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.     

The relative influence of natural selection and geography on gene flow in guppies

Two general processes may influence gene flow among populations. One involves divergent selection, wherein the maladaptation of immigrants and hybrids impedes gene flow between ecological environments (i.e. ecological speciation). The other involves geographic features that limit dispersal. We determined the relative influence of these two processes in natural populations of Trinidadian guppies (Poecilia reticulata). If selection is important, gene flow should be reduced between different selective environments. If geography is important, gene flow should be impeded by geographic distance and physical barriers. We examined how genetic divergence, long-term gene flow, and contemporary dispersal within a watershed were influenced by waterfalls, geographic distance, predation, and habitat features. We found that waterfalls and geographic distance increased genetic divergence and reduced dispersal and long-term gene flow. Differences in predation or habitat features did not influence genetic divergence or gene flow. In contrast, differences in predation did appear to reduce contemporary dispersal. We suggest that the standard predictions of ecological speciation may be heavily nuanced by the mating behaviour and life history strategies of guppies.     

Divergence with gene flow and fine-scale phylogeographical structure in the wedge-billed woodcreeper, Glyphorynchus spirurus, a Neotropical rainforest bird

Determining the relative roles of vicariance and selection in restricting gene flow between populations is of central importance to the evolutionary process of population divergence and speciation. Here we use molecular and morphological data to contrast the effect of isolation (by mountains and geographical distance) with that of ecological factors (altitudinal gradients) in promoting differentiation in the wedge-billed woodcreeper, Glyphorynchus spirurus , a tropical forest bird, in Ecuador. Tarsus length and beak size increased relative to body size with altitude on both sides of the Andes, and were correlated with the amount of moss on tree trunks, suggesting the role of selection in driving adaptive divergence. In contrast, molecular data revealed a considerable degree of admixture along these altitudinal gradients, suggesting that adaptive divergence in morphological traits has occurred in the presence of gene flow. As suggested by mitochondrial DNA sequence data, the Andes act as a barrier to gene flow between ancient subspecific lineages. Genome-wide amplified fragment length polymorphism markers reflected more recent patterns of gene flow and revealed fine-scale patterns of population differentiation that were not detectable with mitochondrial DNA, including the differentiation of isolated coastal populations west of the Andes. Our results support the predominant role of geographical isolation in driving genetic differentiation in G. spirurus , yet suggest the role of selection in driving parallel morphological divergence along ecological gradients.     

Genomic consequences of multiple speciation processes in a stick insect

Diverse geographical modes and mechanisms of speciation are known, and individual speciation genes have now been identified. Despite this progress, genome-wide outcomes of different evolutionary processes during speciation are less understood. Here, we integrate ecological and spatial information, mating trials, transplantation data and analysis of 86 130 single nucleotide polymorphisms (SNPs) in eight populations (28 pairwise comparisons) of Timema cristinae stick insects to test the effects of different factors on genomic divergence in a system undergoing ecological speciation. We find patterns consistent with effects of numerous factors, including geographical distance, gene flow, divergence in host plant use and climate, and selection against maladaptive hybridization (i.e. reinforcement). For example, the number of highly differentiated ‘outlier loci’, allele-frequency clines and the overall distribution of genomic differentiation were recognizably affected by these factors. Although host use has strong effects on phenotypic divergence and reproductive isolation, its effects on genomic divergence were subtler and other factors had pronounced effects. The results demonstrate how genomic data can provide new insights into speciation and how genomic divergence can be complex, yet predictable. Future work could adopt experimental, mapping and functional approaches to directly test which genetic regions are affected by selection and determine their physical location in the genome.     

Panmixia supports divergence with gene flow in Darwin's small ground finch, Geospiza fuliginosa, on Santa Cruz, Galápagos Islands

