Population genomic evidence of selection on structural variants in a natural hybrid zone.

Structural variants (SVs) can promote speciation by directly causing reproductive isolation or by suppressing recombination across large genomic regions. Whereas examples of each mechanism have been documented, systematic tests of the role of SVs in speciation are lacking. Here, we take advantage of long-read (Oxford nanopore) whole-genome sequencing and a hybrid zone between two Lycaeides butterfly taxa (L. melissa and Jackson Hole Lycaeides) to comprehensively evaluate genome-wide patterns of introgression for SVs and relate these patterns to hypotheses about speciation. We found >100,000 SVs segregating within or between the two hybridizing species. SVs and SNPs exhibited similar levels of genetic differentiation between species, with the exception of inversions, which were more differentiated. We detected credible variation in patterns of introgression among SV loci in the hybrid zone, with 562 of 1419 ancestry-informative SVs exhibiting genomic clines that deviated from null expectations based on genome-average ancestry. Overall, hybrids exhibited a directional shift towards Jackson Hole Lycaeides ancestry at SV loci, consistent with the hypothesis that these loci experienced more selection on average than SNP loci. Surprisingly, we found that deletions, rather than inversions, showed the highest skew towards excess ancestry from Jackson Hole Lycaeides. Excess Jackson Hole Lycaeides ancestry in hybrids was also especially pronounced for Z-linked SVs and inversions containing many genes. In conclusion, our results show that SVs are ubiquitous and suggest that SVs in general, but especially deletions, might disproportionately affect hybrid fitness and thus contribute to reproductive isolation.

Genetic traces of hybrid zone movement across a fragmented habitat.

Theoretical and empirical studies suggest that the structure and position of hybrid zones can change over time. Evidence for moving hybrid zones has been directly inferred by repeated sampling over time, or indirectly through the detection of genetic footprints left by the receding species and the resulting asymmetric patterns of introgression across markers. We here investigate a hybrid zone formed by two subspecies of the Iberian golden-striped salamander, Chioglossa lusitanica, using a panel of 35 nuclear loci (31 SNPs and 4 allozymes) and one mitochondrial locus in a transect in central Portugal. We found concordant and coincident clines for most of the nuclear loci (n = 22, 63%), defining a narrow hybrid zone of ca. 6 km wide, with the centre positioned ca. 15 km south of the Mondego River. Asymmetric introgression was observed at another 14 loci. Their clines are displaced towards the north, with positions located either close to the Mondego River (n = 6) or further northwards (n = 8). We interpret these profiles as genetic traces of the southward displacement of C. lusitanica lusitanica by C. l. longipes over the wider Mondego River valley. We noted the absence of significant linkage disequilibrium, and we inferred low levels of effective selection per locus against hybrids, suggesting that introgression in the area of species replacement occurred under a neutral diffusion process. A species distribution model suggests that the C. lusitanica hybrid zone coincides with a narrow corridor of fragmented habitat. From the position of the displaced clines, we infer that patches of locally suitable habitat trapped some genetic variants that became disassociated from the southward moving hybrid zone. This study highlights the influence of habitat availability on hybrid zone movement.

Müllerian mimicry of a quantitative trait despite contrasting levels of genomic divergence and selection.

Hybrid zones, where distinct populations meet and interbreed, give insight into how differences between populations are maintained despite gene flow. Studying clines in genetic loci and adaptive traits across hybrid zones is a powerful method for understanding how selection drives differentiation within a single species, but can also be used to compare parallel divergence in different species responding to a common selective pressure. Here, we study parallel divergence of wing colouration in the butterflies Heliconius erato and H. melpomene, which are distantly related Müllerian mimics which show parallel geographic variation in both discrete variation in pigmentation, and quantitative variation in structural colour. Using geographic cline analysis, we show that clines in these traits are positioned in roughly the same geographic region for both species, which is consistent with direct selection for mimicry. However, the width of the clines varies markedly between species. This difference is explained in part by variation in the strength of selection acting on colour traits within each species, but may also be influenced by differences in the dispersal rate and total strength of selection against hybrids between the species. Genotyping-by-sequencing also revealed weaker population structure in H. melpomene, suggesting the hybrid zones may have evolved differently in each species, which may also contribute to the patterns of phenotypic divergence in this system. Overall, we conclude that multiple factors are needed to explain patterns of clinal variation within and between these species, although mimicry has probably played a central role.

