Evolutionary Divergence of mTOR-mediated Transcriptional Regulation Between Drosophila melanogaster and Drosophila simulans is modulated by sex and tissue

Abstract Evolutionary divergence in gene regulation is a major source of phenotypic novelty, yet understanding how deeply conserved, pleiotropic signaling pathways evolve under selective constraint while maintaining essential cellular functions remains an important challenge. Here, we investigate the evolutionary divergence in transcriptional regulation mediated by the mechanistic target of rapamycin (mTOR) pathway in two related species, Drosophila melanogaster and Drosophila simulans. mTOR is a highly conserved central regulator of cellular growth and metabolism, making it well-suited for the study of regulatory evolution under functional constraint. Using a fully factorial RNA-seq design, we quantified transcriptional responses to mTOR inhibition by rapamycin across three tissues (head, thorax, and abdomen) and both sexes. Despite highly conserved tissue-specific expression – reflecting the close phylogenetic relationship between species – mTOR-mediated transcriptional responses showed clear evidence of evolutionary divergence. Divergence varied across tissues and sexes: heads of both sexes and female thoraces and abdomens showed the most rapid gene-level divergence, suggesting stronger directional selection, whereas male thorax and abdomen exhibited comparatively conserved responses, consistent with stabilizing selection. Gene-level divergence patterns were consistent across multiple metrics and generally mirrored pathway-level divergence, except in the female abdomen, which showed relative pathway-level conservation despite extensive gene-level divergence. Species-by-treatment interaction analyses further revealed divergence in core mTOR-regulated biological processes. Together, our results suggest that regulatory modularity may allow even highly conserved signaling pathways to evolve under context-specific selective pressures while maintaining critical functionality. Significance statement The evolution of gene regulation is increasingly recognized as a major contributor to biological diversification. However, how highly pleiotropic and deeply conserved pathways evolve new regulatory effects without disrupting essential cellular functions remains an open question in evolutionary biology. Using the nutrient-sensing mTOR pathway as a model, we show that gene regulation mediated by this core, selectively-constrained signaling network has diverged between two closely related fruit fly species in a tissue- and sex-specific manner. Our findings suggest that even pleiotropic pathways like mTOR can diverge over short evolutionary timescales, and that regulatory modularity may facilitate the evolution of novel transcriptional effects in specific contexts while preserving essential functions. Competing Interest Statement The authors have declared no competing interest.