RIDGE, a tool tailored to detect gene flow barriers across species pairs.

Characterizing the processes underlying reproductive isolation between diverging lineages is central to understanding speciation. Here, we present RIDGE-Reproductive Isolation Detection using Genomic polymorphisms-a tool tailored for quantifying gene flow barrier proportion and identifying the relevant genomic regions. RIDGE relies on an Approximate Bayesian Computation with a model-averaging approach to accommodate diverse scenarios of lineage divergence. It captures heterogeneity in effective migration rate along the genome while accounting for variation in linked selection and recombination. The barrier detection test relies on numerous summary statistics to compute a Bayes factor, offering a robust statistical framework that facilitates cross-species comparisons. Simulations revealed RIDGE's efficiency in capturing signals of ongoing migration. Model averaging proved particularly valuable in scenarios of high model uncertainty where no migration or migration homogeneity can be wrongly assumed, typically for recent divergence times <0.1 2Ne generations. Applying RIDGE to four published crow data sets, we first validated our tool by identifying a well-known large genomic region associated with mate choice patterns. Second, while we identified a significant overlap of outlier loci using RIDGE and traditional genomic scans, our results suggest that a substantial portion of previously identified outliers are likely false positives. Outlier detection relies on allele differentiation, relative measures of divergence and the count of shared polymorphisms and fixed differences. Our analyses also highlight the value of incorporating multiple summary statistics including our newly developed outlier ones that can be useful in challenging detection conditions.

Spatiotemporal dynamics in butterfly hybrid zones.

Evaluating whether hybrid zones are stable or mobile can provide novel insights for evolution and conservation biology. Butterflies exhibit high sensitivity to environmental changes and represent an important model system for the study of hybrid zone origins and maintenance. Here, we review the literature exploring butterfly hybrid zones, with a special focus on their spatiotemporal dynamics and the potential mechanisms that could lead to their movement or stability. We then compare different lines of evidence used to investigate hybrid zone dynamics and discuss the strengths and weaknesses of each approach. Our goal with this review is to reveal general conditions associated with the stability or mobility of butterfly hybrid zones by synthesizing evidence obtained using different types of data sampled across multiple regions and spatial scales. Finally, we discuss spatiotemporal dynamics in the context of a speciation/divergence continuum, the relevance of hybrid zones for conservation biology, and recommend key topics for future investigation.

Acknowledging more biodiversity without more species.

Delimiting and naming biodiversity is a vital step toward wildlife conservation and research. However, species delimitation must be consistent across biota so that the limited resources available for nature protection can be spent effectively and objectively. To date, newly discovered lineages typically are either left undescribed and thus remain unprotected or are being erroneously proposed as new species despite mixed evidence for completed speciation, in turn contributing to the emerging problem of taxonomic inflation. Inspired by recent conceptual and methodological progress, we propose a standardized workflow for species delimitation that combines phylogenetic and hybrid zone analyses of genomic datasets ("genomic taxonomy"), in which phylogeographic lineages that do not freely admix are ranked as species, while those that have remained fully genetically compatible are ranked as subspecies. In both cases, we encourage their formal taxonomic naming, diagnosis, and description to promote social awareness toward biodiversity. The use of loci throughout the genome overcomes the unreliability of widely used barcoding genes when phylogeographic patterns are complex, while the evaluation of divergence and reproductive isolation unifies the long-opposed concepts of lineage species and biological species. We suggest that a shift in conservation assessments from a single level (species) toward a two-level hierarchy (species and subspecies) will lead to a more balanced perception of biodiversity in which both intraspecific and interspecific diversity are valued and more adequately protected.

Large X-effects are absent in torrent frogs with nascent sex chromosomes.

