Population genetics of freshwater snails

TREE vol. 6, no. 12, December

1991

PopulationGeneticsof Freshwater Snails Freshwater snails have attracted the attention of biologists for a long time, because they are intermediate hosts ofschistosomes, agents of Gilharziases. However, populatio+genetic studies of freshwater snails only during the have been undertaken past decade, covering topics such as the relative roles of genetic drift and gene flow in subdivided populations and the roles of extinction and recolonization events in determining population structure. Other studies in freshwater snails have invesfigated the maintenance of sex and the evolution of selling, widening a debate restricted mainly to plant populations. The possi6le role of parasites in freshwatersnail population genetics has also been investigated. Terrestrial gastropods have long occupied a central position in evolutionary biology: classic studies on shell color and banding patterns of terrestrial snails, followed by thorough studies of population structure by protein electrophoresis, have had a major influence on the development of ecological and population genetics. Freshwater gastropods, by contrast, have received less attention, but recent work has shown that they too have much to offer to evolutionary biology. One of the basic concerns of population genetics is population genetic structure, i.e. the distribution of alleles at different loci within and among populations. This has been mainly investigated using protein electrophoresis’. important determinants of this distribution are movement and incorporation of alleles among populations (gene flow1 and random fixation of alleles within finite populations due to sampling effects (genetic drift12. These parameters have been studied in freshwater-snail populations, which are generally patchily distributed with their limits often clearly defined by wateravailability, depth, flow speed or calcium availability. In such populations, gene flow is promoted by passive dispersal during floods or transport of eggs by birds. Philippe lame is at the Dept of Ecology and Evolution, University of Chicago, II01 E 57th S. Chicago, IL 60637, USA; Bernard Delay is at the lnstitut des Sciences de I’Evolution, Laboratoire CCnhique et Environnement, pellier II. Place E Bataillon, Cedex. France

Universite Mont34095 Montpellier

Philippe Jarne and Bernard Delay Reductions in population size, or even extinction, can be caused by droughts. However, many freshwatersnail species are good recolonizers, as indicated by invasion of newly dug drainage systems’,3,4. Another important topic in population genetics is the evolution of mating systems5,6, particularly sex versus non-sex and selfing versus outcrossing. This question can be addressed in freshwater snails, because some prosobranch species are parthenogenetic and all basommatophoran species studied are self-fertile7,8 (Box I I. Population geneticists have recently analysed the evolution of mating systems and their influence on population genetic structure in freshwater snails. Finally, freshwater snails are intermediate hosts for various paraincluding human-infecting sites, schistosomes3. Recent attention to the possible influence of parasites on population structure and the evolution of mating systems5 has led to studies of snail-parasite interactions at the population level. Population genetic structure Clonal population structure in prosobranchs In the gonochoric Thiara (= Melanoides) balonnensis, studies have shown that populations generally consist of one clone, with genetic distance among clones dependent on geographic distance9. New clones presumably evolve by mutations and loss of alleles after geographic isolation, with the whole species accumulating considerable variation. In contrast, in Melanoides tuberculata. parthenogenetic populations seem to comprise many clones. Bisexual populations also occur in this species, and there is evidence of sexual reproduction. Their intrapopulation genetic diversity is higher than that of parthenogenetic whereas interpopupopulations, lation diversity is lowerlO. The maintenance of such genetic diversity in parthenogenetic populations may be due to mutation, clone hybridization or, more probably, episodic outcrossing as a consequence of the

occurrence of males frequency).

(even in low

Subdivided sexual populations Gene flow is a force generally preventing genetic differentiation of populations, and population geneticists have derived models to analyse the interaction of gene flow with other forces modifying gene frequencies, such as genetic drift or selection2. Various techniques are available to estimate gene flow and the forces acting within populations. Among the most widely used indices are Wright’s F statistics, which can be computed from allele frequencies. Within a group of populations, F statistics can be partitioned into components that give information, under various assumptions, about the forces acting within and among populations. For example, FsST measures the degree to which genetic variance is partitioned among populations. It varies between 0 and I, and can be used to estimate gene flow: low values indicate high population differentiation and generally low gene flow. Alternatively, gene flow can be estimated from the frequency of ‘private’ alleles (alleles present in only one population I?. In freshwater snails, F indices have been widely used”- Ii, because populations are patchily distributed. However, when genetic variability is very low, these indices are not very informative. This is unhappily the case in the most widely studied basommatophorans, the medically important tropical species1’,‘2,i6. An extreme example is Bulinus truncatus3, which shows almost no variability for seven electrophoretic loci among and within 20 populations from Morocco to Sudan’“.

