Host-Size-Matching in a Sperm-Dependent Asexual Fish

Among vertebrates, there are approximately fifty sperm-dependent asexual species, all of which are of hybrid origin. Sperm-dependent asexual vertebrates use sperm from one, or both, of their parental species for reproduction. To address the hypothesis that selection has resulted in asexual phenotypes that resemble their host’s phenotype, I studied size, shape, and genetic variation (using six microsatellite loci) in nine populations of a sperm-dependent asexual fish, Chrosomus eos-neogaeus, in Alberta, Canada. These nine populations differed in the presence or absence of each of the two parental species: three populations coexist with C. eos, two populations coexist with C. neogaeus, and four populations coexist with both parental species. Consistent with my hypothesis, I found that C. eos-neogaeus tended to match the body size of the parental species with which they coexist, and that C. eos-neogaeus that coexist with only C. eos were genetically divergent from C. eos-neogaeus that are syntopic with only C. neogaeus. The genetic divergence among C. eos-neogaeus populations was independent of geographic distance among populations, and estimates of quantitative trait divergence among C. eos-neogaeus populations exceeded neutral expectations. These observations suggest that processes other than migration, mutation, and drift are likely shaping the diversity of C. eos-neogaeus in the lakes sampled for this study. For example, sexual selection leading to host mimicry, or natural selection leading to environmental adaptation, may explain the observed pattern of host-size matching.     

Distribution of Phoxinus eos,Phoxinus neogaeus,and Their Asexually-Reproducing Hybrids (Pisces: Cyprinidae) in Algonquin Provincial Park,Ontario

Hybrid Phoxinus are one of the few asexually reproducing vertebrates species. The distribution of hybrid Phoxinus among lakes in Algonquin Park, Ontario, was evaluated relative to the distribution of parental species and relative to physiochemical lake characteristics. No association between the distribution of hybrids and the distribution of parental species was found, suggesting that the hybrids can successfully coexist with either parental species. In addition, we found no association between hybrid distribution and the physiochemical characteristics of lakes, suggesting that the hybrids are generalists with respect to the ecological niches available in Algonquin Park. There was a difference between the physiochemical characteristics of lakes with and without the parental species P. neogaeus. The lakes containing P. neogaeus were lower elevation than the lakes containing the other parental species, P. eos. The difference in distribution between the parental species may therefore be due to different dispersal abilities, to later arrival following post-glacial dispersal, or to differences in ecology. These results suggest that asexual reproduction is a successful strategy for hybrid Phoxinus in Algonquin Park because these sperm-dependent asexual hybrids are able to survive and persist regardless of which parental species is present, and regardless of the physiochemical characteristics of their habitat.     

CLONAL INHERITANCE OF A DIPLOID NUCLEAR GENOME BY A HYBRID FRESHWATER MINNOW (PHOXINUS EOS-NEOGAEUS,PISCES: CYPRINIDAE)

