BACKGROUND
divergent selection for different habitats has long been proposed as a contributing factor to the evolution of species  <cit> .
regardless of geographic associations, ecological speciation from strong single selective agents, such as predation, or from the additive effects of multiple selective agents  now have empirical support  <cit> .
a remaining theoretical gap is a detailed understanding of the role of the breeding system in speciation  <cit> .
although many eukaryotic taxa reproduce both sexually and asexually  <cit> , there are still few empirical studies of speciation beyond strictly sexual taxa  <cit> .
because many organisms with mixed breeding systems are excellent dispersers  <cit> , breeding systems with even a small amount of sexual reproduction should suffer homogenization of ecologically differentiated lineages  <cit> .
thus, taxa with mixed breeding systems coupled with high dispersal should possess a weak capacity for rapid ecological radiation compared to strict asexuals and sexuals  <cit> .
others, however, have proposed that priority effects where early colonization offers an advantage and divergent selection are frequently strong enough to overcome the homogenizing effects of such mixed breeding systems  <cit> .
cladocerans contain several candidate groups for detailed empirical study of ecological and genetic divergence in a system characterized by both sexual and asexual reproduction .
these microcrustaceans often inhabit insular freshwater systems  with a mosaic of selective regimes through many environmental factors such as ephemerality, temperature, nutrient availability, competition, and predation  <cit> .
for example, a common contrast occurs between lakes with visual vertebrate predators that can select for smaller prey species, and lakes dominated by gape-limited invertebrate predators that favor cladoceran communities dominated by large or helmeted species  <cit> , as prey vulnerability is a function of prey size relative to predator size.
predation has been proposed to drive evolutionary divergence in morphology and behaviour  <cit>  and speciation in cladocerans  <cit> .
still, there is little knowledge of ecological speciation among sister species of lacustrine cladocerans, where sexual reproduction is relatively infrequent.
the cladocerans, daphnia parvula fordyce,  <dig> and daphnia retrocurva forbes,  <dig> , provide an opportunity to study the role of ecological factors in promoting rapid divergence in potentially young lacustrine taxa with a mixed breeding system.
although there is variation in the frequency of sex among populations, both species typically reproduce predominantly by clonal asexual reproduction with a few periodic episodes of sex throughout the season  <cit> .
in north america, d. retrocurva has a more northerly distribution occurring from the central midwest through northern canada, whereas d. parvula occurs from south america through the southern parts of canada.
however, the species overlap largely near the southern extent of the glacial lake zone in north america where rare co-occurrence is documented ,  <cit> .
brooks  <cit>  proposed that more northerly d. retrocurva is a postglacial derivative of d. parvula based on their distribution and d. retrocurva inhabiting more novel post-glacial large lake habitats.
the two species have a sister group relationship, but no multi-population genetic studies or detailed studies of genetic divergence have been carried out  <cit> .
daphnia parvula lacks the anterior retrocurved helmet  that appears to be controlled by polyploid cells in the head region of d. retrocurva  <cit> .
the helmet undergoes cyclomorphosis in d. retrocurva  <cit> , which can be enhanced by cues from invertebrate predators  <cit>  and laboratory studies also show decreased vulnerabilities of d. retrocurva to invertebrate predation with increased helmet size  <cit> .
importantly, multigenerational lab culture experiments failed to convert the head shape of d. retrocurva into the non-helmeted head shape of d. parvula  <cit> .
although the body size and tail spine length in d. parvula can be influenced by predator cues, attempts to induce helmet production have also been unsuccessful  <cit> , suggesting the morphological differences between these two species likely has a genetic component.
additionally, although d. retrocurva is a classic example of seasonal polymorphism, experiments reveal that phenotypic plasticity also fails to explain the morphological differences between the two species  <cit> .
although there are no studies that specifically address ecological differentiation between d. retrocurva and d. parvula, there is substantial evidence that the interaction of predation with helmet phenotypes is evolutionarily and ecologically relevant.
kerfoot and weider  <cit>  for example, show that the abundance of d. retrocurva and larger size classes of helmets in d. retrocurva, are positively associated with the increased abundance and size of the invertebrate cladoceran predator, leptodora, and reduced levels of planktivorous fish over time.
likewise, following the introduction of leptodora in a lake, a shift from d. parvula  was observed to helmeted or large daphnia  <cit> , suggesting a role for predation as a selective agent for helmets.
in contrast, a sixteen-year study  <cit>  found repeated smooth, continuous transitions between large bodied daphnia and small daphnia  associated with planktivorous fish density.
additionally, the abundance of d. retrocurva in glacial lakes is positively correlated with the abundance of invertebrate predators, and the abundance of introduced d. parvula is negatively associated with rates of invertebrate predation during a season  <cit> .
taken together, the translocation and natural experiments are consistent with divergent selection from predation playing an isolating role, which could also affect genetic differentiation.
however, lukaszewski et al.
