Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Male phenotypes in a female framework: Evidence for degeneration in sperm produced by male snails from asexual lineages

How changes in selective regimes affect trait evolution is an important open biological question. We take advantage of naturally occurring and repeated transitions from sexual to asexual reproduction in a New Zealand freshwater snail species, Potamopyrgus antipodarum, to address how evolution in an asexual context—including the potential for relaxed selection on male‐specific traits—influences sperm morphology. The occasional production of male offspring by the otherwise all‐female asexual P. antipodarum lineages affords a unique and powerful opportunity to assess the fate of sperm traits in a context where males are exceedingly rare. These comparisons revealed that the sperm produced by ‘asexual’ males are markedly distinct from sexual counterparts. We also found that the asexual male sperm harboured markedly higher phenotypic variation and was much more likely to be morphologically abnormal. Together, these data suggest that transitions to asexual reproduction might be irreversible, at least in part because male function is likely to be compromised. These results are also consistent with a scenario where relaxed selection and/or mutation accumulation in the absence of sex translates into rapid trait degeneration.     

Parasite resistance predicts fitness better than fecundity in a natural population of the freshwater snail Potamopyrgus antipodarum

The cost of males should give asexual females an advantage when in competition with sexual females. In addition, high‐fecundity asexual genotypes should have an advantage over low‐fecundity clones, leading to reduction in clonal diversity over time. To evaluate fitness components in a natural population, we measured the annual reproductive rate of individual sexual and asexual female Potamopyrgus antipodarum, a New Zealand freshwater snail, in field enclosures that excluded competitors and predators. We used allozyme genotyping to assign the asexual females to particular clonal genotypes. We found that the most fecund asexual clones had similar or higher fecundity as the top 10% of sexual families, suggesting that fecundity selection, even without the cost of males, would lead to replacement of the sexual population by clones. Consequently, we expected that the clones with the highest fecundity would dominate the natural population. Counter to this prediction, we found that high annual reproductive rates did not correlate with the frequency of clones in the natural population. When we exposed the same clones to parasites in the laboratory, we found that resistance to infection was positively correlated with the frequency of clones in the population. The correlation between fecundity and parasite resistance was negative, suggesting a trade‐off between these two traits. Our results thus suggest that parasite resistance is an important short‐term predictor of the success of asexual P. antipodarum in this population.     

Asexuality Associated with Marked Genomic Expansion of Tandemly Repeated rRNA and Histone Genes

How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits—mode of genomic inheritance—influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S–5.8S–28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA–histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction—with potential functional consequences related to the loss of sexual reproduction.     

Ecomorphology of a generalist freshwater gastropod: complex relations of shell morphology,habitat, and fecundity

Evolutionary and ecological situations in a species’ native and invasive ranges can be drastically different. This is the case for Potamopyrgus antipodarum Gray (1843) a morphologically highly variable freshwater snail native to New Zealand, where sexual and asexual individuals coexist and experience selective pressure by sterilizing endoparasites. By contrast, only a few asexual lineages have been established in invaded regions around the globe, where parasite infection is extremely rare. We analyzed the ecomorphology of 996 native P. antipodarum in a geometric morphometric framework, using brood size as proxy for fecundity, and mtDNA and nuclear SNPs to account for relatedness and identify reproductive mode. As expected, we found genetic and morphological diversity to be higher in native than in invasive snails investigated previously, but surprisingly no higher morphological diversity in sexual versus asexual individuals. The relationships between shell morphology, habitat, and fecundity were complex. Shape variation was primarily linked to genetic relatedness but specific environmental factors including flow rate induced similar shell shapes. By contrast, shell size was largely explained by environmental factors. Fecundity was correlated with size but showed trade-offs with shape in increasingly extreme conditions. With increasing flow and toward small springs, the trend of shell shape becoming wider was reversed, i.e., snails with narrower shells were brooding more embryos. We concluded that both genetic and environmental contributions to variation in shell morphology in P. antipodarum likely play an important role in the ability of this species to adapt to a wide spectrum of habitats.     

ENVIRONMENTAL STRESS AND THE MAINTENANCE OF SEX IN A FRESHWATER SNAIL

Synergism among mutations can lead to an advantage to sexual reproduction, provided mutation rates are high enough (the mutational deterministic hypothesis). Here we tested the idea that competition for food can increase the advantage to sexual reproduction, perhaps by increasing the synergism among mutations in asexual individuals. We compared the survivorship of sexual and asexual snails (Potamopyrgus antipodarum) under two treatments: starved and fed. We predicted higher mortality for asexual snails when starved, but found that sexual and asexual individuals survived at the same rate, independent of treatment. These results suggest that the distribution of sex in this snail may not be explained by variation in competition among populations.     

