Variability of mitochondrial ORFans hints at possible differences in the system of doubly uniparental inheritance of mitochondria among families of freshwater mussels (Bivalvia: Unionida)

Abstract

Background: Supernumerary ORFan genes (i.e., open reading frames without obvious homology to other genes) are present in the mitochondrial genomes of gonochoric freshwater mussels (Bivalvia: Unionida) showing doubly uniparental inheritance (DUI) of mitochondria. DUI is a system in which distinct female-transmitted and male-transmitted mitotypes coexist in a single species. In families Unionidae and Margaritiferidae, the transition from dioecy to hermaphroditism and the loss of DUI appear to be linked, and this event seems to affect the integrity of the ORFan genes. These observations led to the hypothesis that the ORFans have a role in DUI and/or sex determination. Complete mitochondrial genome sequences are however scarce for most families of freshwater mussels, therefore hindering a clear localization of DUI in the various lineages and a comprehensive understanding of the influence of the ORFans on DUI and sexual systems. Therefore, we sequenced and characterized eleven new mitogenomes from poorly sampled freshwater mussel families to gather information on the evolution and variability of the ORFan genes and their protein products.

Results: We obtained ten complete plus one almost complete mitogenome sequence from ten representative species (gonochoric and hermaphroditic) of families Margaritiferidae, Hyriidae, Mulleriidae, and Iridinidae. ORFan genes are present only in DUI species from Margaritiferidae and Hyriidae, while non-DUI species from Hyriidae, Iridinidae, and Mulleriidae lack them completely, independently of their sexual system. Comparisons among the proteins translated from the newly characterized ORFans and already known ones provide evidence of conserved structures, as well as family-specific features.

Conclusions: The ORFan proteins show a comparable organization of secondary structures among different families of freshwater mussels, which supports a conserved physiological role, but also have distinctive family-specific features. Given this latter observation and the fact that the ORFans can be either highly mutated or completely absent in species that secondarily lost DUI depending on their respective family, we hypothesize that some aspects of the connection among ORFans, sexual systems, and DUI may differ in the various lineages of unionids.

Keywords: Doubly uniparental inheritance of mitochondrial DNA; Evolution of protein structures and functions; Freshwater mussels; Mitochondria and sexual systems; Mitochondrial ORFan genes; mtDNA sequencing.

Fig. 1

Bayesian inference phylogenetic tree of freshwater mussel mt genomes based on nucleotide sequences from 12 of their protein coding genes (atp8 was excluded). All nodes have posterior probability 1.000, except where indicated. An arrow indicates the split of freshwater mussel M mtDNAs clade. Clades and groups of mtDNAs are color coded according to the family and/or type of mtDNA indicated on the right side of the figure. The new mtDNAs sequenced in this study are in bold character

Fig. 2

Bayesian inference phylogenetic tree of freshwater mussel mt genomes based on protein sequences translated from 12 of their protein coding genes (atp8 was excluded). All nodes have posterior probability 1.000, except where indicated. An arrow indicates the split of freshwater mussel M mtDNAs clade. Clades and groups of mtDNAs are color coded according to the family and/or type of mtDNA indicated on the right side of the figure. The new mtDNAs sequenced in this study are in bold character

Fig. 3

Schematic organization of the atp6-nad4L and nad2-trnE regions of the freshwater mussel (Unionida) based on mt genomes presented in this study and of already published ones. Sample size for each family: 2 Iridinidae (all non-DUI), 5 Mulleriidae (all non-DUI), 7 Hyriidae (2 F, 2 M, 3 non-DUI), 10 Margaritiferidae (6 F, 3 M, 1 H), 38 Unionidae (17 F, 17 M, 4 H). GenBank accession numbers of the mt genomes used are enlisted in Table 1 and Additional file 1: Table S1. Standard mitochondrial genes are in grey, while ORFan genes (see the main text for a complete description of these genes) are colored following this code: green, Anodontites trapesialis specific ORFans; blue, M-orfs; pink, F-orfs; light pink, H-orfs. Genes are pointed according to their relative direction on the mtDNAs. tRNA genes are indicated with the one-letter code of their respective amino acid. Dotted lines represent the segments between the two regions, which are not indicated for simplicity. Sinanodonta woodiana annotation is based on [12] and on the current study

Fig. 4

Percentage amino acid composition of the ORFan proteins considered in this study (their relative nucleotide sequences are enlisted in Additional file 2). The sample size for each boxplot is indicated inside the legends in square parentheses as ‘N’. Amino acid names are indicated with the IUPAC three-letter and one-letter codes

Fig. 5

Summary of the CLANS analysis for F-ORFs and M-ORFs. Because the original CLANS output is a three-dimensional space, here are shown the three two-dimensional faces of the cube (one for each possible couple of axis: X vs Y, Z vs Y, Z vs X) obtainable by rotating the three-dimensional space of each analysis with 90° movements on one axis. The ‘+’ inside each panel represents the center of the cube. Each dot represents a single protein sequence (color code in the legends)

Fig. 6

Unrooted maximum likelihood (ML) trees for F-ORF and M-ORF proteins of freshwater mussels. Color code for each family are indicated inside the panels. Bootstrap values are indicated at each node

Fig. 7

3D models of representative F-ORF proteins of DUI freshwater mussels. The models shown are the first of the top five predicted by I-TASSER for each sequence. Number of amino acids (aa) of each protein and C-score of the models are indicated under the relative species names. C-scores are usually comprised between − 5 and 2: higher values indicate higher confidence of the model. The color shading of each protein goes from the blue of the N-terminus to the red of the C-terminus

Fig. 8

3D models of the proteins encoded by Anodontites trapesialis ORFans and of representative M-ORF proteins of DUI freshwater mussels. The models shown are the first of the top five predicted by I-TASSER for each sequence. Number of amino acids (aa) of each protein and C-score of the models are indicated under the relative species names. C-scores are usually comprised between − 5 and 2: higher values indicate higher confidence of the model. The color shading of each protein goes from the blue of the N-terminus to the red of the C-terminus