<cit>  found evidence that ecological conditions beyond predation can prevent the successful colonization of d. retrocurva into a lake containing d. parvula.
isolation affects population genetic patterns in a time dependent fashion.
omland et al.
<cit>  defined "intermediate divergence" as the stages between panmixis and reciprocal monophyly.
the lineage divergence continuum, as visualized by networks, begins with gene frequencies diverging with only older internal haplotypes shared , followed by a lack of haplotype sharing.
eventually, population-specific clades  are formed.
in post-glacial isolation, which has been proposed by brooks for d. parvula and d. retrocurva  <cit> , such intermediate divergence is often produced and haplotype sharing is limited to the older central haplotypes of the networks  <cit> .
in contrast, if isolation is caused by multiple glacial refugia during the late pleistocene, patterns of multiple geographic subclades along with multiple demographic expansions is predicted as observed in other daphnia  <cit> .
here, we specifically test for ecological and genetic divergence between d. parvula and d. retrocurva.
ecological speciation predicts that populations of d. parvula and d. retrocurva whose ranges overlap will be associated with ecologically different habitats.
if predation is a major selective agent, then we should detect differences in the relative vulnerabilities to habitat-specific predators.
if the morphotypes of "retrocurva" and "parvula" are isolated, then we expect to detect significant genetic differentiation in the zone of overlap.
further, under the postglacial isolation hypothesis, we expect intermediate divergence , a lack of multiple demographic expansions, and divergence times from coalescence modelling to be late or post-glacial.
we test these objectives through data culled from pre-existing databases, laboratory predation experiments, and population genetic analyses.
RESULTS
habitat differentiation
of the  <dig> lakes analyzed from the data collected from the environmental protection agency  of lakes in the northeastern united states,  <dig> contained d. parvula,  <dig> contained d. retrocurva, and one contained both species.
the discriminant analysis  indicated that abiotic variables significantly discriminated between lakes containing d. parvula and lakes containing d. retrocurva , with lake area, lake depth, and lake volume contributing most to this group separation.
the analysis of variance  for lake volume was significant , with d. retrocurva inhabiting lakes of larger volume than those of d. parvula .
canonical variate correlation coefficients derived from the discriminant analysis for each abiotic variable from the epa dataset along with the wilks' lambda p. the variables with the highest correlation with discriminant function  contribute most to the group separation .
of the  <dig> lakes analyzed from the department of natural resources  in wisconsin, there were  <dig> lakes with only d. parvula,  <dig> lakes with only d. retrocurva, and two lakes with both species.
in both co-occurrence lakes, one species dominated while the other was rare or only present in a single sampling period throughout the year.
the da indicated that invertebrate predators significantly discriminated between lakes inhabited by d. parvula and those inhabited by d. retrocurva .
the abundance of two copepod predators, acanothocyclops vernalis and epischura sp.
contributed most to this group separation .
daphnia retrocurva inhabits lakes with significantly greater abundances of both these invertebrate predators relative to habitats where d. parvula resides .
canonical variate correlation coefficients derived from the discriminant analysis for abundances for each invertebrate predator in the dnr dataset and the wilk's lambda p value.
the variables with the highest canonical variate correlations  contribute most to the group separation .
laboratory predation experiments
for the invertebrate predation experiment, there were significant effects of treatment , prey species , and the interaction on proportion of prey missing .
there was no difference between the proportion of prey missing of the two species in the controls; however, in the predator treatments d. parvula suffered significantly higher mortality by leptodora kindti than did d. retrocurva .
the predation rate of l. kindti on d. parvula was also higher  than on d. retrocurva , .
results for anova's on the proportion missing prey following the invertebrate and vertebrate predation experiments.
in the vertebrate predation experiment, there were significant effects of treatment  and treatment by species interaction for the proportion on prey missing .
there was no significant difference between the proportion of prey missing for d. parvula and d. retrocurva in the control treatments, but d. retrocurva suffered higher mortality by emerald shiners in the predator treatments than did d. parvula .
the predation rate of the fish on d. retrocurva was also higher  than that on d. parvula .
for all prey populations used in both predator experiments, the anova's indicated body length, from top of the compound eye to the base of the carapace  and total body length, from highest point of helmet or head to the tip of the tailspine  were both significant by species.
daphnia parvula was significantly smaller than d. retrocurva and this difference is accentuated when including cyclomorphic features .
the nested effect of the source population within species  was also significant for body length  and borderline significant for total body length , indicating that despite much variation across populations, d. parvula was consistently smaller than d. retrocurva.
