Taxonomic and conservation implications of population genetic admixture, mito-nuclear discordance, and male-biased dispersal of a large endangered snake, Drymarchon couperi

We extracted and genotyped microsatellite DNA from 428 tissue samples of Eastern Indigo Snakes (Drymarchon couperi). DNA was extracted from scales, shed skins, or muscle tissue collected from road-killed individuals throughout peninsular Florida and southern Georgia. Twenty-five samples were obtained from the collections of the Florida Museum of Natural History, including 20 samples used in Krysko et al. 2016 Molecular Phylogenetics and Evolution. The samples from Krysko et al. 2016 (op. cit.) included individuals from central Florida that represented both mitochondrial lineages where they occur in close proximity. The remaining Florida samples (N = 170) were collected during field studies of D. couperi in and around Highlands County or opportunistically by authorized project partners. The samples from Georgia (N = 233) were collected by multiple project partners as part of a study of population fragmentation in the state (S. Spear et al., unpublished data). Our samples include similar representation of both mitochondrial lineages (55% Atlantic and 45% Gulf). We extracted DNA using the Qiagen DNeasy blood and tissue extraction kit (Qiagen, Inc., Valencia, CA). We ran 17 microsatellite loci within three multiplexed panels using the Qiagen Multiplex PCR kit. Each reaction contained 1X Qiagen Multiplex PCR Master Mix, 0.2 µM multiplexed primer mix (each primer at equal concentrations), and 1 µl of DNA extract in a total volume of 7 µl. The PCR protocol was modified from Shamblin et al. (2011) Conservation Genetic Resources for multiplex PCR and consisted of an initial denaturation of 95ºC for 15 min, 20 touchdown cycles of 94°C for 30 s, 60°C minus 0.5°C per cycle for 90 s and 72°C for 1 min, followed by 30 cycles of 94°C for 30 s, 50°C for 90 s and 72°C for 1 min, and a final elongation step of 60°C for 30 min. Multiplexed PCR products were run on a 3130xl Applied Biosystems Genetic Analyzer at the University of Idaho’s Laboratory for Ecological, Evolutionary, and Conservation Genetics. We scored fragment sizes using Genemapper 3.7 (Applied Biosystems).