Cultural isolation is greater than genetic isolation across an avian hybrid zone

Elucidating the relationship between genetic and cultural evolution is important in understanding speciation, as learned premating barriers might be involved in maintaining species differences. Here, we test this relationship by examining a widely recognized premating barrier, bird song, in a hybrid zone between black‐throated green (Setophaga virens) and Townsend's warblers (S. townsendi). We use song analysis, genomic techniques and playback experiments to characterize the cultural and genetic backgrounds of individuals in this region, expecting that if song is an important reproductive barrier between these species, there should be a strong relationship between song and genotype. We show that songs in the hybrid zone correspond to the distinctly different songs found in allopatry but that song and genotype are not tightly coupled in sympatry. Allopatric individuals responded only to local songs, indicating that individuals may have learned to respond to songs they commonly hear. We observed discordance between song and genotype clines; a narrower cline suggests that cultural selection on song is stronger than natural selection on genotype. These findings indicate that song is unlikely to play a role in reproductive isolation between these species, and we suggest that spatial variation in song may nonetheless be maintained by frequency‐dependent cultural selection. This decoupling of genes and culture may contribute to hybridization in this region.     

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.     

What can patterns of differentiation across plant genomes tell us about adaptation and speciation?

Genome scans have become a common approach to identify genomic signatures of natural selection and reproductive isolation, as well as the genomic bases of ecologically relevant phenotypes, based on patterns of polymorphism and differentiation among populations or species. Here, we review the results of studies taking genome scan approaches in plants, consider the patterns of genomic differentiation documented and their possible causes, discuss the results in light of recent models of genomic differentiation during divergent adaptation and speciation, and consider assumptions and caveats in their interpretation. We find that genomic regions of high divergence generally appear quite small in comparisons of both closely and more distantly related populations, and for the most part, these differentiated regions are spread throughout the genome rather than strongly clustered. Thus, the genome scan approach appears well-suited for identifying genomic regions or even candidate genes that underlie adaptive divergence and/or reproductive barriers. We consider other methodologies that may be used in conjunction with genome scan approaches, and suggest further developments that would be valuable. These include broader use of sequence-based markers of known genomic location, greater attention to sampling strategies to make use of parallel environmental or phenotypic transitions, more integration with approaches such as quantitative trait loci mapping and measures of gene flow across the genome, and additional theoretical and simulation work on processes related to divergent adaptation and speciation.     

ddRAD‐seq data reveal significant genome‐wide population structure and divergent genomic regions that distinguish the mallard and close relatives in North America

Recently evolved species typically share genetic variation across their genomes due to incomplete lineage sorting and/or ongoing gene flow. Given only subtle allele frequency differences at most loci and the expectation that divergent selection may affect only a tiny fraction of the genome, distinguishing closely related species based on multi‐locus data requires substantial genomic coverage. In this study, we used ddRAD‐seq to sample the genomes of five recently diverged, New World “mallards” (Anas spp.), a group of dabbling duck species characterized by diagnosable phenotypic differences but minimal genetic differentiation. With increased genomic sampling, we aimed to characterize population structure within this group and identify genomic regions that may have experienced divergent selection during speciation. We analyzed 3,017 autosomal ddRAD‐seq loci and 177 loci from the Z‐chromosome. In contrast to previous studies, the ddRAD‐seq data were sufficient to assign individuals to their respective species or subspecies and to generate estimates of gene flow in a phylogenetic framework. We find limited evidence of contemporary gene flow between the dichromatic mallard and several monochromatic taxa, but find evidence for historical gene flow between some monochromatic species pairs. We conclude that the overall genetic similarity of these taxa likely reflects retained ancestral polymorphism rather than recent and extensive gene flow. Thus, despite recurring cases of hybridization in this group, our results challenge the current dogma predicting the genetic extinction of the New World monochromatic dabbling ducks via introgressive hybridization with mallards. Moreover, ddRAD‐seq data were sufficient to identify previously unknown outlier regions across the Z‐chromosome and several autosomal chromosomes that may have been involved in the diversification of species in this recent radiation.     

