged in mating behaviors with conspecific mating partners more frequently and for a greater duration than with heterospecific mating partners. By contrast, asexual females mated at similar frequency and duration as sexual females, but did not mate more often or for longer duration with conspecific vs. heterospecific males. These results are consistent with a scenario where selection acting to maintain mate choice in asexual P. antipodarum is weak or ineffective relative to sexual females and, thus, where asexual reproduction contributes to the evolutionary decay of mating-related traits in this system. Adaptations that were once important for survival but subsequently become useless or impose 25 new fitness costs tend to decay over time in a process known as vestigialization (Darwin 1859; 26 Wiedersheim 1895; Van der Kooi and Schwander 2014) . Many examples of this phenomenon 27 can be seen throughout evolutionary history, including the loss of limbs in snakes (Lande 1978) , 28 eye structures in cave-dwelling fish (Jeffery 2005) , and wings in many insect species (Roff 29 1990) . 30 While the genetic mechanisms underlying vestigialization depend on evolutionary 31 context, traits are most commonly lost by the accumulation of selectively neutral mutations 32 4 evidence for distinct genomic consequences of asexuality in P. antipodarum (Sharbrough et al 70 2018; McElroy et al 2021) suggests that such gene flow is rare at most. Sexual males also show 71 poor ability to discriminate between favorable and unfavorable mates, mating with equal 72 frequency with sexual and asexual females and with parasitically castrated vs. healthy females 73 . Prior emphasis on investigation of male mating behavior is largely a 74 result of the assumption that males are physically in control of mating outcomes (Neiman and 75 Neiman 2011) , despite a lack of studies assessing the female role. These previous results set the 76 stage for a direct comparison between sexual and asexual P. antipodarum females. In a mating 77 paradigm like that demonstrated for P. antipodarum where males seem to lack adequate mate 78 discrimination, differences in mating between sexual and asexual females can be attributed to 79 mating differences between these two female groups. Therefore, we hypothesize that sexual 80 female P. antipodarum are better able to choose a more favorable mate when presented with a 81 choice than asexual counterparts. 82 We evaluated this hypothesis by comparing the mating behavior of sexual and asexual P. 83 antipodarum females with sexual male P. antipodarum and sexual male Potamopyrgus 84 estuarinus. Potamopyrgus estuarinus is a closely related and phenotypically similar species that 85 nevertheless has minimal habitat overlap with P. antipodarum (Haase 2003 We haphazardly selected three sets of 10 sexually mature (>3 mm long; Richards These lineages were separately derived from the lineages from which we drew females. All P. 107 estuarinus males were collected from the Ashley River estuary in New Zealand in February, 108 All snails used in the experiment had been housed in 10-L plastic tanks filled about 75% 110 full with carbon-filtered tap water and fed Spirulina algae, a common Potamopyrgus lab food, 111 three times weekly. These tanks were maintained in our constant-temperature snail room, which 112 is held at 16°C and on a 12:12 hour light:dark cycle. Snails were given powdered chalk as a 113 source of dietary calcium once weekly (feeding and maintenance following Nelson and Neiman 114 (2011) ). We isolated each population used for the experiment in a 0.95 L plastic cup containing 115 6 ~700 mL of water for approximately 14 days prior to behavioral trials to control for previous 116 exposure to males and possible recent mating. We painted female shells with light-colored nail 117 polish to aid in differentiation between females and males when analyzing video footage. 118 Feeding and maintenance for the populations in cups followed our standard snail room protocol. 119 Mating trials occurred in standard four-inch petri dishes. We covered the exterior of each 120 dish in electrical tape to control for external visual stimuli. To begin each trial for the sexual 121 females, we placed one sexual P. antipodarum female, one sexual P. antipodarum male, and one 122 P. estuarinus male equidistant from one another around the edges of the inside of the petri dish 123 ( Fig. 1 ). After the three snails were placed, we filled the petri dish to about 75% total volume 124 with carbon-filtered tap water. We used this same setup for asexual P. antipodarum females, 125 with the asexual female replacing the sexual female. No snail was used in more than one trial. 