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Fig. S1 Overview of experimental setup for acclimation experiments. In the long-term (50 days) experiment 5–6 individuals were sampled in each group (6 reciprocal and 5 controls). In the short-term (1 day) experiment 5 individuals were sampled in both Baltic Sea groups (reciprocal and control) and 4 individuals in both North Sea groups (reciprocal and control).

Fig. S2 Gene expression verification of the 5-aminolevulinic acid synthase gene expression. Samples are identified with North Sea (pooled North Sea samples) and Baltic Sea (pooled Baltic Sea samples) mRNA levels are normalized to the corresponding EF1a abundance from the same sample, and mean (n = 11 for NS and n = 11 for BS) values are expressed in arbitrary units ± S.E. Difference in gene expression is significant between populations (t-test, p = 0.006).

Fig. S3 Gene expression verification of the angiotensinogen gene expression. Samples are identified with a 9ppt (NSR and BS samples) and 33ppt (NS and BSR). mRNA levels are normalized to the corresponding EF1a abundance from the same sample, and mean (n = 11 for 33ppt and n = 11 for 9ppt) values are expressed in arbitrary units ± S.E. Difference in gene expression is significant between treatments (t-test, p = 0.0072).

Table S1 Gene groups statistically significantly expressed between North Sea and Baltic Sea populations based on gene ontology (Welch t-test, p-value cutoff set to 0.01).

Table S2 Gene list of genes differently expressed between North Sea and Baltic Sea flounder (Welch t-test, p-value cutoff set to 0.01) sorted according to gene expression (fold changes). Genes passing Benjamini and Hodges (1995) correction for multiples testing (p < 0.05) are marked with bold in p-value.

Table S3  Gene list of genes differently expressed between salinity acclimation groups (Welch t-test, p-value cutoff set to 0.01) sorted according to gene expression (fold changes).

Table S4  Gene groups statistically significantly expressed between salinity treatment groups based on gene ontology (Welch t-test, p-value cutoff set to 0.01).

Table S5  Gene list of genes differently expressed between North Sea flounders acclimated to natural and reciprocal salinities (Welch t-test, p-value cutoff set to 0.01) sorted according to gene expression (fold changes). Genes passing Benjamini and Hodges (1995) correction for multiples testing (p < 0.05) are marked with bold in p-value.

Table S6 Gene list of genes differently expressed between Baltic Sea flounders acclimated to natural and reciprocal salinities (Welch t-test, p-value cutoff set to 0.01) sorted according to gene expression (fold changes).

Table S7 Parameter association test. The parameter association test estimates the association strength and number of genes supporting the different parameters included in the experiment. Parameter accounts for the different parameters included in the analysis based on the expression of 3144 clones. Origin accounts for origin of flounders (North Sea or Baltic Sea), salinity accounts for acclimation salinity (9ppt or 33ppt) and time accounts for long-term or short-term acclimation – whereas type is a combination of the other three parameters. Association strength is calculated from the Equation: S − (log p-values), so that lower p-values result in higher association strength. Number of Genes is the number of genes that were found to be supported in samples when defined by a specific parameter. Minimum p-value is the minimum observed p-value for a gene in the resulting gene list (GeneSpring 7.2 manual, Silicon Genetics).

Table S8 Microsatellite information. Microsatellite ID, reference, repeat motif, applied annealing temperature and primer sequences for microsatellites.

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