As populations diverge, genetic differences accumulate across the genome. Although genetic differentiation is well documented, evolutionary change in epigenetic modifications is less well understood. Here, we integrate genomic and methylomic data from 168 barn swallows (Hirundo rustica) to examine the relationship between genetic and epigenetic population divergence at the resolution of CpG islands and single CpG sites. Sampling across all six extant subspecies and two hybrid zones, we assess the roles of genetic ancestry and environment in shaping DNA methylation patterns. Genome-wide methylation differentiation (P ST) closely mirrors genetic differentiation (F ST), with secondary contributions from geographic and environmental variation. DNA methylation patterns track the genomic landscape of sequence divergence, especially in CpG islands of nonregulatory regions. Across hybrid zones, DNA methylation levels are associated with nearby cis-acting genetic variation and global genomic ancestry. Moreover, CpG sites exhibit linkage disequilibrium with nearby SNPs, extending over several thousand base pairs within a putative inversion. These results suggest that although environmental effects on genome-wide DNA methylation are present, epigenomic patterns are costructured by the evolutionary processes shaping genetic variation.

Many socially monogamous bird species engage in multiple mating through extra-pair (EP) fertilizations, yet the role of female plumage in EP outcomes is under studied. Sexual selection involving EP fertilization is hypothesized to influence female trait evolution if heritable plumage variation informs EP mating decisions and EP mating decisions affect fitness. Here, we test for 2 key prerequisite patterns of sexual selection through EP fertilizations by asking: (1) is female reproductive success associated with EP fertilizations, and (2) are EP fertilizations associated with female plumage traits? Studying these 2 patterns is essential for better understanding how the costs and benefits of EP fertilizations should translate to sexual selection on female plumage. We provide a conceptual diagram to highlight how plumage traits of both members of a social pair can influence fertilizations within and outside their pair bond from the perspective of each sex. In our sample of 47 Hirundo rustica erythrogaster (North American Barn Swallow) social pairs, females who engaged in EP mating tended to fledge more offspring, and females with longer tails were more likely to mate with EP sires. Furthermore, female traits were more strongly associated with EP fertilizations than male traits for predicting both female and male EP outcomes. Finally, we found that female traits were not correlated with fecundity (total eggs laid) and found no association between fecundity and EP fertilizations. Thus, sexual selection on EP fertilizations may be more important than selection on fecundity for understanding female plumage variation in our study population. Taken together, we provide evidence for multiple conditions necessary for sexual selection on female plumage traits in H. r. erythrogaster that are likely also relevant for understanding female trait evolution in other socially monogamous birds. Understanding how traits evolve in female birds is essential for explaining the diversity of bird species. Fertilizations with birds other than a social mate in a shared nest ("extra-pair" fertilizations) are common in songbirds, yet we know little about how this process relates to the evolution of female plumage traits. We found that female Hirundo rustica (Barn Swallow) with longer tails were more likely to mate with multiple extra-pair males, and females with more extra-pair offspring also raised more total offspring over the course of a single breeding season. These findings mean that selection for extra-pair mating could help us understand how and why female plumage traits change over time. By giving female birds equal attention as male birds, we can gain a better understanding of how the phenotypic traits of birds change in populations and species overall. Muchas especies de aves socialmente mon & oacute;gamas participan en apareamientos m & uacute;ltiples mediante fertilizaciones extrapareja (EP); sin embargo, el papel del plumaje femenino en los resultados EP ha sido poco estudiado. Se plantea que la selecci & oacute;n sexual que involucra fertilizaciones EP puede influir en la evoluci & oacute;n de los rasgos femeninos si la variaci & oacute;n heredable del plumaje influye en las decisiones de apareamiento EP y si dichas decisiones afectan la aptitud biol & oacute;gica. Aqu & iacute; evaluamos dos patrones clave que constituyen prerrequisitos de la selecci & oacute;n sexual a trav & eacute;s de fertilizaciones EP preguntando: (1) & iquest;est & aacute; el & eacute;xito reproductivo femenino asociado con las fertilizaciones EP?, y (2) & iquest;est & aacute;n las fertilizaciones EP asociadas con rasgos del plumaje femenino? El estudio de estos dos patrones es esencial para comprender mejor c & oacute;mo los costos y beneficios de las fertilizaciones EP deber & iacute;an traducirse en selecci & oacute;n sexual sobre el plumaje femenino. Presentamos un diagrama conceptual para resaltar c & oacute;mo los rasgos del plumaje de ambos miembros de una pareja social pueden influir en las fertilizaciones dentro y fuera del v & iacute;nculo de pareja desde la perspectiva de cada sexo. En nuestra muestra de 47 parejas sociales de Hirundo rustica erythrogaster, las hembras que participaron en apareamientos EP tendieron a sacar adelante m & aacute;s cr & iacute;as, y las hembras con colas m & aacute;s largas tuvieron mayor probabilidad de aparearse con machos EP. Adem & aacute;s, los rasgos femeninos estuvieron m & aacute;s fuertemente asociados con las fertilizaciones EP que los rasgos masculinos para predecir tanto los resultados EP femeninos como masculinos. Finalmente, encontramos que los rasgos femeninos no estuvieron correlacionados con la fecundidad (total de huevos puestos) y no hallamos asociaci & oacute;n entre fecundidad y fertilizaciones EP. Por lo tanto, la selecci & oacute;n sexual sobre las fertilizaciones EP puede ser m & aacute;s importante que la selecci & oacute;n sobre la fecundidad para comprender la variaci & oacute;n del plumaje femenino en nuestra poblaci & oacute;n de estudio. En conjunto, aportamos evidencia de m & uacute;ltiples condiciones necesarias para la selecci & oacute;n sexual sobre los rasgos del plumaje femenino en H. r. erythrogaster, que probablemente tambi & eacute;n sean relevantes para comprender la evoluci & oacute;n de rasgos femeninos en otras aves socialmente mon & oacute;gamas.

