Quantifying the constraining influence of gene flow on adaptive divergence in the lake-stream threespine stickleback system

The constraining effect of gene flow on adaptive divergence is often inferred but rarely quantified. We illustrate ways of doing so using stream populations of threespine stickleback (Gasterosteus aculeatus) that experience different levels of gene flow from a parapatric lake population. In the Misty Lake watershed (British Columbia, Canada), the inlet stream population is morphologically divergent from the lake population, and presumably experiences little gene flow from the lake. The outlet stream population, however, shows an intermediate phenotype and may experience more gene flow from the lake. We first used microsatellite data to demonstrate that gene flow from the lake is low into the inlet but high into the outlet, and that gene flow from the lake remains relatively constant with distance along the outlet. We next combined gene flow data with morphological and habitat data to quantify the effect of gene flow on morphological divergence. In one approach, we assumed that inlet stickleback manifest well-adapted phenotypic trait values not constrained by gene flow. We then calculated the deviation between the observed and expected phenotypes for a given habitat in the outlet. In a second approach, we parameterized a quantitative genetic model of adaptive divergence. Both approaches suggest a large impact of gene flow, constraining adaptation by 80-86% in the outlet (i.e., only 14-20% of the expected morphological divergence in the absence of gene flow was observed). Such approaches may be useful in other taxa to estimate how important gene flow is in constraining adaptive divergence in nature.     

Differences in rheotactic responses contribute to divergent habitat use between parapatric lake and stream threespine stickleback

Migration among populations is widely thought to undermine adaptive divergence, assuming gene flow arises from random movement of individuals. If individuals instead differ in dispersal behavior, phenotype‐dependent dispersal can reduce the effective rate of gene flow or even facilitate divergence. For example, parapatric populations of lake and stream stickleback tend to actively avoid dispersing into the adjoining habitat. However, the behavioral basis of this nonrandom dispersal was previously unknown. Here, we show that lake and stream stickleback exhibit divergent rheotactic responses (behavioral response to currents). During the breeding season, wild‐caught inlet stream stickleback were better than lake fish at maintaining position in currents, faced upstream more, and spent more time in low‐current areas. As a result, stream fish expended significantly less energy in currents than did lake fish. These divergent rheotactic responses likely contribute to divergent habitat use by lake and stream stickleback. Although rheotactic differences were absent in nonbreeding fish, divergent behavior of breeding‐season fish may suffice for assortative mating by breeding location. The resulting reproductive isolation between lake and stream fish may explain the fine‐scale evolutionary differentiation in parapatric stickleback populations.     

PHENOTYPE-DEPENDENT NATIVE HABITAT PREFERENCE FACILITATES DIVERGENCE BETWEEN PARAPATRIC LAKE AND STREAM STICKLEBACK

Adaptive divergence between adjoining populations reflects a balance between the diversifying effect of divergent selection and the potentially homogenizing effect of gene flow. In most models of migration-selection balance, gene flow is assumed to reflect individuals' inherent capacity to disperse, without regard to the match between individuals' phenotypes and the available habitats. However, habitat preferences can reduce dispersal between contrasting habitats, thereby alleviating migration load and facilitating adaptive divergence. We tested whether habitat preferences contribute to adaptive divergence in a classic example of migration-selection balance: parapatric lake and stream populations of three-spine stickleback ( Gasterosteus aculeatus ). Using a mark-transplant-recapture experiment on morphologically divergent parapatric populations, we showed that 90% of lake and stream stickleback returned to their native habitat, reducing migration between habitats by 76%. Furthermore, we found that dispersal into a nonnative habitat was phenotype dependent. Stream fish moving into the lake were morphologically more lake-like than those returning to the stream (and the converse for lake fish entering the stream). The strong native habitat preference documented here increases the extent of adaptive divergence between populations two- to fivefold relative to expectations with random movement. These results illustrate the potential importance of adaptive habitat choice in driving parapatric divergence.     