Methods for delimiting species via population genetics and phylogenetics using genotype data

In species delimitation, a formidable goal in the discipline of systematic biology, we identify and describe species morphologically and ecologically based on phenotypic data. Efficient genotyping technologies produce genetic and genomic data with relative ease, which promotes species discovery and validation using genotype data. For the last two decades, we have seen the development of species delimitation methods based on genetic distances and phylogenetic trees using genotype data. However, speciation processes via evolutionary relationship among species were mostly divorced from species delimitation. Recent approaches to drawing species boundaries use multi-locus sequence data to account for evolutionary processes including speciation and gene flow. They allow us to learn of jointly speciation and species delimitation, leveraging computational and statistical techniques developed in population genetics and phylogenetics. Here, we review the recent progress in the development of species delimitation using genotype data and discuss the future outlook for the research of developing species delimitation methods.     

Recombinant hybrids retain heterozygosity at many loci: new insights into the genomics of reproductive isolation in Populus

The maintenance of species barriers in the face of gene flow is often thought to result from strong selection against intermediate genotypes, thereby preserving genetic differentiation. Most speciation genomic studies thus aim to identify exceptionally divergent loci between populations, but divergence will be affected by many processes other than reproductive isolation (RI) and speciation. Through genomic studies of recombinant hybrids sampled in the wild, genetic variation associated with RI can be observed in situ, because selection against incompatible genotypes will leave detectable patterns of variation in the hybrid genomes. To better understand the mechanisms directly involved in RI, we investigated three natural ‘replicate’ hybrid zones between two divergent Populus species via locus‐specific patterns of ancestry across recombinant hybrid genomes. As expected, genomic patterns in hybrids and their parental species were consistent with the presence of underdominant selection at several genomic regions. Surprisingly, many loci displayed greatly increased between‐species heterozygosity in recombinant hybrids despite striking genetic differentiation between the parental genomes, the opposite of what would be expected with selection against intermediate genotypes. Only a limited, reproducible set of genotypic combinations was present in hybrid genomes across localities. In the absence of clearly delimited ‘hybrid habitats’, our results suggest that complex epistatic interactions within genomes play an important role in advanced stages of RI between these ecologically divergent forest trees. This calls for more genomic studies that test for unusual patterns of genomic ancestry in hybridizing species.     

Ring species as bridges between microevolution and speciation

A demonstration of how small changes can lead to species-level differences is provided by ring species, in which two reproductively isolated forms are connected by a chain of intermediate populations. We review proposed cases of ring species and the insights they provide into speciation. Ring species have been viewed both as illustrations of the history of divergence of two species from their common ancestor and as demonstrations that speciation can occur in spite of gene flow between the diverging forms. Theoretical models predict that speciation with gene flow can occur when there is divergent ecological selection, and geographical differentiation increases the likelihood of speciation. Thus ring species are ideal systems for research into the role of both ecological and geographical differentiation in speciation, but few examples have been studied in detail. The Greenish warbler is a ring species in which two northward expansions around the Tibetan plateau have been accompanied by parallel evolution in morphology, ecology, and song length and complexity. However, songs have diverged in structure, resulting in a lack of recognition where the reproductively isolated forms come into contact in Siberia. Our analysis suggests that these differences could have arisen even with gene flow, and that parallel rather than divergent ecological changes have led to divergence in sexually selected traits and subsequent speciation.     

