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.     

Sensory trait variation contributes to biased dispersal of threespine stickleback in flowing water

Gene flow is widely thought to homogenize spatially separate populations, eroding effects of divergent selection. The resulting theory of ‘migration–selection balance’ is predicated on a common assumption that all genotypes are equally prone to dispersal. If instead certain genotypes are disproportionately likely to disperse, then migration can actually promote population divergence. For example, previous work has shown that threespine stickleback (Gasterosteus aculeatus) differ in their propensity to move up‐ or downstream (‘rheotactic response’), which may facilitate genetic divergence between adjoining lake and stream populations of stickleback. Here, we demonstrate that intraspecific variation in a sensory system (superficial neuromast lines) contributes to this variation in swimming behaviour in stickleback. First, we show that intact neuromasts are necessary for a typical rheotactic response. Next, we showed that there is heritable variation in the number of neuromasts and that stickleback with more neuromasts are more likely to move downstream. Variation in pectoral fin shape contributes to additional variation in rheotactic response. These results illustrate how within‐population quantitative variation in sensory and locomotor traits can influence dispersal behaviour, thereby biasing dispersal between habitats and favouring population divergence.     

Microhabitat contributes to microgeographic divergence in threespine stickleback

Since the New Synthesis, most migration-selection balance theory has predicted that there should be negligible differentiation over small spatial scales (relative to dispersal), because gene flow should erode any effect of divergent selection. Nevertheless, there are classic examples of microgeographic divergence, which theory suggests can arise under specific conditions: exceptionally strong selection, phenotypic plasticity in philopatric individuals, or nonrandom dispersal. Here, we present evidence of microgeographic morphological variation within lake and stream populations of threespine stickleback (Gasterosteus aculeatus). It seems reasonable to assume that a given lake or stream population of fish is well-mixed. However, we found this assumption to be untenable. We examined trap-to-trap variation in 34 morphological traits measured on stickleback from 16 lakes and 16 streams. Most traits varied appreciably among traps within populations. Both between-trap distance and microhabitat characteristics such as depth and substrate explained some of the within-population morphological variance. Microhabitat was also associated with genotype at particular loci but there was no genetic isolation by distance, implying that heritable habitat preferences may contribute to microgeographic variation. Our study adds to growing evidence that microgeographic divergence can occur across small spatial scales within individuals’ daily dispersal neighborhood where gene flow is expected to be strong.     

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.     

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.     

VARIABLE PROGRESS TOWARD ECOLOGICAL SPECIATION IN PARAPATRY: STICKLEBACK ACROSS EIGHT LAKE-STREAM TRANSITIONS

Divergent selection between contrasting habitats can sometimes drive adaptive divergence and the evolution of reproductive isolation in the face of initially high gene flow. "Progress" along this ecological speciation pathway can range from minimal divergence to full speciation. We examine this variation for threespine stickleback fish that evolved independently across eight lake-stream habitat transitions. By quantifying stickleback diets, we show that lake-stream transitions usually coincide with limnetic-benthic ecotones. By measuring genetically based phenotypes, we show that these ecotones often generate adaptive divergence in foraging morphology. By analyzing neutral genetic markers (microsatellites), we show that adaptive divergence is often associated with the presence of two populations maintaining at least partial reproductive isolation in parapatry. Coalescent-based simulations further suggest that these populations have diverged with gene flow within a few thousand generations, although we cannot rule out the possibility of phases of allopatric divergence. Finally, we find striking variation among the eight lake-stream transitions in progress toward ecological speciation. This variation allows us to hypothesize that progress is generally promoted by strong divergent selection and limited dispersal across the habitat transitions. Our study thus makes a case for ecological speciation in a parapatric context, while also highlighting variation in the outcome.     

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.     

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.     

Natural selection on MHC IIβ in parapatric lake and stream stickleback: Balancing,divergent, both or neither?

