Parallelism in gene expression patterns across drainages
To test for shared mechanisms mediating parallel phenotypic adaptation across drainages, we asked whether differentially expressed genes within drainages were overlapping in identity and expression direction between drainages. Of the genes that diverged between high- and low-predation populations within a drainage (i.e., population main effect), 174 were overlapping between drainages (Table S6), more than expected by chance (χ2=8.02, p=0.0046). However, the direction of expression divergence among these overlapping genes was not associated across the two drainages (χ2=0.16, p=0.686): 91 genes had concordant expression changes and 83 genes had non-concordant expression changes between drainages (Fig. 5). To control for the influence of false negatives on our conclusions, we repeated this comparison at less stringent significance cut-offs. At a p=0.1, we found marginally significant overlap in gene identify (347 overlapping genes; χ2=3.38, p=0.06), but no association in expression direction (χ2=0.65, p=0.42), and at p=0.2 we found no significant overlap or association in expression direction. As a final, more conservative test, we compared the subset of genes with strong evidence for adaptive population divergence due to selection by restricting our comparison to population DE genes for which PST > FST. We found 534 genes (mean PST = 0.38, FST = 0.225) in the Aripo and 1163 genes (mean PST = 0.496, FST = 0.340) in the Quare drainage that met this criterion. Of these, 53 were overlapping between drainages, again more than expected by chance (χ2=12.7, p=0.0004), but without any association in expression direction (28 genes had concordant expression changes and 25 genes had non-concordant expression changes between drainages). PST values for all genes are in Table S8.
Of the genes that showed significant expression plasticity (i.e. main effect of rearing), 64 transcripts were overlapping between drainages (Table S7), more than expected by chance (χ2=14.06, p=0.0001). Of these 43 had expression changes in the same direction and 21 had expression changes in opposite directions between drainages, a significant association between drainages in the direction of expression plasticity associated with rearing environment (χ2=7.56, p=0.006) (Fig. 5). At less stringent p-value cut-offs for differential expression we found no more overlap in gene identity than expected by chance (p<0.1: 108 overlapping genes; χ2=0.05, p=0.81; p<0.2: 276 overlapping genes; χ2=1.36, p=0.24).