in addition, d. parvula was relatively more transparent than d. retrocurva , which was still the case when only non-parthenogenic or non-gravid females were analyzed .
genetic analyses
the tree was comprised of monophyletic subclades of d. parvula and d. retrocurva , but there is insufficient information to place the root or to determine if the subclades form reciprocally monophyletic groups .
the genealogical sorting index indicates that the species do form two significantly diverged lineages .
mitochondrial and nuclear networks show haplotype sharing but there is differentiation between d. parvula and d. retrocurva with most of the shared haplotypes having a central or presumed ancestral location .
genealogical sorting index along with the probability statistic testing the null hypothesis that the gene copies labelled d. retrocurva and d. parvula form a single intermixed group.
the divergence time  was estimated to be at  <dig>  years ago when the migration prior was set to zero migrants/generation, and  <dig>  years ago with a migration prior set at  <dig> migrants/generation .
when treating the data as a single group, the mismatch distribution was not significantly different from what is expected under a single expansion .
the τ parameter estimating the date initiating the expansion  <cit>  was  <dig>   and the demographic expansion within this group was estimated at  <dig>  years ago .
the mismatch distributions within d. parvula and d. retrocurva were not significantly different from the expected distributions under the expansion model .
the τ parameter estimating the date initiating the expansion was  <dig>   for d. parvula and  <dig>   for d. retrocurva.
demographic expansion was estimated at  <dig>  years ago  for d. parvula and  <dig>  years ago  for d. retrocurva.
the results of the analysis of molecular variance  of both the individual markers and all four markers combined indicate a relatively small proportion of the total genetic variation is found between the two species.
in addition, the fixation indices demonstrate very high differentiation among populations within a species and within populations .
hierarchal amova results between species, among populations within species and within populations across both markers.
discussion
our findings illustrate rare co-occurrence of d. parvula and d. retrocurva in the field with strong spatial segregation in ecologically differentiated lakes supporting ecological isolation.
priority effects and dispersal limitation can contribute to habitat isolation  <cit> , but do not predict a pattern of distribution associated with lake type.
moreover, both species appear to be strong dispersers  <cit>  and unidirectional translocation studies have failed to result in colonization  <cit> .
the rare co-occurrence of parvula-like morphotypes with retrocurva described by brooks  <cit>  remains mysterious.
we failed to detect parvula-like specimens in multiple samples from the same embayment of lake ontario examined by brooks.
the rare parvula-like morphs in glacial lakes could be the result of recent inflow from shallow adjacent waters or transient ex-ephippial clones of parvula.
what are the ecological traits associated with isolation?
kerfoot and weider  <cit>  have elegantly shown that evolution of the helmet size  can be attributed to historical changes in invertebrate predation.
here we show that this process may also contribute to the spatial segregation of these two species in nature, as d. retrocurva inhabit larger lakes that contain higher abundances of invertebrate predators relative to lakes where d. parvula is found.
although the data collected did not permit us to examine if fish, or vertebrate predators discriminated between the habitats encountered by these two species, the abundance of d. parvula has been found to be positively associated with the abundance of planktivorous fish  <cit> .
we also were unable to make a direct association between the abiotic and biotic factors compared in this study since these data were obtained from different sources, yet deeper aquatic habitats can have higher abundances of invertebrate predators of daphnia because depth affords a refuge from visual predators for conspicuous invertebrate predators  <cit> .
unexpectedly, the lake data failed to reveal an association between the abundance of leptodora and daphnia species.
one possible explanation for the lack of association is the difficulty in quantifying abundances of vertically migrating leptodora in daylight hauls  <cit> , yet the abundances of d. retrocurva and leptodora have been found to be positively associated in other studies  <cit> .
we also found that d. parvula has an advantage relative to d. retrocurva in the presence of visual, vertebrate predation which may be a consequence of the smaller body size and greater transparency of d. parvula .
additionally, d. retrocurva has an advantage over d. parvula in the lab to the invertebrate predator tested.
thus, the present study and existing translocation and longitudinal evidence suggest that d. parvula and d. retrocurva have a selective advantage to different habitat-associated predators.
it is possible that predation may promote "immigrant inviability" between these two species, or selection against migrants between locally adapted populations, which is a direct result of natural selection reducing gene flow  <cit> .
the differing predation regimes likely contribute to the spatial segregation of these sister species, but additional local processes may to contribute as well in this system  <cit> .
our study found that abiotic chemical variables  did not discriminate between the two species' habitats, yet other processes that were not included in this study could also contribute to their habitat segregation such as competition, the interactive effects of multiple predators, priority effects, and dispersal  <cit> .
further studies are needed to evaluate the role these other factors may play in facilitating habitat isolation between d. parvula and d. retrocurva, yet evidence suggests that predation is a key factor in determining zooplankton community composition and can reduce the importance of other factors influencing the establishment of daphnia populations  <cit> .