Hybrids between the minnows Phoxinus eos and Phoxinus neogaeus coexist with a population of P. eos in East Inlet Pond, Coos Co., New Hampshire. Chromosome counts and flow cytometric analysis of erythrocyte DNA indicate that these hybrids include diploids, triploids, and diploid-triploid mosaics. The mosaics have both diploid and triploid cells in their bodies, even within the same tissues. All three hybrid types are heterozygous at seven putative loci for which P. eos and P. neogaeus are fixed for different allozymes, indicating that the hybrids carry one eos and one neogaeus haploid genome. The diploid hybrids are therefore P. eos-neogaeus, whereas the triploids and mosaics are derived from P. eos-neogaeus but have an extra eos or neogaeus genome in all or some of their cells. Diploid, triploid, and mosaic hybrids accept tissue grafts from diploid hybrids, indicating that all individuals carry the identical eos-neogaeus diploid genome. Thus, one P. eos-neogaeus clone exists at East Inlet Pond. Grafts among the triploids and mosaics or from these individuals to diploid hybrids are rejected, indicating that the third genome is different in each triploid and mosaic individual. In this study, diploid and mosaic hybrids, carrying the clonal eos-neogaeus genome, were bred in the laboratory with males of P. eos or P. neogaeus. Both diploid and mosaic hybrids produced diploid, triploid, and mosaic offspring, revealing the source of the three hybrid types present at East Inlet Pond. These offspring accepted grafts from P. eos-neogaeus individuals, indicating that they all had inherited the identical eos-neogaeus genome. Most grafts among triploid and mosaic progeny, or from these individuals to their diploid broodmates, were rejected, indicating that the third genome was different in each triploid and mosaic (as was observed in the wild hybrids) and was contributed by sperm from males of P. eos or P. neogaeus. Diploid progeny are produced if sperm serves only to stimulate embryogenesis; triploid or mosaic progeny are produced if the sperm genome is incorporated. Although based on a mode of reproduction that by definition results in a genetically identical community of individuals, i.e., gynogenesis, reproduction in hybrid Phoxinus results in a variety of genetically distinct individuals by the incorporation of sperm into approximately 50% of the diploid ova produced.     

Sperm-dependent parthenogens delay the spatial expansion of their sexual hosts

It has been known for long time that asexual organisms may affect the distribution of sexual taxa. In fact, such phenomenon is inherent in the concept of geographical parthenogenesis. On the other hand, it was generally hypothesized that sperm-dependent asexuals may not exercise the same effect on related sexual population, due to their dependence upon them as sperm-donors. Recently, however, it became clear that sperm-dependent asexuals may directly or indirectly affect the distribution of their sperm-hosts, but rather in a small scale. No study addressed the large-scale biogeographic effect of the coexistence of such asexuals with the sexual species. In our study we were interested in the effect of sexual–asexual coexistence on the speed of spatial expansion of the whole complex. We expand previously published Lotka–Volterra model of the coexistence of sexual and gynogenetic forms of spined loach (Cobitis; Teleostei) hybrid complex by diffusion. We show that presence of sperm-dependent parthenogens is likely to negatively affect the spatial expansion of sexuals, and hence the whole complex, compared to pure sexual population. Given that most of the known sperm-dependent asexual complexes are distributed in areas prone to climate-induced colonization/extinction events, we conclude that such mechanism may be an important agent in determining the biogeography of sexual taxa and therefore requires further attention including empirical tests.     

Equal fecundity in asexual and sexual mollies (<Emphasis Type="Italic">Poecilia</Emphasis>)

The evolution and maintenance of sexual reproduction is still one of the major unresolved problems in evolutionary biology. Sexual reproduction is fraught with a number of costs as compared to asexual reproduction. For example, sexuals have to produce males, which–given a 1:1 sex ratio—results in a two-fold advantage for asexuals that do not produce males. Consequently, asexuals will outperform and replace sexuals over time assuming everything else is equal. Nonetheless, a few cases of closely related asexuals and sexuals have been documented to coexist stably in natural systems. We investigated the presence of a two-fold cost in a unique system of three closely related fish species: the asexual Amazon Molly (Poecilia formosa), and two sexual species, Sailfin Molly (P. latipinna) and Atlantic Molly (P. mexicana). Amazon Molly reproduce gynogenetically (by sperm dependent parthenogenesis) and always coexist with one of the sexual species, which serves as sperm donor. In the laboratory, we compared reproductive output between P. formosa and P. mexicana as well as P. formosa and P. latipinna. We found no differences in the fecundity in either comparison of a sexual and the asexual species. Under the assumption of a 1:1 sex ratio, the asexual Amazon Molly should consequently have a full two-fold advantage and be able to outcompete sexuals over time. Hence, the coexistence of the species pairs in nature presents a paradox still to be solved.     

