Genomic and Morphological Evidence Support Contemporary Three-Way Interspecific Hybridization in Ranid Frogs

Hybridization is increasingly understood as common throughout and beyond the speciation process, rather than an anomaly. Sympatric taxa are expected to exhibit strong reproductive isolation, and although hybridization may occur, it often results in inviable offspring. We investigated hybridization among three ranid frogs (Rana areolata, R. palustris, and R. sphenocephala) in eastern Oklahoma, where their distributions and breeding phenology overlap. Using micro-CT scans of cranial morphology, genomic SNP data, and phenological records, we confirmed two putative hybrids collected in the field-remarkable given the phylogenetic distance between these non-sister taxa. Genomic data show split ancestry from parental populations, representing one R. areolata × R. palustris and one R. areolata × R. sphenocephala. Cranial morphology indicates hybrids exhibit intermediate phenotypes, and our assessment identified a third likely hybrid, a specimen collected two decades earlier from the same area. Both confirmed hybrids were R. areolata backcrosses, but minimal introgression throughout the dataset suggests hybrid fitness may be lower than that of parental populations. Hybridization appears facilitated by overlapping breeding strategies and ecological factors leading to misdirected amplexus. This study provides the first documentation of natural hybridization in R. areolata, a species of conservation concern throughout its range. Our findings emphasize the utility of high-resolution morphological data (micro-CT) in complementing genomic approaches for hybrid diagnosis and suggest cranial morphology may be an effective method for hybrid identification in similar systems. Understanding this atypical three-species hybridization has important implications for conservation, as hybrid fitness and introgression can influence population dynamics and genetic integrity.