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Appendix S1 Supplementary methods.

Table S1 DNA microsatellite loci used in this study and the number of alleles found at each locus within each regional population (BP, Bruce Peninsula; EGB, Eastern Georgian Bay).

Table S2 fragstats results.

Table S3 Results from partial Mantel tests across three study regions after varying relative resistance values assigned to landscape features.

Table S4 Results from partial Mantel tests for sub-samples of 29 individuals south of Parry Sound.

Table S5 Pairwise Fst between diagnosed clusters.

Table S6 Results from the second step of two-step causal modeling for empirical data north and south of Parry Sound in Eastern Georgian Bay.

Table S7 Mantel correlations between forest, water, roads on the Bruce Peninsula (above the diagonal) and eastern Georgian Bay (below the diagonal).

Fig. S1 Barplot representing the q matrix from a global spatial assignment test in tess.

Fig. S2 Plot showing the detection of IBD across simulated generations.

Fig. S3 Plot of partial Mantel r-values across all sampled generations, demonstrating step 2 in the Wasserman et al. (2010)two-step causal modeling approach, where IBRwater|IBRwater + roads indicates a partial Mantel test between genetic distance and IBRwater partialling out the effects of IBRwater + roads, and IBRwater + roads|IBRwater indicates a partial Mantel test between genetic distance and IBRwater + roads partialling out the effects of IBRwater.

Fig. S4 Estimates of Ne with 95% confidence intervals from sibship assignment for 3 sampling locations.

Fig. S5 Correlations between empirical (gen.emp) and simulated (gen.sim) genetic distance matrices for a subset of individuals (= 53) south of Parry Sound.

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