Evidence for extreme sequence divergence between the male‐ and female‐transmitted mitochondrial genomes in the bivalve mollusc,Modiolus modiolus (Mytilidae)

Marine mussels of the family Mytilidae, as well as a number of other bivalves, have a unique system of mitochondrial DNA inheritance called doubly uniparental inheritance (DUI). DUI is characterized by the presence of an ‘F’ mitochondrial genome that is transmitted through mothers to daughters and sons, and an ‘M’ mitochondrial genome that is transmitted only from fathers to sons. In this paper, we demonstrate that DUI exists in the horse mussel, Modiolus modiolus (Linnaeus, 1758) and compare the pattern of molecular evolution of the M and F types in this species. Total DNA was isolated from M. modiolus male and female gonad tissues, as well as from spawned sperm cells. From these DNA samples, partial mitochondrial DNA fragments were amplified from both cytochrome c oxidase subunit I (cox1), and 16S ribosomal RNA (rrnL) genes. Based on cox1 and rrnL sequences, heteroplasmy was observed in M. modiolus and characterized by the resolution of two mitotypes: an F mitotype present in tissues of both males and females, and an M mitotype present in spawned sperm. Using standardized p‐distance and Tamura‐Nei values, M. modiolus is found to display the highest M/F conspecific sequence divergence for any member of the family Mytilidae (i.e. 38% M/F sequence divergence, which is 9% higher than any other intraspecific M/F comparison for the family Mytilidae when standardized using p‐distances across all taxa observed). Sequence analysis also indicated that the M. modiolus M mitotype evolves significantly faster than its conspecific F type. The findings discussed herein broaden the range of mytilid species known to exhibit DUI and they also establish a new threshold for the genetic divergence of male mytilid mitochondrial genomes.     

High fidelity of mitochondrial genome transmission under the doubly uniparental mode of inheritance in freshwater mussels (Bivalvia: Unionoidea)

Abstract.— Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) has been demonstrated in both mytilid and unionid bivalves. Under DUI, females pass on their mtDNA to both sons and daughters, whereas males pass on their mtDNA to only sons. In mytilids, the loss of an original male (or M) mitotype, with its subsequent replacement by that lineage's female (or F) mitotype, has been called a role-reversal or, more specifically, a masculinization event. Multiple masculinization events have been inferred during the evolutionary history of mytilids but not unionids. The perceived lack of role-reversal events in unionids may represent a significant difference in the evolutionary dynamics of DUI between the two bivalve taxa or simply a lack of sufficient taxon sampling in unionids. To evaluate these alternative hypotheses, six additional unionoidean bivalve genera were sampled for DUI including one genus from the sister taxon of the Unionidae, the Hyriidae. Phylogenetic analyses of 619 base pairs of cytochrome c oxidase I (COI) from eight genera (nine species) of unionoidean bivalves, plus the sister taxon to the Unionoida, Neotrigonia , revealed that the M and F unionoidean mitotypes were contained in gender-specific, topologically congruent clades. This supports the hypothesis that either role-reversal events do not occur in unionoideans or, if they do occur, their products are ephemeral in an evolutionary sense. Furthermore, the fact that the mantle-tissue-derived Neotrigonia mitotype is the sister mitotype to the unionoidean F mitotype clade suggests that DUI has been operating with high fidelity in unionoids for at least 200 million years. A relatively low incidence of interspecific hybridization in unionoideans and a possibly obligate role for the M mitotype in unionoidean gender determination are offered as potential explanations for the disparate evolutionary dynamics of DUI observed between mytilid and unionoidean bivalves.     

MULTIPLE ORIGINS OF GENDER-ASSOCIATED MITOCHONDRIAL DNA LINEAGES IN BIVALVES (MOLLUSCA: BIVALVIA)

Previous studies have shown that marine mussels (genus Mytilus) and a freshwater mussel (Pyganodon grandis) contain two distinct gender-associated mitotypes, which is a characteristic feature of the phenomenon of doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA). Here we present evidence for the presence of distinct male (M) and female (F) mitotypes in three other bivalve species, the mytilid Geukensia demissa, and the unionid species P. fragilis and Fusconaia flava. Nucleotide sequences of a segment of the COI gene from the M and F mitotypes from each of the three mytilid species (M. edulis, M. trossulus, G. demissa) and three unionid species (P. grandis, P. fragilis, F. flava) were used for phylogenetic analysis. The analysis suggests three independent origins of M and F mitotypes for the six species examined; one for the three unionid species, one for the two Mytilus species, and one for Geukensia. The first of these F/M divergence events, while of uncertain age, predates the divergence of the two unionid genera and is likely older than either of the two F/M divergence events in the mytilid taxa. The most parsimonious explanation of multiple F/M divergence events is that they represent independent origins of DUI. Another possibility is that, in a given taxon, an F or M mitotype assumes the role of the opposite mitotype (by virtue of a mechanism that remains to be clarified) and subsequently was fixed within its new gender. The fixation of a mtDNA lineage derived from a mitotype of switched function would reset the divergence of the gender-associated lineages to zero, thereby mimicking a de novo split of F and M lineages from a preexisting mtDNA genome that was not gender specific. Further broad-scale taxonomic studies of the occurrence of distinct M and F mitotypes may allow for the evaluation of the latter hypothesis.     