Male mate choice in the gynogenetic–sexual complex of crucian carp,<Emphasis Type="Italic"> Carassius auratus</Emphasis>

The asexual all-female Japanese crucian carp, Carassius auratus langsdorfii (Teleostei: Cypriniformes), reproduces gynogenetically, relying on the sperm of males of the sexual "host," C. auratus subspp. Theoretically, frequency-dependent mating preference of males to conspecific females can lead to the coexistence of asexual and sexual fish, if all else is equal. Our specific questions are whether males prefer conspecific females over asexual females and whether individuals show dominance hierarchies that potentially cause frequency-dependent mating preference. In an individual choice experiment, a tank was partitioned into three compartments with the middle one for a single male and the two outer ones for a sexual and an asexual female. The males of C. auratus bürgeri demonstrated a significant preference for ovulated conspecific females over ovulated asexual females. In contrast, in a group mating experiment, a single experimental tank included two males, a sexual female, and an asexual female together, and males chased and mated with both asexual and sexual females equally. Male mate preference was weak in group mating, which is typical in natural populations. Males and females of crucian carp showed no apparent agonistic behavior to each other in the group mating experiment. This is different from other gynogenetic complexes with the dominance hierarchy of males showing strong frequency-dependent mating preference (e.g., Poeciliopsis). We conclude that male mate preference is unlikely to be a strong frequency-dependent force maintaining the coexistence of asexual–sexual complexes of Japanese crucian carp. Received in revised form: 5 February 2001 Electronic Publication     

Adaptive phenotypic plasticity in a clonal invader

Organisms featuring wide trait variability and occurring in a wide range of habitats, such as the ovoviviparous New Zealand freshwater snail Potamopyrgus antipodarum, are ideal models to study adaptation. Since the mid‐19th century, P. antipodarum, characterized by extremely variable shell morphology, has successfully invaded aquatic areas on four continents. Because these obligately and wholly asexual invasive populations harbor low genetic diversity compared to mixed sexual/asexual populations in the native range, we hypothesized that (1) this phenotypic variation in the invasive range might be adaptive with respect to colonization of novel habitats, and (2) that at least some of the variation might be caused by phenotypic plasticity. We surveyed 425 snails from 21 localities across northwest Europe to attempt to disentangle genetic and environmental effects on shell morphology. We analyzed brood size as proxy for fitness and shell geometric morphometrics, while controlling for genetic background. Our survey revealed 10 SNP genotypes nested into two mtDNA haplotypes and indicated that mainly lineage drove variation in shell shape but not size. Physicochemical parameters affected both shell shape and size and the interaction of these traits with brood size. In particular, stronger stream flow rates were associated with larger shells. Our measurements of brood size suggested that relatively larger slender snails with relatively large apertures were better adapted to strong flow than counterparts with broader shells and relatively small apertures. In conclusion, the apparent potential to modify shell morphology plays likely a key role in the invasive success of P. antipodarum; the two main components of shell morphology, namely shape and size, being differentially controlled, the former mainly genetically and the latter predominantly by phenotypic plasticity.     

A layover in Europe: Reconstructing the invasion route of asexual lineages of a New Zealand snail to North America

Non‐native invasive species are threatening ecosystems and biodiversity worldwide. High genetic variation is thought to be a critical factor for invasion success. Accordingly, the global invasion of a few clonal lineages of the gastropod Potamopyrgus antipodarum is thus both puzzling and has the potential to help illuminate why some invasions succeed while others fail. Here, we used SNP markers and a geographically broad sampling scheme (N = 1617) including native New Zealand populations and invasive North American and European populations to provide the first widescale population genetic assessment of the relationships between and among native and invasive P. antipodarum. We used a combination of traditional and Bayesian molecular analyses to demonstrate that New Zealand populations harbour very high diversity relative to the invasive populations and are the source of the two main European genetic lineages. One of these two European lineages was in turn the source of at least one of the two main North American genetic clusters of invasive P. antipodarum, located in Lake Ontario. The other widespread North American group had a more complex origin that included the other European lineage and two New Zealand clusters. Altogether, our analyses suggest that just a small handful of clonal lineages of P. antipodarum were responsible for invasion across continents. Our findings provide critical information for prevention of additional invasions and control of existing invasive populations and are of broader relevance towards understanding the establishment and evolution of asexual populations and the forces driving biological invasion.     

