Comparing methods for detecting multilocus adaptation with multivariate genotype–environment associations

Identifying adaptive loci can provide insight into the mechanisms underlying local adaptation. Genotype–environment association (GEA) methods, which identify these loci based on correlations between genetic and environmental data, are particularly promising. Univariate methods have dominated GEA, despite the high dimensional nature of genotype and environment. Multivariate methods, which analyse many loci simultaneously, may be better suited to these data as they consider how sets of markers covary in response to environment. These methods may also be more effective at detecting adaptive processes that result in weak, multilocus signatures. Here, we evaluate four multivariate methods and five univariate and differentiation‐based approaches, using published simulations of multilocus selection. We found that Random Forest performed poorly for GEA. Univariate GEAs performed better, but had low detection rates for loci under weak selection. Constrained ordinations, particularly redundancy analysis (RDA), showed a superior combination of low false‐positive and high true‐positive rates across all levels of selection. These results were robust across the demographic histories, sampling designs, sample sizes and weak population structure tested here. The value of combining detections from different methods was variable and depended on the study goals and knowledge of the drivers of selection. Re‐analysis of genomic data from grey wolves highlighted the unique, covarying sets of adaptive loci that could be identified using RDA. Although additional testing is needed, this study indicates that RDA is an effective means of detecting adaptation, including signatures of weak, multilocus selection, providing a powerful tool for investigating the genetic basis of local adaptation.     

Fine‐grained adaptive divergence in an amphibian: genetic basis of phenotypic divergence and the role of nonrandom gene flow in restricting effective migration among wetlands

Adaptive ecological differentiation among sympatric populations is promoted by environmental heterogeneity, strong local selection and restricted gene flow. High gene flow, on the other hand, is expected to homogenize genetic variation among populations and therefore prevent local adaptation. Understanding how local adaptation can persist at the spatial scale at which gene flow occurs has remained an elusive goal, especially for wild vertebrate populations. Here, we explore the roles of natural selection and nonrandom gene flow (isolation by breeding time and habitat choice) in restricting effective migration among local populations and promoting generalized genetic barriers to neutral gene flow. We examined these processes in a network of 17 breeding ponds of the moor frog Rana arvalis, by combining environmental field data, a common garden experiment and data on variation in neutral microsatellite loci and in a thyroid hormone receptor (TRβ) gene putatively under selection. We illustrate the connection between genotype, phenotype and habitat variation and demonstrate that the strong differences in larval life history traits observed in the common garden experiment can result from adaptation to local pond characteristics. Remarkably, we found that haplotype variation in the TRβ gene contributes to variation in larval development time and growth rate, indicating that polymorphism in the TRβ gene is linked with the phenotypic variation among the environments. Genetic distance in neutral markers was correlated with differences in breeding time and environmental differences among the ponds, but not with geographical distance. These results demonstrate that while our study area did not exceed the scale of gene flow, ecological barriers constrained gene flow among contrasting habitats. Our results highlight the roles of strong selection and nonrandom gene flow created by phenological variation and, possibly, habitat preferences, which together maintain genetic and phenotypic divergence at a fine‐grained spatial scale.     

Variable patterns of introgression in two sculpin hybrid zones suggest that genomic isolation differs among populations

Theory predicts that reproductive isolation may be due to intrinsic genetic incompatibilities or extrinsic ecological factors. Therefore, an understanding of the genetic basis of isolation may require analyses of evolutionary processes in situ to include environmental factors. Here we study genetic isolation between populations of sculpins ( Cottus ) at 168 microsatellites. Genomic clines were fit using 480 individuals sampled across independent natural hybrid zones that have formed between one invading species and two separate populations of a resident species. Our analysis tests for deviations from neutral patterns of introgression at individual loci based on expectations given genome-wide admixture. Roughly 51% of the loci analysed displayed significant deviations. An overall deficit of interspecific heterozygotes in 26% and 21% of the loci suggests that widespread underdominance drives genomic isolation. At the same time, selection promotes introgression of almost 30% of the markers, which implies that hybridization may increase the fitness of admixed individuals. Cases of overdominance or epistatic interactions were relatively rare. Despite the similarity of the two hybrid zones in their overall genomic composition, patterns observed at individual loci show little correlation between zones and many fit different genotypic models of fitness. At this point, it remains difficult to determine whether these results are due to differences in external selection pressures or cryptic genetic differentiation of distinct parental populations. In the future, data from mapped genetic markers and on variation of ecological factors will provide additional insights into the contribution of these factors to variation in the evolutionary consequences of hybridization.     