Sex-specific effects of hybridization on reproductive fitness in Mytilus

Blue mussels of the genus Mytilus form extensive hybrid zones in the North Atlantic and elsewhere where the distributions of different species overlap. Mytilus species transmit both maternal and paternal mtDNA through egg and sperm, respectively, a process known as doubly uniparental inheritance (DUI), and some females produce offspring with extremely biased sex ratios. These two traits have been shown to be linked and maternally controlled, with sex determination involving nuclear–cytoplasmic interactions. Hybridization has been shown to disrupt DUI mitochondrial inheritance and sex ratio bias; however, the effect of hybridization on reproductive fitness has not previously been examined. We investigated this effect in M. edulis × M. trossulus crosses through histological examination of mature F1 progeny, and spawning of F1 hybrids to monitor survival of their progeny through to the D stage of larval development. For progeny produced from mothers with a strong bias toward female offspring (often 100%) in pure-bred crosses, there was a clear breakdown in female dominance of progeny and significantly more hermaphrodites in the hybrid crosses produced from sperm with the M-tr1 mitotype. We also found significant sex-specific differences among hybrid progeny, with females producing normal eggs while males and hermaphrodites evidenced impaired gonadal development with significantly greater numbers of Sertoli cells, phagocytic hemocytes, and degenerating germ cells, all associated with gonad resorption. Males from crosses where DUI was disrupted and where male progeny were homoplasmic for the female mtDNA were the most severely compromised. Allelic incongruity between maternal and paternal mitotypes in hybrid crosses was associated with significant disruption of male gonadal development.     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Disruption of doubly uniparental inheritance of mitochondrial DNA in hybrid mussels (Mytilus edulis x M. galloprovincialis)

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA inheritance termed doubly uniparental inheritance (DUI). Females are homoplasmic for the F mitotype which is inherited maternally, whereas males are heteroplasmic for this and the paternally inherited M mitotype. In areas where species distributions overlap a varying degree of hybridization occurs; yet genetic differences between allopatric populations are maintained. Observations from natural populations and previous laboratory experiments suggest that DUI may be disrupted by hybridization, giving rise to heteroplasmic females and homoplasmic males. We carried out controlled laboratory crosses between Mytilus edulis and M. galloprovincialis to produce pure species and hybrid larvae of known parentage. DNA markers were used to follow the fate of the F and M mitotypes through larval development. Disruption of the mechanism which determines whether the M mitotype is retained or eliminated occurred in an estimated 38% of M. edulis x M. galloprovincialis hybrid larvae, a level double that previously observed in adult mussels from a natural M. edulis x M. galloprovincialis hybrid population. Furthermore, reciprocal hybrid crosses exhibited contrasting types of DUI disruption. The results indicate that disruption of DUI in hybrid mussels may be associated with increased mortality and hence could be a factor in the maintenance of genetic integrity for each species.     

Mitochondrial DNA polymorphisms and sperm motility in Mytilus edulis (Bivalvia: Mytilidae)

The system of mitochondrial DNA (mtDNA) inheritance in Mytilus and other bivalves, termed doubly uniparental inheritance (DUI), is novel among animals. Males pass on their male transmitted (M-type) mtDNA from fathers to their sons whereas females pass on their female transmitted (F-type) mtDNA from mothers to both sons and daughters. Thus, Mytilus males contain two distinct types of mtDNA. Interestingly, sperm contains only the paternal mtDNA. Phylogenetic analysis has shown that some female types have been able to switch their route of inheritance. These "recently masculinized" mitochondrial genomes behave as a typical M-type in that they are transmitted from generation to generation through sperm. Because the "recently masculinized" and "standard" male mitotypes in M. edulis exhibit approximately 8.7% amino acid sequence divergence, we hypothesized that these differences could affect mitochondrial, and hence sperm, functions. Furthermore, since recently masculinized mitotypes have been shown to replace standard male types periodically over evolutionary timescales, we tested the hypothesis that sperm swimming speeds would be greater for males with recently masculinized M-type genomes. Sperm activity was videotaped, digitized and tracked. A linear mixed effects model found no significant difference in linear velocities or curvilinear speeds between the mitotypes suggesting that swimming speeds are similar for both in the period shortly after spawning.     