The genetic mosaic suggests a new role for hitchhiking in ecological speciation

Early in ecological speciation, the genomically localized effects of divergent selection cause heterogeneity among loci in divergence between incipient species. We call this pattern of genomic variability in divergence the 'genetic mosaic of speciation'. Previous studies have used F(ST) outliers as a way to identify divergently selected genomic regions, but the nature of the relationship between outlier loci and quantitative trait loci (QTL) involved in reproductive isolation has not yet been quantified. Here, we show that F(ST) outliers between a pair of incipient species are significantly clustered around QTL for traits that cause ecologically based reproductive isolation. Around these key QTL, extensive 'divergence hitchhiking' occurs because reduced inter-race mating and negative selection decrease the opportunity for recombination between chromosomes bearing different locally adapted QTL alleles. Divergence hitchhiking is likely to greatly increase the opportunity for speciation in populations that are sympatric, regardless of whether initial divergence was sympatric or allopatric. Early in ecological speciation, analyses of population structure, gene flow or phylogeography based on different random or arbitrarily chosen neutral markers should be expected to conflict--only markers in divergently selected genomic regions will reveal the evolutionary history of adaptive divergence and ecologically based reproductive isolation. Species retain mosaic genomes for a very long time, and gene exchange in hybrid zones can vary dramatically among loci. However, in hybridizing species, the genomic regions that affect ecologically based reproductive isolation are difficult to distinguish from regions that have diverged for other reasons.     

Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow

Speciation, the evolution of reproductive isolation among populations, is continuous, complex, and involves multiple, interacting barriers. Until it is complete, the effects of this process vary along the genome and can lead to a heterogeneous genomic landscape with peaks and troughs of differentiation and divergence. When gene flow occurs during speciation, barriers restricting gene flow locally in the genome lead to patterns of heterogeneity. However, genomic heterogeneity can also be produced or modified by variation in factors such as background selection and selective sweeps, recombination and mutation rate variation, and heterogeneous gene density. Extracting the effects of gene flow, divergent selection and reproductive isolation from such modifying factors presents a major challenge to speciation genomics. We argue one of the principal aims of the field is to identify the barrier loci involved in limiting gene flow. We first summarize the expected signatures of selection at barrier loci, at the genomic regions linked to them and across the entire genome. We then discuss the modifying factors that complicate the interpretation of the observed genomic landscape. Finally, we end with a road map for future speciation research: a proposal for how to account for these modifying factors and to progress towards understanding the nature of barrier loci. Despite the difficulties of interpreting empirical data, we argue that the availability of promising technical and analytical methods will shed further light on the important roles that gene flow and divergent selection have in shaping the genomic landscape of speciation.     

Divergent selection and heterogeneous genomic divergence

Levels of genetic differentiation between populations can be highly variable across the genome, with divergent selection contributing to such heterogeneous genomic divergence. For example, loci under divergent selection and those tightly physically linked to them may exhibit stronger differentiation than neutral regions with weak or no linkage to such loci. Divergent selection can also increase genome‐wide neutral differentiation by reducing gene flow (e.g. by causing ecological speciation), thus promoting divergence via the stochastic effects of genetic drift. These consequences of divergent selection are being reported in recently accumulating studies that identify: (i) ‘outlier loci’ with higher levels of divergence than expected under neutrality, and (ii) a positive association between the degree of adaptive phenotypic divergence and levels of molecular genetic differentiation across population pairs [‘isolation by adaptation’ (IBA)]. The latter pattern arises because as adaptive divergence increases, gene flow is reduced (thereby promoting drift) and genetic hitchhiking increased. Here, we review and integrate these previously disconnected concepts and literatures. We find that studies generally report 5–10% of loci to be outliers. These selected regions were often dispersed across the genome, commonly exhibited replicated divergence across different population pairs, and could sometimes be associated with specific ecological variables. IBA was not infrequently observed, even at neutral loci putatively unlinked to those under divergent selection. Overall, we conclude that divergent selection makes diverse contributions to heterogeneous genomic divergence. Nonetheless, the number, size, and distribution of genomic regions affected by selection varied substantially among studies, leading us to discuss the potential role of divergent selection in the growth of regions of differentiation (i.e. genomic islands of divergence), a topic in need of future investigation.     