Sex chromosomes are popularized as a special role in driving speciation. However, the empirical evidence from natural population processes has been limited to organisms with degenerated sex chromosomes, where hemizygosity is mainly considered to act as the driver of reproductive isolation. Here, we examined several hybrid zones of torrent frog Amolops mantzorum species complex, using an approach by mapping species-diagnostic loci onto the reference genome to compare sex-linked versus autosomal patterns of introgression. We find little support in sex-linked incompatibilities for large X-effects for these populations in hybrid zones with homomorphic sex chromosomes, due to the absence of the hemizygous effects. As expected, the large X-effects were not found in those with heteromorphic but newly evolved sex chromosomes, owing to the absence of strong genetic differences between X and Y chromosomes. The available data so far on amphibians suggest little role for sex-linked genes in speciation. The large X-effects in those with nascent sex chromosomes may not be as ubiquitous as presumed across the animal kingdom.

Development of microsatellite markers for sister species Linum suffruticosum and Linum tenuifolium in their overlapping ranges.

BACKGROUND: Microsatellite markers were developed for distylous Linum suffruticosum and tested in the monomorphic sister species Linum tenuifolium. These species are perennial herbs endemic to the western and northwestern Mediterranean, respectively, with a partially overlapping distribution area. METHODS AND RESULTS: We developed 12 microsatellite markers for L. suffruticosum using next generation sequencing, and assessed their polymorphism and genetic diversity in 152 individuals from seven natural populations. The markers displayed high polymorphism, with two to 16 alleles per locus and population, and average observed and expected heterozygosities of 0.833 and 0.692, respectively. All loci amplified successfully in the sister species L. tenuifolium, and 150 individuals from seven populations were also screened. The polymorphism exhibited was high, with two to ten alleles per locus and population, and average observed and expected heterozygosities of 0.77 and 0.62, respectively. CONCLUSIONS: The microsatellite markers identified in L. suffruticosum and tested in L. tenuifolium are a powerful tool to facilitate future investigations of the population genetics, mating patterns and hybridization between both Linum species in their contact zone.

An allozyme polymorphism is associated with a large chromosomal inversion in the marine snail Littorina fabalis.

Understanding the genetic targets of natural selection is one of the most challenging goals of population genetics. Some of the earliest candidate genes were identified from associations between allozyme allele frequencies and environmental variation. One such example is the clinal polymorphism in the arginine kinase (Ak) gene in the marine snail Littorina fabalis. While other enzyme loci do not show differences in allozyme frequencies among populations, the Ak alleles are near differential fixation across repeated wave exposure gradients in Europe. Here, we use this case to illustrate how a new sequencing toolbox can be employed to characterize the genomic architecture associated with historical candidate genes. We found that the Ak alleles differ by nine nonsynonymous substitutions, which perfectly explain the different migration patterns of the allozymes during electrophoresis. Moreover, by exploring the genomic context of the Ak gene, we found that the three main Ak alleles are located on different arrangements of a putative chromosomal inversion that reaches near fixation at the opposing ends of two transects covering a wave exposure gradient. This shows Ak is part of a large (3/4 of the chromosome) genomic block of differentiation, in which Ak is unlikely to be the only target of divergent selection. Nevertheless, the nonsynonymous substitutions among Ak alleles and the complete association of one allele with one inversion arrangement suggest that the Ak gene is a strong candidate to contribute to the adaptive significance of the inversion.

Widespread genomic signatures of reproductive isolation and sex-specific selection in the Eastern Yellow Robin, Eopsaltria australis.

How new species evolve is one of the most fundamental questions in biology. Population divergence, which may lead to speciation, may be occurring in the Eastern Yellow Robin, a common passerine that lives along the eastern coast of Australia. This species is composed of 2 parapatric lineages that have highly divergent mitochondrial DNA; however, similar levels of divergence have not been observed in the nuclear genome. Here we re-examine the nuclear genomes of these mitolineages to test potential mechanisms underlying the discordance between nuclear and mitochondrial divergence. We find that nuclear admixture occurs in a narrow hybrid zone, although the majority of markers across the genome show evidence of reproductive isolation between populations of opposing mitolineages. There is an 8 MB section of a previously identified putative neo-sex chromosome that is highly diverged between allopatric but not parapatric populations, which may be the result of a chromosomal inversion. The neo-sex chromosomal nature of this region, as well as the geographic patterns in which it exhibits divergence, suggest it is unlikely to be contributing to reproductive isolation through mitonuclear incompatibilities as reported in earlier studies. In addition, there are sex differences in the number of markers that are differentiated between populations of opposite mitolineages, with greater differentiation occurring in females, which are heterozygous, than males. These results suggest that, despite the absence of previously observed assortative mating, mitolineages of Eastern Yellow Robin experience at least some postzygotic isolation from each other, in a pattern consistent with Haldane's Rule.