Box 1, Freshwater gastropod taxonomy

I

There are two groups of freshwater gastropod. The prosobranchsare characterized by a branchial respiratory system and a gonochoristic reproductive system (separate sexes), and the bssommatq~horaasare pulmonate and hermaphrodite7,*.

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However, some species are variable enough to allow their population history to be traced. In Biomphalaria glabrata and B. pfeifferi, the analysis of allelicfrequency distribution showed high Fs, values, suggesting a prominent role for genetic drift”,‘?,‘“. In B. straminea, individuals were introduced to Hong Kong in the early 1970s and the resulting populations have retained a level of variability comparable to related species. This suggests that the number of immigrants was not very low and/or that populations have increased very quickly after their introduction. Also, the geographic distribution of various alleles indicates that some populations in this study derived from others by colonization, but that others were founded by a new introduction event13. High levels of polymorphism have been retained in some basommatophoran species, both among and within populations, as indicated by studies in North America and Europe in the genus Lymnaea”15. Bulinus cernicus, restricted to Mauritius, also exhibits high genetic variability”, whereas closely related species inhabiting much larger areas are very much less variable16. The main question is why these populations retain such a high level of polymorphism. High effec-

analysis Sex 2. Parent-offspring This analysis allows one to estimate the number of fathers, and also to test for the occurrence of setfing in hermaphrodite species, among the offspring of a given individual, using polymorphic markers (pigment markers, allozyme or even DNA fingerprinting). In freshwater snails, the hypotheses of either pure selfing, or pure outcrossing with only one partner, have been tested. Coupled with population-structure analysis, this analysis could also be used to estimate the selfing ratez2. Individuals sampled from the field are allowed to reproduce in isolation in laboratory condilions. The first offspring are reared until they reach a size compatible with analysis (e.g. analysis can be performed on embryos when using pigment markers). The mother genotype is then compared to that of her offspring. In hermaphrodite species, it is crucial to analyse the first individuals born in tie laboratory, because sperm srorage is a highly variable parameter emong ~indlvidoats.Thus, isolated individuals that usually outcroas may switch to selling after aome time in laboratory conditions.

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tive population size could be an explanation, if neutrality of alleles is assumed. However, L. peregra populations seem to be discrete units of limited area and number of individuals along the shores of Lake Geneva, even though they are connected by a high level of gene flow that maintains genetic homogeneity over the lake15, and the L. e/odes and B. cemicus populations studied are from nonpermanent habitats, probably subject to drastic population-size variations. Although this last effect might lead to fluctuation in allelic frequencies over years due to sampling effects, a considerable amount of stability has been shown in B. cernicus17. Moreover, the presence of private alleles suggests that gene flow among the populations studied is limited, even though they are geographically very close. Dillon’s’a20 analysis of the North American prosobranch Goniobasis proxima is a model study of the role of genetic drift within a group of populations. This species occurs in numerous highly discrete populationsls in the Appalachian creeks. One of its peculiar features is upstream migration into tributary rivers, by crawling against the current, which results in a highly fragmented range. Dillon’s allozyme data, analysed mainly as geneticdistance indices (genetic distances are an estimation of population difference generally computed from allelic frequencies), suggest that there is no current gene flow among most of the populations studied, even between closely neighboring populations, resulting in isolation with distance. However, it is possible that ancestral populations were connected by high gene flow’8. Fragmentation of range, and possibly local environmental pressures, could subsequently have resulted in high morphological divergence between isolated populations. The analysis of transplants from genetically distinct populations four years after introduction reinforced the idea that upstream gene flow is higher than downstream20. Moreover, this experiment - the only one using genetically marked individuals in natural populations of freshwater gastropods - showed that introduced genomes were more fit than local ones, which implies that