Speciation with gene flow poses a central paradox: how do genome-wide barriers to gene exchange accumulate as recombination continually breaks down associations among selected loci? Although theory predicts that together recombination, selection, and genome structure shape reproductive isolation, empirical studies often report conflicting patterns, suggesting that these determinants change across the speciation continuum. Here we compare genomic landscapes of introgression across rattlesnake lineages spanning a range of divergence. We generated a chromosome-level reference genome for the Southwestern Speckled Rattlesnake (Crotalus pyrrhus) and analyzed whole genome data from 181 individuals across two species complexes with a history of gene flow upon secondary contact. We show that reproductive isolation is highly polygenic and dynamically structured. At early divergence, introgression is most reduced in high recombination regions, consistent with increased efficacy of selection against gene flow at few large-effect loci. As divergence progresses, linked selection against gene flow dominates, generating a positive relationship between recombination and introgression expected to occur through the genome-wide coupling of polygenic barrier effects. Introgression landscapes also become increasingly correlated across species pairs as divergence increases due to repeated evolution of barriers in the same genomic regions. Here, we infer that the Z chromosome plays a prominent role in reproductive isolation, harboring a disproportionate number of barrier loci and showing reduced introgression even at early divergence. Together, these results reveal how recombination, selection, and genome organization interact to shape speciation with gene flow upon secondary contact, reconciling empirical patterns with predictions of speciation theory.

Evolutionary potential, the capacity to evolve in response to environmental change, is important for the persistence of threatened species. This potential is diminished by genetic drift in small populations, causing the loss of genetic diversity and reduced efficiency of selection. We introduce an approach to evaluate evolutionary potential that integrates across these components by conducting genome-scale selection analyses of genes of ecological importance. We inferred selection in small populations of the endangered eastern massasauga rattlesnake (Sistrurus catenatus) by comparing summary statistics between background regions and focal adaptive gene families underlying the Major Histocompatibility Complex and venom protein phenotypes. We applied tests sensitive to selection at multiple timescales to populations that have experienced differential declines and compared results with an outbred population of the sister species, S. tergeminus. We detected signatures of selection in focal regions in the recent and distant past, suggesting balancing selection as a dominant force shaping genetic diversity underlying these traits. Recently declined S. catenatus populations exhibited weaker signals of recent selection, consistent with reduced efficiency of selection when effective population size is small. Drift has reduced genome-wide genetic diversity both in relatively large and recently declined S. catenatus populations compared to outbred S. tergeminus, but these reductions were less pronounced in focal adaptive regions. These findings suggest that selection has buffered the loss of adaptive variation, but that small populations of these snakes may be approaching a critical loss of evolutionary potential limiting their capacity to respond to human-induced environmental change.