126 127 We conducted each mating trial at approximately 1500 hours, for three hours. As the maximum 129 copulation duration for P. antipodarum is about two hours , we 130 established this three-hour time frame to allow for multiple mating attempts to occur. We 131 recorded trials using a USB webcam and time-lapse video software VideoVelocity (v3.7.2090, 132 CandyLabs 2019). The three snails per trial were allowed to move and interact freely without 133 physical barriers inside the petri dish. 134 We loosely followed previous studies of mating in P. antipodarum (e.g., Nelson and 135 Neiman 2011) to define attempted mating behavior as any physical interaction resulting in one 136 snail mounting the shell of another. It is otherwise impossible to visually confirm sexual contact 137 (joining of genitalia) in Potamopyrgus because this phenomenon is internal relative to the 138 7 operculum and is thus obscured by the shells. Once this mounting behavior was initiated, we 139 arbitrarily established that any such interaction lasting 15 seconds or longer would be counted as 140 an instance of mate choice; interactions shorter than 15 seconds were potentially too short to be 141 discernible with our time-lapse software and were excluded. Fifteen seconds is also more than 142 enough time for snails to reorient and move on following an incidental -versus mating -143 encounter. Both the frequency (number of mating interactions each female had with conspecific 144 vs. heterospecific males) and duration (length of time (seconds) that each female was in physical 145 contact with each male type) of mating interactions between females and conspecific vs. 146 heterospecific males were recorded. 147 We also used this experimental framework to establish expected outcomes and determine 148 how these outcomes would inform our hypotheses. First, a fair test of the trait decay hypothesis 149 would require that sexual females mate more frequently and for longer with conspecific males 150 than with P. estuarinus males. This outcome is critical in indicating that sexual females seem to 151 possess the ability to discriminate between preferential and poor mating partners in our 152 experimental setting. Absence of a difference in mating behavior across male type for sexual 153 females would not support the trait decay hypothesis, suggesting instead, for example, that these 154 females are unable to differentiate between mates or that observed contact is reflective of 155 behavior outside of mating. Mating-related trait decay in asexual females consistent with the trait 156 decay hypothesis would be reflected in relatively weak or absent mate choice for conspecific vs. 157 heterospecific males relative to choices in sexual female P. antipodarum. 158 159 160 8 We used the Shapiro-Wilk test to determine that both the mating frequency (sexual females: W = 161 0.769, p = 8.18x10 -8 ; asexual females: W = 0.810, p = 1.12x10 -5 ) and the mating duration (sexual 162 females: W = 0.642, p = 1.05x10 -9 ; asexual females: W = 0.493, p = 3.17x10 -8 ) datasets violated 163 normality assumptions of parametric analysis. Accordingly, we used nonparametric approaches 164 to analyze the data. Analyses for mating duration were executed using the total mating duration 165 for each individual (Tables S1, S2). First, we used Mann-Whitney U tests to compare overall 166 mating frequency and duration between individual sexual and asexual females. Second, we used 167 Wilcoxon rank-sum analyses to determine whether mating durations and frequencies of sexual 168 and asexual females, respectively, with conspecific males differed from mating durations and 169 frequencies with heterospecific males. As another means of comparing mating behavior across 170 sexual and asexual females, we calculated discrimination scores -defined as the difference 171 between mating frequency and mating duration, respectively, with conspecific vs. heterospecific 172 males -for each female. We then used Mann-Whitney U tests to determine whether there was a 173 significant difference in the mating frequency and mating duration discrimination scores, 174 respectively, between sexual and asexual females. All analyses were executed with R 3.6. (Fig. 2d) . 