Figures legends
Figure 1 . Study area. a) Map of Norway showing the position of the sampling sites just south of Oslo, b) illustrates the two sampling locations: Glitredammen (freshwater) and Sandspollen (saltwater).
Figure 2 : Experimental design. a) wild caught fish from saltwater and freshwater (see Figure 1) were taken into the lab and placed in holding tanks of their native salinity for a minimum of three weeks. After acclimation, two groups of eight fish from both populations/holding tanks were exposed to either saltwater (30‰; dark grey) or freshwater (0 ‰; light grey) for six hours. The exposure tanks were divided in two by a perforated wall, so both populations could be exposed to the same water quality at the same time (SwC and FwSw shared tank, as did FwC and SwFw). b) the four comparative analytical setups for which we compared the gene expression patterns: comparison 1) saltwater control (SwC ) compared to saltwater fish exposed to freshwater (SwFw); 2) freshwater control compared to freshwater fish exposed to saltwater (FwSw); 3) the two control groups; SwC compared to FwC and 4) ecotype independent of salinity.
Figure 3 . a) MDS plot. Multidimensional scaling (MDS) plot generated with edgeR, where each point represents one sample and the distance between individual samples reflects the leading-fold-change (logFC) of the corresponding RNA samples. The leading logFC is the average (root mean square) of the 500 largest absolute logFCs for genes between those two samples (default plotting parameter) (Two SC individuals are overlapping on the top), b) Principal component analysis (PCA) of gene expression profiles for all genes after filtering and normalization. FC= blue triangles pointing up, FS = orange triangles pointing down, SC= Black circle’s, SF= beige diamonds (see Figure 1 for abbreviations for groups).
Figure 4. Venn diagram illustrating the total number of differentially expressed transcripts for the four different comparisons, see Figure 2 for abbreviations for groups.
Figure 5 . Heatmaps of the differentially expressed annotated genes from the different comparisons (comp), see Figure 2 for experimental comparisons and Figure 4 for the number of overlapping transcripts between the analysed contrasts: a) shows the results of all 10 transcripts that are significantly different in comparison 1, including the transcripts that overlap with comparison 2 and comparison 3, b) shows the 1400 transcripts only significantly expressed in comparison 2, c) shows the 130 transcripts that are significantly differentially expressed between both comparison 2 and comparison 3, d) shows the 675 transcripts differentially regulated between comparison 3 and e) shows the 502 transcripts that are different in comparison 4, the ecogenes. Blue colour indicates less expression, red colour indicates a higher expression.
Figure 6 . Examples of differentially expressed genes for the four different groups (SwC, SwFw, FwC, FwSW) plotted as boxplots, of a) genes differentially regulated in comparison 1, b) genes differentially regulated in comparison 2, c) genes differentially regulated in comparison 3 and d) genes differentially regulated in comparison 4. The boxplots show the 25-75% quantiles (boxes), median (black horizontal line), 95% limits (bars) and outliers (open circles).
Figure 7. Heatmap of ATP6V0 and ATP6V1. For these two gene families, almost all transcripts showed signs of differential expression between the two ecotypes regardless of exposure treatment. The heatmap sums up individual fish on the X-axis; Swc= saltwater control fish (n=3); SwFw =saltwater fish exposed to freshwater (n=5); FwC = freshwater control fish (n=3) and FwSw = freshwater fish exposed to saltwater (n=5).