Phenotypic plasticity under the effects of multiple environmental variables.

Organisms exposed to major environmental change face atypical and stressful conditions across multiple environmental variables, yet studies of phenotypically plastic responses historically focus on one environmental variable at a time. Evaluating multivariate plasticity of traits across different, simultaneously varying environmental variables provides new insights into the fate of populations amidst environmental changes. We aimed to investigate plasticity in multivariate environments by (a) examining the individual and joint effects of two environmental variables and (b) calculating genotype-by-environment interactions and genetic correlations of character states to investigate potential evolutionary constraints. We performed a lab controlled-environment experiment under a full factorial design of low and high temperatures and salinities with multiple maternal lineages of a parthenogenetic freshwater snail, Potamopyrgus antipodarum. Our results revealed that predictions of plastic trait responses among multivariate environments may be unexpected due to nonadditive effects of environmental variables and varying magnitudes and orientations of genetic correlations among fitness-related traits. Considering multivariate environments provides deeper insight and advancement of understanding trait evolution by revealing trait patterns that would otherwise be missed in univariate studies.

Between-Generation Phenotypic and Epigenetic Stability in a Clonal Snail.

Epigenetic variation might play an important role in generating adaptive phenotypes by underpinning within-generation developmental plasticity, persistent parental effects of the environment (e.g., transgenerational plasticity), or heritable epigenetically based polymorphism. These adaptive mechanisms should be most critical in organisms where genetic sources of variation are limited. Using a clonally reproducing freshwater snail (Potamopyrgus antipodarum), we examined the stability of an adaptive phenotype (shell shape) and of DNA methylation between generations. First, we raised three generations of snails adapted to river currents in the lab without current. We showed that habitat-specific adaptive shell shape was relatively stable across three generations but shifted slightly over generations two and three toward a no-current lake phenotype. We also showed that DNA methylation specific to high-current environments was stable across one generation. This study provides the first evidence of stability of DNA methylation patterns across one generation in an asexual animal. Together, our observations are consistent with the hypothesis that adaptive shell shape variation is at least in part determined by transgenerational plasticity, and that DNA methylation provides a potential mechanism for stability of shell shape across one generation.

Regional epigenetic variation in asexual snail populations among urban and rural lakes.

Epigenetic variation has the potential to influence environmentally dependent development and contribute to phenotypic responses to local environments. Environmental epigenetic studies of sexual organisms confirm the capacity to respond through epigenetic variation. An epigenetic response could be even more important in a population when genetic variation is lacking. A previous study of an asexual snail, Potamopyrgus antipodarum, demonstrated that different populations derived from a single clonal lineage differed in both shell phenotype and methylation signature when comparing lake versus river populations. Here, we examine methylation variation among lakes that differ in environmental disturbance and pollution histories. Snails were collected from a more pristine rural Lake 1 (Lake Lytle), and two urban lakes, Lake 2 (Capitol Lake) and Lake 3 (Lake Washington) on the Northwest Pacific coast. DNA methylation was assessed for each sample population using methylated DNA immunoprecipitation, MeDIP, followed by next-generation sequencing. The differential DNA methylation regions (DMRs) identified among the different lake comparisons suggested a higher number of DMRs and variation between rural Lake 1 and one urban Lake 2, and between the two urban Lakes 2 and 3, but limited variation between the rural Lake 1 and urban Lake 3. DMR genomic characteristics and gene associations were investigated. The presence of site-specific differences between each of these lake populations suggest an epigenetic response to varied environmental factors. The results do not support an effect of geographic distance in these populations. The role of dispersal distance among lakes, population history, environmental pollution and stably inherited methylation versus environmentally triggered methylation in producing the observed epigenetic variation are discussed. Observations support the proposal that epigenetic alterations may associate with phenotypic variation and environmental factors and history of the different lakes.

The adaptive value of epigenetic mutation: Limited in large but high in small peripheral populations.

