Microsatellite development and genotyping

In order to isolate species‐specific microsatellite markers, tail clips were taken from two individuals collected from different localities in Southwestern Virginia in Spring 2014 and immediately preserved in RNAlater (Life Technologies, Carlsbad, CA). Whole sample RNA was extracted using a Qiagen RNEasy Mini Kit (Qiagen, Valencia, CA) following manufacturer's suggested protocols and processed in the Genomic Core Facility at the University of Virginia's Department of Biology for the construction of individual paired‐end, non‐normalized cDNA libraries using a NEBNextmRNA library Prep Master Mix Set (New England Biolab, Ipswich, MA). Individual libraries were given barcodes and pooled, and sent to Genewiz (GENEWIZ, South Plainfield, NJ) for sequencing on the Illumina HiSeq 2500 using a PE 2 × 100 bp format. Resulting sequences from each sample were assembled using the program Trinity v.11‐10‐2013 (Grabherr et al. 2011), and a summary of the sequencing results for each library can be found in Table S3 (Supporting Information). A custom BLAST search was used to locate regions of the mitochondrial genes cytb and ND2 sequenced previously from E. lucifuga in order to confirm the identity of the samples. Putative microsatellite loci were identified in each group of transcripts using the program msatcommander v. 1.0.8 (Faircloth 2008), specifying a search for repeats of trinucleotides or greater with at least five repeats. We used BLAST to confirm the presence of primer sequences in both samples, and discarded primers that were not present in both group of transcripts.

Following the method of Schuelke (2000), we screened each primer set for amplification and polymorphism using m13 fluorescently labeled tags. Each test was performed using eight samples collected from across the species range, and confirmational sequencing was performed using fragment analysis on an ABI 3130 Sequencer (Life Technologies). Primer pairs that amplified in all eight test samples and were polymorphic were produced with fluorescent tags incorporated into each forward primer and multiplexed. Thermocycler conditions used throughout the screening process are as follows: 94°C for 15 min, 40 cycles of 94°C for 30 sec, 60°C for 1:30 min, and 72°C for 1:30 min, and finally 72°C for 10 min. After identifying putative microsatellite loci and using BLAST to validate them, we had 220 markers to screen. Twenty‐four of these amplified in all test individuals and were polymorphic (>2 alleles), and these were multiplexed into six multiplexes, each containing four loci Table S4 (Supporting Information).

Loci were amplified in multiplexed reactions using PCR, and products were sent to the DNA Analysis Facility on Science Hill (Yale University, New Haven, CT) for fragment analysis using a 3730xl 96‐Capillary Genetic Analyzer, with the DS‐33 dye set. Finally, we used GeneMarker v2.4 (SoftGenetics, State College, PA) to call alleles at each marker locus for each individual.