Presence of male mitochondria in somatic tissues and their functional importance at the whole animal level in the marine bivalve Arctica islandica

Metazoans normally possess a single lineage of mitochondria inherited from the mother (♀-type mitochondria) while paternal mitochondria are absent or eliminated in fertilized eggs. In doubly uniparental inheritance (DUI), which is specific to the bivalve clade including the ocean quahog, Arctica islandica, ♂-type mitochondria are retained in male gonads and, in a few species, small proportions of ♂-type mitochondria co-exist with ♀-type in somatic tissues. To the best of our knowledge, we report, for the first time in metazoan, the natural occurrence of male and female individuals with exclusively ♂-type mitochondria in somatic tissues of the bivalve A. islandica. Mitochondrial genomes differ by ~5.5% at DNA sequence level. Exclusive presence of ♂-type mitochondria affects mitochondrial complexes partially encoded by mitochondrial genes and leads to a sharp drop in respiratory capacity. Through a combination of whole mitochondrial genome sequencing and molecular assays (gene presence and expression), we demonstrate that 1) 11% of individuals of an Icelandic population appear homoplasmic for ♂-type mitochondria in somatic tissues, 2) ♂-type mitochondrial genes are transcribed and 3) individuals with ♂-type mitochondria in somatic cells lose 30% of their wild-type respiratory capacity. This mitochondrial pattern in A. islandica is a special case of DUI, highlighted in individuals from both sexes with functional consequences at cellular and conceivably whole animal level.

Bioenergetic consequences of sex-specific mitochondrial DNA evolution

Doubly uniparental inheritance (DUI) represents a notable exception to the general rule of strict maternal inheritance (SMI) of mitochondria in metazoans. This system entails the coexistence of two mitochondrial lineages (F- and M-type) transmitted separately through oocytes and sperm, thence providing an unprecedented opportunity for the mitochondrial genome to evolve adaptively for male functions. In this study, we explored the impact of a sex-specific mitochondrial evolution upon gamete bioenergetics of DUI and SMI bivalve species, comparing the activity of key enzymes of glycolysis, fermentation, fatty acid metabolism, tricarboxylic acid cycle, oxidative phosphorylation and antioxidant metabolism. Our findings suggest reorganized bioenergetic pathways in DUI gametes compared to SMI gametes. This generally results in a decreased enzymatic capacity in DUI sperm with respect to DUI oocytes, a limitation especially prominent at the terminus of the electron transport system. This bioenergetic remodelling fits a reproductive strategy that does not require high energy input and could potentially link with the preservation of the paternally transmitted mitochondrial genome in DUI species. Whether this phenotype may derive from positive or relaxed selection acting on DUI sperm is still uncertain.

Expanding the Search for Sperm Transmission Elements in the Mitochondrial Genomes of Bivalve Mollusks

Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) in bivalve mollusks is one of the most notable departures from the paradigm of strict maternal inheritance of mtDNA among metazoans. Recently, work on the Mediterranean mussel Mytilus galloprovincialis suggested that a nucleotide motif in the control region of this species, known as the sperm transmission element (STE), helps protect male-transmitted mitochondria from destruction during spermatogenesis. Subsequent studies found similar, yet divergent, STE motifs in other marine mussels. Here, we extend the in silico search for mtDNA signatures resembling known STEs. This search is carried out for the large unassigned regions of 157 complete mitochondrial genomes from within the Mytiloida, Veneroida, Unionoida, and Ostreoida bivalve orders. Based on a sliding window approach, we present evidence that there are additional putative STE signatures in the large unassigned regions of several marine clams and freshwater mussels with DUI. We discuss the implications of this finding for interpreting the origin of doubly uniparental inheritance in ancestral bivalve mollusks, as well as potential future in vitro and in silico studies that could further refine our understanding of the early evolution of this unusual system of mtDNA inheritance.

Molluscan mitochondrial genomes break the rules

The first animal mitochondrial genomes to be sequenced were of several vertebrates and model organisms, and the consistency of genomic features found has led to a ‘textbook description’. However, a more broad phylogenetic sampling of complete animal mitochondrial genomes has found many cases where these features do not exist, and the phylum Mollusca is especially replete with these exceptions. The characterization of full mollusc mitogenomes required considerable effort involving challenging molecular biology, but has created an enormous catalogue of surprising deviations from that textbook description, including wide variation in size, radical genome rearrangements, gene duplications and losses, the introduction of novel genes, and a complex system of inheritance dubbed ‘doubly uniparental inheritance’. Here, we review the extraordinary variation in architecture, molecular functioning and intergenerational transmission of molluscan mitochondrial genomes. Such features represent a great potential for the discovery of biological history, processes and functions that are novel for animal mitochondrial genomes. This provides a model system for studying the evolution and the manifold roles that mitochondria play in organismal physiology, and many ways that the study of mitochondrial genomes are useful for phylogeny and population biology. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

