Defective splicing of Y-chromosome-linked gigantic genes contributes to hybrid male sterility in Drosophila

The Y chromosome evolves rapidly, often differing dramatically even between closely related species. While such divergence has long been suspected to contribute to hybrid male sterility, leading to reproductive isolation and thus speciation, the underlying mechanisms remain elusive. Here, we identify a molecular basis linking Y chromosome divergence to reproductive isolation in Drosophila. We show that male hybrids between D. simulans and D. mauritiana fail to properly express key Y-linked fertility genes. These genes contain unusually large introns, exceeding megabases and show substantial sequence divergence between species. In the hybrids, these gigantic introns are misprocessed, resulting in widespread splicing defects, including aberrant "back-splicing" events that join later exons to earlier ones. Our findings suggest that sequence divergence within introns can disrupt essential gene expression through defective splicing, providing a mechanistic link between rapid Y chromosome evolution and hybrid sterility. This work highlights the underappreciated role of intronic divergence in speciation.