Comparative single-nucleus transcriptomics reveals asymmetric evolution of the Drosophila male and female germlines

Reproductive organs vary widely across species yet share conserved cell types that produce gametes, sustaining species' perpetuation. However, tissue-level comparisons mask critical differences among cell types, obscuring where evolutionary divergence occurs even between closely related species. We quantified expression divergence at cell-type resolution between two sibling species, Drosophila melanogaster and D. simulans, while disentangling adaptive and nonadaptive evolutionary mechanisms. We built a comparative single-nucleus transcriptomic atlas of over 100,000 nuclei from testes and ovaries of both species. Our analysis revealed sharply heterogeneous divergence across testis cell types, contrasting with a broader conservation across ovary cell types. Notably, in both organs, ~40% of genes showing interspecific differences did so in only one cell type. In the testis, spermatogonia were largely conserved, whereas divergence peaked in primary spermatocytes with extensive rewiring of coexpression modules linked to microtubule and mitochondrial functions. In the ovary, expression was largely conserved, except in early germline and late follicle cells, which showed shifts in oogenesis and cell-cycle-related coexpression modules. Divergent cell types in both tissues were enriched for evolutionarily young genes with narrow expression breadth and faster protein evolution rates. Additionally, the ovary exhibited a faster-X effect consistent with adaptive evolution. These findings reveal a fundamental asymmetry in how male and female germlines evolve, with functional constraints relaxed in specific testis cell types but broadly maintained across the ovary. Our work provides an evolutionary framework explaining how core reproductive functions are safeguarded during species diversification while identifying germline cells that drive evolutionary change.