Response to Phosphorus Limitation Varies among Lake Populations of the Freshwater Snail Potamopyrgus antipodarum

Local adaptation – typically recognized as higher values of fitness-related traits for native vs. non-native individuals when measured in the native environment - is common in natural populations because of pervasive spatial variation in the intensity and type of natural selection. Although local adaptation has been primarily studied in the context of biotic interactions, widespread variation in abiotic characteristics of environments suggests that local adaptation in response to abiotic factors should also be common. Potamopyrgus antipodarum, a freshwater New Zealand snail that is an important model system for invasion biology and the maintenance of sexual reproduction, exhibits local adaptation to parasites and rate of water flow. As an initial step to determining whether P. antipodarum are also locally adapted to phosphorus availability, we examined whether populations differ in their responses to phosphorus limitation. We found that field-collected juvenile P. antipodarum grew at a lower rate and reached an important size threshold more slowly when fed a relatively low vs. a relatively high- phosphorus diet. We also detected significant across-population variation in individual growth rate. A marginally significant population-by-dietary phosphorus interaction along with a two-fold difference across populations in the extent of suppression of growth by low phosphorus suggests that populations of P. antipodarum may differ in their response to phosphorus limitation. Local adaptation may explain this variation, with the implication that snails from lakes with relatively low phosphorus availability should be less severely affected by phosphorus limitation than snails from lakes with higher phosphorus availability.     

Profound Effects of Population Density on Fitness-Related Traits in an Invasive Freshwater Snail

Population density can profoundly influence fitness-related traits and population dynamics, and density dependence plays a key role in many prominent ecological and evolutionary hypotheses. Here, we evaluated how individual-level changes in population density affect growth rate and embryo production early in reproductive maturity in two different asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail that is an important model system for ecotoxicology and the evolution of sexual reproduction as well as a potentially destructive worldwide invader. We showed that population density had a major influence on individual growth rate and early-maturity embryo production, effects that were often apparent even when comparing treatments that differed in population density by only one individual. While individual growth rate generally decreased as population density increased, we detected a hump-shaped relationship between embryo production and density, with females from intermediate-density treatments producing the most embryos and females from low- and high-density treatments producing the fewest embryos. The two lineages responded similarly to the treatments, indicating that these effects of population density might apply more broadly across P. antipodarum. These results indicate that there are profound and complex relationships between population density, growth rate, and early-maturity embryo production in at least two lineages of this important model system, with potential implications for the study of invasive populations, research on the maintenance of sex, and approaches used in ecotoxicology.     

Genetic variation in sexual and clonal lineages of a freshwater snail

Sexual reproduction within natural populations of most plants and animals continues to remain an enigma in evolutionary biology. That the enigma persists is not for lack of testable hypotheses but rather because of the lack of suitable study systems in which sexual and asexual females coexist. Here we review our studies on one such organism, the freshwater snail Potamopyrgus antipodarum (Gray). We also present new data that bear on hypotheses for the maintenance of sex and its relationship to clonal diversity. We have found that sexual populations of the snail are composed of diploid females and males, while clonal populations are composed of a high diversity of triploid apomictic females. Sexual and asexual individuals coexist in stable frequencies in many ‘mixed’ populations; genetic data indicate that clones from these mixed populations originated from the local population of sexual individuals without interspecific hybridization. Field data show that clonal and sexual snails have completely overlapping life histories, but individual clonal genotypes are less variable than individuals from the sympatric sexual population. Field data also show segregation of clones among depth‐specific habitat zones within a lake, but clonal diversity remains high even within habitats. A new laboratory experiment revealed extensive clonal variation in reproductive rate, a result which suggests that clonal diversity would be low in nature without some form of frequency‐dependent selection. New results from a long‐term field study of a natural, asexual population reveal that clonal diversity remained nearly constant over a 10‐year period. Nonetheless, clonal turnover occurs, and it occurs in a manner that is consistent with parasite‐mediated, frequency‐dependent selection. Reciprocal cross‐infection experiments have further shown that parasites are more infective to sympatric host snails than to allopatric snails, and that they are also more infective to common clones than rare clones within asexual host populations. Hence we suggest that sexual reproduction in these snails may be maintained, at least in part, by locally adapted parasites. Parasite‐mediated selection possibly also contributes to the maintenance of local clonal diversity within habitats, while clonal selection may be responsible for the distribution of clones among habitats. © 2003 The Linnean Society of London. Biological Journal of the Linnean Society 2003, 79 , 165–181.     

