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).