195 The discrimination score analysis revealed that sexual but not asexual females with a 196 choice between conspecific and heterospecific males tend to make mating behavior decisions 197 with respect to mating frequency that favor the former (Mann-Whitney U = 175.5, z = 2.02, p = 198 0.043). By contrast, there was no significant difference in discrimination scores for mating 199 duration between sexual and asexual females (Mann-Whitney U = 208, z = 1.32, p = 0.187). Our experiment revealed that sexual female P. antipodarum exhibited significantly higher 203 mating frequency and longer mating duration with conspecific vs. heterospecific males. These 204 results are as predicted if sexual female P. antipodarum are able to discriminate between mates 205 of higher vs. lower quality and are important in demonstrating that discriminatory behavior can 206 occur in the context of our experiment. By contrast, although asexual P. antipodarum females 207 mated at the same frequency and for the same duration as their sexual counterparts, the asexuals 208 showed no evidence for mate discrimination or preference. Together, these findings are 209 consistent with a scenario where sexual but not asexual female P. antipodarum exhibit mate 210 choice. Evolutionary decay of mating-related traits in an asexual context is a plausible 211 explanation for these results and is consistent with a previous study demonstrating evidence for 212 decay of sperm traits in the male offspring occasionally produced by asexual female P. 213 antipodarum (Jalinsky et al 2020) . Another non-mutually exclusive explanation for our results is 214 provided by the possibility that asexual female P. antipodarum are experiencing more general 215 degradation of phenotype associated with the accumulation of harmful mutations that is expected 216 to accompany obligately asexual reproduction (Muller 1964; Hill and Robertson 1966) . While a 217 relatively high rate of accumulation of potentially harmful mutations has been observed in 218 asexual P. antipodarum relative to sexual conspecifics (e.g., Sharbrough et al 2018) , that asexual 219 P. antipodarum females are similar to or even outperform sexual females when it comes to key 220 determinants of fitness like fecundity (Paczesniak et al 2019) and growth rate and age at 221 reproductive maturity (Larkin et al 2016) suggests that these mutations might not be translating 222 into major phenotypic consequences. We also cannot formally exclude the possibility that 223 asexual female snails might demonstrate mating preferences in a different context, though the 224 fact that we observed such preferences in closely related sexual counterparts in the same 225 experiment suggests that this explanation is not likely to entirely explain the different outcomes 226 between sexual and asexual females. Finally, it is important to acknowledge that our study only 227 included female P. antipodarum from one lake and that we only compared one sexual lineage to 228 one asexual lineage. This Covid19-imposed limitation on our experimental design means that 229 11 when possible, it will be important to repeat the experiment using additional lineages to 230 determine whether our results are indeed more broadly generalizable. 231 The exhibition of similar mating behavior to their sexual counterparts combined with the 232 lack of observable discriminatory behavior in asexual females could be explained by a situation 233 where copulatory behavior -even in the absence of a connection between egg fertilization and 234 embryo development -is still necessary and/or beneficial although choice of the mating partner 235 is no longer important. For example, Neiman (2004) speculated that asexual females that have no 236 direct use for sperm contributed by males might still benefit from mating if copulatory stimuli 237 are required for maximization of reproduction, though Neiman (2006) , showed that this scenario 238 is unlikely for P. antipodarum. Alternatively, male ejaculate may provide nutritive benefits to 239 asexual females. This possibility is supported by the fact that sperm storage structures are 240 maintained in asexual P. antipodarum females (Dillon 2000) , though evidence that these 241 structures might also function for the digestion of waste in asexual P. antipodarum suggests the 242 potential for a non-mating-specific or even unique function for this organ in asexuals (Fretter and 243 Graham 1962) . Follow-up studies of whether and how sperm/ejaculate are used in asexual P. 244 antipodarum and other asexual taxa will help clarify the prevalence and persistence of 245 copulatory behavior in asexual females. 246 Another and perhaps simpler explanation is that mating behavior has not fully decayed in 247 P. antipodarum, and that the ability to discriminate is lost before the behavior as a whole 248 disappears. That asexual females nevertheless engage in mating behavior at all could be a 249 function of the relatively recent derivation of most asexual female P. antipodarum from sexual 250 counterparts . With this in mind, it would be valuable to repeat this 251 12 experiment with females from older asexual lineages (sensu Nelson and Neiman 2011) but 252 departing from this prior study so that females vs. males have mate choice capabilities. 