A mitochondrial orphan protein contributes to sperm development in bivalves

Lineage-specific genes, or orphan genes, are increasingly recognized as important drivers of evolutionary innovation, yet their functional characterization remains largely restricted to nuclear genomes of model organisms. Whether lineage-specific genes encoded by mitochondrial DNA contribute to phenotypic diversification remains virtually unexplored. Here we investigate MORF, a rapidly evolving ORFan protein encoded by the paternally transmitted mitochondrial genome of bivalves with doubly uniparental inheritance (DUI). Combining immunolocalization, functional neutralization, protein interaction assays and proteomics in the distantly related bivalves Mytilus edulis and Venustaconcha ellipsiformis, we characterize MORF localization, function and molecular associations during male germ cell development. MORF localizes to developing sperm cells and mature sperm mitochondria in both species and is additionally detected in the acrosome of M. edulis. In cultured M. edulis germ cells, antibody-mediated neutralization of MORF increases the frequency of spermatids displaying four rather than the canonical five mitochondria, providing functional evidence that MORF contributes to mitochondrial organization during spermatogenesis. Comparative proteomic analyses identify MORF-associated proteins involved in cytoskeletal organization, protein homeostasis and extracellular matrix organization. The association between MORF and laminin is independently supported by western blotting in both species. Despite extensive sequence divergence, the two MORF proteins display overlapping functional and molecular features. Together, these findings uncover a functional role for a rapidly evolving mitochondrial orphan protein and point to mitochondrial orphan genes as a potentially overlooked source of evolutionary novelty.