The fate of populations during range expansions, invasions and environmental changes is largely influenced by their ability to adapt to peripheral habitats. Recent models demonstrate that stable epigenetic modifications of gene expression that occur more frequently than genetic mutations can both help and hinder adaptation in panmictic populations. However, these models do not consider interactions between epimutations and evolutionary forces in peripheral populations. Here, we use mainland-island mathematical models and simulations to explore how the faster rate of epigenetic mutation compared to genetic mutations interacts with migration, selection and genetic drift to affect adaptation in peripheral populations. Our model focuses on cases where epigenetic marks are stably inherited. In a large peripheral population, where the effect of genetic drift is negligible, our analyses suggest that epimutations with random fitness impacts that occur at rates as high as 10-3 increase local adaptation when migration is strong enough to overwhelm divergent selection. When migration is weak relative to selection and epimutations with random fitness impacts decrease adaptation, we find epigenetic modifications must be highly adaptively biased to enhance adaptation. Finally, in small peripheral populations, where genetic drift is strong, epimutations contribute to adaptation under a wider range of evolutionary conditions. Overall, our results suggest that epimutations can change outcomes of adaptation in peripheral populations, which has implications for understanding conservation and range expansions and contractions, especially of small populations.

Epigenetics and adaptive phenotypic variation between habitats in an asexual snail.

In neo-Darwinian theory, adaptation results from a response to selection on relatively slowly accumulating genetic variation. However, more rapid adaptive responses are possible if selectable or plastic phenotypic variation is produced by epigenetic differences in gene expression. This rapid path to adaptation may prove particularly important when genetic variation is lacking, such as in small, bottlenecked, or asexual populations. To examine the potential for an epigenetic contribution to adaptive variation, we examined morphological divergence and epigenetic variation in genetically impoverished asexual populations of a freshwater snail, Potamopyrgus antipodarum, from distinct habitats (two lakes versus two rivers). These populations exhibit habitat specific differences in shell shape, and these differences are consistent with adaptation to water current speed. Between these same habitats, we also found significant genome wide DNA methylation differences. The differences between habitats were an order of magnitude greater than the differences between replicate sites of the same habitat. These observations suggest one possible mechanism for the expression of adaptive shell shape differences between habitats involves environmentally induced epigenetic differences. This provides a potential explanation for the capacity of this asexual snail to spread by adaptive evolution or plasticity to different environments.

Identifying coevolving loci using interspecific genetic correlations.

Evaluating the importance of coevolution for a wide range of evolutionary questions, such as the role parasites play in the evolution of sexual reproduction, requires that we understand the genetic basis of coevolutionary interactions. Despite its importance, little progress has been made identifying the genetic basis of coevolution, largely because we lack tools designed specifically for this purpose. Instead, coevolutionary studies are often forced to re-purpose single species techniques. Here, we propose a novel approach for identifying the genes mediating locally adapted coevolutionary interactions that relies on spatial correlations between genetic marker frequencies in the interacting species. Using individual-based multi-locus simulations, we quantify the performance of our approach across a range of coevolutionary genetic models. Our results show that when one species is strongly locally adapted to the other and a sufficient number of populations can be sampled, our approach accurately identifies functionally coupled host and parasite genes. Although not a panacea, the approach we outline here could help to focus the search for coevolving genes in a wide variety of well-studied systems for which substantial local adaptation has been demonstrated.

Quantifying the coevolutionary potential of multistep immune defenses.

Coevolutionary models often assume host infection by parasites depends on a single bout of molecular recognition. As detailed immunological studies accumulate, however, it becomes increasingly apparent that the outcome of host-parasite interactions more generally depends on complex multiple step infection processes. For example, in plant and animal innate immunity, recognition steps are followed by downstream effector steps that kill recognized parasites, with the outcome depending on an escalatory molecular arms race. Here, we explore the consequences of such multistep infection processes for coevolution using a genetically explicit model. Model analyses reveal that polymorphism is much greater at recognition loci than effector loci, that host-genotype by parasite-genotype (Gh × Gp) interactions are larger for the recognition step, and that the recognition step contributes more to local adaptation than the effector step. These results suggest that (1) local adaptation is more likely when fitness measures are related to recognition versus downstream effectors, (2) effector loci, while mechanistically important, are less likely to harbor the Gh × Gp variation that fuels coevolution, and (3) recognition loci are better candidates for genomic hotspots of coevolution.

Identifying the molecular basis of host-parasite coevolution: merging models and mechanisms.

