Song‐based species discrimination in a rapid Neotropical radiation of grassland seedeaters

Acoustic signals among newly diverged taxa have the potential to convey species identity, information that is key to reducing hybridization. Capuchino seedeaters constitute a remarkable example of recently radiated endemic species from the grasslands of South America. They are sexually dimorphic and show striking differences in male plumage coloration and song. Contrasting with this divergence in phenotype most species show extremely low neutral genetic differentiation and lack of reciprocal monophyly, which is interpreted to be a product of recent common ancestry and hybridization. Here we use field‐based playback experiments to test for the first time if males of two species, Sporophila hypoxantha and S. palustris, discriminate between conspecific and heterospecific song. Using various measures of behavior we find that both species react more strongly to their own songs. The response to playback from another southern capuchino cannot be differentiated from that of a control song from a more distantly related Sporophila species. Additionally, we did not find evidence for reinforcement as the response of S. hypoxantha did not differ between individuals that co‐occur with S. palustris and those that do not. Our finding suggests that song, a culturally inherited trait, may help maintain reproductive isolation between species in the rapid and explosive capuchino radiation.     

Speciation genomics and a role for the Z chromosome in the early stages of divergence between Mexican ducks and mallards

Speciation is a continuous and dynamic process, and studying organisms during the early stages of this process can aid in identifying speciation mechanisms. The mallard (Anas platyrhynchos) and Mexican duck (A. [p.] diazi) are two recently diverged taxa with a history of hybridization and controversial taxonomy. To understand their evolutionary history, we conducted genomic scans to characterize patterns of genetic diversity and divergence across the mitochondrial DNA (mtDNA) control region, 3523 autosomal loci and 172 Z‐linked sex chromosome loci. Between the two taxa, Z‐linked loci (Φ_ST = 0.088) were 5.2 times more differentiated than autosomal DNA (Φ_ST = 0.017) but comparable to mtDNA (Φ_ST = 0.092). This elevated Z differentiation deviated from neutral expectations inferred from simulated data that incorporated demographic history and differences in effective population sizes between marker types. Furthermore, 3% of Z‐linked loci, compared to <0.1% of autosomal loci, were detected as outlier loci under divergent selection with elevated relative (Φ_ST) and absolute (d_XY) estimates of divergence. In contrast, the ratio of Z‐linked and autosomal differentiation among the seven Mexican duck sampling locations was close to 1:1 (Φ_ST = 0.018 for both markers). We conclude that between mallards and Mexican ducks, divergence at autosomal markers is largely neutral, whereas greater divergence on the Z chromosome (or some portions thereof) is likely the product of selection that has been important in speciation. Our results contribute to a growing body of literature indicating elevated divergence on the Z chromosome and its likely importance in avian speciation.     

Molecular signatures of divergence and selection in closely related pine taxa

Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo, P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation.     

Population‐genomic approach reveals adaptive floral divergence in discrete populations of a hawk moth‐pollinated violet

Local adaptation to contrasting biotic or abiotic environments is an important evolutionary step that presumably precedes floral diversification at the species level, yet few studies have demonstrated the adaptive nature of intraspecific floral divergence in wild plant populations. We combine a population‐genomic approach with phenotypic information on floral traits to examine whether the differentiation in metric floral traits exhibited by 14 populations of the southern Spanish hawk moth‐pollinated violet Viola cazorlensis reflects adaptive divergence. Screening of many amplified fragment length polymorphism (AFLP) loci using a multiple‐marker‐based neutrality test identified nine outlier loci (2.6% of the total) that departed from neutral expectations and were potentially under selection. Generalized analysis of molecular variance revealed significant relationships between genetic distance and population divergence in three floral traits when genetic distance was based on outlier loci, but not when it was based on neutral ones. Population means of floral traits were closely correlated with population scores on the first principal coordinate axis of the genetic distance matrix using outlier loci, and with the allelic frequencies of four of the outlier loci. Results strongly support the adaptive nature of intraspecific floral divergence exhibited by V. cazorlensis and illustrate the potential of genome scans to identify instances of adaptive divergence when used in combination with phenotypic information.     

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.     