Riverscape properties contribute to the origin and structure of a hybrid zone in a Neotropical freshwater fish.

Understanding the structure of hybrid zones provides valuable insights about species boundaries and speciation, such as the evolution of barriers to gene flow and the strength of selection. In river networks, studying evolutionary processes in hybrid zones can be especially challenging, given the influence of past and current river properties along with biological species-specific traits. Here, we suggest that a natural hybrid zone between two divergent lineages of the sexually dimorphic Neotropical fish Nematocharax venustus was probably established by secondary contact as a result of a river capture event between the Contas and Pardo river basins. This putative river capture is supported by hydrogeological evidence of elbows of capture, wind gaps and geological faults. The morphological (colour pattern) and genetic (mtDNA and RADseq) variation reveal a clinal transition between parental lineages along the main river, with predominance of F2 hybrids at the centre of the hybrid zone, absence of early generation backcrosses and different levels of hybridization in the tributaries. We highlight that different sources of information are crucial for understanding how the riverscape spatial history influences the connectivity between and within river systems and, consequently, the dynamics of gene flow between freshwater lineages/species.

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt.

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south-western and south-eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south-western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south-eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150-km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2-27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population-level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.

High gene flow despite opposite chirality in hybrid zones between enantiomorphic door snails.

We studied differentiation and geneflow patterns between enantiomorphic door-snail species in two hybrid zones in the Bucegi Mountains (Romania) to investigate the effects of intrinsic barriers (complications in copulation) and extrinsic selection by environmental factors. A mitochondrial gene tree confirmed the historical separation of the examined populations into the dextral Alopia livida and the sinistral Alopia straminicollis in accordance with the morphological classification, but also indicated gene flow between the species. By contrast, a network based on amplified fragment length polymorphisms (AFLP) markers revealed local groups of populations as units independent of their species affiliation. Admixture analyses based on AFLP data showed that the genomes of most individuals in the hybrid zones are composed of parts of the genomes of both parental taxa. The introgression patterns of a notable fraction of the examined markers deviated from neutral introgression. However, the patterns of most non-neutral markers were not concordant between the two hybrid zones. There was also no concordance between non-neutral markers in the two genomic clines and markers that were correlated with environmental variables or markers that were correlated with the proportion of dextral individuals in the populations. Neither extrinsic selection by environmental factors nor intrinsic barriers resulting from positive frequency-dependent selection of the prevailing coiling direction were sufficient to maintain the distinctness of A. straminicollis and A. livida. Despite being historically separated units, we conclude that these taxa now merge where they come into contact.

Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads.

Much progress in speciation research stems from documenting patterns of morphological and genetic variation in hybrid zones. Contrasting patterns of marker introgression in different sections of the contact can provide valuable insights on the relative importance of various evolutionary mechanisms maintaining species differences in the face of hybridization and gene flow and on hybrid zone temporal and spatial dynamics. We studied species interactions in the common toads Bufo bufo and B. spinosus in France and northwestern Italy using morphological and molecular data from the mitochondrial and nuclear genomes in an extensive survey, including two independent transects west and east of the Alps. At both, we found sharp, coincident and concordant nuclear genetic transitions. However, morphological clines were wider or absent and mtDNA introgression was asymmetric. We discuss alternative, nonexclusive hypotheses about evolutionary processes generating these patterns, including drift, selection, long-distance dispersal and spatial shifts in hybrid zone location and structure. The distribution of intraspecific mtDNA lineages supports a scenario in which B. bufo held a local refugium during the last glacial maximum. Present-day genetic profiles are best explained by an advance of B. spinosus from a nearby Iberian refugium, largely superseding the local B. bufo population, followed by an advance of B. bufo from the Balkans, with prongs north and south of the Alps, driving B. spinosus southwards. A pendulum moving hybrid zone, first northwards and then southwards, explains the wide areas of introgression at either side of the current position of the contact zones.

Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France.