Genomic coupling theory predicts that progress towards speciation involves a transition from the dominant effects of selection on individual barrier loci to the aggregate effects of direct and indirect selection across loci that collectively produce stronger barriers to gene flow through genetic associations. However, our ability to test this prediction and to understand the factors that lead to the buildup and maintenance of these associations has been limited by a lack of methods to estimate variation in coupling across the genome. Here we develop approaches to quantify coupling using window-based estimates of Barton's coupling coefficient and apply these to a dataset of 118 genomes from a rattlesnake hybrid zone. Our results provide empirical evidence for genomic coupling that is consistent with the predicted relationships of coupling with recombination, linkage, and inferences of selection. Applying these approaches, we find evidence for coupling within and among chromosomes, and highlight the roles of coupling in complex barrier effects, including the Large-Z effect, cytonuclear incompatibilities, and incompatibilities related to venom resistance. Together, our findings demonstrate the mechanism by which coupling is predicted to lead to speciation, and highlight how genome-wide quantification of coupling presents a promising framework for understanding progress towards speciation and the processes that underlie this progress.

While female mate choice is well established, mutual choice may play a larger role in mate selection than currently recognized. Assortative mating is a common form of non-random mating in animals that can result from mutual choice. However, few studies address assortative patterns beyond the social pair, potentially overlooking assortativity in the mating pair and in the social environment that shapes reproductive decisions. We asked whether North American barn swallows (Hirundo rustica erythrogaster) breeding in a large colony form pairs, mate (through both within-pair and extra-pair fertilizations), and interact assortatively by ventral plumage color, wing length, and age. Social interactions were tracked using proximity loggers, which recorded close contact between tagged individuals when birds were mating and laying eggs. Barn swallows paired and mated assortatively by their ventral plumage color; however, the assortative patterns in mating pairs were not as strong as they were in social pairs. Barn swallows also interacted assortatively, associating more often with individuals of both sexes who had similar phenotypes relative to the other birds in the colony. Finally, older males and females with darker ventral plumage achieved the highest reproductive success. Investigation of assortative behavior beyond the level of the social pair provides a more complete understanding of mate choice and suggests a mechanism that may maintain the large variation in ventral plumage color in North American barn swallows.

Extra-pair mating is common in avian species and can modulate the strength of sexual selection. Mate searching behavior of female birds may be an important predictor of mating opportunities and extra-pair mating, yet important knowledge is lacking as we have little data on fine-scale movement of females during the peak fertilization period. Accordingly, much is still unknown about whether and how female phenotypes contribute to extra-pair mating. Here, we examined how female space use and female plumage color are associated with extra-pair mating outcomes in wild barn swallows (Hirundo rustica erythrogaster). We tracked 10 females breeding in Colorado, USA with GPS backpack tags for two hours each morning during their fertile period following an experimental nest failure. We then used low-coverage whole-genome sequencing to determine offspring paternity and to quantify extra-pair mating in the removed clutch and the replacement clutch. Plumage and movement did not correlate with changes in paternity between successive clutches, but movement did correlate with paternity in the replacement clutch. Females that spent more time away from the nest had a higher proportion and number of extra-pair offspring in the clutch laid immediately after the tracking period. These results suggest that differences in female space use contribute to differences in extra-pair fertilizations. In contrast to the historic emphasis on male traits, our study highlights female movement behavior as an important variable associated with mating outcomes in natural populations. Mate choice is a critical step in reproduction, but variation in how extensively individuals in the wild search for and sample potential mates is not well understood. We measured movement behavior of female barn swallows during their fertile period to assess variation in mate sampling and linked this to variation in mating outcomes. We expected that females that flew farther would encounter more potential mates and produce offspring with multiple males. By tracking females’ movement after removing their first clutch of eggs, we were able to correlate female movements with paternity in the collected and replacement clutches. We found that females that spent more time away from the nest, but didn’t necessarily cover a larger distance, were more likely to have offspring with mixed paternity in their replacement clutch.

