Comparative mitochondrial genomics of freshwater mussels (Bivalvia: Unionoida) with doubly uniparental inheritance of mtDNA: gender-specific open reading frames and putative origins of replication

Abstract

Doubly uniparental inheritance (DUI) of mitochondrial DNA in marine mussels (Mytiloida), freshwater mussels (Unionoida), and marine clams (Veneroida) is the only known exception to the general rule of strict maternal transmission of mtDNA in animals. DUI is characterized by the presence of gender-associated mitochondrial DNA lineages that are inherited through males (male-transmitted or M types) or females (female-transmitted or F types), respectively. This unusual system constitutes an excellent model for studying basic aspects of mitochondrial DNA inheritance and the evolution of mtDNA genomes in general. Here we compare published mitochondrial genomes of unionoid bivalve species with DUI, with an emphasis on characterizing unassigned regions, to identify regions of the F and M mtDNA genomes that could (i) play a role in replication or transcription of the mtDNA molecule and/or (ii) determine whether a genome will be transmitted via the female or the male gamete. Our results reveal the presence of one F-specific and one M-specific open reading frames (ORFs), and we hypothesize that they play a role in the transmission and/or gender-specific adaptive functions of the M and F mtDNA genomes in unionoid bivalves. Three major unassigned regions shared among all F and M unionoid genomes have also been identified, and our results indicate that (i) two of them are potential heavy-strand control regions (O(H)) for regulating replication and/or transcription and that (ii) multiple and potentially bidirectional light-strand origins of replication (O(L)) are present in unionoid F and M mitochondrial genomes. We propose that unassigned regions are the most promising candidate sequences in which to find regulatory and/or gender-specific sequences that could determine whether a mitochondrial genome will be maternally or paternally transmitted.

Figure 1.—

Gene maps of the gender-associated mitochondrial genomes of unionoid mussels. Gene identities: nd1–6 and nd4l, NADH dehydrogenase subunits 1–6 and 4L (complex I in green); cytb, cytochrome b (complex III in light blue); cox1-3, cytochrome c oxidase subunits I–III (complex IV in blue); atp6 and atp8, ATP synthase subunits 6 and 8 (complex V in light purple); 12SrRNA and 16SrRNA, small and large subunits of ribosomal RNA (in purple). Transfer RNA genes (in gray) are depicted by one-letter amino acid codes; L1, L2, S1, and S2 are differentiated by their anticodon sequences CUA, UAA, AGA and UCA, respectively. F ORF, F-specific open reading frame (orange); M ORF, M-specific open reading frame (red). Genes positioned inside the white circle are encoded on the light strand and genes outside the circle are encoded on the heavy strand. Arrows A, B, and C indicate shared unassigned regions >20 bp between F and M unionoid genomes [i.e., A, nd5–trnQ (this region contains trnH in M genomes); B, trnF–nd5; and C, between nd3–trnA (this region contains trnH in P. grandis, Q. quadrula, and V. ellipsiformis F genomes)].

Figure 2.—

Amino acid sequences of the potential peptides encoded by gender-specific open reading frames (F and M ORFs). The amino acids that constitute the putative transmembrane helix are indicated in boldface type and bigger characters. Positively charged amino acids are in red. The synthesized antigenic peptides used to generate antibodies for the Western blots are underlined.

Figure 3.—

Expression of the F and M ORFs in female and male gonads, respectively, from V. ellipsiformis. Western blots are revealed with anti-F ORF and anti-M ORF antibodies. The origins of the tissue samples for each lane are indicated. The positions of marker proteins (kDa) are shown.

Figure 4.—

AT-skew values in 12 mitochondrial genes (excluding atp8) of unionoid mussels and the deduced locations of the putative O_H and O_L in each genome. Because they share a similar pattern, V. ellipsiformis and Q. quadrula have been analyzed together. The big black arrow indicates the unassigned region located between “F nd5–trnQ” and “M nd5–trnQ+H” and the potential direction of DNA synthesis, whereas the small black arrow indicates the unassigned region located between “M nd3–trnA” and “F trnH–trnA” and the potential direction of DNA synthesis. (A) Inversidens japanensis, (B) Quadrula quadrula and Venustaconcha ellipsiformis, and (C) Pyganodon grandis.