Discordance between nuclear and mitochondrial genomes in sexual and asexual lineages of the freshwater snail Potamopyrgus antipodarum

The presence and extent of mitonuclear discordance in coexisting sexual and asexual lineages provides insight into 1) how and when asexual lineages emerged, and 2) the spatial and temporal scales at which the ecological and evolutionary processes influencing the evolution of sexual and asexual reproduction occur. Here, we used nuclear single‐nucleotide polymorphism (SNP) markers and a mitochondrial gene to characterize phylogeographic structure and the extent of mitonuclear discordance in Potamopyrgus antipodarum. This New Zealand freshwater snail is often used to study the evolution and maintenance of sex because obligately sexual and obligately asexual individuals often coexist. While our data indicate that sexual and asexual P. antipodarum sampled from the same lake population are often genetically similar, suggesting recent origin of these asexuals from sympatric sexual P. antipodarum, we also found significantly more population structure in sexuals vs. asexuals. This latter result suggests that some asexual lineages originated in other lakes and/or in the relatively distant past. When comparing mitochondrial and nuclear population genetic structure, we discovered that one mitochondrial haplotype (‘1A’) was rare in sexuals, but common and widespread in asexuals. Haplotype 1A frequency and nuclear genetic diversity were not associated, suggesting that the commonness of this haplotype cannot be attributed entirely to genetic drift and pointing instead to a role for selection.     

Effects of polyploidy and reproductive mode on life history trait expression

Ploidy elevation is increasingly recognized as a common and important source of genomic variation. Even so, the consequences and biological significance of polyploidy remain unclear, especially in animals. Here, our goal was to identify potential life history costs and benefits of polyploidy by conducting a large multiyear common garden experiment in Potamopyrgus antipodarum, a New Zealand freshwater snail that is a model system for the study of ploidy variation, sexual reproduction, host–parasite coevolution, and invasion ecology. Sexual diploid and asexual triploid and tetraploid P. antipodarum frequently coexist, allowing for powerful direct comparisons across ploidy levels and reproductive modes. Asexual reproduction and polyploidy are very often associated in animals, allowing us to also use these comparisons to address the maintenance of sex, itself one of the most important unresolved questions in evolutionary biology. Our study revealed that sexual diploid P. antipodarum grow and mature substantially more slowly than their asexual polyploid counterparts. We detected a strong negative correlation between the rate of growth and age at reproductive maturity, suggesting that the relatively early maturation of asexual polyploid P. antipodarum is driven by relatively rapid growth. The absence of evidence for life history differences between triploid and tetraploid asexuals indicates that ploidy elevation is unlikely to underlie the differences in trait values that we detected between sexual and asexual snails. Finally, we found that sexual P. antipodarum did not experience discernable phenotypic variance‐related benefits of sex and were more likely to die before achieving reproductive maturity than the asexuals. Taken together, these results suggest that under benign conditions, polyploidy does not impose obvious life history costs in P. antipodarum and that sexual P. antipodarum persist despite substantial life history disadvantages relative to their asexual counterparts.     

How to go extinct by mating too much: population consequences of male mate choice and efficiency in a sexual–asexual species complex

Selection acting on individuals is not predicted to maximize population persistence, yet examples that explicitly quantify conflicts between individual and population level benefits are scarce. One such conflict occurs over sexual reproduction because of the cost of sex: sexual populations that suffer the cost of producing males have only half the growth rate compared to asexuals. Male behaviour can additionally impact population dynamics in a variety of ways, and here we study an example where the impact is unusually clear: the riddle of persistence of sperm‐dependent sexual–asexual species complexes. Here, a sexually reproducing host species coexists with an ameiotically reproducing all‐female sperm parasite. Sexual–asexual coexistence should not be stable because the proportion of asexually reproducing females will rapidly increase and the relative abundance of the sexually reproducing host species will decline. A severe shortage of males will lead to sperm limitation for sexual and asexual females and the system collapses. Male mate choice could reduce the reproductive potential of the asexual species and thus potentially prevent the collapse. In the gynogenetic (sperm‐dependent parthenogenetic) Amazon molly Poecilia formosa and its host (P. latipinna or P. mexicana), males discriminate against asexual females to some extent. Using a population‐dynamical model, we examine the population dynamics of this species complex with varying strengths of male discrimination ability and efficiency with which they locate females and produce sperm. The sexual species would benefit from stronger discrimination, thus preventing being displaced by the asexual females. However, males would be required to evolve preferences that are probably too strong to be purely based upon selection acting on individuals. We conclude that male behaviour does not fully prevent but delays extinction, yet this is highly relevant because low local extinction rates strongly promote coexistence as a metapopulation.     