The divergence‐with‐gene‐flow model of speciation has a strong theoretical basis with a growing number of plausible examples in nature, but remains hotly debated. Darwin’s finches of the Galápagos Archipelago have played an important role in our understanding of speciation processes. Recent studies suggest that this group may also provide insights into speciation via divergence with gene flow. On the island of Santa Cruz, recent studies found evidence for adaptive divergence in Darwin’s small ground finch, Geospiza fuliginosa, between ecologically contrasting arid and humid zones. Despite the short geographical distance between these zones, strong disruptive selection during low rainfall periods is expected to generate and maintain adaptive divergence. Conversely, during high rainfall periods, when disruptive selection is predicted to be weakened, population divergence in adaptive traits is expected to break down. Because periods of low and high rainfall irregularly alternate, the geographical pattern of adaptive divergence can be assumed to break down and, importantly, regenerate in situ. Here, we use microsatellite allele frequency data to assess the genetic population structure of G. fuliginosa on Santa Cruz. We sample 21 sites and four ecological zones across the island. We reject hypotheses of population substructure linked to ecological and geographical differences among sites in favour of a single panmictic population. Panmixia implies high levels of gene flow within Santa Cruz, which favours selection over genetic drift as a valid process generating phenotypic divergence in G. fuliginosa on Santa Cruz. We discuss how our findings may support classic adaptation, phenotypic plasticity, matching habitat choice or any combination of these three processes.     

Comparative landscape genetics and the adaptive radiation of Darwin's finches: the role of peripheral isolation

We use genetic divergence at 16 microsatellite loci to investigate how geographical features of the Galápagos landscape structure island populations of Darwin's finches. We compare the three most genetically divergent groups of Darwin's finches comprising morphologically and ecologically similar allopatric populations: the cactus finches (Geospiza scandens and Geospiza conirostris), the sharp-beaked ground finches (Geospiza difficilis) and the warbler finches (Certhidea olivacea and Certhidea fusca). Evidence of reduced genetic diversity due to drift was limited to warbler finches on small, peripheral islands. Evidence of low levels of recent interisland migration was widespread throughout all three groups. The hypothesis of distance-limited dispersal received the strongest support in cactus and sharp-beaked ground finches as evidenced by patterns of isolation by distance, while warbler finches showed a weaker relationship. Support for the hypothesis that gene flow constrains morphological divergence was only found in one of eight comparisons within these groups. Among warbler finches, genetic divergence was relatively high while phenotypic divergence was low, implicating stabilizing selection rather than constraint due to gene flow. We conclude that the adaptive radiation of Darwin's finches has occurred in the presence of ongoing but low levels of gene flow caused by distance-dependent interisland dispersal. Gene flow does not constrain phenotypic divergence, but may augment genetic variation and facilitate evolution due to natural selection. Both microsatellites and mtDNA agree in that subsets of peripheral populations of two older groups are genetically more similar to other species that underwent dramatic morphological change. The apparent decoupling of morphological and molecular evolution may be accounted for by a modification of Lack's two-stage model of speciation: relative ecological stasis in allopatry followed by secondary contact, ecological interactions and asymmetric phenotypic divergence.     

Patterns of gene flow define species of thermophilic Archaea

Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.     

Gene flow in Dubautia arborea and D. ciliolata: the roles of ecology and isolation by distance in maintaining species boundaries despite ongoing hybridization

The relative roles of gene flow and natural selection in maintaining species differentiation have been a subject of debate for some time. The traditional view is that gene flow constrains adaptive divergence and maintains species cohesiveness. Alternatively, ecological speciation posits that the reverse is true: that adaptive ecological differentiation constrains gene flow. In this study, we examine gene flow and population differentiation among populations of two species of the Hawaiian silversword alliance, Dubautia arborea and D. ciliolata. We compare divergence in putatively neutral microsatellite markers with divergence in leaf morphometric traits, which may be selectively important or physiologically linked to selectively important traits. Gene flow between populations was found to be significant in only one of the two species, D. arborea. Leaf morphometric differentiation between species was significant, though not among populations within species. No evidence of effective genetic introgression was observed between apparently 'pure' populations of these species. Gene flow as measured by microsatellites was not correlated with geographic distance between populations, but was correlated with the linear placement of the widest part of the leaf. Because these two species are interfertile, as demonstrated by the presence of active hybrid zone, the lack of genetic introgression and the maintenance of species boundaries may be associated with natural selection on differential habitat.     

Evaluation of genetic isolation within an island flora reveals unusually widespread local adaptation and supports sympatric speciation

It is now recognized that speciation can proceed even when divergent natural selection is opposed by gene flow. Understanding the extent to which environmental gradients and geographical distance can limit gene flow within species can shed light on the relative roles of selection and dispersal limitation during the early stages of population divergence and speciation. On the remote Lord Howe Island (Australia), ecological speciation with gene flow is thought to have taken place in several plant genera. The aim of this study was to establish the contributions of isolation by environment (IBE) and isolation by community (IBC) to the genetic structure of 19 plant species, from a number of distantly related families, which have been subjected to similar environmental pressures over comparable time scales. We applied an individual-based, multivariate, model averaging approach to quantify IBE and IBC, while controlling for isolation by distance (IBD). Our analyses demonstrated that all species experienced some degree of ecologically driven isolation, whereas only 12 of 19 species were subjected to IBD. The prevalence of IBE within these plant species indicates that divergent selection in plants frequently produces local adaptation and supports hypotheses that ecological divergence can drive speciation in sympatry.     