BACKGROUND: Hybrid zones are regions where individuals of two species meet and produce hybrid progeny, and are often regarded as natural laboratories to understand the process of species formation. Two microevolutionary processes can take place in hybrid zones, with opposing effects on population differentiation. Hybridization tends to produce genetic homogenization, reducing species differences, whereas the presence of mechanisms of reproductive isolation result in barriers to gene flow, maintaining or increasing differences between taxa. RESULTS: Here we study a contact zone between two hybridizing toad species, Bufo bufo and B. spinosus, through a combination of molecular (12 polymorphic microsatellites, four nuclear and two mitochondrial SNP markers) and morphological data in a transect in the northwest of France. The results show largely concordant clines across markers, defining a narrow hybrid zone of ca. 30 km wide. Most hybrids in the centre of the contact zone are classified as F2 or backcrossed individuals, with no individuals assigned to the F1 hybrid class. CONCLUSIONS: We discuss the implications of these results for our understanding of the evolutionary history of these species. We anticipate that the toad contact zone here described will become an important asset in the study of hybrid zone dynamics and evolutionary biology because of its easy access and the abundance of the species involved.

Revisiting the Iberian honey bee (Apis mellifera iberiensis) contact zone: maternal and genome-wide nuclear variations provide support for secondary contact from historical refugia.

Dissecting diversity patterns of organisms endemic to Iberia has been truly challenging for a variety of taxa, and the Iberian honey bee is no exception. Surveys of genetic variation in the Iberian honey bee are among the most extensive for any honey bee subspecies. From these, differential and complex patterns of diversity have emerged, which have yet to be fully resolved. Here, we used a genome-wide data set of 309 neutrally tested single nucleotide polymorphisms (SNPs), scattered across the 16 honey bee chromosomes, which were genotyped in 711 haploid males. These SNPs were analysed along with an intergenic locus of the mtDNA, to reveal historical patterns of population structure across the entire range of the Iberian honey bee. Overall, patterns of population structure inferred from nuclear loci by multiple clustering approaches and geographic cline analysis were consistent with two major clusters forming a well-defined cline that bisects Iberia along a northeastern-southwestern axis, a pattern that remarkably parallels that of the mtDNA. While a mechanism of primary intergradation or isolation by distance could explain the observed clinal variation, our results are more consistent with an alternative model of secondary contact between divergent populations previously isolated in glacial refugia, as proposed for a growing list of other Iberian taxa. Despite current intense honey bee management, human-mediated processes have seemingly played a minor role in shaping Iberian honey bee genetic structure. This study highlights the complexity of the Iberian honey bee patterns and reinforces the importance of Iberia as a reservoir of Apis mellifera diversity.

Strong selection on male plumage in a hybrid zone between a hybrid bird species and one of its parents.

Homoploid hybrid speciation (HHS) requires reproductive barriers between hybrid and parent species, despite incomplete reproductive isolation (RI) between the parents. Novel secondary sexual trait values in hybrids may cause prezygotic isolation from both parents, whereas signals inherited by the hybrid from one parent species may cause prezygotic isolation with the other. Here we investigate whether differences in male plumage function as a premating barrier between the hybrid Italian sparrow and one of its parent species, the house sparrow, in a narrow Alpine hybrid zone. Italian sparrow male plumage is a composite mosaic of the parental traits, with its head plumage most similar to its other parent, the Spanish sparrow. We use geographical cline analysis to examine selection on three plumage traits, 75 nuclear single nucleotide polymorphisms (SNPs) and hybrid indices based on these SNPs. Several SNPs showed evidence of restricted introgression in the Alps, supporting earlier findings. Crown colour exhibited the narrowest plumage cline, representing a 37% (range 4-65%) drop in fitness. The cline was too narrow to be due to neutral introgression. Only crown colour was significantly bimodal in the hybrid zone. Bimodality may be due to RI or a major QTL, although fitness estimates suggest that selection contributes to the pattern. We discuss the implications with respect to HHS and the species status of the Italian sparrow.

Hybridization and mitochondrial genome introgression between Rana chensinensis and R. kukunoris.

Mitochondrial genome (mito-genome) introgression among metazoans is commonplace, and several biological processes may promote such introgression. We examined two proposed processes for the mito-genome introgression between Rana chensinensis and R. kukunoris: natural hybridization and sex-biased dispersal. We sampled 477 individuals from 28 sites in the potential hybrid zone in the western Tsinling Mountains. Mitochondrial gene (cyt-b) trees were used to examine the introgression events. Microsatellite DNA loci, cyt-b and morphological data were used to identify hybrids and to examine the extent of natural hybridization. We detected rampant bidirectional introgressions, both ancient and recent, between the two species. Furthermore, we found a wide hybrid zone, and frequent and asymmetric hybridization. The hybrid zone cline analysis revealed a clear mitochondrial-nuclear discordance; while most nuclear markers displayed similar and steep clines, cyt-b had a displaced cline centre and a more gradual and wider cline. We also detected strong and asymmetric historical maternal gene flow across the hybrid zone. This widespread hybridization and detected low mito-nuclear conflicts may, at least partially, explain the high frequency of introgression. Lastly, microsatellite data and population genetic methods were used to assess sex-biased dispersal. A weak pattern of female-biased dispersal was detected in both species, suggesting it may not play an important role in the observed introgression. Our data are consistent with the hybridization hypothesis, but support for the sex-biased dispersal hypothesis is weak. We further suggest that selective advantages of the R. kukunoris-type mito-genome in thermal adaptation may also contribute to the introgression between the two species.

