Hybrid zones act as natural laboratories where divergent genomes interact, providing powerful systems for examining the evolutionary processes underlying biological diversity. In this study, we characterized patterns of genomic and phenotypic variation resulting from hybridization between divergent intraspecific lineages of the Neotropical red‐eyed treefrog (Agalychnis callidryas). We found genetic evidence of a newly discovered contact zone and phenotypic novelty in leg colour—a trait suspected to play a role in mediating assortative mating in this species. Analysis of hybrid ancestry revealed an abundance of later‐generation Fn individuals, suggesting persistence of hybrids in the contact zone. Hybrids are predominantly of southern ancestry but are phenotypically more similar to northern populations. Genome‐wide association mapping revealed QTL with measurable effects on leg‐colour variation, but further work is required to dissect the architecture of this trait and establish causal links. Further, genomic cline analyses indicated substantial variation in patterns of introgression across the genome. Directional introgression of loci associated with different aspects of leg colour are inherited from each parental lineage, creating a distinct hybrid colour pattern. We show that hybridization can generate new phenotypes, revealing the evolutionary processes that potentially underlie patterns of phenotypic diversity in this iconic polytypic frog. Our study is consistent with a role of hybridization and sexual selection in lineage diversification, evolutionary processes that have been implicated in accelerating divergence in the most phenotypically diverse species.

Patterns of phenotypic variation across a geographic range provide important insights into evolutionary processes underlying diversification and speciation. Most evolutionary studies use putatively neutral markers to examine evolutionary diversification. However, functional phenotypes such as gene-encoded host-defense polypeptides (HDPs) could provide key insights into the processes of population differentiation, yet they are rarely included in population analyses. The red-eyed treefrog, Agalychnis callidryas Cope, 1862, exhibits regional variation in multiple traits, including color pattern and body size across a narrow geographic range. This treefrog produces bioactive peptides exuded onto the skin surface, presumably for pathogen and predator defense. However, the geographic patterns of variation in peptides and the factors that mediate intraspecific peptide variation across the range of this species remain untested. Here, we examine the roles of phylogenetic history, geographic barriers, geographic distance, and color-pattern variation as determinants of skin peptide diversity in 54 individuals among 11 populations across Costa Rica and Panama. Each of the five distinct Agalychnis color morphs are represented in our sample. We performed peptide mass fingerprinting and compared mass spectral data from skin peptide secretions to quantify divergence in peptide profiles among individuals, both within and among regions. We used two metrics to estimate genetic variation: Genetic distance estimated from microsatellites and patristic distance estimated from mtDNA haplotype diversity. Matrix correspondence tests revealed that skin peptide variation is best predicted by differences in leg color pattern across all regions. In addition, we found that flank color pattern and phylogeny also explain differences in peptide diversity. Patterns of peptide differentiation and phylogenetic topology were incongruent in two regions, indicating a possible role of localized selection on peptide variation. Skin peptide profiles are useful in population differentiation studies of August 2016Davis et al. Skin Peptide Variation in Agalychnis callidryasa polymorphic species as well as studies of selection and phenotype co-variation among closely related species. Our results highlight the use of skin peptides as characters for future studies of population differentiation and contribute to our understanding of biogeography in Central America.

Intraspecific color variation has long fascinated evolutionary biologists. In species with bright warning coloration, phenotypic diversity is particularly compelling because many factors, including natural and sexual selection, contribute to intraspecific variation. To better understand the causes of dramatic phenotypic variation in Malagasy poison frogs, we quantified genetic structure and color and pattern variation across three closely related species, Mantella aurantiaca , Mantella crocea , and Mantella milotympanum . Although our restriction site‐associated DNA (RAD) sequencing approach identified clear genetic clusters, they do not align with current species designations, which has important conservation implications for these imperiled frogs. Moreover, our results suggest that levels of intraspecific color variation within this group have been overestimated, while species diversity has been underestimated. Within major genetic clusters, we observed distinct patterns of variation including: populations that are phenotypically similar yet genetically distinct, populations where phenotypic and genetic breaks coincide, and populations that are genetically similar but have high levels of within‐population phenotypic variation. We also detected admixture between two of the major genetic clusters. Our study suggests that several mechanisms—including hybridization, selection, and drift—are contributing to phenotypic diversity. Ultimately, our work underscores the need for a reevaluation of how polymorphic and polytypic populations and species are classified, especially in aposematic organisms.