7997

the genetic differences between populations may in some cases be due to random processes. Breeding systems TCle maintenance of sex Sexual reproduction can be favored over parthenogenesis only if it confers an immediate fitness advantage greater than the twofold advantage of non-sex over sex?. Prosobranchs offer the opportunity issue, parto address this thenogenesis having evolved in some genera. One hypothesis explaining the maintenance of sex postulates the importance of parasites, which are supposed to create an advantage to sex in parasitized populations, because of the production of variable offspring*‘. Experimental evidence has been provided in favor of this hypothesis in the New Zealand snail species Potamopyrgus antipodarum - sexual populations tend to have higher levels of infection by parasitic trematodes, and strong local adaptations to parasites have been demonstrated2’. In Thiara balonnensis (see above), sexual reproduction might alternatively be maintained by a fluctuating environment, promoting cycles of population extinction and recolonization’). A further issue is related to the maintenance of males. The percentage of males in parthenogenetic species is highly variable, ranging from 0% in Thiara ba/onnensisq to up to 33% in some populations of Melanoides tubercu/ata’O. In sexual populations of the latter species, mother-offspring (Box 2) and allele frequency analyses indicate that males participate in reproduction. However, if outcrossing is the only mating system, it is unclear why the sex ratio should remain biased. On the other hand, the maintenance of outcrossing and parthenogenesis in the same populations implies that parthenogenesis must have a cost; otherwise only pure parthenogenetic populations would be produced?. Selfing versus oulcrossing Basommatophorans are simultaneous hermaphrodites, able to reproduce by both selfing and outcrossings. Outcrossing generally occurs as soon as copulation is possible (Fig. I I. Sperm storage

TREE vol. 6, no. 12, December

1991

after copulation with one or many partners allows isolated individuals to reproduce by outcrossing for up to five months in some species8,21,24. These phenomena have been investigated using various methods: population structure analysis; mother-offspring analysis; and crossing between strains with pigment or protein electrophoresis markers’3-‘7,23,24. Even if outcrossing seems to be the rule, some proteinelectrophoresis studies have shown a clear tendency for a deficiency of heterozygote genotypes at particular loci, which suggests that selfing could play a role in some species’*, although spatial subdivision of populations could explain these results. Inbreeding depression, a direct consequence of selfing, is likely to be one of the major selective forces in favor of outcrossing (Box 31. In snails, other factors, such as the difference of fecundity of selfing and outcrossing snails of the same inbreeding coefficient, or the earlier egg laying of outcrossing individuals, must be taken into account. Combined with inbreeding depression, these factors have been referred to as self-fertilization depression25, and have been explicitly studied in Bulinus globosus25 and in Lymnaea peregraXb. Both studies have shown that outcrossing is the main breeding system in the population studied, in agreement with previous results. Under what conditions can selfing be selected? Possibilities include low population density, adaptation to local environmental conditions or even parasitism (see below). However, none of these arguments have received any experimental support in studies of basommatophorans. A particular situation promoting seifing might be aphally (Box 41**. Partial selfing was suggested in Buhus crossing expertruncatus from iments27, and recent empirical and theoretical studies indicate the same conclusion2*, although selffertilization depression and selfing rate must be measured before any definite conclusions can be drawn. Snails and parasites Parasites can increase mortality and decrease reproduction2’,30,3’, or even eliminate it (‘parasitic castration”‘\. Studies in natural popu-

lations of freshwater snails have indicated that parasite prevalence is highly variable in time and space4. Parasites could indirectly influence the evolution of population genetic structure in some prosobranch species if they have a role in maintaining sex2’. By analogy, it can be argued that parasites might select for outcrossing in basommatophorans, because outcrossing generally maintains more heterozygosity than selfing. On the other hand, parasites could select for selfing by modifying snail copulatory behavior: parasitized snails could reduce their reproductive activity by reducing their mating activity and carrying on reproducing by selfing, which clearly has a lower cost compared with outcrossing. However, no evidence of selection by parasite pressure for either outcrossing or selfing has yet been provided. The influence of parasites on population structure may result from parasite resistance having a genetic basis, as shown in some snail laboratory stocksj2. For such a trait to evolve in natural populations, its cost must be less than the fitness cost of parasite infection. However, experimental results have shown that resistant snails were selectively disadvantaged in the presence of both susceptible snails schistosome parasitesj’. and Another possibility is that more polymorphic genomes may be more able to withstand infection. Infected Helisoma anceps from natural populations in a highly infected pond have indeed been reported to have a lower protein electrophoretic variability than uninfected snailsj3. However, genetic differences between infected and uninfected snails could also reflect spatial heterogeneity of the snail distribution, for example local subdivision of uninfected and infected snail groups. Similarly, the patchy distribution of snail susceptibility to schistosomes could be a consequence of genetic drift and differentiation within snail populationsr3. This hypothesis has been proposed on the basis of snail population structure, and requires further analysis 01 compatibility between snails and parasites, population genetic structure with polymorphic markers and