Natural selection drives patterns of lake-stream divergence in stickleback foraging morphology

To what extent are patterns of biological diversification determined by natural selection? We addressed this question by exploring divergence in foraging morphology of threespine stickleback fish inhabiting lake and stream habitats within eight independent watersheds. We found that lake fish generally displayed more developed gill structures and had more streamlined bodies than did stream fish. Diet analysis revealed that these morphological differences were associated with limnetic vs. benthic foraging modes, and that the extent of morphological divergence within watersheds reflected differences in prey resources utilized by lake and stream fish. We also found that patterns of divergence were unrelated to patterns of phenotypic trait (co)variance within populations (i.e. the ‘line of least resistance’). Instead, phenotypic (co)variances were more likely to have been shaped by adaptation to lake vs. stream habitats. Our study thus implicates natural selection as a strong deterministic force driving morphological diversification in lake–stream stickleback. The strength of this inference was obtained by complementing a standard analysis of parallel divergence in means between discrete habitat categories (lake vs. stream) with quantitative estimates of selective forces and information on trait (co)variances.     

Partitioning the effects of isolation by distance,environment, and physical barriers on genomic divergence between parapatric threespine stickleback

Genetic divergence between populations is shaped by a combination of drift, migration, and selection, yielding patterns of isolation‐by‐distance (IBD) and isolation‐by‐environment (IBE). Unfortunately, IBD and IBE may be confounded when comparing divergence across habitat boundaries. For instance, parapatric lake and stream threespine stickleback (Gasterosteus aculeatus) may have diverged due to selection against migrants (IBE), or mere spatial separation (IBD). To quantitatively partition the strength of IBE and IBD, we used recently developed population genetic software (BEDASSLE) to analyze partial genomic data from three lake‐stream clines on Vancouver Island. We find support for IBD within each of three outlet streams (unlike prior studies of lake‐stream stickleback). In addition, we find evidence for IBE (controlling for geographic distance): the genetic effect of habitat is equivalent to geographic separation of ～1.9 km of IBD. Remarkably, of our three lake‐stream pairs, IBE is strongest where migration between habitats is easiest. Such microgeographic genetic divergence would require exceptionally strong divergent selection, which multiple experiments have failed to detect. Instead, we propose that nonrandom dispersal (e.g., habitat choice) contributes to IBE. Supporting this conclusion, we show that the few migrants between habitats are a nonrandom subset of the phenotype distribution of the source population.     

Can gene flow have negative demographic consequences? Mixed evidence from stream threespine stickleback

Dispersal and gene flow can have both positive and negative effects on population size, but little empirical support from nature exists for the negative effects. We test for such effects in a stream population of threespine stickleback (Gasterosteus aculeatus L.) that is subject to high gene flow from a lake and is thus maladapted to stream conditions. In this system, maladaptation increases with distance along the stream, and this increase is associated with decreasing population densities until stickleback are no longer present (2.5 km from the lake). We conducted field experiments to inform whether this association might reflect a negative role for gene flow in constraining population size and therefore causing a local range limit. We specifically tested predictions deriving from theory: peripheral populations should show partial local adaptation, be under strong selection and not simply be maintained by dispersal. First, a transplant experiment suggested a weak home-site advantage in the peripheral population. Second, a mark–recapture study showed directional selection for a stream-adapted phenotype in 1 of 2 years. Third, another mark–recapture experiment showed that dispersal is limited to the point that positive demographic effects of dispersal are probably minimal. We conclude that, although gene flow does constrain morphological maladaptation in the outlet stream population, the evidence for its contribution to population size and range limits is mixed. We discuss the implications of our work for the study of factors influencing the evolution of species'' ranges.     