Molecular divergence in tropical tree populations occupying environmental mosaics

Unveiling the genetic basis of local adaptation to environmental variation is a major goal in molecular ecology. In rugged landscapes characterized by environmental mosaics, living populations and communities can experience steep ecological gradients over very short geographical distances. In lowland tropical forests, interspecific divergence in edaphic specialization (for seasonally flooded bottomlands and seasonally dry terra firme soils) has been proven by ecological studies on adaptive traits. Some species are nevertheless capable of covering the entire span of the gradient; intraspecific variation for adaptation to contrasting conditions may explain the distribution of such ecological generalists. We investigated whether local divergence happens at small spatial scales in two stands of Eperua falcata (Fabaceae), a widespread tree species of the Guiana Shield. We investigated Single Nucleotide Polymorphisms (SNP) and sequence divergence as well as spatial genetic structure (SGS) at four genes putatively involved in stress response and three genes with unknown function. Significant genetic differentiation was observed among sub‐populations within stands, and eight SNP loci showed patterns compatible with disruptive selection. SGS analysis showed genetic turnover along the gradients at three loci, and at least one haplotype was found to be in repulsion with one habitat. Taken together, these results suggest genetic differentiation at small spatial scale in spite of gene flow. We hypothesize that heterogeneous environments may cause molecular divergence, possibly associated to local adaptation in E. falcata.     

Ecological genomics of local adaptation in Cornus florida L. by genotyping by sequencing

Discovering local adaptation, its genetic underpinnings, and environmental drivers is important for conserving forest species. Ecological genomic approaches coupled with next‐generation sequencing are useful means to detect local adaptation and uncover its underlying genetic basis in nonmodel species. We report results from a study on flowering dogwood trees (Cornus florida L.) using genotyping by sequencing (GBS). This species is ecologically important to eastern US forests but is severely threatened by fungal diseases. We analyzed subpopulations in divergent ecological habitats within North Carolina to uncover loci under local selection and associated with environmental–functional traits or disease infection. At this scale, we tested the effect of incorporating additional sequencing before scaling for a broader examination of the entire range. To test for biases of GBS, we sequenced two similarly sampled libraries independently from six populations of three ecological habitats. We obtained environmental–functional traits for each subpopulation to identify associations with genotypes via latent factor mixed modeling (LFMM) and gradient forests analysis. To test whether heterogeneity of abiotic pressures resulted in genetic differentiation indicative of local adaptation, we evaluated F_st per locus while accounting for genetic differentiation between coastal subpopulations and Piedmont‐Mountain subpopulations. Of the 54 candidate loci with sufficient evidence of being under selection among both libraries, 28–39 were Arlequin–BayeScan F_st outliers. For LFMM, 45 candidates were associated with climate (of 54), 30 were associated with soil properties, and four were associated with plant health. Reanalysis of combined libraries showed that 42 candidate loci still showed evidence of being under selection. We conclude environment‐driven selection on specific loci has resulted in local adaptation in response to potassium deficiencies, temperature, precipitation, and (to a marginal extent) disease. High allele turnover along ecological gradients further supports the adaptive significance of loci speculated to be under selection.     

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.     

Indirect Evolution of Hybrid Lethality Due to Linkage with Selected Locus in Mimulus guttatus

Most species are superbly and intricately adapted to the environments in which they live. Adaptive evolution by natural selection is the primary force shaping biological diversity. Differences between closely related species in ecologically selected characters such as habitat preference, reproductive timing, courtship behavior, or pollinator attraction may prevent interbreeding in nature, causing reproductive isolation. But does ecological adaptation cause reproductive incompatibilities such as hybrid sterility or lethality? Although several genes causing hybrid incompatibilities have been identified, there is intense debate over whether the genes that contribute to ecological adaptations also cause hybrid incompatibilities. Thirty years ago, a genetic study of local adaptation to copper mine soils in the wildflower Mimulus guttatus identified a locus that appeared to cause copper tolerance and hybrid lethality in crosses to other populations. But do copper tolerance and hybrid lethality have the same molecular genetic basis? Here we show, using high-resolution genome mapping, that copper tolerance and hybrid lethality are not caused by the same gene but are in fact separately controlled by two tightly linked loci. We further show that selection on the copper tolerance locus indirectly caused the hybrid incompatibility allele to go to high frequency in the copper mine population because of hitchhiking. Our results provide a new twist on Darwin''s original supposition that hybrid incompatibilities evolve as an incidental by-product of ordinary adaptation to the environment.     

