Pliocene-Pleistocene lineage diversifications in the Eastern Indigo Snake (Drymarchon couperi) in the Southeastern United States

Indigo Snakes (Drymarchon; with five currently recognized species) occur from northern Argentina, northward to the United States in southern Texas and eastward in disjunct populations in Florida and Georgia. Based on this known allopatry and a difference in supralabial morphology the two United States taxa previously considered as subspecies within D. corais (Boie 1827), the Western Indigo Snake, D. melanurus erebennus (Cope 1860), and Eastern Indigo Snake, D. couperi (Holbrook 1842), are currently recognized as separate species. Drymarchon couperi is a Federally-designated Threatened species by the United States Fish and Wildlife Service under the Endangered Species Act, and currently being incorporated into a translocation program. This, combined with its disjunct distribution makes it a prime candidate for studying speciation and genetic divergence. In this study, we (1) test the hypothesis that D. m. erebennus and D. couperi are distinct lineages by analyzing 2411 base pairs (bp) of two mitochondrial (mtDNA) loci and one single copy nuclear (scnDNA) locus; (2) estimate the timing of speciation using a relaxed phylogenetics method to determine if Milankovitch cycles during the Pleistocene might have had an influence on lineage diversifications; (3) examine historical population demography to determine if identified lineages have undergone population declines, expansions, or remained stable during the most recent Milankovitch cycles; and (4) use this information to assist in an effective and scientifically sound translocation program. Our molecular data support the initial hypothesis that D. melanurus and D. couperi should be recognized as distinct species, but further illustrate that D. couperi is split into two distinct genetic lineages that correspond to historical biogeography and sea level changes in peninsular Florida. These two well-supported genetic lineages (herein termed Atlantic and Gulf lineages) illustrate a common biogeographic distributional break previously identified for other plants and animals, suggesting that these organisms might have shared a common evolutionary history related to historic sea level changes caused by Milankovitch cycles. Our estimated divergence times suggest that the most recent common ancestor (MRCA) between D. melanurus and southeastern United States Drymarchon occurred ca. 5.9Ma (95% HPD=2.5-9.8Ma; during the late Blancan of the Pleistocene through the Hemphillian of the Miocene), whereas the MRCA between the Atlantic and Gulf lineages in the southeastern United States occurred ca. 2.0Ma (95% HPD=0.7-3.7Ma; during the Irvingtonian of the Pleistocene through the Blancan of the Pliocene). During one or more glacial intervals within these times, these two lineages must have become separated and evolved independently. Despite numerous Milankovitch cycles along with associated forming of physical barriers (i.e., sea level fluctuations, high elevation sand ridges, clayey soils, and/or insufficient habitats) since their initial lineage diversification, these two lineages have likely come in and out of contact with each other many times, yet today they still illustrate near discrete geographic distributions. Although the Atlantic and Gulf lineages appear to be cryptic, a thorough study examining morphological characters should be conducted. We believe that our molecular data is crucial and should be incorporated in making conscious decisions in the management of a translocation program. We suggest that source populations for translocations include maintaining the integrity of the known genetic lineages found herein, as well as those coming from the closest areas that currently support sizable Drymarchon populations.