Disentangling the drivers of genomic divergence during speciation is essential to our broader understanding of the generation of biological diversity. Genetic changes accumulate at variable rates across the genome as populations diverge, leading to heterogenous landscapes of genetic differentiation. The 'islands of differentiation' that characterise these landscapes harbour genetic signatures of the evolutionary processes that led to their formation, providing insight into the roles of these processes in adaptation and speciation. Here, we study swallows in the genus Hirundo to investigate genomic landscapes of differentiation between species spanning a continuum of evolutionary divergence. Genomic differentiation spans a wide range of values (FST = 0.01-0.8) between species, with substantial heterogeneity in genome-wide patterns. Genomic landscapes are strongly correlated among species (ρ = 0.46-0.99), both at shallow and deep evolutionary timescales, with broad evidence for the role of linked selection together with recombination rate in shaping genomic differentiation. Further dissection of genomic islands reveals patterns consistent with a model of 'recurrent selection', wherein differentiation increases due to selection in the same genomic regions in ancestral and descendant populations. Finally, we use measures of the site frequency spectrum to differentiate between alternative forms of selection, providing evidence that genetic hitchhiking due to positive selection has contributed substantially to genomic divergence. Our results demonstrate the pervasive role of recurrent linked selection in shaping genomic divergence despite a history of gene flow and underscore the importance of non-neutral evolutionary processes in predictive frameworks for genomic divergence in speciation genomics studies.

Species tree inference is often assumed to be more accurate as datasets increase in size, with whole genomes representing the best-case-scenario for estimating a single, most-likely speciation history with high confidence. However, genomes may harbor a complex mixture of evolutionary histories among loci, which amplifies the opportunity for model misspecification and impacts phylogenetic inference. Accordingly, multiple distinct and well-supported phylogenetic trees are often recovered from genome-scale data, and approaches for biologically interpreting these distinct signatures are a major challenge for evolutionary biology in the age of genomics. Here, we analyze 32 whole genomes of nine taxa and two outgroups from the Western Rattlesnake species complex. Using concordance factors, topology weighting, and concatenated and species tree analyses with a chromosome-level reference genome, we characterize the distribution of phylogenetic signal across the genomic landscape. We find that concatenated and species tree analyses of autosomes, the Z (sex) chromosome, and mitochondrial genome yield distinct, yet strongly supported phylogenies. Analyses of site-specific likelihoods show additional patterns consistent with rampant model misspecification, a likely consequence of several evolutionary processes. Together, our results suggest that a combination of historic and recent introgression, along with natural selection, recombination rate variation, and cytonuclear co-evolution of nuclear-encoded mitochondrial genes, underlie genome-wide variation in phylogenetic signal. Our results highlight both the power and complexity of interpreting whole genomes in a phylogenetic context and illustrate how patterns of phylogenetic discordance can reveal the impacts of different evolutionary processes that contribute to genome-wide variation in phylogenetic signal.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

