File S1 - AFLPs and Mitochondrial Haplotypes Reveal Local Adaptation to Extreme Thermal Environments in a Freshwater Gastropod

Supporting Information. Supporting information contains detailed description of the AFLPs reaction together with the three figures (Fig. A-C) and eight tables (Table A-H). Figures: Fig. A. Histograms of assignment probabilities calculated by TESS for the six populations genotyped at 117 neutral loci after averaging the 20% best runs for K = 6 with CLUMPP [5]. Each vertical bar represents an individual and its assignment proportion into six clusters. Sites are ordered in the barplot according to the N-S directions that corresponds to decreasing habitat temperature. Fig. B. Graphs of the AFLPs genetic networks created with a) six loci under directional selection detected by BayeScan with log10(BF)>2 (“very strong selection”); b) 60 loci under directional selection detected by SAM, and c) six loci under directional selection detected by Mcheza. Black circles represent populations in high temperature sites (24°C), grey circles depict intermediate temperatures (12–19°C) and white circles represent sites at low temperature (6–8°C). Site pairs in a network connected by lines are considered to exchange migrants exhibiting significant conditional genetic covariance. Solid lines indicate connections assuming an IBD process where genetic distances and spatial distances are proportional. Dotted lines (…) represent compressed edges with relatively higher conditional genetic distance (cGD) in respect to spatial distance (suggesting geographical or ecological barriers), whereas dashed lines (---) depict extended edges indicating long distance migration processes. Fig. C. Median-joining network of COI haplotypes. Each haplotype is represented by a circle, and its area is proportional to its relative frequency; shared haplotypes are represented as frequency diagrams. Smaller black circle (mv) represents an unsampled hypothetical haplotype. Numbers correspond to mutational positions in the studied 643-bp fragment. Colors in the diagrams follow a decreasing thermal trend (i.e. warmest populations are depicted in the darkest colors). Tables: Table A. Diversity statistics by population for 376 repeatable AFLP markers, using Bayesian estimation of allele frequencies with non-uniform priors [6]. Numbers in parenthesis correspond to the values for the 117 neutral loci found simultaneously with BayeScan, Mcheza and SAM. Table B. Shaded cells with numbers in bold depict loci detected to be under directional selection by: SAM (P values for G and Wald Beta 1 with a significance threshold set to 95% corresponding to P10(BF)>1.5 corresponding to “very strong selection”) and MCHEZA at a significance P value of 0.01. Table C. Analysis of four populations at extreme temperatures.- Shaded cells with numbers in bold depict loci detected to be under directional selection by: SAM (P values for G and Wald Beta 1 with a significance threshold set to 95% corresponding to P10(BF)>1.5 corresponding to “very strong selection”) and MCHEZA at a significance P value of 0.01. The markers selected in the analyses with the total six populations are depicted in bold italics. Table D. Distribution of frequencies (%) per population for the four markers under directional selection detected with the three approaches (BayeScan, Mcheza and SAM) simultaneously. Table E. Pairwise FST between populations calculated with AFLPSurv for the 117 neutral data (below diagonal) and pairwise FST for the four loci under directional selection (above diagonal). Pairwise FST for loci under stabilizing selection took value of 0.000NS for every pair of populations. Significance was based on 10000 permutations. Table F. mtDNA. Distribution of frequencies (%) per population for the haplotypes showing correlation with temperature according to SAM. Table G. Pairwise ΦST between populations calculated with the 22 haplotypes not correlated with temperature (below diagonal) and with the 3 correlated haplotypes (above) computed from haplotype frequencies frequencies with Arlequin. Significance was based on 10000 permutations. Table H. Results on Mantel and partial Mantel tests comparing matrices of geographic distance (Geo), temperature (Temp) and genetic distance (estimated as pairwise Graph Distance with GeneStudio) for AFLPs and COI, and assessed for: neutral markers (NGD); loci under directional selection (DirGD) and loci under stabilizing selection (StaGD). Boldface type indicates significant values after 9999 permutations. (DOCX)