Mathematical models of the coevolutionary process have uncovered consequences of host-parasite interactions that go well beyond the traditional realm of the Red Queen, potentially explaining several important evolutionary transitions. However, these models also demonstrate that the specific consequences of coevolution are sensitive to the structure of the infection matrix, which is embedded in models to describe the likelihood of infection in encounters between specific host and parasite genotypes. Traditional cross-infection approaches to estimating infection matrices might be unreliable because evolutionary dynamics and experimental sampling lead to missing genotypes. Consequently, our goal is to identify the likely structure of infection matrices by synthesizing molecular mechanisms of host immune defense and parasite counterdefense with coevolutionary models. This synthesis reveals that the molecular mechanisms of immune reactions, although complex and diverse, conform to two basic models commonly used within coevolutionary theory: matching infection and targeted recognition. Our synthesis also overturns conventional wisdom, revealing that the general models are not taxonomically restricted but are applicable to plants, invertebrates, and vertebrates. Finally, our synthesis identifies several important areas for future research that should improve the explanatory power of coevolutionary models. The most important among these include empirical studies to identify the molecular hotspots of genotypic specificity and theoretical studies examining the consequences of matrices that more accurately represent multistep infection processes and quantitative defenses.

Phenotypic plasticity of the introduced New Zealand mud snail, Potamopyrgus antipodarum, compared to sympatric native snails.

Phenotypic plasticity is likely to be important in determining the invasive potential of a species, especially if invasive species show greater plasticity or tolerance compared to sympatric native species. Here in two separate experiments we compare reaction norms in response to two environmental variables of two clones of the New Zealand mud snail, Potamopyrgus antipodarum, isolated from the United States, (one invasive and one not yet invasive) with those of two species of native snails that are sympatric with the invader, Fossaria bulimoides group and Physella gyrina group. We placed juvenile snails in environments with high and low conductivity (300 and 800 mS) in one experiment, and raised them at two different temperatures (16 °C and 22 °C) in a second experiment. Growth rate and mortality were measured over the course of 8 weeks. Mortality rates were higher in the native snails compared to P. antipodarum across all treatments, and variation in conductivity influenced mortality. In both experiments, reaction norms did not vary significantly between species. There was little evidence that the success of the introduced species is a result of greater phenotypic plasticity to these variables compared to the sympatric native species.

Consumer-resource interactions and the evolution of migration.

Theoretical studies have demonstrated that selection will favor increased migration when fitnesses vary both temporally and spatially, but it is far from clear how pervasive those theoretical conditions are in nature. Although consumer-resource interactions are omnipresent in nature and can generate spatial and temporal variation, it is unknown even in theory whether these dynamics favor the evolution of migration. We develop a mathematical model to address whether and how migration evolves when variability in fitness is determined at least in part by consumer-resource coevolutionary interactions. Our analyses show that such interactions can drive the evolution of migration in the resource, consumer, or both species and thus supplies a general explanation for the pervasiveness of migration. Over short time scales, we show the direction of change in migration rate is determined primarily by the state of local adaptation of the species involved: rates increase when a species is locally maladapted and decrease when locally adapted. Our results reveal that long-term evolutionary trends in migration rates can differ dramatically depending on the strength or weakness of interspecific interactions and suggest an explanation for the evolutionary divergence of migration rates among interacting species.

Adaptive responses and invasion: the role of plasticity and evolution in snail shell morphology.

Invasive species often exhibit either evolved or plastic adaptations in response to spatially varying environmental conditions. We investigated whether evolved or plastic adaptation was driving variation in shell morphology among invasive populations of the New Zealand mud snail (Potamopyrgus antipodarum) in the western United States. We found that invasive populations exhibit considerable shell shape variation and inhabit a variety of flow velocity habitats. We investigated the importance of evolution and plasticity by examining variation in shell morphological traits 1) between the parental and F1 generations for each population and 2) among populations of the first lab generation (F1) in a common garden, full-sib design using Canonical Variate Analyses (CVA). We compared the F1 generation to the parental lineages and found significant differences in overall shell shape indicating a plastic response. However, when examining differences among the F1 populations, we found that they maintained among-population shell shape differences, indicating a genetic response. The F1 generation exhibited a smaller shell morph more suited to the low-flow common garden environment within a single generation. Our results suggest that phenotypic plasticity in conjunction with evolution may be driving variation in shell morphology of this widespread invasive snail.