Diets of syntopic finescale dace,Phoxinus neogaeus,and northern redbelly dace,Phoxinus eos: a reflection of trophic morphology

Synopsis We examined relative abundance, distribution, and gut contents of Phoxinus eos and P. neogaeus in a small northern Michigan bog lake. P. eos were usually 5–100 x more abundant than P. neogaeus, but both species were more abundant near the lake margin than offshore. Both species fed primarily on algae and invertebrates associated with the bog mat, but differences in diet were associated with differences in trophic morphology. As expected, because of its relatively longer intestine and smaller mouth, P. eos consumed relatively more plant matter, especially green algae, and fewer macroinvertebrates than P. neogaeus. These dietary conclusions were supported by analysis of two independent data sets. Further work is needed to discriminate between the effects of body size and trophic morphology on diet.     

Juvenile survival in a unisexual/sexual complex of mollies

Unisexual species like the gynogenetic Amazon molly, Poecilia formosa, enjoy a twofold advantage over sexual species, because they do not produce males. Therefore, unisexuals should be able to outcompete and consequently, replace sexual species. For sperm-dependent (gynogenetic) unisexuals this creates a paradox: they cannot replace their sexual hosts without eradicating themselves. Thus, mechanisms must be in place to stabilize such mating systems. We assessed juvenile survivorship between asexual P. formosa and sexual Poecilia latipinna as a possible factor allowing for persistence and coexistence between the two sympatric species. Offspring of gynogenetic Amazon mollies did not differ significantly in survivorship compared to their sexual host, the Sailfin molly, P. latipinna. The presence of an adult female significantly reduced survival in both species, suggesting that filial cannibalisms operates in this system, but does not appear to play a role in stabilizing mixed sexual/asexual populations. Clark Hubbs, who spent 59 years at the University of Texas and was widely regarded as one of the state’s foremost researchers in the field of ichthyology, the study of fish, passed away February 3rd of 2008 after a long battle with colorectal cancer. He was 86.     

THE DISTRIBUTION OF LIFE-HISTORY VARIATION IN THE DAPHNIA PULEX COMPLEX

In the midwestern United States the Daphnia pulex complex consists of a mosaic of sexual and asexual populations, providing a useful model system for studying the evolutionary forces underlying the maintenance of sex. One asexual and two sexual populations were surveyed for genetic variation for isozymes, mitochondrial DNA, and life-history characters. While the sexual populations exhibited substantial levels of genetic variance for fitness characters, no variation was detected in the asexual population at any level. However, a parallel survey among asexual clones derived from other ponds revealed large amounts of quantitative variation among clones, even among those with the same molecular profile. As a group, the asexuals are more variable for life histories than are the sexual populations. The molecular data indicate a relatively recent origin for the extant asexual D. pulex. The polyphyletic origin of these clones, combined with their microevolutionary potential, provides an explanation for their broad geographic distribution. The distribution of sex in the complex cannot be explained with the standard models that assume an invariant asexual population in reproductive isolation from the parental species. Although the frequency of asexuality may be driven by the spread of a sex-limited meiosis suppressor through sexual populations, the complete displacement of sexuality may be prevented by ecological distinctions between the two classes of individuals. On average, the asexuals are larger but produce smaller clutches than the sexuals.     

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.     

VARIATION IN THE STRENGTH OF MALE MATE CHOICE ALLOWS LONG‐TERM COEXISTENCE OF SPERM‐DEPENDENT ASEXUALS AND THEIR SEXUAL HOSTS

In several asexual taxa, reproduction requires mating with related sexual species to stimulate egg development, even though genetic material is not incorporated from the sexuals (gynogenesis). In cases in which gynogens do not invest in male function, they can potentially have a twofold competitive advantage over sexuals because the asexuals avoid the cost of producing males. If unmitigated, however, the competitive success of the asexuals would ultimately lead to their own demise, following the extinction of the sexual species that stimulate egg development. We have studied a model of mate choice among sexual individuals and asexual gynogens, where males of the sexual species preferentially mate with sexual females over gynogenetic females, to determine if such mating preferences can stably maintain both gynogenetic and sexual individuals within a community. Our model shows that stable coexistence of gynogens and their sexual hosts can occur when there is variation among males in the degree of preference for mating with sexual females and when pickier males pay a higher cost of preference.     