Swallows (Hirundinidae) are a globally distributed family of passerine birds that exhibit remarkable similarity in body shape but tremendous variation in plumage, sociality, nesting behavior, and migratory strategies. As a result, swallow species have become models for empirical behavioral ecology and evolutionary studies, and variation across the Hirundinidae presents an excellent opportunity for comparative analyses of trait evolution. Exploiting this potential requires a comprehensive and well -resolved phylogenetic tree of the family. To address this need, we estimated swallow phylogeny using genetic data from thousands of ultraconserved element (UCE) loci sampled from nearly all recognized swallow species. Maximum likelihood, coalescent -based, and Bayesian approaches yielded a well -resolved phylogenetic tree to the generic level, with minor disagreement among inferences at the species level, which likely reflect ongoing population genetic processes. The UCE data were particularly useful in helping to resolve deep nodes, which previously confounded phylogenetic reconstruction efforts. Divergence time estimates from the improved swallow tree support a Miocene origin of the family, roughly 13 million years ago, with subsequent diversification of major groups in the late Miocene and Pliocene. Our estimates of historical biogeography support the hypothesis that swallows originated in the Afrotropics and have subsequently expanded across the globe, with major in situ diversification in Africa and a secondary major radiation following colonization of the Neotropics. Initial examination of nesting and sociality indicates that the origin of mud nesting - a relatively rare nest construction phenotype in birds - was a major innovation coincident with the origin of a clade giving rise to over 40% of extant swallow diversity. In contrast, transitions between social and solitary nesting appear less important for explaining patterns of diversification among swallows.

Both the metabolic theory of ecology and dynamic energy budget theory predict that climate influences body size through its effects on first-order determinants of energetics: reactive temperatures, carbon resources and oxygen availability. Although oxygen is seldom limiting in terrestrial systems, temperature and resources vary spatially. We used redundancy analyses and variation partitioning to evaluate the influence of climatic temperature, precipitation and their seasonalities on multivariate body size across the distributions of four species of the western rattlesnake group in North America (Crotalus pyrrhus, C. scutulatus, C. oreganus and C. viridis). Most species showed a pattern of increased body size in cooler, mesic climates and decreased body size in warmer, xeric climates. Exceptions to the pattern provided additional context through climatic idiosyncrasies in the distributions of each species. For example, the general pattern of a negative influence of temperature on body size was not apparent for C. oreganus, which ranges across the mildest climates overall among the four species. In contrast to previous studies, we found that seasonality had negligible effects on body size. We suggest that precipitation gradients correlate positively with resource availability in driving intraspecific body size and that temperature compounds this gradient by increasing baseline metabolic demands and restricting activity in particularly warm or otherwise extreme climates.

Despite the well-known effects of sexual selection on phenotypes, links between this evolutionary process and reproductive isolation, genomic divergence, and speciation have been difficult to establish. We unravel the genetic basis of sexually selected plumage traits to investigate their effects on reproductive isolation in barn swallows. The genetic architecture of sexual traits is characterized by 12 loci on two autosomes and the Z chromosome. Sexual trait loci exhibit signatures of divergent selection in geographic isolation and barriers to gene flow in secondary contact. Linkage disequilibrium between these genes has been maintained by selection in hybrid zones beyond what would be expected under admixture alone. Our findings reveal that selection on coupled sexual trait loci promotes reproductive isolation, providing key empirical evidence for the role of sexual selection in speciation.

BACKGROUND:Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom.RESULTS:Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition.CONCLUSIONS:Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

Despite the increasing feasibility of sequencing whole genomes from diverse taxa, a persistent problem in phylogenomics is the selection of appropriate genetic markers or loci for a given taxonomic group or research question. In this review, we aim to streamline the decision-making process when selecting specific markers to use in phylogenomic studies by introducing commonly used types of genomic markers, their evolutionary characteristics, and their associated uses in phylogenomics. Specifically, we review the utilities of ultraconserved elements (including flanking regions), anchored hybrid enrichment loci, conserved nonexonic elements, untranslated regions, introns, exons, mitochondrial DNA, single nucleotide polymorphisms, and anonymous regions (nonspecific regions that are evenly or randomly distributed across the genome). These various genomic elements and regions differ in their substitution rates, likelihood of neutrality or of being strongly linked to loci under selection, and mode of inheritance, each of which are important considerations in phylogenomic reconstruction. These features may give each type of marker important advantages and disadvantages depending on the biological question, number of taxa sampled, evolutionary timescale, cost effectiveness, and analytical methods used. We provide a concise outline as a resource to efficiently consider key aspects of each type of genetic marker. There are many factors to consider when designing phylogenomic studies, and this review may serve as a primer when weighing options between multiple potential phylogenomic markers.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone.