A genomic island linked to ecotype divergence in Atlantic cod

The genomic architecture underlying ecological divergence and ecological speciation with gene flow is still largely unknown for most organisms. One central question is whether divergence is genome‐wide or localized in ‘genomic mosaics’ during early stages when gene flow is still pronounced. Empirical work has so far been limited, and the relative impacts of gene flow and natural selection on genomic patterns have not been fully explored. Here, we use ecotypes of Atlantic cod to investigate genomic patterns of diversity and population differentiation in a natural system characterized by high gene flow and large effective population sizes, properties which theoretically could restrict divergence in local genomic regions. We identify a genomic region of strong population differentiation, extending over approximately 20 cM, between pairs of migratory and stationary ecotypes examined at two different localities. Furthermore, the region is characterized by markedly reduced levels of genetic diversity in migratory ecotype samples. The results highlight the genomic region, or ‘genomic island’, as potentially associated with ecological divergence and suggest the involvement of a selective sweep. Finally, we also confirm earlier findings of localized genomic differentiation in three other linkage groups associated with divergence among eastern Atlantic populations. Thus, although the underlying mechanisms are still unknown, the results suggest that ‘genomic mosaics’ of differentiation may even be found under high levels of gene flow and that marine fishes may provide insightful model systems for studying and identifying initial targets of selection during ecological divergence.     

Mutation‐order divergence by sexual selection: diversification of sexual signals in similar environments as a first step in speciation

The origin of species remains a central question, and recent research focuses on the role of ecological differences in promoting speciation. Ecological differences create opportunities for divergent selection (i.e. ‘ecological’ speciation), a Darwinian hypothesis that hardly requires justification. In contrast, ‘mutation‐order’ speciation proposes that, instead of adapting to different environments, populations find different ways to adapt to similar environments, implying that speciation does not require ecological differences. This distinction is critical as it provides an alternative hypothesis to the prevailing view that ecological differences drive speciation. Speciation by sexual selection lies at the centre of debates about the importance of ecological differences in promoting speciation; here, we present verbal and mathematical models of mutation‐order divergence by sexual selection. We develop three general cases and provide a two‐locus population genetic model for each. Results indicate that alternative secondary sexual traits can fix in populations that initially experience similar natural and sexual selection and that divergent traits and preferences can remain stable in the face of low gene flow. This stable divergence can facilitate subsequent divergence that completes or reinforces speciation. We argue that a mutation‐order process could explain widespread diversity in secondary sexual traits among closely related, allopatric species.     

Sexual selection drives speciation in an Amazonian frog

One proposed mechanism of speciation is divergent sexual selection, whereby divergence in female preferences and male signals results in behavioural isolation. Despite the appeal of this hypothesis, evidence for it remains inconclusive. Here, we present several lines of evidence that sexual selection is driving behavioural isolation and speciation among populations of an Amazonian frog (Physalaemus petersi). First, sexual selection has promoted divergence in male mating calls and female preferences for calls between neighbouring populations, resulting in strong behavioural isolation. Second, phylogenetic analysis indicates that populations have become fixed for alternative call types several times throughout the species' range, and coalescent analysis rejects genetic drift as a cause for this pattern, suggesting that this divergence is due to selection. Finally, gene flow estimated with microsatellite loci is an average of 30 times lower between populations with different call types than between populations separated by a similar geographical distance with the same call type, demonstrating genetic divergence and incipient speciation. Taken together, these data provide strong evidence that sexual selection is driving behavioural isolation and speciation, supporting sexual selection as a cause for speciation in the wild.     