Hybrid zone analysis confirms cryptic species of banded newt and does not support competitive displacement since secondary contact.

When two putatively cryptic species meet in nature, hybrid zone analysis can be used to estimate the extent of gene flow between them. Two recently recognized cryptic species of banded newt (genus Ommatotriton) are suspected to meet in parapatry in Anatolia, but a formal hybrid zone analysis has never been conducted. We sample populations throughout the range, with a focus on the supposed contact zone, and genotype them for 31 nuclear DNA SNP markers and mtDNA. We determine the degree of genetic admixture, introgression, and niche overlap. We reveal an extremely narrow hybrid zone, suggesting strong selection against hybrids, in line with species status. The hybrid zone does not appear to be positioned at an ecological barrier, and there is significant niche overlap. Therefore, the hybrid zone is best classified as a tension zone, maintained by intrinsic selection against hybrids. While the two banded newt species can evidently backcross, we see negligible introgression and the pattern is symmetric, which we interpret as supporting the fact that the hybrid zone has been practically stationary since its origin (while extensive hybrid zone movement has been suggested in other newt genera in the region). Our study illustrates the use of hybrid zone analysis to test cryptic species status.

Genotype-environment associations across spatial scales reveal the importance of putative adaptive genetic variation in divergence.

Identifying areas of high evolutionary potential is a judicious strategy for developing conservation priorities in the face of environmental change. For wide-ranging species occupying heterogeneous environments, the evolutionary forces that shape distinct populations can vary spatially. Here, we investigate patterns of genomic variation and genotype-environment associations in the hermit thrush (Catharus guttatus), a North American songbird, at broad (across the breeding range) and narrow spatial scales (at a hybrid zone). We begin by building a genoscape or map of genetic variation across the breeding range and find five distinct genetic clusters within the species, with the greatest variation occurring in the western portion of the range. Genotype-environment association analyses indicate higher allelic turnover in the west than in the east, with measures of temperature surfacing as key predictors of putative adaptive genomic variation rangewide. Since broad patterns detected across a species' range represent the aggregate of many locally adapted populations, we investigate whether our broadscale analysis is consistent with a finer scale analysis. We find that top rangewide temperature-associated loci vary in their clinal patterns (e.g., steep clines vs. fixed allele frequencies) across a hybrid zone in British Columbia, suggesting that the environmental predictors and the associated candidate loci identified in the rangewide analysis are of variable importance in this particular region. However, two candidate loci exhibit strong concordance with the temperature gradient in British Columbia, suggesting a potential role for temperature-related barriers to gene flow and/or temperature-driven ecological selection in maintaining putative local adaptation. This study demonstrates how patterns identified at the broad (macrogeographic) scale can be validated by investigating genotype-environment correlations at the local (microgeographic) scale. Furthermore, our results highlight the importance of considering the spatial distribution of putative adaptive variation when assessing population-level sensitivity to climate change and other stressors.

Asymmetric allelic introgression across a hybrid zone of the coal tit (Periparus ater) in the central Himalayas.

In the Himalayas, a number of secondary contact zones have been described for vicariant vertebrate taxa. However, analyses of genetic divergence and admixture are missing for most of these examples. In this study, we provide a population genetic analysis for the coal tit (Periparus ater) hybrid zone in Nepal. Intermediate phenotypes between the distinctive western "spot-winged tit" (P. a. melanolophus) and Eastern Himalayan coal tits (P. a. aemodius) occur across a narrow range of <100 km in western Nepal. As a peculiarity, another distinctive cinnamon-bellied form is known from a single population so far. Genetic admixture of western and eastern mitochondrial lineages was restricted to the narrow zone of phenotypically intermediate populations. The cline width was estimated 46 km only with a center close to the population of the cinnamon-bellied phenotype. In contrast, allelic introgression of microsatellite loci was asymmetrical from eastern P. a. aemodius into far western populations of phenotypic P. a. melanolophus but not vice versa. Accordingly, the microsatellite cline was about 3.7 times wider than the mitochondrial one.