Revisiting a classic hybrid zone: Movement of the northern flicker hybrid zone in contemporary times.

Natural hybrid zones have provided important insights into the evolutionary process, and their geographic dynamics over time can help to disentangle the underlying biological processes that maintain them. Here, we leverage replicated sampling of an identical transect across the hybrid zone between yellow-shafted and red-shafted flickers in the Great Plains to assess its stability over ∼60 years (1955-1957 to 2016-2018). We identify a ∼73-km westward shift in the hybrid zone center toward the range of the red-shafted flicker, but find no associated changes in width over our sampling period. In fact, the hybrid zone remains remarkably narrow, suggesting some kind of selective pressure maintains the zone. By comparing to previous work in the same geographic region, it appears likely that the movement in the hybrid zone has occurred in the years since the early 1980s. This recent movement may be related to changes in climate or land management practices that have allowed westward movement of yellow-shafted flickers into the Great Plains.

Candidate-species delimitation in Desmognathus salamanders reveals gene flow across lineage boundaries, confounding phylogenetic estimation and clarifying hybrid zones.

Dusky Salamanders (genus Desmognathus) currently comprise only 22 described, extant species. However, recent mitochondrial and nuclear estimates indicate the presence of up to 49 candidate species based on ecogeographic sampling. Previous studies also suggest a complex history of hybridization between these lineages. Studies in other groups suggest that disregarding admixture may affect both phylogenetic inference and clustering-based species delimitation. With a dataset comprising 233 Anchored Hybrid Enrichment (AHE) loci sequenced for 896 Desmognathus specimens from all 49 candidate species, we test three hypotheses regarding (i) species-level diversity, (ii) hybridization and admixture, and (iii) misleading phylogenetic inference. Using phylogenetic and population-clustering analyses considering gene flow, we find support for at least 47 candidate species in the phylogenomic dataset, some of which are newly characterized here while others represent combinations of previously named lineages that are collapsed in the current dataset. Within these, we observe significant phylogeographic structure, with up to 64 total geographic genetic lineages, many of which hybridize either narrowly at contact zones or extensively across ecological gradients. We find strong support for both recent admixture between terminal lineages and ancient hybridization across internal branches. This signal appears to distort concatenated phylogenetic inference, wherein more heavily admixed terminal specimens occupy apparently artifactual early-diverging topological positions, occasionally to the extent of forming false clades of intermediate hybrids. Additional geographic and genetic sampling and more robust computational approaches will be needed to clarify taxonomy, and to reconstruct a network topology to display evolutionary relationships in a manner that is consistent with their complex history of reticulation.

Morphometry of two cryptic tree frog species at their hybrid zone reveals neither intermediate nor transgressive morphotypes.

Under incomplete reproductive isolation, secondary contact of diverged allopatric lineages may lead to the formation of hybrid zones that allow to study recombinants over several generations as excellent systems of genomic interactions resulting from the evolutionary forces acting on certain genes and phenotypes. Hybrid phenotypes may either exhibit intermediacy or, alternatively, transgressive traits, which exceed the extremes of their parents due to epistasis and segregation of complementary alleles. While transgressive morphotypes have been examined in fish, reptiles, birds, and mammals, studies in amphibians are rare. Here, we associate microsatellite-based genotypes with morphometrics-based morphotypes of two tree frog species of the Hyla arborea group, sampled across a hybrid zone in Poland, to understand whether the genetically differentiated parental species also differ in morphology between each other and their hybrids and whether secondary contact leads to the evolution of intermediate or transgressive morphotypes. Using univariate approaches, explorative multivariate methods (principal component analyses) as well as techniques with prior grouping (discriminant function analyses), we find that morphotypes of both parental species and hybrids differ from each other. Importantly, hybrid morphotypes are neither intermediate nor transgressive but found to be more similar to H. orientalis than to H. arborea.