Differential divergence in autosomes and sex chromosomes is associated with intra‐island diversification at a very small spatial scale in a songbird lineage

Recently diverged taxa showing marked phenotypic and ecological diversity provide optimal systems to understand the genetic processes underlying speciation. We used genome‐wide markers to investigate the diversification of the Reunion grey white‐eye (Zosterops borbonicus) on the small volcanic island of Reunion (Mascarene archipelago), where this species complex exhibits four geographical forms that are parapatrically distributed across the island and differ strikingly in plumage colour. One form restricted to the highlands is separated by a steep ecological gradient from three distinct lowland forms which meet at narrow hybrid zones that are not associated with environmental variables. Analyses of genomic variation based on single nucleotide polymorphism data from genotyping‐by‐sequencing and pooled RAD‐seq approaches show that signatures of selection associated with elevation can be found at multiple regions across the genome, whereas most loci associated with the lowland forms are located on the Z sex chromosome. We identified TYRP1, a Z‐linked colour gene, as a likely candidate locus underlying colour variation among lowland forms. Tests of demographic models revealed that highland and lowland forms diverged in the presence of gene flow, and divergence has progressed as gene flow was restricted by selection at loci across the genome. This system holds promise for investigating how adaptation and reproductive isolation shape the genomic landscape of divergence at multiple stages of the speciation process.     

Speciation along a depth gradient in a marine adaptive radiation

Oceans are home to much of the world''s biodiversity, but we know little about the processes driving speciation in marine ecosystems with few geographical barriers to gene flow. Ecological speciation resulting from divergent natural selection between ecological niches can occur in the face of gene flow. Sister species in the young and ecologically diverse rockfish genus Sebastes coexist in the northeast Pacific, implying that speciation may not require geographical isolation. Here, I use a novel phylogenetic comparative analysis to show that rockfish speciation is instead associated with divergence in habitat depth and depth-associated morphology, consistent with models of parapatric speciation. Using the same analysis, I find no support for alternative hypotheses that speciation involves divergence in diet or life history, or that speciation involves geographic isolation by latitude. These findings support the hypothesis that rockfishes undergo ecological speciation on an environmental gradient.     

Population genetic structure across dissolved oxygen regimes in an African cichlid fish

Ecological isolation is a process whereby gene flow between selective environments is reduced due to selection against maladapted dispersers, migrant alleles, or hybrids. Although ecological isolation has been documented in several systems, gene flow can often be high among selective regimes, and more studies are thus needed to better understand the conditions under which ecological gradients or divergent selective regimes should influence population structure. We test for ecological isolation in a system in which high plasticity occurs with respect to traits that are adaptive in alternate forms under different environmental conditions. Pseudocrenilabrus multicolor victoriae is a widespread haplochromine cichlid fish in East Africa that exploits both normoxic (normal oxygen) rivers/lakes and hypoxic (low oxygen) swamps. Here, we examine population structure, using mitochondrial DNA and microsatellites, to determine if genetic divergence is significantly increased between dissolved oxygen regimes relative to within them, while controlling for geographical structure. Our results indicate that geographical separation influences population structure, while no effects of divergent selection with respect to oxygen regimes were detected. Specifically, we document (i) genetic clustering according to geographical region, but no clustering according to oxygen regime; (ii) higher genetic variation among than within regions, but no effect of oxygen regime on genetic variation; (iii) isolation by distance within one region; and (iv) decreasing genetic variability with increasing geographical distance from Lake Victoria. We speculate that plasticity may be facilitating gene flow between oxygen regimes in this system.     

Assessing models of speciation under different biogeographic scenarios; an empirical study using multi‐locus and RNA‐seq analyses

Evolutionary biology often seeks to decipher the drivers of speciation, and much debate persists over the relative importance of isolation and gene flow in the formation of new species. Genetic studies of closely related species can assess if gene flow was present during speciation, because signatures of past introgression often persist in the genome. We test hypotheses on which mechanisms of speciation drove diversity among three distinct lineages of desert tortoise in the genus Gopherus. These lineages offer a powerful system to study speciation, because different biogeographic patterns (physical vs. ecological segregation) are observed at opposing ends of their distributions. We use 82 samples collected from 38 sites, representing the entire species' distribution and generate sequence data for mtDNA and four nuclear loci. A multilocus phylogenetic analysis in *BEAST estimates the species tree. RNA‐seq data yield 20,126 synonymous variants from 7665 contigs from two individuals of each of the three lineages. Analyses of these data using the demographic inference package ?a?i serve to test the null hypothesis of no gene flow during divergence. The best‐fit demographic model for the three taxa is concordant with the *BEAST species tree, and the ?a?i analysis does not indicate gene flow among any of the three lineages during their divergence. These analyses suggest that divergence among the lineages occurred in the absence of gene flow and in this scenario the genetic signature of ecological isolation (parapatric model) cannot be differentiated from geographic isolation (allopatric model).     

