Differential gene expression and GO term enrichment
Before testing our specific hypotheses, we first summarized differential expression patterns in both datasets. In the Aripo drainage, 766 genes were differentially expressed (DE) between high- and low-predation populations, 1,118 genes were differentially expressed between pred- and pred+ fish, and 825 genes had interaction effects (Fig. 2; Table S2). DE genes were enriched for GO categories including DNA integration, immune function, and morphogenesis. In the Quare drainage, 3,831 genes were differentially expressed between high- and low-predation populations, 791 genes were differentially expressed between pred- and pred+ fish, and 586 genes had interaction effects (Fig. 2; Table S3). DE genes were enriched for GO categories including immune function, developmental processes, and neurogenesis. Complete results of GO enrichment analyses for population of origin, rearing, and interaction effects are in the Supplemental Materials (Tables S4 & S5).
Given the marked difference in the total number of DE genes between datasets, we looked for evidence that this pattern might arise from technical variation or expression bias. While we cannot rule out some influence of technical variation, we found no evidence for differences in sequence quality, read alignment statistics, or variance in transcript abundance to suggest that variation in the number of DE transcripts between drainages was of technical origin. Nor did we find evidence that identification of DE transcripts was biased by transcript expression level: mean and median gene expression were significantly lower in DE (population, rearing, and/or interaction effects) as compared to non-DE transcripts in both the Aripo (Fig. S1; mean: DE=263, non-DE=344, p<0.0001; median: DE=65, non-DE=107, p<0.0001), and Quare (Fig. S1; mean: DE=264, non-DE=334, p<0.0001; median: DE=86, non-DE=101, p<0.0001) datasets. This suggests that identification of DE transcripts was not simply a side effect of relatively greater expression of these transcripts.