Species Delimitation of Slimy Salamanders, Plethodon kisatchie and Plethodon mississippi, across the Lower Mississippi River

Brock Hunter Stevenson, Louisiana Tech University


Species are fundamental units of biodiversity yet delimiting species can be challenging. Slimy Salamanders of the Plethodon glutinosus species complex are a classic example of cryptic species for which species boundaries and relationships have proved difficult to determine. Once thought to be a single species ranging across the eastern United States, protein analysis revealed high genetic divergences among geographically distinct groups of populations, leading to 16 species being recognized within the group. Two of these species, the Louisiana Slimy Salamander (Plethodon kisatchie) and the Mississippi Slimy Salamander (Plethodon mississippi), are closely related but occur on opposite sides of the Mississippi River, a strong barrier to gene flow in many organisms. Previous phylogenetic studies of Plethodon have only included 1–2 samples of each of these species, thus a rigorous test of their validity has never been conducted. To investigate the evolutionary relationships of P. kisatchie and P. mississippi, I obtained tissue samples from throughout their distributions, extracted DNA, and then amplified and sequenced the mitochondrial ND2 gene and three nuclear loci. Sequence data were then analyzed using coalescent-based species delimitation methods to test the hypothesis that P. kisatchie and P. mississippi are independently evolving and thus, valid species under the general lineage concept. Results supported P. kisatchie and P. mississippi are species distinct from one another. However, I also found evidence that P. mississippi is hybridizing with P. glutinosus in Alabama. Furthermore, little genetic diversity occurs within P. kisatchie, likely due to recent separation from populations of P. mississippi (~520,000 years ago), which raises concern for the species’ long-term conservation. Based on the results of this study, I recommend both P. kisatchie and P. mississippi continue to be recognized.