Range-wide Genomics Reveals Deep Divergence in Peripheral Populations of the Streamside Salamander (Ambystoma barbouri)

Peripheral populations often harbor unique evolutionary diversity yet face disproportionate conservation risk. The streamside salamander ( Ambystoma barbouri ) occurs primarily in the Ohio River basin in Kentucky, Indiana, and Ohio, but persists as isolated, disjunct populations in central Tennessee and western West Virginia, where rapid urbanization threatens remaining habitat. We combined mitochondrial DNA sequencing (~ 1,200 bp of the D-loop) and genome-wide SNP data to assess range-wide phylogeographic structure and fine-scale population differentiation across 89 populations throughout the contemporary geographic range of the species. Mitochondrial analyses revealed multiple deeply divergent lineages, whereas SNP-based analyses demonstrated strong nuclear differentiation between Tennessee populations and the species’ northern distribution in Kentucky, Indiana, Ohio, and West Virginia. Within Tennessee, genomic data identified three well-defined genetic clusters structured by watershed boundaries, with hierarchical AMOVA supporting significant differentiation among watersheds and among populations within watersheds. Together, these results indicate that Tennessee populations of A. barbouri represent a distinct and evolutionarily significant component of the species’ range and support their recognition as distinct conservation units.