our results support postglacial genetic divergence between d. parvula and d. retrocurva despite a mixed breeding system and high dispersal.
the networks, single demographic expansion patterns, and divergence estimates based on nuclear and mitochondrial dna are consistent with intermediate divergence and postglacial isolation.
this postglacial timing of divergence is consistent with the origins of the deep temperate glacial lakes  <cit>  that constitute the main habitat of d. retrocurva.
we note that the findings of intermediate divergence do not rule out actual or potential gene flow between d. retrocurva and d. parvula.
the amova reveals a relatively low proportion of the total genetic variation is found between the two species and genetic divergence associated with ecological processes is often characterized by intermediate divergence with ongoing gene flow  <cit> .
although daphnia are known to have high dispersal and colonization rates  <cit> , the fixation indices suggest strong genetic differentiation among intraspecific populations , suggesting natural selection may also be operating within species as well.
therefore, selection against migrants may lead to a reduction in gene flow in this system on two scales, between two species and the two larger classes of habitat types they reside in, and within a species and the individual local habitat each population resides in.
according to island population models, as long as the island population size increases more rapidly than the number of migrants, the per-generation genetic contribution of migrants should decrease and an equilibrium is reached  <cit>  which is likely in daphnia.
their cyclic parthenogenic reproduction, rapid population growth, and priority effects supported by a large resting bank coupled with local adaptations may allow residents to maintain large populations relative to incoming migrants.
many studies illustrate strong, local adaptations in daphnia populations and the reduced success of invaders  <cit> , this high genetic differentiation among intraspecific daphnia populations often leads to a monopolization effect  <cit>  in which predation, competition, reproduction, ontogeny, and tolerance to eutrophication have been implicated to promote  <cit> .
conflicting models have been proposed whereby mixed breeding systems either slow  <cit>  or accelerate divergence  <cit> .
in our study, despite their young age, d. retrocurva and d. parvula illustrate ecological divergence with respect to their habitats, morphology, and predator vulnerabilities suggesting possible selective advantages to each in their respective environments.
both species typically have a mixed breeding system with relatively infrequent sex  <cit>  and illustrate strong dispersal capabilities  <cit> .
although some believe that populations with mixed breeding systems are less likely to yield discrete or distinct groups relative to populations with strictly sexual or asexual reproduction  <cit> , this study illustrates two groups that have diverged into two distinct lineages and maintained this divergence with a mixed breeding system coupled with high dispersal, and more importantly over a relatively short period of time.
lynch and gabriel  <cit>  propose that a single generation of sex can reveal 50-75% of the genetic variance hidden by asexual reproduction.
therefore, reproduction marked by cyclic parthenogenesis could allow such rapid divergence through the combined benefits of rapid population growth rates that asexual reproduction offers along with the release of genetic variation in the few episodes of sexual reproduction  <cit> .
we postulate this mixed breeding system permits such rapid postglacial divergence of d. parvula and d. retrocurva, with selection against migrants  maintaining this divergence by reducing the homogenizing effects of dispersal.
CONCLUSIONS
we present evidence for rapid ecological, morphological, and genetic divergence of sister species of lacustrine cyclic parthenogens.
the species differ in genetically-based defensive structures and predation regimes.
predation has been implicated as a selective force promoting divergence among taxa in only a few animal lineages.
the present study suggests a role for predation in facilitating habitat isolation in young sister species with a mixed breeding system.
however, multifarious selection can bring about reproductive isolation more effectively than selection from a single factor, and the habitats of d. retrocurva and d. parvula differ in numerous ways that could affect selection.
although predation has been implicated as a key factor in determining community composition and establishment of daphnia  <cit> , other factors need to be further evaluated.
our results do indicate that a better understanding of the evolution of the numerous presumed intraspecific defensive-morphotypes of cladocerans is needed.
intermediate divergence of aquatic cyclic parthenogens is likely more common than previously thought.