Isolation and characterization of di‐ and trinucleotide microsatellites in the freshwater snail Potamopyrgus antipodarum

The freshwater prosobranch snail Potamopyrgus antipodarum is an important model system for studying the maintenance of sexual reproduction in its native New Zealand. Since introduction to the UK in the 1850s, the species has spread throughout Europe and recently the USA. We present the first microsatellites for P. antipodarum. Using two isolation protocols, which are qualitatively compared, we have developed PCR primers for three di‐ and four trinucleotide microsatellites. All loci proved to be polymorphic, when screened for variability in several UK populations. These markers should be of considerable utility in future population and ecological genetics studies of this species.     

Radical amino acid mutations persist longer in the absence of sex

Harmful mutations are ubiquitous and inevitable, and the rate at which these mutations are removed from populations is a critical determinant of evolutionary fate. Closely related sexual and asexual taxa provide a particularly powerful setting to study deleterious mutation elimination because sexual reproduction should facilitate mutational clearance by reducing selective interference between sites and by allowing the production of offspring with different mutational complements than their parents. Here, we compared the rate of removal of conservative (i.e., similar biochemical properties) and radical (i.e., distinct biochemical properties) nonsynonymous mutations from mitochondrial genomes of sexual versus asexual Potamopyrgus antipodarum, a New Zealand freshwater snail characterized by coexisting and ecologically similar sexual and asexual lineages. Our analyses revealed that radical nonsynonymous mutations are cleared at higher rates than conservative changes and that sexual lineages eliminate radical changes more rapidly than asexual counterparts. These results are consistent with reduced efficacy of purifying selection in asexual lineages allowing harmful mutations to remain polymorphic longer than in sexual lineages. Together, these data illuminate some of the population‐level processes contributing to mitochondrial mutation accumulation and suggest that mutation accumulation could influence the outcome of competition between sexual and asexual lineages.     

Can phosphorus limitation contribute to the maintenance of sex? A test of a key assumption

Why sex is so common remains unclear; what is certain is that the predominance of sex despite its profound costs means that it must confer major advantages. Here, we use elemental and nucleic acid assays to evaluate a key element of a novel, integrative hypothesis considering whether sex might be favoured because of differences in body composition between sexuals and asexuals. We found that asexual Potamopyrgus antipodarum, a New Zealand snail, have markedly higher bodily phosphorus and nucleic acid content per unit mass than sexual counterparts. These differences coincide with and are almost certainly linked to the higher ploidy of the asexuals. Our results are the first documented body composition differences between sexual and asexual organisms, and the first detected phenotypic difference between sexual and asexual P. antipodarum, an important natural model system for the study of the maintenance of sex. These findings also verify a central component of our hypothesis that competition between diploid sexuals and polyploid asexuals could be influenced by phosphorus availability.     

Sex ratio,sex‐specific pattern in vegetative growth and gemma production in an aquatic liverwort,Scapania undulata (Marchantiophyta: Scapaniaceae)

Scapania undulata is an aquatic dioicous liverwort growing in shallow streams in boreal to subtropical zones. We studied the expressed sex ratio, sex‐specific differences in shoot architecture and possible trade‐off between sexual and asexual reproduction in ten populations of S. undulata by surveying 100 plots in ten streams in southern Finland. The expressed sex ratio was male biased, in contrast with the sex ratio in most dioicous bryophytes. It was also highly variable between the streams, but individual plots frequently comprised shoots from only one sex. The overproduction of males might be a strategy to overcome sperm dilution and ensure fertilization over longer distances in water. No size differences between females and males were detected, but they differed in branching patterns. Evidence for a higher cost of sexual reproduction in females than males can be seen from the following: the male‐biased sex ratio; low number of sex‐expressing female shoots in female‐only plots; no co‐occurrence of gemmae and female sex organs on a single branch, and no more than one sexual branch per female shoot. In contrast, high gemma production of male and female sex‐expressing shoots indicates a minimal trade‐off between sexual and asexual reproduction. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 229–241.