253 "SexF") and asexual (b, d; "AsexF") females with P. antipodarum ("PAM") and P. estuarinus ("PEM") males. Individual data points are indicated with black dots. Studies on the reproduction of cirripedes. I. Introduction: copulation, release of oocytes, and formation of the egg lamellae VideoVelocity Time-Lapse Capture Studio Decay of female sexual behavior under parthenogenesis The origin of species by means of natural selection or, the preservation of favoured races in the struggle for life The ecology of freshwater molluscs Sexual selection by cryptic female choice on male seminal products-a new bridge between sexual selection and reproductive physiology British prosobranch molluscs. Their functional anatomy and ecology Vestigialization and loss of nonfunctional characters Clinal variation in shell morphology of the freshwater gastropod Potamopyrgus antipodarum along two hill-country streams in New Zealand The radiation of hydrobiid gastropods in New Zealand: a revision including the description of new species based on morphology and mtDNA sequence information Sexual selection by the handicap mechanism The effect of linkage on limits to artificial selection Male phenotypes in a female framework: Evidence for degeneration in sperm produced by male snails from asexual lineages Adaptive evolution of eye degeneration in the Mexican blind cavefish Male sexual trait decay in two asexual springtail populations follows neutral mutation accumulation ggpubr: 'ggplot2' Based Publication Ready Plots Sexual selection and the evolution of female choice The evolution of mate choice and mating biases Decay of sexual trait genes in an asexual parasitoid wasp Relaxed selection in the wild Effects of polyploidy and reproductive mode on life history trait expression Evolutionary mechanisms of limb loss in tetrapods Evidence from a New Zealand snail for the maintenance of sex by parasitism Predation risk as a cost of reproduction Asexuality associated with marked genetic expansion of tandemly repeated rDNA and histone genes environmental factors associated with fecundity of a parthenogenic invader The relation of recombination to mutational advance Physiological dependence on copulation in parthenogenetic females can reduce the cost of sex Embryo production in a parthenogenetic snail (Potamopyrgus antipodarum) is negatively affected by the presence of other parthenogenetic females Male New Zealand mud snails (Potamopyrgus antipodarum) persist in copulating with asexual and parasitically castrated females Variation in asexual lineage age in Potamopyrgus antipodarum, a New Zealand snail Wide variation in ploidy level and genome size in a New Zealand freshwater snail with coexisting sexual and asexual lineages Persistent copulation in asexual female Potamopyrgus antipodarum: evidence for male control with size-based preferences Parasite resistance predicts fitness better than fecundity in a natural population of the freshwater snail Potamopyrgus antipodarum R: A language and environment for statistical computing Intraspecific competition and development of size structure in the invasive snail Potamopyrgus antipodarum (Gray, 1853) The evolution of flightlessness in insects Neutral and selection-driven decay of sexual traits in asexual stick insects Radical amino acid mutations persist longer in the absence of sex Documentation of copulatory behavior in triploid male freshwater snails On the fate of sexual traits under asexuality ggplot2: Elegant graphics for data analysis using the grammar of graphics Parthenogenesis, sex and chromosomes in Potamopyrgus 2021) dplyr: A Grammar of Data Manipulation Wiedersheim R (1895) The Structure of Man: an Index to His Past History Natural selection, the costs of reproduction, and a refinement of Lack's principle Mate selection-a selection for a handicap We thank the Iowa Center for Research by Undergraduates for contributing to mentorship and training for SS, Dr. Lori Adams for her input and guidance through the Honors Program for SS; John Logsdon, Mike Winterbourn, and Mary Morgan-Richards for help in snail collection; andMarissa Roseman for setting up, characterizing, and maintaining the snail lineages that we used.Josephine Bliss also contributed to snail care. This work was supported by the Iowa Center for Research by Undergraduates, by Linda and Rick Maxson, and via NSF grant DEB-1753851. We also acknowledge very constructive and helpful critiques from Prof. Ken Kraajiveld, Prof. Ingo Schlupp, and an anonymous reviewer that greatly improved the paper.