Heterogeneity and concordance in locus‐specific differentiation and introgression between species of towhees

The maintenance or breakdown of reproductive isolation is an observable outcome of secondary contact between species. In cases where hybrids beyond the F1 are formed, the representation of each species' ancestry can vary dramatically among genomic regions. This genomic heterogeneity in ancestry and introgression can offer insight into evolutionary processes, particularly if introgression is compared in multiple hybrid zones. Similarly, considerable heterogeneity exists across the genome in the extent to which populations and species have diverged, reflecting the combined effects of different evolutionary processes on genetic variation. We studied hybridization across two hybrid zones of two phenotypically well‐differentiated bird species in Mexico (Pipilo maculatus and P. ocai), to investigate genomic heterogeneity in differentiation and introgression. Using genotyping‐by‐sequencing (GBS) and hierarchical Bayesian models, we genotyped 460 birds at over 41 000 single nucleotide polymorphism (SNP) loci. We identified loci exhibiting extreme introgression relative to the genome‐wide expectation using a Bayesian genomic cline model. We also estimated locus‐specific F_ST and identified loci with exceptionally high genetic divergence between the parental species. We found some concordance of locus‐specific introgression in the two independent hybrid zones (6–20% of extreme loci shared across zones), reflecting areas of the genome that experience similar gene flow when the species interact. Additionally, heterogeneity in introgression and divergence across the genome revealed another subset of loci under the influence of locally specific factors. These results are consistent with a history in which reproductive isolation has been influenced by a common set of loci in both hybrid zones, but where local environmental and stochastic factors also lead to genomic differentiation.     

Genomic regions repeatedly involved in divergence among plant‐specialized pea aphid biotypes

Understanding the genetic bases of biological diversification is a long‐standing goal in evolutionary biology. Here, we investigate whether replicated cases of adaptive divergence involve the same genomic regions in the pea aphid, Acyrthosiphon pisum, a large complex of genetically differentiated biotypes, each specialized on different species of legumes. A previous study identified genomic regions putatively involved in host‐plant adaptation and/or reproductive isolation by performing a hierarchical genome scan in three biotypes. This led to the identification of 11 F_ST outliers among 390 polymorphic microsatellite markers. In this study, the outlier status of these 11 loci was assessed in eight biotypes specialized on other host plants. Four of the 11 previously identified outliers showed greater genetic differentiation among these additional biotypes than expected under the null hypothesis of neutral evolution (α < 0.01). Whether these hotspots of genomic divergence result from adaptive events, intrinsic barriers or reduced recombination is discussed.     

Hybridization and divergence in multi‐species oak (Quercus) communities

Oaks (Quercus: Fagaceae) commonly interbreed yet retain their morphological, genetic and ecological distinctiveness. Post‐zygotic isolation mechanisms, such as ecologically dependent selection on adaptive loci, may therefore limit introgression. To test this hypothesis, we quantified hybridization and genetic divergence across the contact zone of four red oaks (Quercus section Lobatae) in the Great Lakes region of North America using a suite of 259 amplified fragment length polymorphisms and 27 genic and genomic microsatellite markers. First, we identified hybrids using genetic structure analysis and confirmed the reliability of our assignments via simulations. Then, we identified candidate loci for species maintenance with three complementary tests for selection and obtained partial gene sequences linked to an outlier locus and three other loci. We detected evidence of recent hybridization among all species and considerable gene flow between Q. ellipsoidalis and Q. velutina. Overall, c. 20% of Q. velutina had recent ancestry from Q. ellipsoidalis, whereas nearly 30% of Q. ellipsoidalis had a Q. velutina ancestor. Most loci were negligibly to weakly differentiated among species, but two gene‐linked microsatellites deviated significantly from neutral expectations in multiple, complementary outlier tests. Both outlier loci were located in the same 15‐cM bin on an existing Q. robur linkage map, a region under divergent selection in other oak species. Adaptive loci in this highly differentiated genomic region may contribute to ecological divergence among species and limit introgression.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION DURING SPECIATION‐WITH‐GENE‐FLOW IN LAKE WHITEFISH SPECIES PAIRS ASSESSED BY RAD SEQUENCING