Genomic Adaptations to Salinity Resist Gene Flow in the Evolution of Floridian Watersnakes

The migration-selection balance often governs the evolution of lineages, and speciation with gene flow is now considered common across the tree of life. Ecological speciation is a process that can facilitate divergence despite gene flow due to strong selective pressures caused by ecological differences; however, the exact traits under selection are often unknown. The transition from freshwater to saltwater habitats provides strong selection targeting traits with osmoregulatory function. Several lineages of North American watersnakes (Nerodia spp.) are known to occur in saltwater habitat and represent a useful system for studying speciation by providing an opportunity to investigate gene flow and evaluate how species boundaries are maintained or degraded. We use double digest restriction-site associated DNA sequencing to characterize the migration-selection balance and test for evidence of ecological divergence within the Nerodia fasciata-clarkii complex in Florida. We find evidence of high intraspecific gene flow with a pattern of isolation-by-distance underlying subspecific lineages. However, we identify genetic structure indicative of reduced gene flow between inland and coastal lineages suggesting divergence due to isolation-by-environment. This pattern is consistent with observed environmental differences where the amount of admixture decreases with increased salinity. Furthermore, we identify significantly enriched terms related to osmoregulatory function among a set of candidate loci, including several genes that have been previously implicated in adaptation to salinity stress. Collectively, our results demonstrate that ecological differences, likely driven by salinity, cause strong divergent selection which promotes divergence in the N. fasciata-clarkii complex despite significant gene flow.     

GENOME DIVERGENCE AND THE GENETIC ARCHITECTURE OF BARRIERS TO GENE FLOW BETWEEN LYCAEIDES IDAS AND L. MELISSA

Genome divergence during speciation is a dynamic process that is affected by various factors, including the genetic architecture of barriers to gene flow. Herein we quantitatively describe aspects of the genetic architecture of two sets of traits, male genitalic morphology and oviposition preference, that putatively function as barriers to gene flow between the butterfly species Lycaeides idas and L. melissa. Our analyses are based on unmapped DNA sequence data and a recently developed Bayesian regression approach that includes variable selection and explicit parameters for the genetic architecture of traits. A modest number of nucleotide polymorphisms explained a small to large proportion of the variation in each trait, and average genetic variant effects were nonnegligible. Several genetic regions were associated with variation in multiple traits or with trait variation within‐ and among‐populations. In some instances, genetic regions associated with trait variation also exhibited exceptional genetic differentiation between species or exceptional introgression in hybrids. These results are consistent with the hypothesis that divergent selection on male genitalia has contributed to heterogeneous genetic differentiation, and that both sets of traits affect fitness in hybrids. Although these results are encouraging, we highlight several difficulties related to understanding the genetics of speciation.     

Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa

Sexual isolation has been considered one of the primary causes of speciation and its genetic study has the potential to reveal the genetics of speciation. In Drosophila, the importance of courtship songs in sexual isolation between closely related species has been well investigated, but studies analysing the genetic basis of the difference in the courtship songs associated with sexual isolation are less well documented. Drosophila ananassae and Drosophila pallidosa are useful for studies of sexual isolation, because of their sympatric distribution and absence of postmating isolation. Courtship songs are known to play a crucial role in sexual isolation between these two species, and the female discrimination behaviour against the courting male has been revealed to be controlled by a very narrow region on the second chromosome. In this study we investigated the genetic basis controlling the song differences associated with their sexual isolation, using intact and wingless males with chromosomes substituted between species. The results obtained from F₁ hybrid males between these species indicate the dominance of the song characters favoured by D. pallidosa females. In addition, the results obtained from backcross F₂ males indicate that chromosome 2 had a major effect on the control of the song characters associated with sexual isolation.     

生态物种形成及其研究进展

物种形成是基本的进化过程, 也是生物多样性形成的基础。自然选择可以导致新物种的产生。生态物种形成是指以生态为基础的歧化选择使不同群体分化产生生殖隔离的物种形成过程。本文首先回顾了生态物种形成的研究历史, 并详细介绍了生态物种形成的3个要素, 即歧化选择的来源、生殖隔离的形式以及关联歧化选择与生殖隔离的遗传机制。歧化选择的来源主要包括不同的环境或生态位、不同形式的性选择, 以及群体间的相互作用。生殖隔离的形式多种多样, 我们总结了合子前和合子后隔离的遗传学机制以及在生态物种形成中起到的作用。控制适应性性状的基因与导致生殖隔离的基因可以通过基因多效性或连锁不平衡相互关联起来。借助于第二代测序技术, 研究者可以对生态物种形成的遗传学与基因组学基础进行研究。此外, 本文还总结了生态物种形成领域最新的研究进展, 包括平行进化的全基因组基础, 以及基因流影响群体分化的理论基础。通过归纳比较由下至上和由上至下这两种不同的研究思路, 作者认为这两种思路的结合可以为生态物种形成基因的筛选提供更有力也更精确的方法。同时, 作者还提出生态物种形成的研究应该基于更好的表型描述以及更完整的基因组信息, 研究的物种也应该具有更广泛的代表性。     