 divergent selection for different habitats has long been proposed as a contributing factor to the evolution of species. regardless of geographic associations, ecological speciation from strong single selective agents, such as predation, or from the additive effects of multiple selective agents now have empirical support. a remaining theoretical gap is a detailed understanding of the role of the breeding system in speciation. although many eukaryotic taxa reproduce both sexually and asexually, there are still few empirical studies of speciation beyond strictly sexual taxa. because many organisms with mixed breeding systems are excellent dispersers, breeding systems with even a small amount of sexual reproduction should suffer homogenization of ecologically differentiated lineages. thus, taxa with mixed breeding systems coupled with high dispersal should possess a weak capacity for rapid ecological radiation compared to strict asexuals and sexuals. others, however, have proposed that priority effects where early colonization offers an advantage and divergent selection are frequently strong enough to overcome the homogenizing effects of such mixed breeding systems. cladocerans contain several candidate groups for detailed empirical study of ecological and genetic divergence in a system characterized by both sexual and asexual reproduction. these microcrustaceans often inhabit insular freshwater systems with a mosaic of selective regimes through many environmental factors such as ephemerality, temperature, nutrient availability, competition, and predation. for example, a common contrast occurs between lakes with visual vertebrate predators that can select for smaller prey species, and lakes dominated by gape-limited invertebrate predators that favor cladoceran communities dominated by large or helmeted species, as prey vulnerability is a function of prey size relative to predator size. predation has been proposed to drive evolutionary divergence in morphology and behaviour and speciation in cladocerans. still, there is little knowledge of ecological speciation among sister species of lacustrine cladocerans, where sexual reproduction is relatively infrequent. the cladocerans, daphnia parvula fordyce, and daphnia retrocurva forbes,, provide an opportunity to study the role of ecological factors in promoting rapid divergence in potentially young lacustrine taxa with a mixed breeding system. although there is variation in the frequency of sex among populations, both species typically reproduce predominantly by clonal asexual reproduction with a few periodic episodes of sex throughout the season. in north america, d. retrocurva has a more northerly distribution occurring from the central midwest through northern canada, whereas d. parvula occurs from south america through the southern parts of canada. however, the species overlap largely near the southern extent of the glacial lake zone in north america where rare co-occurrence is documented,. brooks proposed that more northerly d. retrocurva is a postglacial derivative of d. parvula based on their distribution and d. retrocurva inhabiting more novel post-glacial large lake habitats. the two species have a sister group relationship, but no multi-population genetic studies or detailed studies of genetic divergence have been carried out. daphnia parvula lacks the anterior retrocurved helmet that appears to be controlled by polyploid cells in the head region of d. retrocurva. the helmet undergoes cyclomorphosis in d. retrocurva, which can be enhanced by cues from invertebrate predators and laboratory studies also show decreased vulnerabilities of d. retrocurva to invertebrate predation with increased helmet size. importantly, multigenerational lab culture experiments failed to convert the head shape of d. retrocurva into the non-helmeted head shape of d. parvula. although the body size and tail spine length in d. parvula can be influenced by predator cues, attempts to induce helmet production have also been unsuccessful, suggesting the morphological differences between these two species likely has a genetic component. additionally, although d. retrocurva is a classic example of seasonal polymorphism, experiments reveal that phenotypic plasticity also fails to explain the morphological differences between the two species. although there are no studies that specifically address ecological differentiation between d. retrocurva and d. parvula, there is substantial evidence that the interaction of predation with helmet phenotypes is evolutionarily and ecologically relevant. kerfoot and weider for example, show that the abundance of d. retrocurva and larger size classes of helmets in d. retrocurva, are positively associated with the increased abundance and size of the invertebrate cladoceran predator, leptodora, and reduced levels of planktivorous fish over time. likewise, following the introduction of leptodora in a lake, a shift from d. parvula was observed to helmeted or large daphnia, suggesting a role for predation as a selective agent for helmets. in contrast, a sixteen-year study found repeated smooth, continuous transitions between large bodied daphnia and small daphnia associated with planktivorous fish density. additionally, the abundance of d. retrocurva in glacial lakes is positively correlated with the abundance of invertebrate predators, and the abundance of introduced d. parvula is negatively associated with rates of invertebrate predation during a season. taken together, the translocation and natural experiments are consistent with divergent selection from predation playing an isolating role, which could also affect genetic differentiation. however, lukaszewski et al. found evidence that ecological conditions beyond predation can prevent the successful colonization of d. retrocurva into a lake containing d. parvula. isolation affects population genetic patterns in a time dependent fashion. omland et al. defined "intermediate divergence" as the stages between panmixis and reciprocal monophyly. the lineage divergence continuum, as visualized by networks, begins with gene frequencies diverging with only older internal haplotypes shared, followed by a lack of haplotype sharing. eventually, population-specific clades are formed. in post-glacial isolation, which has been proposed by brooks for d. parvula and d. retrocurva, such intermediate divergence is often produced and haplotype sharing is limited to the older