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Fig. S1 Example of a scatter plot of SNP readings for Contig16466_1044 for Stewiacke River salmon using Invader™ Plus chemistry by Third Wave Technologies Inc. RED (Redmond Red, excitation 560 nm, emission 620 nm) and FAM (excitation 486 nm, emission 530 nm) are fluorescent dyes, each associated with a different base. FOZ or Fold Over Zero is the signal at a given wavelength for a particular sample, divided by the average signal at that wavelength for the blanks (no template control samples). The genotype for each sample is based on the ratio of FAM FOZ to RED FOZ. Cutoff values associated with each genotype were deduced by visual inspection of the scatter plot. For example, samples with low FAM FOZ to RED FOZ ratios are assigned TT genotypes (triangles), high FAM FOZ to RED FOZ ratios GG genotypes (diamonds), and mid-range values (squares) GT genotypes. For Contig16466_1044 in Stewiacke River samples, for example, the cutoff values for TT are 0–1.0, 1.5–7.0 for GT, and 10.5 and above for GG. Anything with ratios between these cutoff values, 1.0–1.5 and 7.0–10.5, are considered equivocal and a genotype was not assigned.

Fig. S2 Pairwise FST values using the mean number of pairwise differences calculated in Arlequin 3.5 using the 252 loci that were scorable and had less than 10% missing data. Big Salmon River (BSR), Economy River (ECO), Great Village River (GRV), Point Wolfe River (PWF), Stewiacke River (STW), and Upper Salmon River (USR) are inner Bay of Fundy (iBoF) populations. Canaan River (CAN), Hammond River (HAM), Nashwaak River (NSH), Serpentine River (SER), and Tobique River (TOB) are outer Bay of Fundy (oBoF) populations.

Fig. S3 Outlier loci detected in a total of 252 scorable loci using the Arlequin 3.5 hierarchical island model (Excoffier et al. 2009) comparing four inner Bay of Fundy populations (BSR, PWF, STW, USR) and four outer Bay of Fundy populations (CAN, HAM, SER, TOB). For full names of population refer to Fig.  1. All loci above the upper 1% quantile would be classified as outlier loci under diversifying selection by Beaumont and Nichols (1996). (A) FCT outliers comparing the inner and outer populations: 18 = Contig12869_0119, 73 = Contig14711_157, 154 = Contig16129_0239, 165 = Contig16260_0757, 179 = Contig16466_1044. (B) FST outliers (Table  S5, Supporting information): 18 = Contig12869_0119, 73 = Contig14711_157, 155 = Contig16129_0239, 165 = Contig16260_0757, 179 = Contig16466_1044, 192 = Contig16688_0128, 251 = Contig17368_0088.

Fig. S4 FST outlier loci detected in a total of 221 polymorphic SNP loci in eight Bay of Fundy populations using BayeScan (Foll & Gaggiotti 2008). FST outliers under diversifying selection (Table  S5, Supporting information): 14 = Contig12869_0119, 157 = Contig16475_1011, 167 = Contig16688_0128, 214 = Contig17368_0088. Details in Table  4. Bayes factor (BF) >1.5 is very strong with 0.97 < P > 0.99, BF > 2.0 is decisive with P > 0.99.

Table S1 Dilution of PCR product and incubation time (min) for each single nucleotide polymorphism and population genotyped using the Invader™ chemistry2

Table S2 Dilution of archived DNA and number of PCR cycles for each single nucleotide polymorphism and population genotyped using Invader™ Plus1 method by Third Wave Technologies Inc. For explanation of plate names refer to Table   S1 (Supporting information)

Table S3 Loci from MassARRAY™ 320 SNP dataset genotyped at the Centre for Integrative Genetics in Norway that exhibited significant deviations from Hardy Weinberg Equilibrium after traditional Bonferroni corrections (α < 0.000191) are marked with an ‘x’. For full population names refer to Fig.  1

Table S4 Pairwise FST values using the mean number of pairwise differences calculated in Arlequin 3.1 using 252 scorable loci from the MassARRAY™ 320 SNP dataset that had less than 10% missing data for a particular locus. Values above the diagonal are the P-values from exact tests with 110 permutations and below diagonal are the FST values. *Significant after a traditional Bonferroni correction (α < 0.001786). For full population names refer to Fig.  1

Table S5 Contig Numbers and Associated SNP Names. Full locus name, SNP name, female linkage group (map units of SNPs in same EST), and EST protein homology for all loci discussed in this manuscript are outlined in Moen et  al. (2008 in Supplemental Material 1). Loci in bold font were the nine chosen non-neutral single nucleotide polymorphisms and were genotyped on wild Bay of Fundy salmon using Invader™ chemistry. Loci in italic font are one of outlier loci at P < 0.01 as defined in this manuscript (Table  4)

Table S6 FST values and the minor allele frequencies from FSTAT 2.93 for the eight outlier (in italic font) plus 3 additional SNP loci chosen for subsequent Invader™ analysis (in bold font) from the MassARRAY™ 320 SNP dataset for the eight BoF Atlantic salmon populations. See Table  4 for FST outlier analysis and Table  S5 (Supporting information) for details of SNPs. N/A, no estimate obtained

Table S7 Samples genotyped using Invader™ chemistry by Third Wave Technologies Inc. and the total number of homozygotes and heterozygotes called for each SNP locus in each population. RED and FAM are dyes what the samples are labelled with during the Invader™ reaction. For full population names refer to Fig.  1

Table S8 Locus by locus AMOVA among all eight populations for nine non-neutral single nucleotide polymorphisms genotyped using Invader™ chemistry and allowing a 50% level of missing data for a particular locus. Populations were grouped into inner Bay of Fundy (BSR, ECO, GRV, PWF, STW and USR) and outer Bay of Fundy (NSH and TOB). For full population names refer to Fig.  1. *P < 0.0005, **P < 0.00005.

Table S9 Pairwise FST values using the mean number of pairwise differences calculated in Arlequin 3.1 for seven populations genotyped at nine non-neutral single nucleotide polymorphisms using Invader™ chemistry. Values above the diagonal are the P-values with 16 002 permutations from exact tests and below diagonal are the FST values that are shaded green when significant. *Significant after a traditional Bonferroni correction (α < 0.002381). For full population names refer to Fig.  1

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