During speciation‐with‐gene‐flow, effective migration varies across the genome as a function of several factors, including proximity of selected loci, recombination rate, strength of selection, and number of selected loci. Genome scans may provide better empirical understanding of the genome‐wide patterns of genetic differentiation, especially if the variance due to the previously mentioned factors is partitioned. In North American lake whitefish (Coregonus clupeaformis), glacial lineages that diverged in allopatry about 60,000 years ago and came into contact 12,000 years ago have independently evolved in several lakes into two sympatric species pairs (a normal benthic and a dwarf limnetic). Variable degrees of reproductive isolation between species pairs across lakes offer a continuum of genetic and phenotypic divergence associated with adaptation to distinct ecological niches. To disentangle the complex array of genetically based barriers that locally reduce the effective migration rate between whitefish species pairs, we compared genome‐wide patterns of divergence across five lakes distributed along this divergence continuum. Using restriction site associated DNA (RAD) sequencing, we combined genetic mapping and population genetics approaches to identify genomic regions resistant to introgression and derive empirical measures of the barrier strength as a function of recombination distance. We found that the size of the genomic islands of differentiation was influenced by the joint effects of linkage disequilibrium maintained by selection on many loci, the strength of ecological niche divergence, as well as demographic characteristics unique to each lake. Partial parallelism in divergent genomic regions likely reflected the combined effects of polygenic adaptation from standing variation and independent changes in the genetic architecture of postzygotic isolation. This study illustrates how integrating genetic mapping and population genomics of multiple sympatric species pairs provide a window on the speciation‐with‐gene‐flow mechanism.     

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.     

Correlated patterns of genetic diversity and differentiation across an avian family

Comparative studies of closely related taxa can provide insights into the evolutionary forces that shape genome evolution and the prevalence of convergent molecular evolution. We investigated patterns of genetic diversity and differentiation in stonechats (genus Saxicola), a widely distributed avian species complex with phenotypic variation in plumage, morphology and migratory behaviour, to ask whether similar genomic regions have become differentiated in independent, but closely related, taxa. We used whole‐genome pooled sequencing of 262 individuals from five taxa and found that levels of genetic diversity and divergence are strongly correlated among different stonechat taxa. We then asked whether these patterns remain correlated at deeper evolutionary scales and found that homologous genomic regions have become differentiated in stonechats and the closely related Ficedula flycatchers. Such correlation across a range of evolutionary divergence and among phylogenetically independent comparisons suggests that similar processes may be driving the differentiation of these independently evolving lineages, which in turn may be the result of intrinsic properties of particular genomic regions (e.g. areas of low recombination). Consequently, studies employing genome scans to search for areas important for reproductive isolation or adaptation should account for corresponding regions of differentiation, as these regions may not necessarily represent speciation islands or evidence of local adaptation.     

Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species

Genome scans in recently separated species can inform on molecular mechanisms and evolutionary processes driving divergence. Large‐scale polymorphism data from multiple species pairs are also key to investigate the repeatability of divergence—whether radiations tend to show parallel responses to similar selection pressures and/or underlying molecular forces. Here, we used whole‐genome resequencing data from six wood white (Leptidea sp.) butterfly populations, representing three closely related species with karyomorph variation, to infer the species' demographic history and characterize patterns of genomic diversity and differentiation. The analyses supported previously established species relationships, and there was no evidence for postdivergence gene flow. We identified significant intraspecific genetic structure, in particular between karyomorph extremes in the wood white (L. sinapis)—a species with a remarkable chromosome number cline across the distribution range. The genomic landscapes of differentiation were erratic, and outlier regions were narrow and dispersed. Highly differentiated (F_ST) regions generally had low genetic diversity (θ_π), but increased absolute divergence (D_XY) and excess of rare frequency variants (low Tajima's D). A minority of differentiation peaks were shared across species and population comparisons. However, highly differentiated regions contained genes with overrepresented functions related to metabolism, response to stimulus and cellular processes, indicating recurrent directional selection on a specific set of traits in all comparisons. In contrast to the majority of genome scans in recently diverged lineages, our data suggest that divergence landscapes in Leptidea have been shaped by directional selection and genetic drift rather than stable recombination landscapes and/or introgression.     

Sequence differentiation in regions identified by a genome scan for local adaptation

Genome scans using large numbers of randomly selected markers have revealed a small proportion of loci that deviate from neutral expectations and so may mark genomic regions that contribute to local adaptation. Measurements of sequence differentiation and identification of genes in these regions is important but difficult, especially in organisms with limited genetic information available. We have followed up a genome scan in the marine gastropod, Littorina saxatilis, by searching a bacterial artificial chromosome library with differentiated and undifferentiated markers, sequencing four bacterial artificial chromosomes and then analysing sequence variation in population samples for fragments at, and close to the original marker polymorphisms. We show that sequence differentiation follows the patterns expected from the original marker frequencies, that differentiated markers identify independent and highly localized sites and that these sites fall outside coding regions. Two differentiated loci are characterized by insertions of putative transposable elements that appear to have increased in frequency recently and which might influence expression of downstream genes. These results provide strong candidate loci for the study of local adaptation in Littorina. They demonstrate an approach that can be applied to follow up genome scans in other taxa and they show that the genome scan approach can lead rapidly to candidate genes in nonmodel organisms.     