Genome scans reveal candidate regions involved in the adaptation to host plant in the pea aphid complex

A major goal in evolutionary biology is to uncover the genetic basis of adaptation. Divergent selection exerted on ecological traits may result in adaptive population differentiation and reproductive isolation and affect differentially the level of genetic divergence along the genome. Genome‐wide scan of large sets of individuals from multiple populations is a powerful approach to identify loci or genomic regions under ecologically divergent selection. Here, we focused on the pea aphid, a species complex of divergent host races, to explore the organization of the genomic divergence associated with host plant adaptation and ecological speciation. We analysed 390 microsatellite markers located at variable distances from predicted genes in replicate samples of sympatric populations of the pea aphid collected on alfalfa, red clover and pea, which correspond to three common host‐adapted races reported in this species complex. Using a method that accounts for the hierarchical structure of our data set, we found a set of 11 outlier loci that show higher genetic differentiation between host races than expected under the null hypothesis of neutral evolution. Two of the outliers are close to olfactory receptor genes and three other nearby genes encoding salivary proteins. The remaining outliers are located in regions with genes of unknown functions, or which functions are unlikely to be involved in interactions with the host plant. This study reveals genetic signatures of divergent selection across the genome and provides an inventory of candidate genes responsible for plant specialization in the pea aphid, thereby setting the stage for future functional studies.     

THE EFFICACY OF DIVERGENCE HITCHHIKING IN GENERATING GENOMIC ISLANDS DURING ECOLOGICAL SPECIATION

Genes under divergent selection flow less readily between populations than other loci. This observation has led to verbal “divergence hitchhiking” models of speciation in which decreased interpopulation gene flow surrounding loci under divergent selection can generate large regions of differentiation within the genome (genomic islands). The efficacy of this model in promoting speciation depends on the size of the region affected by divergence hitchhiking. Empirical evidence is mixed, with examples of both large and small genomic islands. To address these empirical discrepancies and to formalize the theory, we present mathematical models of divergence hitchhiking, which examine neutral differentiation around selected sites. For a single locus under selection, regions of differentiation do not extend far along a chromosome away from a selected site unless both effective population sizes and migration rates are low. When multiple loci are considered, regions of differentiation can be larger. However, with many loci under selection, genome‐wide divergence occurs and genomic islands are erased. The results show that divergence hitchhiking can generate large regions of differentiation, but that the conditions under which this occurs are limited. Thus, speciation may often require multifarious selection acting on many, isolated and physically unlinked genes. How hitchhiking promotes further adaptive divergence warrants consideration.     

Ecological speciation in sympatric palms: 3. Genetic map reveals genomic islands underlying species divergence in Howea

Although it is now widely accepted that speciation can occur in the face of continuous gene flow, with little or no spatial separation, the mechanisms and genomic architectures that permit such divergence are still debated. Here, we examined speciation in the face of gene flow in the Howea palms of Lord Howe Island, Australia. We built a genetic map using a novel method applicable to long‐lived tree species, combining it with double digest restriction site–associated DNA sequencing of multiple individuals. Based upon various metrics, we detected 46 highly differentiated regions throughout the genome, four of which contained genes with functions that are particularly relevant to the speciation scenario for Howea, specifically salt and drought tolerance.     