Spatial variation in gene flow across a hybrid zone reveals causes of reproductive isolation and asymmetric introgression in wall lizards.

Hybrid zones provide insights into the evolution of reproductive isolation. Sexual selection can contribute to the evolution of reproductive barriers, but it remains poorly understood how sexual traits impact gene flow in secondary contact. Here, we show that a recently evolved suite of sexual traits that function in male-male competition mediates gene flow between two lineages of wall lizards (Podarcis muralis). Gene flow was relatively low and asymmetric in the presence of exaggerated male morphology and coloration compared to when the lineages share the ancestral phenotype. Putative barrier loci were enriched in genomic regions that were highly differentiated between the two lineages and showed low concordance between the transects. The exception was a consistently low genetic exchange around ATXN1, a gene that modulates social behavior. We suggest that this gene may contribute to the male mate preferences that are known to cause lineage-assortative mating in this species. Although female choice modulates the degree of reproductive isolation in a variety of taxa, wall lizards demonstrate that both male-male competition and male mate choice can contribute to the extent of gene flow between lineages.

Tracing the footprints of a moving hybrid zone under a demographic history of speciation with gene flow.

A lack of optimal gene combinations, as well as low levels of genetic diversity, is often associated with the formation of species range margins. Conservation efforts rely on predictive modelling using abiotic variables and assessments of genetic diversity to determine target species and populations for controlled breeding, germplasm conservation and assisted migration. Biotic factors such as interspecific competition and hybridization, however, are largely ignored, despite their prevalence across diverse taxa and their role as key evolutionary forces. Hybridization between species with well-developed barriers to reproductive isolation often results in the production of offspring with lower fitness. Generation of novel allelic combinations through hybridization, however, can also generate positive fitness consequences. Despite this possibility, hybridization-mediated introgression is often considered a threat to biodiversity as it can blur species boundaries. The contribution of hybridization towards increasing genetic diversity of populations at range margins has only recently gathered attention in conservation studies. We assessed the extent to which hybridization contributes towards range dynamics by tracking spatio-temporal changes in the central location of a hybrid zone between two recently diverged species of pines: Pinus strobiformis and P. flexilis. By comparing geographic cline centre estimates for global admixture coefficient with morphological traits associated with reproductive output, we demonstrate a northward shift in the hybrid zone. Using a combination of spatially explicit, individual-based simulations and linkage disequilibrium variance partitioning, we note a significant contribution of adaptive introgression towards this northward movement, despite the potential for differences in regional population size to aid hybrid zone movement. Overall, our study demonstrates that hybridization between recently diverged species can increase genetic diversity and generate novel allelic combinations. These novel combinations may allow range margin populations to track favourable climatic conditions or facilitate adaptive evolution to ongoing and future climate change.

The geography of divergence with gene flow facilitates multitrait adaptation and the evolution of pollinator isolation in Mimulus aurantiacus.

Ecological adaptation is the driving force during divergence with gene flow and generates reproductive isolation early in speciation. Although gene flow opposes divergence, local adaptation can be facilitated by factors that prevent the breakup of favorable allelic combinations. We investigated how selection, genetic architecture, and geography have contributed to the maintenance of floral trait divergence and pollinator isolation between parapatric ecotypes of Mimulus aurantiacus. Combining greenhouse, field, and genomic studies, we show that sharp clines in floral traits are maintained by spatially varying selection. Although adaptation breaks down where the ecotypes co-occur, leading to the formation of a hybrid zone, the largely non-overlapping distributions of the ecotypes shield them from immigrant genes, facilitating divergence across most of the range. In contrast to the sharp genetic discontinuities observed across most hybrid zones, we observed a gradual cline in genome-wide divergence and a pattern of isolation by distance across the landscape. Thus, contrary to a long period of allopatry followed by recent re-contact, our data suggest that floral trait divergence in M. aurantiacus may have evolved with locally restricted, but ongoing gene flow. Therefore, our study reveals how the geographic distribution of an organism can contribute to the evolution of premating isolation in the early stages of divergence with gene flow.