Invasive genotypes are opportunistic specialists not general purpose genotypes.

It is not clear which forms of plasticity in fitness-related traits are associated with invasive species. On one hand, it may be better to have a robust performance across environments. On the other, it may be beneficial to take advantage of limited favorable conditions. We chose to study a worldwide invasive species, Potamopyrgus antipodarum, and compare the plasticity of life-history traits of a sample of invasive genotypes to a sample of ancestral-range genotypes. We examined the responses to salinity in this freshwater snail because it varies spatially and temporally in the introduced range and contributes to variation in fitness in our system. We used a recently developed statistical method that quantifies aspects of differences in the shape among reaction norms. We found that the invasive lineages survived and reproduced with an increased probability at the higher salinities, and were superior to ancestral-range lineages in only two traits related to reproduction. Moreover, we found that in terms of traits related to growth, the invasive lineages have a performance optimum that is shifted to higher salinities than the ancestral-range lineages as well as having a narrower niche breadth. Contrary to the prediction of the general purpose genotype hypothesis, we found that invasive lineages tended to be opportunistic specialists.

The maintenance of sex, clonal dynamics, and host-parasite coevolution in a mixed population of sexual and asexual snails.

Sexual populations should be vulnerable to invasion and replacement by ecologically similar asexual females because asexual lineages have higher per capita growth rates. However, as asexual genotypes become common, they may also become disproportionately infected by parasites. The Red Queen hypothesis postulates that high infection rates in the common asexual clones could periodically favor the genetically diverse sexual individuals and promote the short-term coexistence of sexual and asexual populations. Testing this idea requires comparison of competing sexual and asexual lineages that are attacked by natural parasites. To date no such data have been available. Here, we report on long-term dynamics and parasite coevolution in a "mixed" (sexual and asexual) population of snails (Potamopyrgus antipodarum). We found that, within 7-10 years, the most common clones were almost completely replaced by initially rare clones in two different habitats, while sexuals persisted throughout the study period. The common clones, which were initially more resistant to infection, also became more susceptible to infection by sympatric (but not allopatric) parasites over the course of the study. These results are consistent with the Red Queen hypothesis and show that the coevolutionary dynamics predicted by the theory may also favor sexual reproduction in natural populations.

Hybrid fitness in a locally adapted parasite.

The parasite (Red Queen) hypothesis for the maintenance of sexual reproduction and genetic diversity assumes that host-parasite interactions result from tight genetic specificity. Hence, hybridization between divergent parasite populations would be expected to disrupt adaptive gene combinations, leading to reduced infectivity on exposure to parental sympatric hosts, as long as gene effects are nonadditive. In contrast, hybridization would not cause reduced infectivity on allopatric hosts unless the divergent parasite populations possess alleles that are intrinsically incompatible when they are combined. In three different experiments, we compared the infectivity of locally adapted parasite (trematode) populations with that of F(1) hybrid parasites when exposed to host (snail) populations that were sympatric to one of the two parasite populations. We tested for intrinsic genetic incompatibilities in two experiments by including one host population that was allopatric to both parasite populations. As predicted, when the target host populations were sympatric to the parasite populations, the hybrids were significantly less infective than the parental average, while hybrid parasites on allopatric hosts were not, thereby ruling out intrinsic genetic incompatibilities. The results are consistent with nonadditive gene effects and tightly specific host-driven selection underlying parasite divergence, as envisioned by coevolutionary theory and the Red Queen hypothesis.

Extremely high secondary production of introduced snails in rivers.