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.     

Signal Divergence is Correlated with Genetic Distance and not Environmental Differences in Darters (Percidae: Etheostoma)

Speciation research focuses on the evolutionary mechanisms responsible for the origin of species, and recent treatments have distinguished ecological and mutation-order speciation as distinct evolutionary processes. Using a research framework that considers ??speciation phenotypes?? (sensu Shaw and Mullen in Genet 139(5):649?C661, 2011) and a modified hierarchy of speciation models, we address whether speciation in benthic fishes commonly called darters proceeds under divergent ecological selection or a mutation-order process. We examined neutral genetic divergence, sexual signal (male color) divergence, environmental differences, and geographic distance in 66 species pair comparisons. Modified Mantel tests detected significant relationships between genetic distance and overall male color differences, as well as geographic distance and overall male color differences; however, after accounting for the correlation of male color and geographic distance with genetic distance using a partial Mantel test, no relationship was observed between male color and geographic distance. Neither microhabitat nor climatic measures of environmental differences correlated with overall male color differences. Color difference scores for discrete color categories (i.e., red/orange/yellow, black, and blue/green) differed in their correlations with explanatory variables, implying different selection regimes may be influencing each component of darter color patterns. Our results do not support a primary role for divergent ecological selection shaping early divergence of darter sexual signals. Instead, a model of mutation-order speciation may best explain the clock-like manner of changes in male color among darter species.     

Ecological divergence combined with ancient allopatry in lizard populations from a small volcanic island

Population divergence and speciation are often explained by geographical isolation, but may also be possible under high gene flow due to strong ecology‐related differences in selection pressures. This study combines coalescent analyses of genetic data (11 microsatellite loci and 1 Kbp of mtDNA) and ecological modelling to examine the relative contributions of isolation and ecology to incipient speciation in the scincid lizard Chalcides sexlineatus within the volcanic island of Gran Canaria. Bayesian multispecies coalescent dating of within‐island genetic divergence of northern and southern populations showed correspondence with the timing of volcanic activity in the north of the island 1.5–3.0 Ma ago. Coalescent estimates of demographic changes reveal historical size increases in northern populations, consistent with expansions from a volcanic refuge. Nevertheless, ecological divergence is also supported. First, the two morphs showed non‐equivalence of ecological niches and species distribution modelling associated the northern morph with mesic habitat types and the southern morph with xeric habitat types. It seems likely that the colour morphs are associated with different antipredator strategies in the different habitats. Second, coalescent estimation of gene copy migration (based on microsatellites and mtDNA) suggest high rates from northern to southern morphs demonstrating the strength of ecology‐mediated selection pressures that maintain the divergent southern morph. Together, these findings underline the complexity of the speciation process by providing evidence for the combined effects of ecological divergence and ancient divergence in allopatry.     

Genetic divergence with ongoing gene flow is maintained by the use of different hosts in phytophagous ladybird beetles genus Henosepilachna

Adaptation to different environments can promote population divergence via natural selection even in the presence of gene flow – a phenomenon that typically occurs during ecological speciation. To elucidate how natural selection promotes and maintains population divergence during speciation, we investigated the population genetic structure, degree of gene flow and heterogeneous genomic divergence in three closely related Japanese phytophagous ladybird beetles: Henosepilachna pustulosa, H. niponica and H. yasutomii. These species act as a generalist, a wild thistle (Cirsium spp.) specialist and a blue cohosh (Caulophyllum robustum) specialist, respectively, and their ranges differ accordingly. The two specialist species widely co‐occur but are reproductively isolated solely due to their high specialization to a particular host plant. Genomewide amplified fragment‐length polymorphism (AFLP) markers and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences demonstrated obvious genomewide divergence associated with both geographic distance and ecological divergence. However, a hybridization assessment for both AFLP loci and the mitochondrial sequences revealed a certain degree of unidirectional gene flow between the two sympatric specialist species. Principal coordinates analysis (PCoA) based on all of the variable AFLP loci demonstrated that there are genetic similarities between populations from adjacent localities irrespective of the species (i.e. host range). However, a further comparative genome scan identified a few fractions of loci representing approximately 1% of all loci as different host‐associated outliers. These results suggest that these three species had a complex origin, which could be obscured by current gene flow, and that ecological divergence can be maintained with only a small fraction of the genome is related to different host use even when there is a certain degree of gene flow between sympatric species pairs.