Cis-regulatory divergence and misexpression of spermatogenesis genes underlie hybrid male sterility in the Drosophila bipectinata species complex

Hybrid male sterility (HMS) is one of the most common and earliest forms of postzygotic reproductive isolation in Drosophila, often arising from defects in spermatogenesis linked to regulatory divergence of rapidly evolving male-biased genes. The Drosophila bipectinata species complex (D. bipectinata, D. malerkotliana, D. parabipectinata, and D. pseudoananassae) provides a valuable system to study the speciation paradigm, since, all the twelve F₁ hybrid males are sterile, the precise stage of spermatogenic failure differs among crosses. Here, we integrate cytological, transcriptional, and regulatory sequence analyses to examine the basis of sterility in this sub-complex. Cytological assays revealed that hybrid testes exhibit developmental arrest at different spermatogenesis stages, covering from reduced primary spermatocytes to absence of individualized sperm. Quantitative RT-PCR of seven spermatogenesis genes—aly, bam, sa, dj, topi, can, and Mst98Ca—showed significant downregulation among hybrid males relative to parental species, linking transcriptional misregulation with observed phenotypic defects in spermatogenesis. To test whether regulatory divergence underlies this misexpression, we analyzed ∼1–2 kb promoter regions of five genes (aly, bam, sa, dj, and topi). Motif scans using FIMO identified several lineage-specific turnover of transcription factor binding sites, also PWM-delta analysis revealed substantial interspecific differences in predicted binding strength for many key testis transcription factors. These results demonstrate that HMS in the D. bipectinata complex is consistently associated with spermatogenic arrest and misregulation of genes essential for germline proliferation, meiotic progression, and spermiogenesis. The concordance between expression changes and promoter divergence supports a role for cis-regulatory evolution in hybrid dysfunction, while also leaving open the contribution of trans-acting factors. This study situates the D. bipectinata complex within the broader framework of Drosophila hybrid sterility, extending and highlighting the evolutionary sensitivity of spermatogenesis and regulatory divergence in speciation.