The genic view of hybridization in the Anthropocene.

Human impact is noticeable around the globe, indicating that a new era might have begun: the Anthropocene. Continuing human activities, including land-use changes, introduction of non-native species and rapid climate change, are altering the distributions of countless species, often giving rise to human-mediated hybridization events. While the interbreeding of different populations or species can have detrimental effects, such as genetic extinction, it can be beneficial in terms of adaptive introgression or an increase in genetic diversity. In this paper, I first review the different mechanisms and outcomes of anthropogenic hybridization based on literature from the last five years (2016-2020). The most common mechanisms leading to the interbreeding of previously isolated taxa include habitat change (51% of the studies) and introduction of non-native species (34% intentional and 19% unintentional). These human-induced hybridization events most often result in introgression (80%). The high incidence of genetic exchange between the hybridizing taxa indicates that the application of a genic view of speciation (and introgression) can provide crucial insights on how to address hybridization events in the Anthropocene. This perspective considers the genome as a dynamic collection of genetic loci with distinct evolutionary histories, giving rise to a heterogenous genomic landscape in terms of genetic differentiation and introgression. First, understanding this genomic landscape can lead to a better selection of diagnostic genetic markers to characterize hybrid populations. Second, describing how introgression patterns vary across the genome can help to predict the likelihood of negative processes, such as demographic and genetic swamping, as well as positive outcomes, such as adaptive introgression. It is especially important to not only quantify how much genetic material introgressed, but also what has been exchanged. Third, comparing introgression patterns in pre-Anthropocene hybridization events with current human-induced cases might provide novel insights into the likelihood of genetic swamping or species collapse during an anthropogenic hybridization event. However, this comparative approach remains to be tested before it can be applied in practice. Finally, the genic view of introgression can be combined with conservation genomic studies to determine the legal status of hybrids and take appropriate measures to manage anthropogenic hybridization events. The interplay between evolutionary and conservation genomics will result in the constant exchange of ideas between these fields which will not only improve our knowledge on the origin of species, but also how to conserve and protect them.

ClineHelpR: an R package for genomic cline outlier detection and visualization.

BACKGROUND: Patterns of multi-locus differentiation (i.e., genomic clines) often extend broadly across hybrid zones and their quantification can help diagnose how species boundaries are shaped by adaptive processes, both intrinsic and extrinsic. In this sense, the transitioning of loci across admixed individuals can be contrasted as a function of the genome-wide trend, in turn allowing an expansion of clinal theory across a much wider array of biodiversity. However, computational tools that serve to interpret and consequently visualize 'genomic clines' are limited, and users must often write custom, relatively complex code to do so. RESULTS: Here, we introduce the ClineHelpR R-package for visualizing genomic clines and detecting outlier loci using output generated by two popular software packages, bgc and Introgress. ClineHelpR bundles both input generation (i.e., filtering datasets and creating specialized file formats) and output processing (e.g., MCMC thinning and burn-in) with functions that directly facilitate interpretation and hypothesis testing. Tools are also provided for post-hoc analyses that interface with external packages such as ENMeval and RIdeogram. CONCLUSIONS: Our package increases the reproducibility and accessibility of genomic cline methods, thus allowing an expanded user base and promoting these methods as mechanisms to address diverse evolutionary questions in both model and non-model organisms. Furthermore, the ClineHelpR extended functionality can evaluate genomic clines in the context of spatial and environmental features, allowing users to explore underlying processes potentially contributing to the observed patterns and helping facilitate effective conservation management strategies.