Low genome‐wide divergence between two lizard populations with high adaptive phenotypic differentiation

Usually, adaptive phenotypic differentiation is paralleled by genetic divergence between locally adapted populations. However, adaptation can also happen in a scenario of nonsignificant genetic divergence due to intense gene flow and/or recent differentiation. While this phenomenon is rarely published, findings on incipient ecologically driven divergence or isolation by adaptation are relatively common, which could confound our understanding about the frequency at which they actually occur in nature. Here, we explore genome‐wide traces of divergence between two populations of the lacertid lizard Psammodromus algirus separated by a 600 m elevational gradient. These populations seem to be differentially adapted to their environments despite showing low levels of genetic differentiation (according to previously studies of mtDNA and microsatellite data). We performed a search for outliers (i.e., loci subject to selection) trying to identify specific loci with F_ST statistics significantly higher than those expected on the basis of overall, genome‐wide estimates of genetic divergence. We find that local phenotypic adaptation (in terms of a wide diversity of characters) was not accompanied by genome‐wide differentiation, even when we maximized the chances of unveiling such differentiation at particular loci with F_ST‐based outlier detection tests. Instead, our analyses confirmed the lack of genome‐wide differentiation on the basis of more than 70,000 SNPs, which is concordant with a scenario of local adaptation without isolation by environment. Our results add evidence to previous studies in which local adaptation does not lead to any kind of isolation (or early stages of ecological speciation), but maintains phenotypic divergence despite the lack of a differentiated genomic background.     

Incipient ecological speciation between successional varieties of a dominant tree involves intrinsic postzygotic isolating barriers

Whereas disruptive selection imposed by heterogeneous environments can lead to the evolution of extrinsic isolating barriers between diverging populations, the evolution of intrinsic postzygotic barriers through divergent selection is less certain. Long‐lived species such as trees may be especially slow to evolve intrinsic isolating barriers. We examined postpollination reproductive isolating barriers below the species boundary, in an ephemeral hybrid zone between two successional varieties of the landscape‐dominant Hawaiian tree, Metrosideros polymorpha, on volcanically active Hawai'i Island. These archipelago‐wide sympatric varieties show the weakest neutral genetic divergence of any taxon pair on Hawai'i Island but significant morphological and ecological differentiation consistent with adaptation to new and old lava flows. Cross‐fertility between varieties was high and included heterosis of F₁ hybrids at the seed germination stage, consistent with a substantial genetic load apparent within varieties through low self‐fertility and a lack of self‐pollen discrimination. However, a partial, but significant, barrier was observed in the form of reduced female and male fertility of hybrids, especially backcross hybrids, consistent with the accumulation of genetic incompatibilities between varieties. These results suggest that partial intrinsic postzygotic barriers can arise through disruptive selection acting on large, hybridizing populations of a long‐lived species.     

Patterns of divergence across the geographic and genomic landscape of a butterfly hybrid zone associated with a climatic gradient

Hybrid zones are a valuable tool for studying the process of speciation and for identifying the genomic regions undergoing divergence and the ecological (extrinsic) and nonecological (intrinsic) factors involved. Here, we explored the genomic and geographic landscape of divergence in a hybrid zone between Papilio glaucus and Papilio canadensis. Using a genome scan of 28,417 ddRAD SNPs, we identified genomic regions under possible selection and examined their distribution in the context of previously identified candidate genes for ecological adaptations. We showed that differentiation was genomewide, including multiple candidate genes for ecological adaptations, particularly those involved in seasonal adaptation and host plant detoxification. The Z chromosome and four autosomes showed a disproportionate amount of differentiation, suggesting genes on these chromosomes play a potential role in reproductive isolation. Cline analyses of significantly differentiated genomic SNPs, and of species‐diagnostic genetic markers, showed a high degree of geographic coincidence (81%) and concordance (80%) and were associated with the geographic distribution of a climate‐mediated developmental threshold (length of the growing season). A relatively large proportion (1.3%) of the outliers for divergent selection were not associated with candidate genes for ecological adaptations and may reflect the presence of previously unrecognized intrinsic barriers between these species. These results suggest that exogenous (climate‐mediated) and endogenous (unknown) clines may have become coupled and act together to reinforce reproductive isolation. This approach of assessing divergence across both the genomic and geographic landscape can provide insight about the interplay between the genetic architecture of reproductive isolation and endogenous and exogenous selection.     

