A major driver generating amphibian diversity in the Appalachian Mountains is the complex paleogeography of the zone. Although the Appalachian Mountains have been widely studied, much of its amphibian phylogeography remains poorly known. The Mountain Chorus Frog (Pseudacris brachyphona) is one example of an understudied organism due to its elusiveness, patchy distribution, and short breeding seasons. Phylogenetic studies have suggested the existence of divergent lineages within P. brachyphona; however, insufficient sampling and the lack of diagnostic morphological traits have prevented further assessment of their taxonomic status. Using a genome-wide nuclear data set obtained via anchored hybrid enrichment, acoustic data, and ecological modeling, we tested for the existence of cryptic species within P. brachyphona. Our species tree estimation supports previous mitochondrial-based phylogenetic hypotheses that separate P. brachyphona into the Northern and Southern clades. Population genetic clustering also shows a sharp genetic break, which is concordant with these clades. Admixture was observed between the Northern clade and another chorus frog species (P. feriarum). Analysis of advertisement calls shows a divergent, faster pulse rate, and higher dominant frequency call for the Southern clade in comparison to the Northern clade and other trilling chorus frogs. Furthermore, species distribution models showed that habitat suitability for the Southern clade included pine-dominated and drier areas compared to the Northern clade. In light of the genetic, acoustic, and ecological divergence between the clades, we propose to elevate the taxonomic status of the Southern clade and name this new species the Collinses' Mountain Chorus Frog (P. collinsorum, new species). The new species ranges across northern Alabama, and extends into Georgia, eastern Mississippi, and southern Tennessee. Pseudacris collinsorum, new species, is geographically separated from its sister species, P. brachyphona, by the Tennessee River and surrounding Blue Ridge Escarpment. This study highlights the need for genus-wide, population-level genetic assessments and integrative data sets to uncover diversity within anurans.