central haplotypes of the networks. in contrast, if isolation is caused by multiple glacial refugia during the late pleistocene, patterns of multiple geographic subclades along with multiple demographic expansions is predicted as observed in other daphnia. here, we specifically test for ecological and genetic divergence between d. parvula and d. retrocurva. ecological speciation predicts that populations of d. parvula and d. retrocurva whose ranges overlap will be associated with ecologically different habitats. if predation is a major selective agent, then we should detect differences in the relative vulnerabilities to habitat-specific predators. if the morphotypes of "retrocurva" and "parvula" are isolated, then we expect to detect significant genetic differentiation in the zone of overlap. further, under the postglacial isolation hypothesis, we expect intermediate divergence, a lack of multiple demographic expansions, and divergence times from coalescence modelling to be late or post-glacial. we test these objectives through data culled from pre-existing databases, laboratory predation experiments, and population genetic analyses.  habitat differentiation of the lakes analyzed from the data collected from the environmental protection agency of lakes in the northeastern united states, contained d. parvula, contained d. retrocurva, and one contained both species. the discriminant analysis indicated that abiotic variables significantly discriminated between lakes containing d. parvula and lakes containing d. retrocurva, with lake area, lake depth, and lake volume contributing most to this group separation. the analysis of variance for lake volume was significant, with d. retrocurva inhabiting lakes of larger volume than those of d. parvula. canonical variate correlation coefficients derived from the discriminant analysis for each abiotic variable from the epa dataset along with the wilks' lambda p. the variables with the highest correlation with discriminant function contribute most to the group separation. of the lakes analyzed from the department of natural resources in wisconsin, there were lakes with only d. parvula, lakes with only d. retrocurva, and two lakes with both species. in both co-occurrence lakes, one species dominated while the other was rare or only present in a single sampling period throughout the year. the da indicated that invertebrate predators significantly discriminated between lakes inhabited by d. parvula and those inhabited by d. retrocurva. the abundance of two copepod predators, acanothocyclops vernalis and epischura sp. contributed most to this group separation. daphnia retrocurva inhabits lakes with significantly greater abundances of both these invertebrate predators relative to habitats where d. parvula resides. canonical variate correlation coefficients derived from the discriminant analysis for abundances for each invertebrate predator in the dnr dataset and the wilk's lambda p value. the variables with the highest canonical variate correlations contribute most to the group separation. laboratory predation experiments for the invertebrate predation experiment, there were significant effects of treatment, prey species, and the interaction on proportion of prey missing. there was no difference between the proportion of prey missing of the two species in the controls; however, in the predator treatments d. parvula suffered significantly higher mortality by leptodora kindti than did d. retrocurva. the predation rate of l. kindti on d. parvula was also higher than on d. retrocurva,. results for anova's on the proportion missing prey following the invertebrate and vertebrate predation experiments. in the vertebrate predation experiment, there were significant effects of treatment and treatment by species interaction for the proportion on prey missing. there was no significant difference between the proportion of prey missing for d. parvula and d. retrocurva in the control treatments, but d. retrocurva suffered higher mortality by emerald shiners in the predator treatments than did d. parvula. the predation rate of the fish on d. retrocurva was also higher than that on d. parvula. for all prey populations used in both predator experiments, the anova's indicated body length, from top of the compound eye to the base of the carapace and total body length, from highest point of helmet or head to the tip of the tailspine were both significant by species. daphnia parvula was significantly smaller than d. retrocurva and this difference is accentuated when including cyclomorphic features. the nested effect of the source population within species was also significant for body length and borderline significant for total body length, indicating that despite much variation across populations, d. parvula was consistently smaller than d. retrocurva. in addition, d. parvula was relatively more transparent than d. retrocurva, which was still the case when only non-parthenogenic or non-gravid females were analyzed. genetic analyses the tree was comprised of monophyletic subclades of d. parvula and d. retrocurva, but there is insufficient information to place the root or to determine if the subclades form reciprocally monophyletic groups. the genealogical sorting index indicates that the species do form two significantly diverged lineages. mitochondrial and nuclear networks show haplotype sharing but there is differentiation between d. parvula and d. retrocurva with most of the shared haplotypes having a central or presumed ancestral location. genealogical sorting index along with the probability statistic testing the null hypothesis that the gene copies labelled d. retrocurva and d. parvula form a single intermixed group. the divergence time was estimated to be at years ago when the migration prior was set to zero migrants/generation, and years ago with a migration prior set at migrants/generation. when treating the data as a single group, the mismatch distribution was not significantly different from what is expected under a single expansion. the τ parameter estimating the date initiating the expansion was and the demographic expansion within this group was estimated at years ago. the mismatch distributions within d. parvula and d. retrocurva were not significantly different from the expected distributions under the expansion model. the τ parameter estimating the date initiating the expansion was for d. parvula and for d. retrocurva. demographic