Sculpin hybrid zones: natural laboratories for the early stages of speciation

Firmly rooted as we are in the genomic era, it can seem incredible that as recently as 1974, Lewontin declared, 'we know virtually nothing about the genetic changes that occur in species formation'. To the contrary, we now know the genetic architecture of phenotypic differences and reproductive isolation between species for many diverse groups of plants, animals, and fungi. In recent years, detailed genetic analyses have produced a small but growing list of genes that cause reproductive isolation, several of which appear to have diverged by natural selection. Yet, a full accounting of the speciation process requires that we understand the reproductive and ecological properties of natural populations as they begin to diverge genetically, as well as the dynamics of newly evolved barriers to gene flow. One promising approach to this problem is the study of natural hybrid zones, where gene exchange between divergent populations can produce recombinant genotypes in situ . In such individuals, genomic variation might be shaped by introgression at universally adaptive or neutral loci, even as regions associated with local adaptation or reproductive isolation remain divergent. In Nolte et   al . (2009) , the authors take advantage of two independent, recently formed hybrid zones between sculpin species to investigate genome-wide patterns of reproductive isolation. Using a recently developed genomic clines method, the authors identify marker loci that are associated with isolation, and those that show evidence for adaptive introgression. Remarkably, Nolte et   al . (2009) find little similarity between the two hybrid zones in patterns of introgression, a fact that might reflect genetic variation within species or heterogeneous natural selection. In either case, their study system has the potential to provide insight into the early stages of speciation.     

When invaders meet locally adapted types: rapid moulding of hybrid zones between sculpins (Cottus, Pisces) in the Rhine system

Hybrid zones are commonly studied to dissect the processes that drive divergence among lineages, which have incomplete barriers of reproduction. Most hybrid zones have existed for an extended time making inferences on the initial mode of formation difficult. It is a priori unclear how fast a nascent hybrid zone would form as a response to endogenous and exogenous factors. We have studied several hybrid zones between two lineages of sculpins (Cottus spp.), which emerged due to a recent range expansion of one of the lineages along the river Rhine in the early 1990s. Applying a dense sampling across two contact areas and using a highly informative set of 45 microsatellite markers we found pronounced genetic structure. Steep genetic clines suggest that strong selective forces have shaped the respective hybrid zones from the beginning. We find that the zones are coupled to ecological transitions from small streams to larger rivers. The width of these zones is much smaller than estimates of annual individual dispersal distances, as estimated outside of the hybrid zones. The pattern is apparently not strongly affected by pre- or postzygotic reproductive isolation because numerous backcross hybrids occur within the zones. This suggests that strong natural selection acts against immigrant genotypes. The study exemplifies how local adaptation can play a key role in preventing admixture in dependence of the ecological context.     

Environment-specific heterozygote deficiency and developmental instability in hybrid Mytilus

The multiple discrete hybrid zones that characterize Mytilus blue mussels allow a novel, non-manipulative, examination of the selective pressures that create and maintain species. If endogenous genetic incompatibility is solely responsible for post-zygotic isolation, then individuals of a specified hybrid genotype are expected to show similar average fitness across environments. However, if hybrid fitness differs across environments, then exogenous selection is implicated, either via ecological selection or environment-specific expression of intrinsic genetic incompatibilities. Correspondence between developmental instability of hybrids and heterozygote deficiency, estimated in two M. trossulus×M. galloprovincialis hybrid zones on the coast of North America, indicates that environment-dependent selection against hybrids may contribute to reproductive isolation among Pacific Mytilus species.     

Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky–Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus‐specific TE, the minisatellite‐like Cla‐element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population‐specific TE insertion patterns with many private insertions. A significant correlation of the pairwise F_ST estimated from genomewide single‐nucleotide polymorphisms (SNPs) and the F_ST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla‐element F_ST level due to a high proportion of differentially fixed Cla‐element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla‐elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla‐element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.     

A genome scan to detect candidate regions influenced by local natural selection in human populations

As human populations dispersed throughout the world, they were subjected to new selective forces, which must have led to local adaptation via natural selection and hence altered patterns of genetic variation. Yet, there are very few examples known in which such local selection has clearly influenced human genetic variation. A potential approach for detecting local selection is to screen random loci across the genome; those loci that exhibit unusually large genetic distances between human populations are then potential markers of genomic regions under local selection. We investigated this approach by genotyping 332 short tandem repeat (STR) loci in Africans and Europeans and calculating the genetic differentiation for each locus. Patterns of genetic diversity at these loci were consistent with greater variation in Africa and with local selection operating on populations as they moved out of Africa. For 11 loci exhibiting the largest genetic differences, we genotyped an additional STR locus located nearby; the genetic distances for these nearby loci were significantly larger than average. These genomic regions therefore reproducibly exhibit larger genetic distances between populations than the "average" genomic region, consistent with local selection. Our results demonstrate that genome scans are a promising means of identifying candidate regions that have been subjected to local selection.     