Major histocompatibility complex (MHC) genes encode proteins that play a central role in vertebrates' adaptive immunity to parasites. MHC loci are among the most polymorphic in vertebrates' genomes, inspiring many studies to identify evolutionary processes driving MHC polymorphism within populations and divergence between populations. Leading hypotheses include balancing selection favouring rare alleles within populations, and spatially divergent selection. These hypotheses do not always produce diagnosably distinct predictions, causing many studies of MHC to yield inconsistent or ambiguous results. We suggest a novel strategy to distinguish balancing vs. divergent selection on MHC, taking advantage of natural admixture between parapatric populations. With divergent selection, individuals with immigrant alleles will be more infected and less fit because they are susceptible to novel parasites in their new habitat. With balancing selection, individuals with locally rare immigrant alleles will be more fit (less infected). We tested these contrasting predictions using three‐spine stickleback from three replicate pairs of parapatric lake and stream habitats. We found numerous positive and negative associations between particular MHC IIβ alleles and particular parasite taxa. A few allele–parasite comparisons supported balancing selection, and others supported divergent selection between habitats. But, there was no overall tendency for fish with immigrant MHC alleles to be more or less heavily infected. Instead, locally rare MHC alleles (not necessarily immigrants) were associated with heavier infections. Our results illustrate the complex relationship between MHC IIβ allelic variation and spatially varying multispecies parasite communities: different hypotheses may be concurrently true for different allele–parasite combinations.     

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.     

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.     

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.     

Divergent Macroparasite Infections in Parapatric Swiss Lake-Stream Pairs of Threespine Stickleback (Gasterosteus aculeatus)

Spatial heterogeneity in diversity and intensity of parasitism is a typical feature of most host-parasite interactions, but understanding of the evolutionary implications of such variation is limited. One possible outcome of infection heterogeneities is parasite-mediated divergent selection between host populations, ecotypes or species which may facilitate the process of ecological speciation. However, very few studies have described infections in population-pairs along the speciation continuum from low to moderate or high degree of genetic differentiation that would address the possibility of parasite-mediated divergent selection in the early stages of the speciation process. Here we provide an example of divergent parasitism in freshwater fish ecotypes by examining macroparasite infections in threespine stickleback (Gasterosteus aculeatus) of four Swiss lake systems each harbouring parapatric lake-stream ecotype pairs. We demonstrate significant differences in infections within and between the pairs that are driven particularly by the parasite taxa transmitted to fish from benthic invertebrates. The magnitude of the differences tended to correlate positively with the extent of neutral genetic differentiation between the parapatric lake and stream populations of stickleback, whereas no such correlation was found among allopatric populations from similar or contrasting habitats. This suggests that genetic differentiation is unrelated to the magnitude of parasite infection contrasts when gene flow is constrained by geographical barriers while in the absence of physical barriers, genetic differentiation and the magnitude of differences in infections tend to be positively correlated.     

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.     

Adaptive divergence and the balance between selection and gene flow: lake and stream stickleback in the Misty system

We investigated the interplay between natural selection and gene flow in the adaptive divergence of threespine stickleback (Gasterosteus aculeatus) that reside parapatrically in lakes and streams. Within the Misty Lake system (Vancouver Island, British Columbia), stickleback from the inlet stream (flowing into the lake) have fewer gill rakers and deeper bodies than stickleback from the lake--differences thought to facilitate foraging (benthic macroinvertebrates in the stream vs. zooplankton in the open water of the lake). Common-garden experiments demonstrated that these differences have a genetic basis. Reciprocal transplant enclosure experiments showed that lake and inlet stickleback grow best in their home environments (although differences were subtle and often not significant). Release-recapture experiments in the inlet showed that lake fish are less well-suited than inlet fish for life in the stream (higher mortality or emigration in lake fish). Morphological divergence in the wild and under common rearing was greater between the lake and the inlet than between the lake and the outlet. Genetic divergence (mitochondrial DNA and microsatellites) was greatest between the lake and the upper inlet (1.8 km upstream from the lake), intermediate between the lake and the lower inlet (0.9 km upstream), and least between the lake and the outlet stream (1.2 km downstream). Relative levels of gene flow estimated from genetic data showed the inverse pattern. The negative association between morphological divergence and gene flow is consistent with the expectation that gene flow can constrain adaptation. Estimated absolute levels of gene flow also implied a constraint on adaptation in the outlet but not the inlet. Our results suggest that natural selection promotes the adaptive divergence of lake and stream stickleback. but that the magnitude of divergence can be constrained by gene flow.     