Gradual speciation in a global hotspot of plant diversity

The speciation process that underlies recent, rapid radiations of plants is controversial, and suggested mechanisms range from pollinator or ecological niche differentiation to allopatry and nonadaptive divergence. Phylogenetic approaches to locating the most appropriate speciation models have been constrained by the low levels of molecular divergence between recently diverged species, which are typical of recent, rapid radiations. In this issue of Molecular Ecology, Rymer et al. (2010) used coalescence analyses of sequence data and genome scans of Amplified Fragment Length Polymorphism (AFLP) loci to demonstrate that in a species complex in the irid genus Gladiolus, a member of the hyper diverse Cape flora of southern Africa, speciation is a gradual process. Older divergences are genetically more differentiated, and show a greater difference in flowering time and floral morphology, than taxa that diverged more recently. There is no evidence of any abrupt events. Gene flow is limited by shifts in flowering time and floral morphology; thus, by pre-zygotic rather than by post-zygotic mechanisms, these evolved together with the occupation of somewhat different habitats. This research gives the first critical insight into how the remarkable diversity in a diversity hotspot could have arisen. More importantly, it demonstrates that the speciation process in recent, rapid radiations is tractable and can be investigated with suitable genetic tools.     

Evolution of population structure in an estuarine‐dependent marine fish

Restriction site‐associated DNA (RAD) sequencing was used to characterize neutral and adaptive genetic variation among geographic samples of red drum, Sciaenops ocellatus, an estuarine‐dependent fish found in coastal waters along the southeastern coast of the United States (Atlantic) and the northern Gulf of Mexico (Gulf). Analyses of neutral and outlier loci revealed three genetically distinct regional clusters: one in the Atlantic and two in the northern Gulf. Divergence in neutral loci indicated gradual genetic change and followed a linear pattern of isolation by distance. Divergence in outlier loci was at least an order of magnitude greater than divergence in neutral loci, and divergence between the regions in the Gulf was twice that of divergence between other regions. Discordance in patterns of genetic divergence between outlier and neutral loci is consistent with the hypothesis that the former reflects adaptive responses to environmental factors that vary on regional scales, while the latter largely reflects drift processes. Differences in basic habitat, initiated by glacial retreat and perpetuated by contemporary oceanic and atmospheric forces interacting with the geomorphology of the northern Gulf, followed by selection, appear to have led to reduced gene flow among red drum across the northern Gulf, reinforcing differences accrued during isolation and resulting in continued divergence across the genome. This same dynamic also may pertain to other coastal or nearshore fishes (18 species in 14 families) where genetically or morphologically defined sister taxa occur in the three regions.     

High degree of genetic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus)

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene‐based genome‐scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection—as determined with several outlier detection methods—was low (F_ST = 0.033 or 0.011, respectively), whereas average F_ST for directionally selected markers was significantly higher (F_ST = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.     

Evolution of putative barrier loci at an intermediate stage of speciation with gene flow in campions (Silene)

Understanding the origin of new species is a central goal in evolutionary biology. Diverging lineages often evolve highly heterogeneous patterns of genetic differentiation; however, the underlying mechanisms are not well understood. We investigated evolutionary processes governing genetic differentiation between the hybridizing campions Silene dioica (L.) Clairv. and S. latifolia Poiret. Demographic modelling indicated that the two species diverged with gene flow. The best‐supported scenario with heterogeneity in both migration rate and effective population size suggested that a small proportion of the loci evolved without gene flow. Differentiation (F _ST) and sequence divergence (d _XY) were correlated and both tended to peak in the middle of most linkage groups, consistent with reduced gene flow at highly differentiated loci. Highly differentiated loci further exhibited signatures of selection. In between‐species population pairs, isolation by distance was stronger for genomic regions with low between‐species differentiation than for highly differentiated regions that may contain barrier loci. Moreover, differentiation landscapes within and between species were only weakly correlated, suggesting that linked selection due to shared recombination and gene density landscapes is not the dominant determinant of genetic differentiation in these lineages. Instead, our results suggest that divergent selection shaped the genomic landscape of differentiation between the two Silene species, consistent with predictions for speciation in the face of gene flow.