expansion was estimated at years ago for d. parvula and years ago for d. retrocurva. the results of the analysis of molecular variance of both the individual markers and all four markers combined indicate a relatively small proportion of the total genetic variation is found between the two species. in addition, the fixation indices demonstrate very high differentiation among populations within a species and within populations. hierarchal amova results between species, among populations within species and within populations across both markers. discussion our findings illustrate rare co-occurrence of d. parvula and d. retrocurva in the field with strong spatial segregation in ecologically differentiated lakes supporting ecological isolation. priority effects and dispersal limitation can contribute to habitat isolation, but do not predict a pattern of distribution associated with lake type. moreover, both species appear to be strong dispersers and unidirectional translocation studies have failed to result in colonization. the rare co-occurrence of parvula-like morphotypes with retrocurva described by brooks remains mysterious. we failed to detect parvula-like specimens in multiple samples from the same embayment of lake ontario examined by brooks. the rare parvula-like morphs in glacial lakes could be the result of recent inflow from shallow adjacent waters or transient ex-ephippial clones of parvula. what are the ecological traits associated with isolation? kerfoot and weider have elegantly shown that evolution of the helmet size can be attributed to historical changes in invertebrate predation. here we show that this process may also contribute to the spatial segregation of these two species in nature, as d. retrocurva inhabit larger lakes that contain higher abundances of invertebrate predators relative to lakes where d. parvula is found. although the data collected did not permit us to examine if fish, or vertebrate predators discriminated between the habitats encountered by these two species, the abundance of d. parvula has been found to be positively associated with the abundance of planktivorous fish. we also were unable to make a direct association between the abiotic and biotic factors compared in this study since these data were obtained from different sources, yet deeper aquatic habitats can have higher abundances of invertebrate predators of daphnia because depth affords a refuge from visual predators for conspicuous invertebrate predators. unexpectedly, the lake data failed to reveal an association between the abundance of leptodora and daphnia species. one possible explanation for the lack of association is the difficulty in quantifying abundances of vertically migrating leptodora in daylight hauls, yet the abundances of d. retrocurva and leptodora have been found to be positively associated in other studies. we also found that d. parvula has an advantage relative to d. retrocurva in the presence of visual, vertebrate predation which may be a consequence of the smaller body size and greater transparency of d. parvula. additionally, d. retrocurva has an advantage over d. parvula in the lab to the invertebrate predator tested. thus, the present study and existing translocation and longitudinal evidence suggest that d. parvula and d. retrocurva have a selective advantage to different habitat-associated predators. it is possible that predation may promote "immigrant inviability" between these two species, or selection against migrants between locally adapted populations, which is a direct result of natural selection reducing gene flow. the differing predation regimes likely contribute to the spatial segregation of these sister species, but additional local processes may to contribute as well in this system. our study found that abiotic chemical variables did not discriminate between the two species' habitats, yet other processes that were not included in this study could also contribute to their habitat segregation such as competition, the interactive effects of multiple predators, priority effects, and dispersal. further studies are needed to evaluate the role these other factors may play in facilitating habitat isolation between d. parvula and d. retrocurva, yet evidence suggests that predation is a key factor in determining zooplankton community composition and can reduce the importance of other factors influencing the establishment of daphnia populations. our results support postglacial genetic divergence between d. parvula and d. retrocurva despite a mixed breeding system and high dispersal. the networks, single demographic expansion patterns, and divergence estimates based on nuclear and mitochondrial dna are consistent with intermediate divergence and postglacial isolation. this postglacial timing of divergence is consistent with the origins of the deep temperate glacial lakes that constitute the main habitat of d. retrocurva. we note that the findings of intermediate divergence do not rule out actual or potential gene flow between d. retrocurva and d. parvula. the amova reveals a relatively low proportion of the total genetic variation is found between the two species and genetic divergence associated with ecological processes is often characterized by intermediate divergence with ongoing gene flow. although daphnia are known to have high dispersal and colonization rates, the fixation indices suggest strong genetic differentiation among intraspecific populations, suggesting natural selection may also be operating within species as well. therefore, selection against migrants may lead to a reduction in gene flow in this system on two scales, between two species and the two larger classes of habitat types they reside in, and within a species and the individual local habitat each population resides in. according to island population models, as long as the island population size increases more rapidly than the number of migrants, the per-generation genetic contribution of migrants should decrease and an equilibrium is reached which is likely in daphnia. their cyclic parthenogenic reproduction, rapid population growth, and priority effects supported by a large resting bank coupled with local adaptations may allow residents to maintain large populations relative to incoming migrants. many studies illustrate strong, local adaptations in daphnia populations and the reduced success of invaders, this high genetic differentiation among intraspecific