In silico analyses of mitochondrial ORFans in freshwater mussels (Bivalvia: Unionoida) provide a framework for future studies of their origin and function

Alyssa Mitchell, Davide Guerra, Donald Stewart, Sophie Breton

Many species of bivalves exhibit a unique system of mtDNA transmission named Doubly Uniparental Inheritance (DUI). Under this system, species have two distinct, sex-linked mitochondrial genomes: the M-type mtDNA, which is transmitted by males to male offspring and found in spermatozoa, and the F-type mtDNA, which is transmitted by females to all offspring, and found in all tissues of females and in somatic tissues of males. Bivalves with DUI also have sex-specific mitochondrial ORFan genes, (M-orf in the M mtDNA, F-orf in the F mtDNA), which are open reading frames having no detectable homology and no known function. DUI ORFan proteins have previously been characterized in silico in a taxonomically broad array of bivalves including four mytiloid, one veneroid and one unionoid species. However, the large evolutionary distance among these taxa prevented a meaningful comparison of ORFan properties among these divergent lineages. The present in silico study focuses on a suite of more closely-related Unionoid freshwater mussel species to provide more reliably interpretable information on patterns of conservation and properties of DUI ORFans. Unionoid species typically have separate sexes, but hermaphroditism also occurs, and hermaphroditic species lack the M-type mtDNA and possess a highly mutated version of the F-orf in their maternally transmitted mtDNA (named H-orf in these taxa). In this study, H-orfs and their respective proteins are analysed for the first time. Despite a rapid rate of evolution, strong structural and functional similarities were found for M-ORF proteins compared among species, and among the F-ORF and H-ORF proteins across the studied species. In silico analyses suggest that M-ORFs have a role in transport and cellular processes such as signalling, cell cycle and division, and cytoskeleton organisation, and that F-ORFs may be involved in cellular traffic and transport, and in immune response. H-ORFs appear to be structural glycoproteins, which may be involved in signalling, transport and transcription. Our results also support either a viral or a mitochondrial origin for the ORFans. Our findings reveal striking structural and functional similarities among proteins encoded by mitochondrial ORFans in freshwater mussels, and strongly support a role for these genes in the DUI mechanism. Our analyses also support the possibility of DUI systems with elements of different sources/origins and different mechanisms of action in the distantly-related DUI taxa. Parallel situations to the novel mitochondrially-encoded functions of freshwater mussel ORFans present in some other eukaryotes are also discussed.