Quantitative genetic inheritance of morphological divergence in a lake-stream stickleback ecotype pair: implications for reproductive isolation

Ecological selection against hybrids between populations occupying different habitats might be an important component of reproductive isolation during the initial stages of speciation. The strength and directionality of this barrier to gene flow depends on the genetic architecture underlying divergence in ecologically relevant phenotypes. We here present line cross analyses of inheritance for two key foraging-related morphological traits involved in adaptive divergence between stickleback ecotypes residing parapatrically in lake and stream habitats within the Misty Lake watershed (Vancouver Island, Canada). One main finding is the striking genetic dominance of the lake phenotype for body depth. Selection associated with this phenotype against first- and later-generation hybrids should therefore be asymmetric, hindering introgression from the lake to the stream population but not vice versa. Another main finding is that divergence in gill raker number is inherited additively and should therefore contribute symmetrically to reproductive isolation. Our study suggests that traits involved in adaptation might contribute to reproductive isolation qualitatively differently, depending on their mode of inheritance.     

Parallel and nonparallel ecological,morphological and genetic divergence in lake–stream stickleback from a single catchment

Parallel phenotypic evolution in similar environments has been well studied in evolutionary biology; however, comparatively little is known about the influence of determinism and historical contingency on the nature, extent and generality of this divergence. Taking advantage of a novel system containing multiple lake–stream stickleback populations, we examined the extent of ecological, morphological and genetic divergence between three‐spined stickleback present in parapatric environments. Consistent with other lake–stream studies, we found a shift towards a deeper body and shorter gill rakers in stream fish. Morphological shifts were concurrent with changes in diet, indicated by both stable isotope and stomach contents analysis. Performing a multivariate test for shared and unique components of evolutionary response to the distance gradient from the lake, we found a strong signature of parallel adaptation. Nonparallel divergence was also present, attributable mainly to differences between river locations. We additionally found evidence of genetic substructuring across five lake–stream transitions, indicating that some level of reproductive isolation occurs between populations in these habitats. Strong correlations between pairwise measures of morphological, ecological and genetic distance between lake and stream populations supports the hypothesis that divergent natural selection between habitats drives adaptive divergence and reproductive isolation. Lake–stream stickleback divergence in Lough Neagh provides evidence for the deterministic role of selection and supports the hypothesis that parallel selection in similar environments may initiate parallel speciation.     

Sexual isolation promotes divergence between parapatric lake and stream stickleback

Speciation can be initiated by adaptive divergence between populations in ecologically different habitats, but how sexually based reproductive barriers contribute to this process is less well understood. We here test for sexual isolation between ecotypes of threespine stickleback fish residing in adjacent lake and stream habitats in the Lake Constance basin, Central Europe. Mating trials exposing females to pairings of territorial lake and stream males in outdoor mesocosms allowing for natural reproductive behaviour reveal that mating occurs preferentially between partners of the same ecotype. Compared to random mating, this sexual barrier reduces gene flow between the ecotypes by some 36%. This relatively modest strength of sexual isolation is surprising because comparing the males between the two ecotypes shows striking differentiation in traits generally considered relevant to reproductive behaviour (body size, breeding coloration, nest size). Analysing size differences among the individuals in the mating trials further indicates that assortative mating is not related to ecotype differences in body size. Overall, we demonstrate that sexually based reproductive isolation promotes divergence in lake–stream stickleback along with other known reproductive barriers, but we also caution against inferring strong sexual isolation from the observation of strong population divergence in sexually relevant traits.     

Repeated and predictable patterns of ecotypic differentiation during a biological invasion: lake–stream divergence in parapatric Swiss stickleback

The relative importance of ecological selection and geographical isolation in promoting and constraining genetic and phenotypic differentiation among populations is not always obvious. Interacting with divergent selection, restricted opportunity for gene flow may in some cases be as much a cause as a consequence of adaptation, with the latter being a hallmark of ecological speciation. Ecological speciation is well studied in parts of the native range of the three‐spined stickleback. Here, we study this process in a recently invaded part of its range. Switzerland was colonized within the past 140 years from at least three different colonization events involving different stickleback lineages. They now occupy diverse habitats, ranging from small streams to the pelagic zone of large lakes. We use replicated systems of parapatric lake and stream populations, some of which trace their origins to different invasive lineages, to ask (i) whether phenotypic divergence occurred among populations inhabiting distinct habitats, (ii) whether trajectories of phenotypic divergence follow predictable parallel patterns and (iii) whether gene flow constrains divergent adaptation or vice versa. We find consistent phenotypic divergence between populations occupying distinct habitats. This involves parallel evolution in several traits with known ecological relevance in independent evolutionary lineages. Adaptive divergence supersedes homogenizing gene flow even at a small spatial scale. We find evidence that adaptive phenotypic divergence places constraints on gene flow over and above that imposed by geographical distance, signalling the early onset of ecological speciation.     