daphnia populations often leads to a monopolization effect in which predation, competition, reproduction, ontogeny, and tolerance to eutrophication have been implicated to promote. conflicting models have been proposed whereby mixed breeding systems either slow or accelerate divergence. in our study, despite their young age, d. retrocurva and d. parvula illustrate ecological divergence with respect to their habitats, morphology, and predator vulnerabilities suggesting possible selective advantages to each in their respective environments. both species typically have a mixed breeding system with relatively infrequent sex and illustrate strong dispersal capabilities. although some believe that populations with mixed breeding systems are less likely to yield discrete or distinct groups relative to populations with strictly sexual or asexual reproduction, this study illustrates two groups that have diverged into two distinct lineages and maintained this divergence with a mixed breeding system coupled with high dispersal, and more importantly over a relatively short period of time. lynch and gabriel propose that a single generation of sex can reveal 50-75% of the genetic variance hidden by asexual reproduction. therefore, reproduction marked by cyclic parthenogenesis could allow such rapid divergence through the combined benefits of rapid population growth rates that asexual reproduction offers along with the release of genetic variation in the few episodes of sexual reproduction. we postulate this mixed breeding system permits such rapid postglacial divergence of d. parvula and d. retrocurva, with selection against migrants maintaining this divergence by reducing the homogenizing effects of dispersal.  we present evidence for rapid ecological, morphological, and genetic divergence of sister species of lacustrine cyclic parthenogens. the species differ in genetically-based defensive structures and predation regimes. predation has been implicated as a selective force promoting divergence among taxa in only a few animal lineages. the present study suggests a role for predation in facilitating habitat isolation in young sister species with a mixed breeding system. however, multifarious selection can bring about reproductive isolation more effectively than selection from a single factor, and the habitats of d. retrocurva and d. parvula differ in numerous ways that could affect selection. although predation has been implicated as a key factor in determining community composition and establishment of daphnia, other factors need to be further evaluated. our results do indicate that a better understanding of the evolution of the numerous presumed intraspecific defensive-morphotypes of cladocerans is needed. intermediate divergence of aquatic cyclic parthenogens is likely more common than previously thought. 
we propose that predation may have facilitated the rapid divergence between d. parvula and d. retrocurva and that intermediate divergence of aquatic cyclic parthenogens is likely more common than previously thought. lakes inhabited by d. parvula were smaller in size and contained lower densities of invertebrate predators compared to lakes containing d. retrocurva. in the laboratory, d. retrocurva was less vulnerable to invertebrate predation, whereas d. parvula was less vulnerable to vertebrate predation and was smaller and more transparent than d. retrocurva. despite their young age and mixed breeding system, d. parvula and d. retrocurva exhibit significant ecological and genetic divergence that is coincident with the formation of deep temperate glacial lakes. daphnia parvula and d. retrocurva showed strong habitat separation with rare co-occurrence. coalescence and population genetic modelling indicate a late or post glacial time of divergence with a demographic expansion. however, the role of ecological speciation in animals that reproduce predominantly asexually with periodic sex and strong dispersal, such as lacustrine cladocerans, is poorly understood. ecological shifts can promote rapid divergence and speciation. the species are significantly differentiated at mitochondrial and nuclear loci and form an intermediate genetic divergence pattern between panmixia and reciprocal monophyly. we tested this hypothesis with ecological, morphological, and multilocus coalescence analyses in the putative lacustrine sister species, daphnia parvula and daphnia retrocurva. proponents of the postglacial ecological isolation hypothesis for daphnia suggest that some species have formed postglacially in adjacent, but ecologically different habitats.
 ecological shifts can promote rapid divergence and speciation. however, the role of ecological speciation in animals that reproduce predominantly asexually with periodic sex and strong dispersal, such as lacustrine cladocerans, is poorly understood. these life history traits may slow or prevent ecological lineage formation among populations. proponents of the postglacial ecological isolation hypothesis for daphnia suggest that some species have formed postglacially in adjacent, but ecologically different habitats. we tested this hypothesis with ecological, morphological, and multilocus coalescence analyses in the putative lacustrine sister species, daphnia parvula and daphnia retrocurva.  daphnia parvula and d. retrocurva showed strong habitat separation with rare co-occurrence. lakes inhabited by d. parvula were smaller in size and contained lower densities of invertebrate predators compared to lakes containing d. retrocurva. in the laboratory, d. retrocurva was less vulnerable to invertebrate predation, whereas d. parvula was less vulnerable to vertebrate predation and was smaller and more transparent than d. retrocurva. the species are significantly differentiated at mitochondrial and nuclear loci and form an intermediate genetic divergence pattern between panmixia and reciprocal monophyly. coalescence and population genetic modelling indicate a late or post glacial time of divergence with a demographic expansion.  despite their young age and mixed breeding system, d. parvula and d. retrocurva exhibit significant ecological and genetic divergence that is coincident with the formation of deep temperate glacial lakes. we propose that predation may have facilitated the rapid divergence between d. parvula and d. retrocurva and that intermediate divergence of aquatic cyclic parthenogens is likely more common than previously thought. 