Hybridization facilitates recombination between divergent genetic lineages and can be shaped by both neutral and selective processes. Upon hybridization, loci with no net fitness effects introgress randomly from parental species into the genomes of hybrid individuals. Conversely, alleles from one parental species at some loci may provide a selective advantage to hybrids, resulting in patterns of introgression that do not conform to random expectations. We investigated genomic patterns of differential introgression in natural hybrids of two species of Caribbean anoles, Anolis pulchellus and A. krugi in Puerto Rico. Hybrids exhibit A. pulchellus phenotypes but possess A. krugi mitochondrial DNA, originated from multiple, independent hybridization events, and appear to have replaced pure A. pulchellus across a large area in western Puerto Rico. Combining genome-wide SNP datasets with bioinformatic methods to identify signals of differential introgression in hybrids, we demonstrate that the genomes of hybrids are dominated by pulchellus-derived alleles and show only 10%-20% A. krugi ancestry. The majority of A. krugi loci in hybrids exhibit a signal of non-random differential introgression and include loci linked to genes involved in development and immune function. Three of these genes (delta like canonical notch ligand 1, jagged1 and notch receptor 1) affect cell differentiation and growth and interact with mitochondrial function. Our results suggest that differential non-random introgression for a subset of loci may be driven by selection favouring the inheritance of compatible mitochondrial and nuclear-encoded genes in hybrids.

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance.

Sex chromosomes diverge after the establishment of recombination suppression, resulting in differential sex-linkage of genes involved in genetic sex determination and dimorphic traits. This process produces systems of male or female heterogamety wherein the Y and W chromosomes are only present in one sex and are often highly degenerated. Sex-limited Y and W chromosomes contain valuable information about the evolutionary transition from autosomes to sex chromosomes, yet detailed characterizations of the structure, composition, and gene content of sex-limited chromosomes are lacking for many species. In this study, we characterize the female-specific W chromosome of the prairie rattlesnake (Crotalus viridis) and evaluate how recombination suppression and other processes have shaped sex chromosome evolution in ZW snakes. Our analyses indicate that the rattlesnake W chromosome is over 80% repetitive and that an abundance of GC-rich mdg4 elements has driven an overall high degree of GC-richness despite a lack of recombination. The W chromosome is also highly enriched for repeat sequences derived from endogenous retroviruses and likely acts as a "refugium" for these and other retroelements. We annotated 219 putatively functional W-linked genes across at least two evolutionary strata identified based on estimates of sequence divergence between Z and W gametologs. The youngest of these strata is relatively gene-rich, however gene expression across strata suggests retained gene function amidst a greater degree of degeneration following ancient recombination suppression. Functional annotation of W-linked genes indicates a specialization of the W chromosome for reproductive and developmental function since recombination suppression from the Z chromosome.

Migratory divides are contact zones between breeding populations with divergent migratory strategies during the nonbreeding season. These locations provide an opportunity to evaluate the role of seasonal migration in the maintenance of reproductive isolation, particularly the relationship between population structure and features associated with distinct migratory strategies. We combine light-level geolocators, genomic sequencing, and stable isotopes to investigate the timing of migration and migratory routes of individuals breeding on either side of a migratory divide coinciding with genomic differentiation across a hybrid zone between barn swallow (Hirundo rustica) subspecies in China. Individuals west of the hybrid zone, with H. r. rustica ancestry, had comparatively enriched stable-carbon and hydrogen isotope values and overwintered in eastern Africa, whereas birds east of the hybrid zone, with H. r. gutturalis ancestry, had depleted isotope values and migrated to southern India. The two subspecies took divergent migratory routes around the high-altitude Karakoram Range and arrived on the breeding grounds over 3 weeks apart. These results indicate that assortative mating by timing of arrival and/or selection against hybrids with intermediate migratory traits may maintain reproductive isolation between the subspecies, and that inhospitable geographic features may have contributed to the diversification of Asian avifauna by influencing migratory patterns.