Rapid promoter evolution of male accessory gland genes is accompanied by divergent expression in closely related Drosophila species.

Seminal fluid proteins (Sfps) are essential for reproductive success and evolve fast on average, possibly driven by post-copulatory sexual selection (PCSS) originating from sperm competition and cryptic female choice. Counterintuitively, however, the coding region only in few Sfps evolves adaptively. Hence, additional genomic and functional factors must play a role in Sfp evolution independent of the protein coding region. To shed light on drivers of Sfp evolution we focus on those Sfps predominantly expressed in male accessory glands of Drosophila to examine their evolution in the tissue which produces the majority of Sfps. Unlike the testis, the accessory glands are known to develop normally in hybrids, allowing us to control for cellular environment differences arising during speciation. Here, we identify hotspots of rapid evolution in accessory gland protein genes (Acp) promoters, driven by base changes and insertions/deletions (indels). We further show that changes in promoter sequences are accompanied by gene expression divergence among closely related species. Using hybrids, we demonstrate that species-specific expression divergence is maintained for some Acps, while others exhibit dominance of one allele. These results indicate that regulatory evolution, rather than genome background variation, drives Acp expression changes and promotes their rapid evolution.