The functional importance of invasive animals may be measured as the degree to which they dominate secondary production, relative to native animals. We used this approach to examine dominance of invertebrate secondary production by invasive New Zealand mudsnails (Potamopyrgus antipodarum) in rivers. We measured secondary production of mudsnails and native invertebrates in three rivers in the Greater Yellowstone Area (Wyoming, USA): Gibbon River, Firehole River, and Polecat Creek. Potamopyrgus production was estimated by measuring in situ growth rates and multiplying by monthly biomass; native invertebrate production was estimated using size frequency and instantaneous growth methods. Mudsnail growth rates were high (up to 0.06 d(-1)) for juvenile snails and much lower for adult females (0.003 d(-1)). Potamopyrgus production in Polecat Creek (194 g x m(-2) x yr(-1)) was one of the highest values ever reported for a stream invertebrate. Native invertebrate production ranged from 4.4 to 51 g x m(-2) x yr(-1). Potamopyrgus was the most productive taxon and constituted 65-92% of total invertebrate productivity. Native invertebrate production was low in all streams. Based on a survey of production measures from uninvaded rivers, the distribution of secondary production across taxa was much more highly skewed toward the invasive dominant Potamopyrgus in the three rivers. We suggest that this invasive herbivorous snail is sequestering a large fraction of the carbon available for invertebrate production and altering food web function.

Host sex and local adaptation by parasites in a snail-trematode interaction.

One of the leading theories for the evolutionary stability of sex in eukaryotes relies on parasite-mediated selection against locally common host genotypes (the Red Queen hypothesis). As such, parasites would be expected to be better at infecting sympatric host populations than allopatric host populations. Here we examined all published and unpublished infection experiments on a snail-trematode system (Potamopyrgus antipodarum and Microphallus sp., respectively). A meta-analysis demonstrated significant local adaptation by the parasite, and a variance components analysis showed that the variance due to the host-parasite interaction far exceeded the variance due to the main effects of host source and parasite source. The meta-analysis also indicated that asexual host populations were more resistant to allopatric sources of parasites than were (mostly) sexual host populations, but we found no significant differences among parasite populations in the strength of local adaptation. This result suggests that triploid asexual snails are more resistant to remote sources of parasites, but the parasite has, through coevolution, overcome the difference. Finally, we found that the degree of local adaptation did not depend on the genetic distance among host populations. Taken together, the results demonstrate that the parasites are adapted, on average, to infecting their local host populations and suggest that they may be a factor in selecting against common host genotypes in natural populations.

HOST-PARASITE COEVOLUTION: EVIDENCE FOR RARE ADVANTAGE AND TIME-LAGGED SELECTION IN A NATURAL POPULATION.

In theory, parasites can create time-lagged, frequency-dependent selection in their hosts, resulting in oscillatory gene-frequency dynamics in both the host and the parasite (the Red Queen hypothesis). However, oscillatory dynamics have not been observed in natural populations. In the present study, we evaluated the dynamics of asexual clones of a New Zealand snail, Potamopyrgus antipodarum, and its trematode parasites over a five-year period. During the summer of each year, we determined host-clone frequencies in random samples of the snail to track genetic changes in the snail population. Similarly, we monitored changes in the parasite population, focusing on the dominant parasite, Microphallus sp., by calculating the frequency of clones in samples of infected individuals from the same collections. We then compared these results to the results of a computer model that was designed to examine clone frequency dynamics for various levels of parasite virulence. Consistent with these simulations and with ideas regarding dynamic coevolution, parasites responded to common clones in a time-lagged fashion. Finally, in a laboratory experiment, we found that clones that had been rare during the previous five years were significantly less infectible by Microphallus when compared to the common clones. Taken together, these results confirm that rare host genotypes are more likely to escape infection by parasites; they also show that host-parasite interactions produce, in a natural population, some of the dynamics anticipated by the Red Queen hypothesis.

FLAT REACTION NORMS AND "FROZEN" PHENOTYPIC VARIATION IN CLONAL SNAILS (POTAMOPYRGUS ANTIPODARUM).

