The Rough Greensnake, Opheodrys aestivus, is a moderately sized, semi-arboreal snake broadly distributed throughout eastern North America. Although numerous taxa with similar distributions have been shown to comprise multiple species, O. aestivus has not been examined in a detailed phylogeographic context. Here, we use Sanger-sequence data (one mitochondrial, three nuclear loci) for samples from throughout the distribution of O. aestivus to elucidate phylogeographic patterns in this species. We combine this with ddRADseq data for a subset of samples to test patterns on a more genomically comprehensive scale. In both data sets, we find strong support for three deeply divergent clades within O. aestivus: peninsular Florida, central Texas, and a Main clade comprising the rest of the distribution. Estimates of divergence time suggest that the central Texas and Main clades diverged ∼1.3 million years ago (mya), while the peninsular Florida clade diverged from other lineages ∼2.9 mya, and these lineages diverged from the sister taxon, O. vernalis, ∼6.4 mya. The divergence of peninsular Florida or central Texas populations is not unique among squamates, nor is low levels of divergence from the Atlantic coast to eastern Texas, but this combination of patterns is unusual and yields important insight into the biogeography of North American biota. Further, our approach helps illustrate how dense geographic sampling with limited genomic sequencing can be used as a guide for the selection of samples to test phylogeographic patterns comprehensively. We conclude that elevating O. a. carinatus to species status may better describe the diversity of this genus.

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