Making it on their own: sperm-dependent hybrid fishes (Cobitis) switch the sexual hosts and expand beyond the ranges of their original sperm donors

Interspecific hybridization may result in asexual hybrid lineages that reproduce via parthenogenesis. Contrary to true parthenogens, sperm-dependent asexuals (gynogens and hybridogens) are restricted to the range of bisexual species, generally the parental taxa, by their need for a sperm donor. It has been documented that asexual lineages may rarely use sperm from a non-parental species or even switch a host. The available literature reports do not allow distinguishing, between whether such host switches arise by the expansion of asexuals out of their parental's range (and into that of another's) or by the local extinction of a parental population followed by a host switch. The present study combines new and previously collected data on the distribution and history of gynogenetic spined loaches (Cobitis) of hybrid origin. We identified at least three clonal lineages that have independently switched their sperm dependency to different non-parental Cobitis species, and in cases incorporated their genomes. Our current knowledge of European Cobitis species and their hybrids suggests that this pattern most probably results from the expansion of gynogenetic lineages into new areas. Such expansion was independent of the original parental species. This suggests that sperm dependence is not as restrictive to geographical expansion when compared with true parthenogenesis as previously thought.     

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.     

Female sperm limitation in natural populations of a sexual/asexual mating complex (Poecilia latipinna, Poecilia formosa)

In sperm-dependent sexual/asexual mating systems, male mate choice is critical for understanding the mechanisms behind apparent stability observed in natural populations. The gynogenetic Amazon molly (Poecilia formosa) requires sperm from sexual males (e.g. Poecilia latipinna) to trigger embryogenesis, but inheritance is strictly maternal. Consequently, males should try to avoid or reduce the cost of mating with asexuals. We investigated male mate choice by documenting the presence of sperm in natural populations and found that a higher proportion of sexual females had sperm than asexuals. In addition, among those females that had sperm, sexuals had more sperm than asexuals. Our results hint at a role for male mate choice as a stabilizing factor in such systems.     

Can punishment maintain sex?

Individuals who reproduce asexually have a two-fold advantage over their sexually-reproducing counterparts as they are able to reproduce twice as fast. Explaining why sexual reproduction is favoured over asexual reproduction therefore remains an important challenge in evolutionary biology. Various mechanisms involving resistance to parasites, adaptation to novel environments and helping to purge the genome of deleterious mutations have all been proposed as potential mechanisms which could promote the evolution of sex. A recent article has suggested that spiteful males may help to reduce the two-fold advantage of asexual females. Here I discuss this idea, and further ask whether punishment of asexual females by sexual females could be one way in which sexual reproduction could be maintained in groups of animals; in light of recent research on the repression of competition, it could be possible that asexual females which reproduce faster than their sexual counterparts will be punished for using group resources. It may therefore be possible that the behaviour of sexual individuals towards asexual females could have fitness consequences which could potentially reduce the two-fold advantage they gain from reproducing parthenogenetically.     