A proposed method for analyzing molecular signatures to detect hermaphroditism in freshwater mussels: a case study using the Eastern Floater (Pyganodon cataracta)

Many freshwater mussels (Order Unionida) have an unusual system of doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA. In species with DUI, males possess a female-transmitted (F-type) mt genome and a male-transmitted (M-type) mt genome. These genomes contain non-canonical open reading frame (orf) genes referred to as f-orf and m-orf, present in F and M mt genomes, respectively. These genes have been implicated in sexual development in Unionida. When gonochoric species become hermaphroditic, which has happened several times in Unionida, they lose their M-type mt genome, and f-orf genes evolve dramatically. Resulting F-ORF proteins are highly divergent in terms of primary nucleotide sequence, inferred amino acids, and hydrophobic properties; these genes (and proteins) are referred to as hermaphroditic orfs or h-orfs (and H-ORFs). We investigated patterns of hydrophobicity divergence for H-ORF proteins in hermaphrodites versus F-ORF proteins in closely related gonochoric species against cytochrome c oxidase subunit 1 (cox1) divergences. This approach was used to assess whether cryptic hermaphrodites can be detected. Although we did not detect evidence for the recent transition of any populations of Eastern Floaters, Pyganodon cataracta (Say, 1817) to hermaphroditism, our analyses demonstrate that molecular signatures in mtDNA can be used to detect hermaphroditism in freshwater mussels.

A high-quality reference genome for a parasitic bivalve with doubly uniparental inheritance (Bivalvia: Unionida)

From a genomics perspective, bivalves (Mollusca: Bivalvia) have been poorly explored with the exception for those of high economic value. The bivalve order Unionida, or freshwater mussels, has been of interest in recent genomic studies due to their unique mitochondrial biology and peculiar life cycle. However, genomic studies have been hindered by the lack of a high-quality reference genome. Here, I present a genome assembly of Potamilus streckersoni using Pacific Bioscience single-molecule real-time long reads and 10X Genomics linked read sequencing. Further, I use RNA sequencing from multiple tissue types and life stages to annotate the reference genome. The final assembly was far superior to any previously published freshwater mussel genome and was represented by 2,368 scaffolds (2,472 contigs) and 1,776,755,624 bp, with a scaffold N50 of 2,051,244 bp. A high proportion of the assembly was comprised of repetitive elements (51.03%), aligning with genomic characteristics of other bivalves. The functional annotation returned 52,407 gene models (41,065 protein, 11,342 tRNAs), which was concordant with the estimated number of genes in other freshwater mussel species. This genetic resource, along with future studies developing high-quality genome assemblies and annotations, will be integral toward unraveling the genomic bases of ecologically and evolutionarily important traits in this hyper-diverse group.

A naturally heteroplasmic clam provides clues about the effects of genetic bottleneck on paternal mtDNA

Mitochondrial DNA (mtDNA) is present in multiple copies within an organism. Since these copies are not identical, a single individual carries a heterogeneous population of mtDNAs, a condition known as heteroplasmy. Several factors play a role in the dynamics of the within-organism mtDNA population: among them genetic bottlenecks, selection, and strictly maternal inheritance are known to shape the levels of heteroplasmy across mtDNAs. In Metazoa, the only evolutionarily stable exception to the strictly maternal inheritance of mitochondria is the doubly uniparental inheritance (DUI), reported in 100+ bivalve species. In DUI species there are two highly divergent mtDNA lineages, one inherited through oocyte mitochondria (F-type) and the other through sperm mitochondria (M-type). Having both parents contributing to the mtDNA pool of the progeny makes DUI a unique system to study the dynamics of mtDNA populations. Since in bivalves the spermatozoon has few mitochondria (4-5), M-type mtDNA faces a tight bottleneck during embryo segregation, one of the narrowest mitochondrial bottlenecks investigated so far. Here, we analyzed the F- and M-type mtDNA variability within individuals of the DUI species Ruditapes philippinarum, and we investigated for the first time the effects of such a narrow bottleneck affecting mtDNA populations. As a potential consequence of this narrow bottleneck, the M-type mtDNA shows a large variability in different tissues, a condition so pronounced that it leads to genotypes from different tissues of the same individual not to cluster together. We believe such results may help understanding the effect of low population size on mtDNA bottleneck.