Fig. I. Lymnaea peregra IMiillerl. Copulation is unilateral in basommatophorans, one individual lwith a white dot) acting as male, another as female. The devaginated penial complex is apparent under the tentacle of the male-acting individual. These individuals are about I5mm in length

influence of interaction on life history traits of both snail and parasite. Conclusion The discrete nature of freshwatersnail populations is a key factor in the evolution of their population structure. In the well-documented case of Goniabasis proxima, gene flow is likely to be limited as there is little passive dispersal of individuals. Instead, individuals crawl actively upstream, hence increasing the spatial isolation of populations. This raises the possibility that genetic drift is an important force shaping population genetic structure’%?“. By contrast, inI tropical basommatophoran populations, gene flow promoted by passive dispersal might be more common. Thus, the evolution of population

Box3. Inbreeding depression andseMrt$ The potential advantages of selfing over outcrossing are manifold. Selfing allows an individual to father offspring by a combination of selfing and outcrossing, to reproduce even when the access to partners is limited and to produce locally adapted offspring. Moreover, there is no cost of copulation associated with selfing. However, a major disadvantage of selfing is inbreeding depression. Inbreeding depression is probably the main force behind the evolution of traits reducing or preventing selfing. In real situations, the inbreeding depression, d, can be estimated by: d=

1 - WJW,

with W, and WC being fitness in selffertilizatron and cross-fertilization respectively. There is generally selection for selfing when d< ‘12. Repeated generations of selfing may purge a population from inbreeding depression; thus, highly selfing populations are expected to have low inbreeding depression. On the other hand, outcrossing populations are assumed to have a high inbreeding depression.

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1991

Box4. Aphally Aphally27-2s in hermaphrodite snails is characterized by the lack of the male copulatory organ, which prevents outcrosaing as a male. However, aphallic individuals can outcross as female, or self-fertilize. Aphally has been reported in numerous species, but studied only in Bolinus truncafus. Pure aphallic, pure euphallic and mixed populations occur in this species. No strong evidence of a heritable component to aphally has been shown, although some results suggest that aphally has a genetic basis that appears to be palygenie. A constant ratio of aphallic:euphallic individuals can be maintained over many generations in selfing strains, whatever the sexual phenotype of the parent. This suggests a situation in which an individual has a given probability of giving birth to

an approximately fixed ratio of aphallic: euphallic offspring. The ratio is then expected to follow approximately a binomial distribution. However, some results indicate more variation in the observed proportion of aphallic offspring than expected, which suggests that nongenetic influence could also have a role in some populations.

genetic structure might be interpreted at the level of a group of populations’“. Freshwater snails are particularly suited to study at this level, because the influence of parasites and different mating systems I particularly selfing in basommatophoransl could be analysed. However, the number and origin of individuals founding populations, extinction rate and number of habitat patches remain to be estimated. The use of polymorphic markers is therefore necessary, and could also be used to further the understanding of the function and evolution of breeding systems. These markers could be provided by DNA fingerprinting; for instance, human minisatellite probes can be used to analyse mating systems in Bulinus globosus35. Finally, there are several other lines of research on freshwater snails that deserve mention. Polyploidy (up to octoploidyl has evolved in some species, apparently always in conjunction with uniparental reproduction; snails could be a model to study the evolution of polyploidy among animals. Freshwater-snail shells vary widely among populations within species [see Ref. 3). Genetic studies of shell variation may be helpful in resolving debate over evolutionary modes: many of the species studied in the Turkana Cenozoic mollusc fossil sequence, a ‘prominent case’ in favor of a punctuated pattern of evolution, are still extant36,37. The central questions concern the factors causing shell variation, and the

386

unknown correspondence between shell variation and speciation j7. Freshwater snails are suited for testing general models of breedingsystem evolution, especially in hermaphrodites, an area traditionally studied by plant population biologists. The evolution of selffertilization depression over many generations could be analysed, which could help the understanding of the genetic causes of inbreeding6.25.M. Aphally in B. truncatus could be used to understand the evolution from hermaphroditism to gonochorism. Eventually, sex allocation could also be studied. Acknowledgements This paper was written

while PI was a postdoctoral research associate at the Dept of Ecology and Evolution, University of Chicago and funded by the French Government lprogramme Lavoisierl. Thanks are due to B. Charlesworth for laboratory facilities, to D Charlesworth and S. Schrag for access to unpublished papers and to M. Morgan, M. Raymond, P. Sniegowski and M. Vianey-Liaud for critical reading. Our own research described in this paper has been funded by CNRS I PIREN I and ORSTOM.

References

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A.R. and Slatkin, M. Trends

Ecol. Evol. 6, 221-224

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