Evolution of cis- and trans-regulation. (A) Overall differences (1 − ρ) in total expression between genotypes (blue) and allele-specific expression in F1 hybrids (red) are shown for each comparison, with data used for these calculations shown in Supplemental Figure S13. Relative allelic expression in F1 hybrids provides a readout of relative cis-regulatory activity. (B) For each comparison, the proportions of genes with evidence of significant differences in total expression (blue), cis-regulation (red), and trans-regulation (green) are shown. Data used to determine these proportions are shown in Supplemental Figure S16. Significance tests used to identify differences in trans-regulation had a different power than those used to identify differences in total expression and cis-regulation, thus only the evolutionary trends, not the proportions of significant genes, should be compared among these classes. Power was comparable, however, in the tests for differences in total expression and relative cis-regulatory activity summarized in this figure. (C,D) The line plots show expression differences (C) and differences in relative cis-regulatory activity (D) for individual genes in the mel-mel, sim-sech, and mel-sim comparisons plotted according to divergence time, with the 4851 genes included in Data set 2 classified into nine groups depending on whether they showed increased, decreased, or similar expression differences between mel-mel and sim-sech and between sim-sech and mel-sim. The red line in each plot shows the median expression difference for genes in that class for each comparison. The pie charts show the relative frequency of genes in each class. (E) The proportion of genes with evidence of significant cis- and trans-regulatory changes (red) is compared with the proportion of genes with evidence of cis- or trans-regulatory changes (blue). (F) For genes with evidence of both cis- and trans-regulatory changes, the frequency of genes with cis- and trans-regulatory changes affecting gene expression in the same (“cis + trans,” red) and opposite (“cis × trans,” blue) directions are compared. (G) The relative frequencies of genes with cis- and trans-regulatory changes in opposite directions that do (blue) and do not (red) show evidence of misexpression in F1 hybrids are compared. Error bars in panel A show the 95% quantiles from 10,000 bootstrap replicates in which differences in 1 − ρ between mel-mel and sim-sech as well as between sim-sech and mel-sim were calculated for each bootstrap replicate. The significance of the observed deviation from zero was determined by comparing the observed value to the distribution of bootstrap values. Significance was determined using Fisher’s exact tests in panels B, E, and G and using binomial exact tests in panel F. Significance of each comparison: (NS) Nonsignificant, P > 0.05; (*) P ≤ 0.05; (**) P ≤ 0.001; (***) P ≤ 1 × 10−4. Comparable analyses for reciprocal hybrids are shown in Supplemental Figures S12, S15.