Evidence for asymmetric migration load in a pair of ecologically divergent stickleback populations

Gene flow between ecologically divergent populations can prevent local adaptation, resulting in lower mean fitness and directional selection within a population. Such maladaptation should tend to be stronger in populations receiving a relatively larger fraction of immigrants. We test this expectation by comparing the strength of selection in a pair of three-spine stickleback populations in adjoining but unequal-sized lake basins in British Columbia. A larger deeper basin is connected to a smaller shallower basin by a short channel that allows extensive migration between populations. The two basins contain distinct habitats and prey communities, and stickleback stomach contents and isotope ratios differ accordingly. Trophic morphology is correlated with diet, so we would expect these ecological differences to be accompanied by morphological divergence. However, high gene flow appears to constrain adaptive divergence: microsatellites indicate that the two basins represent a single panmictic gene pool, and phenotypic divergence is very subtle. As a result, fish in the smaller lake basin are subject to persistent directional selection towards a more benthic phenotype, whereas the larger population exhibits no significant selection. The results illustrate the potentially asymmetrical effect of migration-selection balance, and its effect on fitness within populations.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 273–287.     

When maladaptive gene flow does not increase selection

Populations receiving high maladaptive gene flow are expected to experience strong directional selection—because gene flow pulls mean phenotypes away from local fitness peaks. We tested this prediction by means of a large and replicated mark‐recapture study of threespine stickleback (Gasterosteus aculeatus) in two stream populations. One of the populations (outlet) experiences high gene flow from the lake population and its morphology is correspondingly poorly adapted. The other population (inlet) experiences very low gene flow from the lake population and its morphology is correspondingly well adapted. Contrary to the above prediction, selection was not stronger in the outlet than in the inlet, a result that forced us to consider potential reasons for why maladaptive gene flow might not increase selection. Of particular interest, we show by means of a simple population genetic model that maladaptive gene flow can—under reasonable conditions—decrease the strength of directional selection. This outcome occurs when immigrants decrease mean fitness in the resident population, which decreases the strength of selection against maladapted phenotypes. We argue that this previously unrecognized effect of gene flow deserves further attention in theoretical and empirical studies.     

Spatial phenotypic and genetic structure of threespine stickleback (Gasterosteus aculeatus) in a heterogeneous natural system,Lake Mývatn,Iceland

Eco‐evolutionary responses of natural populations to spatial environmental variation strongly depend on the relative strength of environmental differences/natural selection and dispersal/gene flow. In absence of geographic barriers, as often is the case in lake ecosystems, gene flow is expected to constrain adaptive divergence between environments – favoring phenotypic plasticity or high trait variability. However, if divergent natural selection is sufficiently strong, adaptive divergence can occur in face of gene flow. The extent of divergence is most often studied between two contrasting environments, whereas potential for multimodal divergence is little explored. We investigated phenotypic (body size, defensive structures, and feeding morphology) and genetic (microsatellites) structure in threespine stickleback (Gasterosteus aculeatus) across five habitat types and two basins (North and South) within the geologically young and highly heterogeneous Lake Mývatn, North East Iceland. We found that (1) North basin stickleback were, on average, larger and had relatively longer spines than South basin stickleback, whereas (2) feeding morphology (gill raker number and gill raker gap width) differed among three of five habitat types, and (3) there was only subtle genetic differentiation across the lake. Overall, our results indicate predator and prey mediated phenotypic divergence across multiple habitats in the lake, in face of gene flow.     