An asexual strain of Lysiphlebus fabarum (Hymenoptera: Braconidae) gains fitness in superparasitised hosts,but a sexual strain pays costs

1. The fitness impacts of two levels of superparasitism were compared in a sexual and an asexual strain of Lysiphlebus fabarum (Marshall) developing in black bean aphids. 2. Asexual females were larger than sexuals, had higher eggs loads, and better adult emergence in two of three treatments, but parasitised fewer available aphids in 24 h (80% fewer when foraging alone). 3. Superparasitism resulted in wasps that were larger than those emerging from singly parasitised aphids, and asexual females had larger ovaries, without delayed development. In contrast, sexual wasps took about 2 days longer to develop in superparasitised hosts, and females had smaller ovaries with lower egg loads. 4. The growth of host aphids bearing asexual larvae was not reduced relative to healthy aphids, except when heavily superparasitised, whereas sexual larvae reduced aphid growth in all treatments. 5. Elimination of supernumerary larvae was virtually complete at 4.5 days after parasitism by sexual females, but no elimination occurred among asexual larvae. The evolution of diminished aggression in asexual larvae may be facilitated by greater genetic similarity, without necessarily leading to gregarious development, assuming the extended life of supernumeraries somehow improves survivor fitness. 6. Sons of virgin sexual females developed faster than sons of mated females, implicating parental effects that slowed development of the latter. 7. It is concluded that asexual females of this strain use superparasitism to improve host quality, and thus the fitness of their daughters, whereas females of the sexual strain prioritise offspring number.     

Evolution of sexual mimicry in sperm-dependent all-female forms of Poeciliopsis (Pisces: Poeciliidae)

The twofold advantage of all-female reproduction is limited in many asexual lineages because females are sperm-dependent. Males of a related sexual-host species typically prefer conspecific females as mates. According to the “sexual mimicry” hypothesis, an all-female lineage that closely resembles females of the sexual-host species should have enhanced mating success. Examination of mating success in all-female fish of the genus Poeciliopsis supported this hypothesis. The excellent sexual mimicry of some all-female strains could have evolved through mutations within clonal lineages subsequent to their origins as interspecific hybrids. Alternatively, this mimicry may have been “frozen” from variation in the sexual gene pool when new unisexual lineages first arose. To test the latter hypothesis, we examined laboratory synthesized strains of the hybridogenetic fish P. monacha-lucida. The frozen variation hypothesis was supported by the present results.     

Population models of sperm-dependent parthenogenesis

Organisms that reproduce by sperm-dependent parthenogenesis are asexual clones that require sperm of a sexual host to initiate egg production, without the genome of the sperm contributing genetic information to the zygote. Although sperm-dependent parthenogenesis has some of the disadvantages of sex (requiring a mate) without the counterbalancing advantages (mixing of parental genotypes), it appears amongst a wide variety of species. We develop initial models for the density-dependent dynamics of animal populations with sperm-dependent parthenogenesis (pseudogamy or gynogenesis), based on the known biology of the common Enchytraeid worm Lumbricillus lineatus. Its sperm-dependent parthenogenetic populations are reproductive parasites of the hermaphrodite sexual form. Our logistic models reveal two alternative requirements for coexistence at density-dependent equilibria: (i) If the two forms differ in competitive ability, the form with the lower intrinsic birth rate must be compensated by a more than proportionately lower competitive impact from the other, relative to intraspecific competition, (ii) If the two forms differ in their intrinsic capacity to exploit resources, the sperm-dependent parthenogen must be superior in this respect and must have a lower intrinsic birth rate. In general for crowded environments we expect a sperm-dependent parthenogen to compete strongly for limiting resources with the sexual sibling species. Its competitive impact is likely to be weakened by its genetic uniformity, however, and this may suffice to cancel any advantage of higher intrinsic growth rate obtained from reproductive investment only in egg production. We discuss likely thresholds of coexistence for other sperm-dependent parthenogens. The fish Poeciliopsis monacha-lucida likewise obtains an intrinsic growth advantage from reduced investment in male gametes, and so its persistence is likely to depend on it being a poor competitor. The planarian Schmidtea polychroa obtains no such intrinsic benefit because it produces fertile sperm, and its persistence may depend on superior resource exploitation.     