A holocenic and dynamic hybrid zone between two cactophilic Drosophila species in a coastal lowland plain of the Brazilian Atlantic Forest.

Hybridization and introgression are processes that contribute to shaping biological diversity. The factors promoting the formation of these processes are multiples but poorly explored in a biogeographical and ecological context. In the southeast coastal plain of the Brazilian Atlantic Forest, a hybrid zone was described between two closely related cactophilic species, Drosophila antonietae and D. serido. Here, we revisited and analysed specimens from this hybrid zone to evaluate its temporal and spatial dynamic. We examined allopatric and sympatric populations of the flies using independent sources of data such as mitochondrial and nuclear sequences, microsatellite loci, morphometrics of wings and male genitalia, and climatic niche models. We also verified the emergence of the flies from necrotic tissues of collected cacti to verify the role of host association for the population dynamics. Our results support the existence of a hybrid zone due to secondary contact and limited to the localities where the two species are currently in contact. Furthermore, we detected asymmetric bidirectional introgression and the maintenance of the species integrity, ecological association and morphological characters, suggesting selection and limited introgression. Considering our paleomodels, probably this hybrid zone is recent and the contact occurred during the Holocene to the present day, favoured by range expansion of their populations due to expansion of open and dry areas in eastern South America during palaeoclimatic and geomorphological events.

Demographic history shapes genomic ancestry in hybrid zones.

Demographic factors such as migration rate and population size can impede or facilitate speciation. In hybrid zones, reproductive boundaries between species are tested and demography mediates the opportunity for admixture between lineages that are partially isolated. Genomic ancestry is a powerful tool for revealing the history of admixed populations, but models and methods based on local ancestry are rarely applied to structured hybrid zones. To understand the effects of demography on ancestry in hybrids zones, we performed individual-based simulations under a stepping-stone model, treating migration rate, deme size, and hybrid zone age as parameters. We find that the number of ancestry junctions (the transition points between genomic regions with different ancestries) and heterogenicity (the genomic proportion heterozygous for ancestry) are often closely connected to demographic history. Reducing deme size reduces junction number and heterogenicity. Elevating migration rate increases heterogenicity, but migration affects junction number in more complex ways. We highlight the junction frequency spectrum as a novel and informative summary of ancestry that responds to demographic history. A substantial proportion of junctions are expected to fix when migration is limited or deme size is small, changing the shape of the spectrum. Our findings suggest that genomic patterns of ancestry could be used to infer demographic history in hybrid zones.

Isolation and reconnection: Demographic history and multiple contact zones of the green odorous frog (Odorrana margaretae) around the Sichuan Basin.

The green odorous frog (Odorrana margaretae) displays a circular distribution around the Sichuan Basin of western China and possesses multiple replicate hybrid zones between lineages with high levels of divergence. To gain an understanding of the speciation process, we obtained 1540 SNPs from 29 populations and 227 individuals using ddRAD sequencing. Population structure analysis revealed three groups within the species: the West, North & South, and East groups. Demographic inference showed that they were initially isolated at ~2 million years ago, and subsequent post-glacial expansion produced the current circular distribution with four secondary contact zones. Hybridization in those zones involved lineages with various levels of divergence and produced greatly different patterns of introgression. Contact zones between the East and North & South groups (E-S and E-N) had contrast admixture levels but both showed a general lack of potential barrier loci. Meanwhile, the reconnection of the West and North & South groups produced two contact zones along the rim of the Basin. The S-W zone had extensive admixture while the N-W zone had limited admixture within a narrow geographic distance. Both showed substantial barrier effects, and a large number of potential barrier loci were shared. We also detected strong coupling among these loci. The N-W hybrid zone involved two highly-diverged lineages (FST = 0.704) and many loci have reached fixation around the hybrid zone. This study system offers a unique opportunity to understand the dynamics of introgression in contact zones and the architecture of reproductive isolation at different stages of speciation.

Disentangling the components of triploid block and its fitness consequences in natural diploid-tetraploid contact zones of Arabidopsis arenosa.

