Sex-Specific ORFs in Marine Clams (Bivalvia: Veneridae) with Doubly Uniparental Inheritance: Exploring Their Origin...

In DUI animals, two sex-specific mitochondrial lineages are present: one transmitted maternally (F-type) and the other paternally (M-type), each containing a sex-specific mitochondrial ORFan gene. Comprehensive in silico characterization of DUI ORFan proteins has been conducted in freshwater mussels, with partial studies in marine mussels; however, research in other DUI species remains scarce. In this regard, we newly identified two DUI species, Antigona lamellaris (Schumacher, 1817) and Ezocallista brevisiphonata (P. P. Carpenter, 1864), and analyzed the major unassigned regions of the mitochondrial genomes in 169 specimens from eight species of Veneridae, verifying the presence of novel mitochondrial ORFs. Structural and functional analyses were performed on 14 ORFans, and the conservation patterns and properties of DUI ORFans in Veneridae were further explored by integrating in silico characterization results from freshwater and marine mussels. Strong structural and functional similarities were found between F-ORF and M-ORF proteins, both across different bivalve species and within the studied species. In contrast to freshwater and marine mussels, the F and M-ORF sequences in Veneridae show remarkable similarity. Additionally, apart from sex-specific ORFs, reverse ORFs (ORFs encoded on the reverse strand) were identified in Antigona lamellaris. Predicted transmembrane (TMs) number and topology analysis suggest that ORFs in Veneridae exhibit lower conservation compared to those in freshwater mussels. Our findings support the hypothesis that ORFans may originate from viruses or from proteins involved in mitochondrial energy production, providing further evidence for the multiple origins of ORFs. We propose that Veneridae, freshwater mussels, and marine mussels may harbor DUI systems with distinct origins and modes of action. The connection between ORFan proteins and DUI systems may differ across these taxa.