The Frozen Niche-Variation hypothesis (FNV) suggests that clones randomly sample and "freeze" the genotypes of their ancestral sexual populations. Hence, each clone expresses only a fraction of the total niche-use variation observed in the sexual population, which may lead to selection for ecological specialization and coexistence of clones. A generalized form of the FNV model suggests that the same is true for life-history (as well as other) traits that have important fitness consequences, but do not relate directly to niche use. We refer to the general form of the model as the Frozen Phenotypic Variation (FPV) model. A mixed population of sexual and parthenogenetic snails (Potamopyrgus antipodarum) in a New Zealand lake allowed us to examine the phenotypic variation expressed by coexisting clones in two benthic habitats, and to compare that variation to the sexual population. Three clones were found primarily in an aquatic macrophyte zone composed of Isoetes kirkii (1.5-3.0 m deep), and three additional clones were found in a deeper macrophyte zone composed of Elodea canadensis (4.0-6.0 m deep). These clones showed significant variation between habitats, which mirrored that observed in the sexual population. Specifically, clones and sexuals from the deeper habitat matured at a larger size and had larger broods. There was also significant among-clone variation within habitats; and as expected under the FPV model, the within-clone coefficients of variation for size at maturity were low in both habitats when compared to the sexual population. In addition, we found four clones that were common in both macrophyte zones. The reaction norms of these clones were flat across habitats, suggesting little phenotypic plasticity for morphology or life-history traits. Flat reaction norms, high among-clone variation, and low coefficients of variation (relative to the sexual population) are in accordance with the FPV model for the origin of clonal lineages. We also measured the prevalence of infection by trematode larvae to determine whether clones are inherently more or less infectable, or whether they are freezing phenotypic variation for resistance from the sexual population. We did this in the deep habitats of the lake where recycling of the parasite by the vertebrate host is unlikely, thereby reducing the complications raised by frequency-dependent responses of parasites to host genotypes. We found no indication that clones are either more or less infectable than the resident sexual population. Taken together, our results suggest that phenotypic variation for both life-history traits and resistance to parasites is frozen by clones from the local sexual population.

THE GEOGRAPHY OF COEVOLUTION: COMPARATIVE POPULATION STRUCTURES FOR A SNAIL AND ITS TREMATODE PARASITE.

Gene flow and the genetic structure of host and parasite populations are critical to the coevolutionary process, including the conditions under which antagonistic coevolution favors sexual reproduction. Here we compare the genetic structures of different populations of a freshwater New Zealand snail (Potamopyrgus antipodarum) with its trematode parasite (Microphallus sp.) using allozyme frequency data. Allozyme variation among snail populations was found to be highly structured among lakes; but for the parasite there was little allozyme structure among lake populations, suggesting much higher levels of parasite gene flow. The overall pattern of variation was confirmed with principal component analysis, which also showed that the organization of genetic differentiation for the snail (but not the parasite) was strongly related to the geographic arrangement of lakes. Some snail populations from different sides of the Alps near mountain passes were more similar to each other than to other snail populations on the same side of the Alps. Furthermore, genetic distances among parasite populations were correlated with the genetic distances among host populations, and genetic distances among both host and parasite populations were correlated with "stepping-stone" distances among lakes. Hence, the host snail and its trematode parasite seem to be dispersing to adjacent lakes in a stepping-stone fashion, although parasite dispersal among lakes is clearly greater. High parasite gene flow should help to continuously reintroduce genetic diversity within local populations where strong selection might otherwise isolate "host races." Parasite gene flow can thereby facilitate the coevolutionary (Red Queen) dynamics that confer an advantage to sexual reproduction by restoring lost genetic variation.

GENETIC STRUCTURE OF COEXISTING SEXUAL AND CLONAL SUBPOPULATIONS IN A FRESHWATER SNAIL (POTAMOPYRGUS ANTIPODARUM).

We examined clonal diversity and the distribution of both clonal and sexual genotypes in a single population of freshwater snails (Potamopyrgus antipodarum) in which diploid sexual individuals and triploid parthenogens coexist. A genetic analysis of individuals from three habitat zones in Lake Alexandrina, New Zealand revealed extremely high clonal diversity: 165 genotypes among 605 clonal individuals. The frequency of triploid clonal individuals increased with increasing depth in the lake, and most of the individual clones were habitat specific, suggesting that differences among habitats are important in structuring the clonal subpopulation. There were also high levels of clonal diversity within habitats, suggesting frequent origins of habitat-specific clones. In contrast, diploid sexual individuals were proportionately more common in the shallow regions of the lake (where infection by trematode larvae is highest), and there was no significant spatial structure in the sexual subpopulation. We suggest that habitat specialization by clones, as well as parasite-mediated selection against common clones, are important factors affecting the structure of this mixed population of sexual and clonal snails.