How do species barriers decay? Concordance and local introgression in mosaic hybrid zones of mussels

The Mytilus complex of marine mussel species forms a mosaic of hybrid zones, found across temperate regions of the globe. This allows us to study ‘replicated’ instances of secondary contact between closely related species. Previous work on this complex has shown that local introgression is both widespread and highly heterogeneous, and has identified SNPs that are outliers of differentiation between lineages. Here, we developed an ancestry‐informative panel of such SNPs. We then compared their frequencies in newly sampled populations, including samples from within the hybrid zones, and parental populations at different distances from the contact. Results show that close to the hybrid zones, some outlier loci are near to fixation for the heterospecific allele, suggesting enhanced local introgression, or the local sweep of a shared ancestral allele. Conversely, genomic cline analyses, treating local parental populations as the reference, reveal a globally high concordance among loci, albeit with a few signals of asymmetric introgression. Enhanced local introgression at specific loci is consistent with the early transfer of adaptive variants after contact, possibly including asymmetric bi‐stable variants (Dobzhansky‐Muller incompatibilities), or haplotypes loaded with fewer deleterious mutations. Having escaped one barrier, however, these variants can be trapped or delayed at the next barrier, confining the introgression locally. These results shed light on the decay of species barriers during phases of contact.     

The challenge of separating signatures of local adaptation from those of isolation by distance and colonization history: The case of two white pines

Accurately detecting signatures of local adaptation using genetic‐environment associations (GEAs) requires controlling for neutral patterns of population structure to reduce the risk of false positives. However, a high degree of collinearity between climatic gradients and neutral population structure can greatly reduce power, and the performance of GEA methods in such case is rarely evaluated in empirical studies. In this study, we attempted to disentangle the effects of local adaptation and isolation by environment (IBE) from those of isolation by distance (IBD) and isolation by colonization from glacial refugia (IBC) using range‐wide samples in two white pine species. For this, SNPs from 168 genes, including 52 candidate genes for growth and phenology, were genotyped in 133 and 61 populations of Pinus strobus and P. monticola, respectively. For P. strobus and using all 153 SNPs, climate (IBE) did not significantly explained among‐population variation when controlling for IBD and IBC in redundancy analyses (RDAs). However, 26 SNPs were significantly associated with climate in single‐locus GEA analyses (Bayenv2 and LFMM), suggesting that local adaptation took place in the presence of high gene flow. For P. monticola, we found no evidence of IBE using RDAs and weaker signatures of local adaptation using GEA and F_ST outlier tests, consistent with adaptation via phenotypic plasticity. In both species, the majority of the explained among‐population variation (69 to 96%) could not be partitioned between the effects of IBE, IBD, and IBC. GEA methods can account differently for this confounded variation, and this could explain the small overlap of SNPs detected between Bayenv2 and LFMM. Our study illustrates the inherent difficulty of taking into account neutral structure in natural populations and the importance of sampling designs that maximize climatic variation, while minimizing collinearity between climatic gradients and neutral structure.     

Adaptation of redband trout in desert and montane environments

Natural populations that evolve under extreme climates are likely to diverge because of selection in local environments. To explore whether local adaptation has occurred in redband trout (Oncorhynchus mykiss gairdneri) occupying differing climate regimes, we used a limited genome scan approach to test for candidate markers under selection in populations occurring in desert and montane streams. An environmental approach to identifying outlier loci, spatial analysis method and linear regression of minor allele frequency with environmental variables revealed six candidate markers (P < 0.01). Putatively neutral markers identified high genetic differentiation among desert populations relative to montane sites, likely due to intermittent flows in desert streams. Additionally, populations exhibited a highly significant pattern of isolation by temperature (P< 0.0001) and those adapted to the same environment had similar allele frequencies across candidate markers, indicating selection for differing climates. These results imply that many genes are involved in the adaptation of redband trout to differing environments, and selection acts to reinforce localization. The potential to predict genetic adaptability of individuals and populations to changing environmental conditions may have profound implications for species that face extensive anthropogenic disturbances.