Supplemental Information for Editorial Manuscript Number: COGR-D-15-00061.0

Supplemental Information for Editorial manuscript number: COGR-D-15-00061.0

Sample collections and DNA extraction

Field collection of samples and corresponding locality information was provided by personnel from the West Virginia Department of Natural Resources (Elkins office). To develop and characterize markers, DNA was isolated from fiveTriodopsisplatysayoides‘snail trails’embedded inFTA® cards (Whatman, Inc., Piscataway, NJ, USA) collected from the wild following the methods of King and Eackles (unpublished; King et al. 2006). Genomic DNA was extracted from the FTA cards using the QIAGEN DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, U.S.A) using the manufacturer’s recommended protocol.

Demographic analyses

Expected average individual unbiased heterozygosity per locus (GenAlEx 6.5; Peakall and Smouse 2006)for all T.platysayoides from the Cheat River Gorge pooled was 66.9% (S.E. 4.4%) and ranged from 43.34% (TplB3) to 79.7% (TplA9). Average expected heterozygosities for individuals sampled from the left (descending) and right (ascending) banks of the Gorge weresimilar (63.8% and 59.9%, respectively). Tests for conformance to Hardy-Weinberg equilibrium (GENEPOP, v. 4.0; Raymond and Rousset 1995) among all 73 individuals indicated that all loci deviated from expectations (α=0.05, P<0.004; Bonferroni correction, Rice 1989). When individuals from the left bank were pooled and analysed separately, only TplA9 deviated from expectations (α=0.05, P<0.0001; Rice 1989). However, when the individuals from the right bank were pooled five of seven loci deviated from expectations (α=0.05, P<0.0001) (Table 1). Three pairs of loci exhibited statistically significant linkage disequilibrium (LD) (GENEPOP) among the 21 possible pair-wise comparisons for the pooled samples (overall α=0.05, P<0.0024). No LD was observed among the 42 pair-wise comparisons in the 2-bank (pooled) comparison (overall α=0.05, P<0.0012).

NeEstimator(Peel et al. 2004) was used to approximate (using LD) the effective spawning number (Ne) for all individuals pooled and for the 2-bank comparison. Ne was estimated to be 26.5 (95% CL, 21.2 and 34.1) for all collections pooled. Ne was estimated to be 7.4 (5.7-9.7) and 16.9 (12.9-23.0) for the left and right banks, respectively.

References

King TL, Switzer JF, Morrison CL, Eackles MS, Young CC, Lubinski BA, CryanP (2006) Comprehensive genetic analyses reveal evolutionary distinction of a mouse (Zapushudsoniuspreblei) proposed for delisting from the US Endangered Species Act. Molecular Ecology 15:4331-4359.

King TL and Eackles MS (In Preparation) ‘Snail trails’ as a source of noninvasive tissue collection for DNA analysis. Conservation Genetics Resources (In Preparation)

Peakall R, SmousePE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6, 288-295.

Peel D, Ovenden JR, Peel SL (2004) NEESTIMATOR: software forestimating effective population size, Version 1.3. QueenslandGovernment, Department of Primary Industries and Fisheries.Available at

Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. Journal of Heredity 86:248-249.

Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223-225.