Patterns of genetic,phenotypic, and acoustic variation across a chiffchaff (Phylloscopus collybita abietinus/tristis) hybrid zone

Characterizing patterns of evolution of genetic and phenotypic divergence between incipient species is essential to understand how evolution of reproductive isolation proceeds. Hybrid zones are excellent for studying such processes, as they provide opportunities to assess trait variation in individuals with mixed genetic background and to quantify gene flow across different genomic regions. Here, we combine plumage, song, mtDNA and whole‐genome sequence data and analyze variation across a sympatric zone between the European and the Siberian chiffchaff (Phylloscopus collybita abietinus/tristis) to study how gene exchange between the lineages affects trait variation. Our results show that chiffchaff within the sympatric region show more extensive trait variation than allopatric birds, with a large proportion of individuals exhibiting intermediate phenotypic characters. The genomic differentiation between the subspecies is lower in sympatry than in allopatry and sympatric birds have a mix of genetic ancestry indicating extensive ongoing and past gene flow. Patterns of phenotypic and genetic variation also vary between regions within the hybrid zone, potentially reflecting differences in population densities, age of secondary contact, or differences in mate recognition or mate preference. The genomic data support the presence of two distinct genetic clades corresponding to allopatric abietinus and tristis and that genetic admixture is the force underlying trait variation in the sympatric region—the previously described subspecies (“fulvescens”) from the region is therefore not likely a distinct taxon. In addition, we conclude that subspecies identification based on appearance is uncertain as an individual with an apparently distinct phenotype can have a considerable proportion of the genome composed of mixed alleles, or even a major part of the genome introgressed from the other subspecies. Our results provide insights into the dynamics of admixture across subspecies boundaries and have implications for understanding speciation processes and for the identification of specific chiffchaff individuals based on phenotypic characters.     

Incomplete reproductive isolation following host shift in brood parasitic indigobirds

Behavioural and molecular studies suggest that brood parasitic indigobirds (Vidua spp.) rapidly diversified through a process of speciation by host shift. However, behavioural imprinting on host song, the key mechanism promoting speciation in this system, may also lead to hybridization and gene flow among established indigobird species when and if female indigobirds parasitize hosts already associated with other indigobird species. It is therefore not clear to what extent the low level of genetic differentiation among indigobird species is due to recent common ancestry versus ongoing gene flow. We tested for reproductive isolation among three indigobird species in Cameroon, one of which comprises two morphologically indistinguishable host races. Mimicry of host songs corresponded with plumage colour in 184 male indigobirds, suggesting that females rarely parasitize the host of another indigobird species. Paternity analyses, however, suggest that imperfect specificity in host and/or mate choice allows for continuing gene flow between recently formed host races of the Cameroon Indigobird Vidua camerunensis; while 63 pairs of close relatives were associated with the same host, two strongly supported father-son pairs included males mimicking the songs of the two different hosts of V. camerunensis. Thus, complete reproductive isolation is not necessarily an automatic consequence of host shifts, a result that suggests an important role for natural and/or sexual selection in indigobird speciation.     

A role for migration‐linked genes and genomic islands in divergence of a songbird

Next‐generation sequencing has made it possible to begin asking questions about the process of divergence at the level of the genome. For example, recently, there has been a debate around the role of ‘genomic islands of divergence’ (i.e. blocks of outlier loci) in facilitating the process of speciation‐with‐gene‐flow. The Swainson's thrush, Catharus ustulatus, is a migratory songbird with two genetically distinct subspecies that differ in a number of traits known to be involved in reproductive isolation in birds (plumage coloration, song and migratory behaviour), despite contemporary gene flow along a secondary contact zone. Here, we use RAD‐PE sequencing to test emerging hypotheses about the process of divergence at the level of the genome and identify genes and gene regions involved in differentiation in this migratory songbird. Our analyses revealed distinct genomic islands on 15 of the 23 chromosomes and an accelerated rate of divergence on the Z chromosome, one of the avian sex chromosomes. Further, an analysis of loci linked to traits known to be involved in reproductive isolation in songbirds showed that genes linked to migration are significantly more differentiated than expected by chance, but that these genes lie primarily outside the genomic islands. Overall, our analysis supports the idea that genes linked to migration play an important role in divergence in migratory songbirds, but we find no compelling evidence that the observed genomic islands are facilitating adaptive divergence in migratory behaviour.