Gene expression is stable despite widespread cis and trans regulatory divergence in Saccharomyces yeasts.

Regulatory evolution can alter phenotypes, but cis- and trans-regulatory mechanisms may also diverge extensively while total transcript abundance remains stable. Comparisons of parental expression with allele-specific expression in F1 hybrids provide a framework for separating cis- and trans-regulatory effects because both parental alleles are measured in a shared trans-regulatory environment. Here, we analyzed RNA sequencing data from Saccharomyces cerevisiae, Saccharomyces paradoxus, and their F1 hybrid. Among the 4,164 genes with sufficient allele-specific support for strict classification, 2,134 (51.2%) showed detectable cis and/or trans regulatory divergence. However, hybrid expression remained largely conserved, with 81.5% of genes not significantly different from either parent. Compensatory cis-trans divergence predominated over reinforcing divergence; cross-replicate estimation reduced the apparent magnitude of this excess, but opposite-sign effects remained predominant in all 20 non-overlapping replicate comparisons. To connect gene expression to genome sequence, we analyzed the strongly cis-diverged locus LYS2 and found species differences in promoter architecture, including an S. cerevisiae-specific AT-rich insertion, altered spacing among candidate regulatory features, and a promoter-proximal TATA-like element unique to S. cerevisiae. Sequence-based nucleosome prediction suggests that these differences create a broader promoter-proximal nucleosome-depleted region in S. cerevisiae than in S. paradoxus. We also quantified allele-resolved intron retention and found that allele-resolved intron retention was broadly conserved, with only rare locus-specific hybrid-associated shifts. Together, these results show that regulatory divergence is widespread but often buffered in the hybrid, whereas intron-retention divergence is comparatively limited.