Fitness differences between parapatric lake and stream stickleback revealed by a field transplant

Molecular comparisons of populations diverging into ecologically different environments often reveal strong differentiation in localized genomic regions, with the remainder of the genome being weakly differentiated. This pattern of heterogeneous genomic divergence, however, is rarely connected to direct measurements of fitness differences among populations. We here do so by performing a field enclosure experiment in threespine stickleback fish residing in a lake and in three replicate adjoining streams, and displaying weak yet heterogeneous genomic divergence between these habitats. Tracking survival over 29 weeks, we consistently find that lake genotypes transplanted into the streams suffer greatly reduced viability relative to local stream genotypes and that the performance of F1 hybrid genotypes is intermediate. This observed selection against migrants and hybrids combines to a total reduction in gene flow from the lake into streams of around 80%. Overall, our study identifies a strong reproductive barrier between parapatric stickleback populations, and cautions against inferring weak fitness differences between populations exhibiting weak overall genomic differentiation.     

Evidence for morphological and adaptive genetic divergence between lake and stream habitats in European minnows (Phoxinus phoxinus, Cyprinidae)

Natural selection drives local adaptation, potentially even at small temporal and spatial scales. As a result, adaptive genetic and phenotypic divergence can occur among populations living in different habitats. We investigated patterns of differentiation between contrasting lake and stream habitats in the cyprinid fish European minnow (Phoxinus phoxinus) at both the morphological and genomic levels using geometric morphometrics and AFLP markers, respectively. We also used a spatial correlative approach to identify AFLP loci associated with environmental variables representing potential selective forces responsible for adaptation to divergent habitats. Our results identified different morphologies between lakes and streams, with lake fish presenting a deeper body and caudal peduncle compared to stream fish. Body shape variation conformed to a priori predictions concerning biomechanics and swimming performance in lakes vs. streams. Moreover, morphological differentiation was found to be associated with several environmental variables, which could impose selection on body and caudal peduncle shape. We found adaptive genetic divergence between these contrasting habitats in the form of 'outlier' loci (2.9%) whose genetic divergence exceeded neutral expectations. We also detected additional loci (6.6%) not associated with habitat type (lake vs. stream), but contributing to genetic divergence between populations. Specific environmental variables related to trophic dynamics, landscape topography and geography were associated with several neutral and outlier loci. These results provide new insights into the morphological divergence and genetic basis of adaptation to differentiated habitats.     

Repeated Lake-Stream Divergence in Stickleback Life History within a Central European Lake Basin

Life history divergence between populations inhabiting ecologically distinct habitats might be a potent source of reproductive isolation, but has received little attention in the context of speciation. We here test for life history divergence between threespine stickleback inhabiting Lake Constance (Central Europe) and multiple tributary streams. Otolith analysis shows that lake fish generally reproduce at two years of age, while their conspecifics in all streams have shifted to a primarily annual life cycle. This divergence is paralleled by a striking and consistent reduction in body size and fecundity in stream fish relative to lake fish. Stomach content analysis suggests that life history divergence might reflect a genetic or plastic response to pelagic versus benthic foraging modes in the lake and the streams. Microsatellite and mitochondrial markers further reveal that life history shifts in the different streams have occurred independently following the colonization by Lake Constance stickleback, and indicate the presence of strong barriers to gene flow across at least some of the lake-stream habitat transitions. Given that body size is known to strongly influence stickleback mating behavior, these barriers might well be related to life history divergence.     

Gene expression stasis and plasticity following migration into a foreign environment

Selection against migrants is key to maintaining genetic differences between populations linked by dispersal. However, migrants may mitigate fitness costs by proactively choosing among available habitats, or by phenotypic plasticity. We previously reported that a reciprocal transplant of lake and stream stickleback (Gasterosteus aculeatus) found little support for divergent selection. Here, we revisit that experiment to test whether phenotypic plasticity in gene expression may have helped migrants adjust to unfamiliar habitats. We measured gene expression profiles in stickleback via TagSeq and tested whether migrants between lake and stream habitats exhibited a plastic response to their new environment that allowed them to converge on the expression profile of adapted natives. We report extensive gene expression differences between genetically divergent lake and stream stickleback, despite gene flow . But for many genes, expression was highly plastic. Fish transplanted into the adjoining habitat partially converged on the expression profile typical of natives from their new habitat. This suggests that expression plasticity may soften the impact of migration. Nonetheless, lake and stream fish differed in survival rates and parasite infection rates in our study, implying that expression plasticity is not fast or extensive enough to fully homogenize fish performance.     