Novel distribution pattern between coexisting sexual and obligate asexual variants of the true estuarine macroalga Ulva prolifera

Where sexual and asexual forms coexist within a species, the asexuals are often found to be prevalent in marginal habitats. This asexual distribution pattern has received evolutionary attention linked to the paradox of sex. In many marine species, there is a distributional trend of asexual modes being more common in lower salinity waters regarded as the ecogeographic marginal, being explained by negative effects of low salinities on sexual reproductive success. However, the distribution pattern of estuarine species recently adapted to low salinity waters has remained unknown. The brackish macroalga Ulva prolifera being a major benthic component of estuarine ecosystems includes a sexual variant and obligate asexual variants by means of motile spores. We examined the sexual–asexual distribution pattern of this alga along a salinity gradient in river estuaries. Surprisingly, opposite to the distributional trend of marine organisms, the results clearly showed the persistent predominance of sexuals in the lower salinity reaches than the asexuals. In addition, a frequent alternating of dioecious gametophytes and sporophytes in the sexual population was observed, suggesting the sexual reproductive process would be robustly performed in the low salinity waters. Considering U. prolifera had evolved from the ancestral marine species to become a true estuarine species of which the core habitat is the low salinity reaches, in a broad sense its sexual–asexual distribution pattern would be involved in asexual marginal occupations of the species range previously reported in other organisms. Based on the frozen niche variation model, we can give a concise explanation for the evolutionary process of this pattern; multiple asexuals with frozen genotypic variation had arisen from sexual ancestors undergoing low salinity adaptation and share the estuarine habitat with the sexuals at present.     

The coevolution of parasites with host-acquired immunity and the evolution of sex

Abstract. Here I present a deterministic model of the coevolution of parasites with the acquired immunity of their hosts, a system in which coevolutionary oscillations can be maintained. These dynamics can confer an advantage to sexual reproduction within the parasite population, but the effect is not strong enough to outweigh the twofold cost of sex. The advantage arises primarily because sexual reproduction impedes the response to fluctuating epistasis and not because it facilitates the response to directional selection—in fact, sexual reproduction often slows the response to directional selection. Where the cost of sexual reproduction is small, a polymorphism can be maintained between the sexuals and the asexuals. A polymorphism is maintained in which the advantage gained due to recombination is balanced by the cost of sex. At much higher costs of sex, a polymorphism between the asexual and sexual populations can still be maintained if the asexuals do not have a full complement of genotypes available to them, because the asexuals only outcompete those sexuals with which they share the same selected alleles. However, over time we might expect the asexuals to amass the full array of genotypes, thus permanently eliminating sexuals from the population. The sexuals may avoid this fate if the parasite population is finite. Although the model presented here describes the coevolution of parasites with the acquired immune responses of their hosts, it can be compared with other host-parasite models that have more traditionally been used to investigate Red Queen theories of the evolution of sex.     

Across-population variation in sex ratio in invasive Japanese Potamopyrgus antipodarum (Caenogastropoda: Rissooidea: Hydrobiidae)

Potamopyrgus antipodarum, a well-known invasive species, has both sexual and asexual forms in its native area, and is generally asexual in exotic areas. Potamopyrgus antipodarum was sampled from ten locations in central and western Japan, and the sex distribution was analyzed. Males were found in two of the ten localities; the percentages of males in these localities were 7.2 and 7.3 %, which is within the range of that observed in asexual populations in native and introduced areas. A flow cytometry analysis showed that populations with a low percentage of males (Kanro) and populations consisting only of females (Banyu Park, Hadano and Yugawara) and perhaps the population reported as having a high male percentage in a previous study (Chiba; 29.8 %) were triploid. Males from Chiba and Kanro produced sperm. Most of the females from Chiba stored sperm in their seminal receptacle, showing that they had copulated, but no females from Kanro stored sperm. Fertilization by males, normal development of fertilized eggs, and the fecundity of juveniles should be evaluated in further studies to demonstrate the sexual reproduction of P. antipodarum in Japan. The risk of introduction of sexual P. antipodarum individuals is further discussed in the article.