Hybrid seed inviability (HSI) is an important mechanism of reproductive isolation and speciation. HSI varies in strength among populations of diploid species but it remains to be tested whether similar processes affect natural variation in HSI within ploidy-variable species (triploid block). Here we used extensive endosperm, seed and F1 -hybrid phenotyping to explore HSI variation within a diploid-autotetraploid species. By leveraging 12 population pairs from three ploidy contact zones, we tested for the effect of interploidy crossing direction (parent of origin), ploidy divergence and spatial arrangement in shaping reproductive barriers in a naturally relevant context. We detected strong parent-of-origin effects on endosperm development, F1 germination and survival, which was also reflected in the rates of triploid formation in the field. Endosperm cellularization failure was least severe and F1 -hybrid performance was slightly better in the primary contact zone, with genetically closest diploid and tetraploid lineages. We demonstrated overall strong parent-of-origin effects on HSI in a ploidy variable species, which translate to fitness effects and contribute to interploidy reproductive isolation in a natural context. Subtle intraspecific variation in these traits suggests the fitness consequences of HSI are predominantly a constitutive property of the species regardless of the evolutionary background of its populations.

Consequences of Hybridization in Mammals: A Systematic Review.

Hybridization, defined as breeding between two distinct taxonomic units, can have an important effect on the evolutionary patterns in cross-breeding taxa. Although interspecific hybridization has frequently been considered as a maladaptive process, which threatens species genetic integrity and survival via genetic swamping and outbreeding depression, in some cases hybridization can introduce novel adaptive variation and increase fitness. Most studies to date focused on documenting hybridization events and analyzing their causes, while relatively little is known about the consequences of hybridization and its impact on the parental species. To address this knowledge gap, we conducted a systematic review of studies on hybridization in mammals published in 2010-2021, and identified 115 relevant studies. Of 13 categories of hybridization consequences described in these studies, the most common negative consequence (21% of studies) was genetic swamping and the most common positive consequence (8%) was the gain of novel adaptive variation. The total frequency of negative consequences (49%) was higher than positive (13%) and neutral (38%) consequences. These frequencies are biased by the detection possibilities of microsatellite loci, the most common genetic markers used in the papers assessed. As negative outcomes are typically easier to demonstrate than positive ones (e.g., extinction vs hybrid speciation), they may be over-represented in publications. Transition towards genomic studies involving both neutral and adaptive variation will provide a better insight into the real impacts of hybridization.

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

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

Salinity, not genetic incompatibilities, limits the establishment of the invasive hybrid cattail Typha × glauca in coastal wetlands.

Hybrids of a single pair of parent species can be much more common in some geographical regions than in others. The reasons for this are not well understood, but could help explain processes such as species diversification or the range expansion of invasive hybrids. The widespread cattails Typha latifolia and T. angustifolia seldom hybridize in some parts of their range, but in other areas produce the dominant hybrid T. × glauca. We used a combination of field and greenhouse experiments to investigate why T. × glauca has invaded wetlands in the Laurentian Great Lakes region of southern Ontario, Canada, but is much less common in the coastal wetlands of Nova Scotia (NS) in eastern Canada. One potentially important environmental difference between these two regions is salinity. We therefore tested three hypotheses: (1) T. latifolia and T. angustifolia in NS are genetically incompatible; (2) the germination or growth of T. × glauca is reduced by salinity; and (3) T. latifolia, a main competitor of T. × glauca, is locally adapted to saline conditions in NS. Our experiments showed that NS T. latifolia and T. angustifolia are genetically compatible, and that saline conditions do not impede growth of hybrid plants. However, we also found that under conditions of high salinity, germination rates of hybrid seeds were substantially lower than those of NS T. latifolia. In addition, germination rates of NS T. latifolia were higher than those of Ontario T. latifolia, suggesting local adaptation to salinity in coastal wetlands. This study adds to the growing body of literature which identifies the important roles that local habitat and adaptation can play in the distributions and characteristics of hybrid zones.

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.