HOW MUCH OF THE VARIATION IN ADAPTIVE DIVERGENCE CAN BE EXPLAINED BY GENE FLOW? AN EVALUATION USING LAKE‐STREAM STICKLEBACK PAIRS

Abstract How much of the variation in adaptive divergence can be explained by gene flow? The answer to this question should objectively reveal whether gene flow generally places a substantial constraint on evolutionary diversification. We studied multiple independent lake‐stream population pairs of threespine stickleback (Gasterosteus acu‐leatus). For each pair, we quantified adaptive divergence based on morphological traits that have a genetic basis and are subject to divergent selection. We then estimated gene flow based on variation at five unlinked microsatellite loci. We found a consistent and significant pattern for morphological divergence to be positively correlated with genetic divergence and negatively correlated with gene flow. Statistical significance and the amount of variation explained varied within and among traits: 36.1–74.1% for body depth and 11.8–51.7% for gill raker number. Variation within each trait was the result of differences among methods for estimating genetic divergence and gene flow. Variation among traits likely reflects different strengths of divergent selection. We conclude that gene flow has a substantial effect on adaptive divergence in nature but that the magnitude of this effect varies among traits. An alternative explanation is that cause and effect are reversed: adaptive divergence is instead constraining gene flow. This effect seems relatively unimportant for our system because genetic divergence and gene flow were not correlated with ecologically relevant habitat features of lakes (surface area) or streams (width, depth, flow, canopy openness).     

Genetic divergence in morphology-performance mapping between Misty Lake and inlet stickleback

Different environments should select for different aspects of organismal performance, which should lead to correlated divergence in morphological traits that influence performance. The result should be genetic divergence in aspects of performance, morphology and associations ('maps') between morphology and performance. Testing this hypothesis requires quantifying performance and morphology in multiple populations after controlling for environmental differences, but this is rarely attempted. We used a common-garden experiment to examine morphology and several aspects of swimming performance within and between the lake and inlet populations of threespine stickleback (Gasterosteus aculeatus) from the Misty system, Vancouver Island, Canada. Controlling for body size, lake stickleback had shallower bodies, larger caudal fins and smaller pelvic girdles. With or without morphological covariates, lake stickleback showed greater performance in both sustained and burst swimming. In contrast, inlet stickleback showed greater manoeuverability than did lake stickleback in some analyses. Morphology-performance relationships were decoupled when considering variation within vs. between populations. Moreover, morphology-performance mapping differed between the two populations. Based on these observations, we advance a hypothesis for why populations adapting to different environments should show adaptive genetic divergence in morphology-performance mapping.     

The association of feeding behaviour with the resistance and tolerance to parasites in recently diverged sticklebacks

Divergent natural selection regimes can contribute to adaptive population divergence, but can be sensitive to human‐mediated environmental change. Nutrient loading of aquatic ecosystems, for example, might modify selection pressures by altering the abundance and distribution of resources and the prevalence and infectivity of parasites. Here, we used a mesocosm experiment to test for interactive effects of nutrient loading and parasitism on host condition and feeding ecology. Specifically, we investigated whether the common fish parasite Gyrodactylus sp. differentially affected recently diverged lake and stream ecotypes of three‐spined stickleback (Gasterosteus aculeatus). We found that the stream ecotype had a higher resistance to Gyrodactylus sp. infections than the lake ecotype, and that both ecotypes experienced a cost of parasitism, indicated by negative relationships between parasite load and both stomach fullness and body condition. Overall, our results suggest that in the early stages of adaptive population divergence of hosts, parasites can affect host resistance, body condition and diet.