Overall gene expression
When comparing all the 16.211 transcripts that were kept after filtration and normalization, the first axis of the Multidimensional scaling (MDS) plot separated saltwater fish (SW) from freshwater fish (FW), whereas the second axis in part separated the exposure groups from their native salinity (Figure 3a ). The Principal Component Analysis (PCA) on logged values of all expressed genes after filtration and normalization also separated the groups based on original salinity and treatment, where PC1 explained a total of 88.29% of the variation, and PC2 explained 1.88% (Figure 3b ). Both the MDS and PCA separated the samples according to their original ecotype indicating that the overall gene expression pattern is highly linked to “original” environment.
In total, we identified 2717 unique transcripts that were significantly differentially expressed in all the analyzed contrasts (Table 1 , Figure 4, Figure 5, Supplementary Table 1 ). Interestingly, comparing saltwater fish in salt- and freshwater only reported 10 differentially expressed transcripts (Figure 4, Figure 5a ). The contrast between freshwater fish in fresh- and saltwater gave the highest number of differentially expressed transcripts (~1500, Figure 4, Figure 5b,c ), followed by a comparison of the two controls (~1300, Figure 4, Figure 5c,d ) and differences in eco-transcripts in salt and freshwater (~500; also being differentially expressed between the controls in comp. 3, Figure 4, Figure 5e ).