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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.

Geographic variation in courtship behavior can affect reproductive success of divergent phenotypes via mate choice. Over time, this can lead to reproductive isolation and ultimately to speciation. The Neotropical red-eyed treefrog (Agalychnis callidryas) exhibits high levels of phenotypic variation among populations in Costa Rica and Panama, including differences in color pattern, body size, and skin peptides. To test the extent of behavioral premating isolation among differentiated populations, we quantified male advertisement calls from six sites and female responses to male stimuli (acoustic and visual signals) from four sites. Our results show that both male advertisement calls and female behavior vary among populations: Discriminant function analyses can predict the population of origin for 99.3% ± 0.7 of males based on male call (dominant frequency and bandwidth) and 76.1% ± 6.6 of females based on female response behavior (frequency and duration of visual displays). Further, female mate choice trials (n = 69) showed that population divergence in male signals is coupled with female preference for local male stimuli. Combined, these results suggest that evolved differences among populations in male call properties and female response signals could have consequences for reproductive isolation. Finally, population variation in male and female behavior was not well explained by geographic or genetic distance, indicating a role for localized selection and/or drift. The interplay between male courtship and female responses may facilitate the evolution of local variants in courtship style, thus accelerating premating isolation via assortative mating.

Aim  Understanding the patterns and processes underlying phenotype in a polytypic species provides key insights into microevolutionary mechanisms of diversification. The red‐eyed treefrog, Agalychnis callidryas, exhibits strong regional differentiation in colour pattern, corresponding to five admixed mitochondrial DNA clades. We evaluated spatial diversity patterns across multiple, putative barriers to examine the fine‐scale processes that mediate phenotypic divergence between some regions while maintaining homogeneity between others. Location  We examined patterns of phenotypic diversification among 17 sites that span five putative biogeographic barriers in lower Central America (Costa Rica and Panama). Methods  We tested the extent to which genetic distance (FST) derived from six multilocus nuclear genotypes covaried with measures of phenotypic distance (leg coloration) within and between biogeographic regions. We used linear regression analyses to determine the role of geographic and genetic factors in structuring spatial patterns of phenotypic diversity. Results  The factors that best explained patterns of phenotypic diversity varied among biogeographic regions. We identified one geographic barrier that impeded gene exchange and resulted in concordant phenotypic divergence across the Continental Divide, isolating Caribbean and Pacific populations. Across Caribbean Costa Rican populations, one barrier structured phenotypic but not genetic diversity patterns, indicating a role for selection. In other regions, the putative barriers had no determining effect on either genetic or leg colour structure. Main conclusions  The processes mediating the distribution and diversification of colour pattern in this polytypic, wide‐ranging treefrog varied among biogeographic regions. Spatially varying selection combined with the isolating effects of geographic factors probably resulted in the patchy distribution of colour diversity across Costa Rican and Panamanian populations.

Investigating the spatial distribution of genetic and phenotypic variation can provide insights into the evolutionary processes that shape diversity in natural systems. We characterized patterns of genetic and phenotypic diversity to learn about drivers of color-pattern diversification in red-eyed treefrogs (Agalychnis callidryas) in Costa Rica. Along the Pacific coast, red-eyed treefrogs have conspicuous leg color patterning that transitions from orange in the north to purple in the south. We measured phenotypic variation of frogs across Pacific sites, with increased sampling at sites where the orange-to-purple transition occurs. At the transition zone, we discovered the co-occurrence of multiple color-pattern morphs. To explore possible causes of this variation, we generated a SNP dataset with RAD sequencing to analyze population genetic structure, measure genetic diversity, and infer the processes that mediate genotype-phenotype dynamics. We investigated how patterns of genetic relatedness correspond with individual measures of color pattern along the coast, including testing for the role of hybridization in geographic regions where orange and purple phenotypic groups co-occur. We found no evidence that color-pattern polymorphism in the transition zone arose through recent hybridization or introgression. Instead, a strong pattern of genetic isolation by distance (IBD) indicates that color-pattern variation was retained through other processes such as ancestral color polymorphisms, ancient secondary contact or generated by novel mutations. We found that color phenotype changes along the Pacific coast more than would be expected from geographic distance alone. Combined, our results suggest the possibility of selective pressures acting on color pattern at a small geographic scale.

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.

Four species of the genus Rhagoletis are native to Chile: R. nova (Schiner), R. conversa, (Brèthes), R. penela Foote and R. tomatis Foote. Currently, identification of these species is based on morphological criteria, but their strong similarity makes precise recognition difficult. To clarify species separation for quarantine purposes, a reliable method based on a PCR-RFLP procedure is reported. A DNA region containing mitochondrial NADH dehydrogenase genes was selected as a target sequence for the analysis. The amplification products (c. 1 kb) were digested with either SspI or DdeI, yielding specific patterns that differentiated each of the endemic species. Complete nucleotide sequences were determined, confirming empirical restriction maps. This report updates information on the geographical distribution of Rhagoletis species in Chile.

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