ASYMMETRIC RESPONSE PATTERNS TO SUBSPECIES‐SPECIFIC SONG DIFFERENCES IN ALLOPATRY AND PARAPATRY IN THE GRAY‐BREASTED WOOD‐WREN

Song divergence between closely related taxa may play a critical role in the evolutionary processes of speciation and hybridization. We explored song variation between two Ecuadorian subspecies of the gray‐breasted wood‐wren (Henicorhina leucophrys) and tested the impact of song divergence on response behaviors. Songs were significantly different between the two subspecies, even between two parapatric populations 10 km apart. Playback experiments revealed an asymmetric response pattern to these divergent subspecies specific songs; one subspecies responded more to songs of its own subspecies than to the other subspecies’ songs, whereas the second responded equally strongly to songs of both subspecies. While song parameters revealed a mixed pattern of divergence between allopatric and parapatric populations, the majority of spectral characteristics showed increased divergence in parapatry, suggestive of character displacement. This increased song divergence in parapatry appeared to affect behavioral responses to playback as discriminating responses were most prominent in parapatry and against parapatric songs. The clear behavioral impact of subspecies‐specific song differences supports a potential role for song as an acoustic barrier to gene flow. The asymmetric nature of the responses suggests that song divergence could affect the direction of gene flow and the position of the subspecies‐specific transition.     

Demography and genome divergence of lake and stream populations of an East African cichlid fish

Disentangling the processes and mechanisms underlying adaptive diversification is facilitated by the comparative study of replicate population pairs that have diverged along a similar environmental gradient. Such a setting is realized in a cichlid fish from southern Lake Tanganyika, Astatotilapia burtoni, which occurs within the lake proper as well as in various affluent rivers. Previously, we demonstrated that independent lake and stream populations show similar adaptations to the two habitat regimes. However, little is known about the evolutionary and demographic history of the A. burtoni populations in question and the patterns of genome divergence among them. Here, we apply restriction site‐associated DNA sequencing (RADseq) to examine the evolutionary history, the population structure and genomic differentiation of lake and stream populations in A. burtoni. A phylogenetic reconstruction based on genome‐wide molecular data largely resolved the evolutionary relationships among populations, allowing us to re‐evaluate the independence of replicate lake–stream population clusters. Further, we detected a strong pattern of isolation by distance, with baseline genomic divergence increasing with geographic distance and decreasing with the level of gene flow between lake and stream populations. Genome divergence patterns were heterogeneous and inconsistent among lake‐stream population clusters, which is explained by differences in divergence times, levels of gene flow and local selection regimes. In line with the latter, we only detected consistent outlier loci when the most divergent lake–stream population pair was excluded. Several of the thus identified candidate genes have inferred functions in immune and neuronal systems and show differences in gene expression between lake and stream populations.     

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.     

Quick divergence but slow convergence during ecotype formation in lake and stream stickleback pairs of variable age

When genetic constraints restrict phenotypic evolution, diversification can be predicted to evolve along so‐called lines of least resistance. To address the importance of such constraints and their resolution, studies of parallel phenotypic divergence that differ in their age are valuable. Here, we investigate the parapatric evolution of six lake and stream threespine stickleback systems from Iceland and Switzerland, ranging in age from a few decades to several millennia. Using phenotypic data, we test for parallelism in ecotypic divergence between parapatric lake and stream populations and compare the observed patterns to an ancestral‐like marine population. We find strong and consistent phenotypic divergence, both among lake and stream populations and between our freshwater populations and the marine population. Interestingly, ecotypic divergence in low‐dimensional phenotype space (i.e. single traits) is rapid and seems to be often completed within 100 years. Yet, the dimensionality of ecotypic divergence was highest in our oldest systems and only there parallel evolution of unrelated ecotypes was strong enough to overwrite phylogenetic contingency. Moreover, the dimensionality of divergence in different systems varies between trait complexes, suggesting different constraints and evolutionary pathways to their resolution among freshwater systems.     

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.