Mitochondrial phylogenomics of the Bivalvia (Mollusca): searching for the origin and mitogenomic correlates of doubly uniparental inheritance of mtDNA

Background  

Doubly uniparental inheritance (DUI) is an atypical system of animal mtDNA inheritance found only in some bivalves. Under DUI, maternally (F genome) and paternally (M genome) transmitted mtDNAs yield two distinct gender-associated mtDNA lineages. The oldest distinct M and F genomes are found in freshwater mussels (order Unionoida). Comparative analyses of unionoid mitochondrial genomes and a robust phylogenetic framework are necessary to elucidate the origin, function and molecular evolutionary consequences of DUI. Herein, F and M genomes from three unionoid species, Venustaconcha ellipsiformis, Pyganodon grandis and Quadrula quadrula have been sequenced. Comparative genomic analyses were carried out on these six genomes along with two F and one M unionoid genomes from GenBank (F and M genomes of Inversidens japanensis and F genome of Lampsilis ornata).     

Identification of mitochondrial genome rearrangements unique to novel cytoplasmic male sterility in radish (Raphanus sativus L.)

A novel cytoplasmic male-sterility (CMS) radish (Raphanus sativus L.) and its associated mitotype (DCGMS) were previously identified; however, no mtDNA fragments flanking the atp6 gene were found in the DCGMS mitotype. Unlike three other mitotypes in this study, a unique mtDNA organization, atp6–nad3–rps12, was found to be the major mtDNA structure associated with this mitotype. This organization may have arisen from short repeat sequence-mediated recombination events. The short repeat clusters involved in the mtDNA rearrangement around the atp6 gene also exist as repetitive sequences in the complete mitochondrial genomes of other members of the Brassicaceae family, including rapeseed and Arabidopsis. These sequences do not exist as repetitive elements in the mtDNA of tobacco, sugar beet, or rice. While studying the regions flanking atp6, we identified a truncated atp6 mtDNA fragment which consists of the 5′ part of the atp6 gene linked to an unidentified sequence. This mtDNA structure was present in all mitotypes; however, a single nucleotide insertion mutation leading to a frame-shift was identified only in the DCGMS mitotype. Although this truncated atp6 organization was transcribed, there was no significantly different expression between male-sterile and fertile segregating individuals from the BC₁F₁ population originating from a cross between male-sterile and restorer parents. Comprehensive survey of the single base-pair insertion showed that it was maternally inherited and unique to the DCGMS mitotype. Therefore, this single nucleotide polymorphism (SNP) in the coding sequence of the mtDNA will be a useful molecular marker for the detection of the DCGMS mitotype. Young-Pyo Lee and Sunggil Kim equally contributed to this work.     

Early replication dynamics of sex-linked mitochondrial DNAs in the doubly uniparental inheritance species Ruditapes philippinarum (Bivalvia Veneridae)

Mitochondrial homoplasmy, which is maintained by strictly maternal inheritance and a series of bottlenecks, is thought to be an adaptive condition for metazoans. Doubly uniparental inheritance (DUI) is a unique mode of mitochondrial transmission found in bivalve species, in which two distinct mitochondrial genome (mtDNA) lines are present, one inherited through eggs (F) and one through sperm (M). During development, the two lines segregate in a sex- and tissue-specific manner: females lose M during embryogenesis, whereas males actively segregate it in the germ line. These two pivotal events are still poorly characterized. Here we investigated mtDNA replication dynamics during embryogenesis and pre-adulthood of the venerid Ruditapes philippinarum using real-time quantitative PCR. We found that both mtDNAs do not detectably replicate during early embryogenesis, and that the M line might be lost from females around 24 h of age. A rise in mtDNA copy number was observed before the first reproductive season in both sexes, with the M mitochondrial genome replicating more than the F in males, and we associate these boosts to the early phase of gonad production. As evidence indicates that DUI relies on the same molecular machine of mitochondrial maternal inheritance that is common in most animals, our data are relevant not only to DUI but also to shed light on how differential segregations of mtDNA variants, in the same nuclear background, may be controlled during development.     

Tissue-specific expression of male-transmitted mitochondrial DNA and its implications for rates of molecular evolution in Mytilus mussels (Bivalvia: Mytilidae).

Mytilus and other bivalves exhibit an unusual system of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Specifically, males transmit the mtDNA they have received from their fathers to their sons. Females transmit their mother's mtDNA to both sons and daughters. Males are normally heteroplasmic and females are normally homoplasmic, but not exclusively. This system is associated with an unusual pattern of molecular evolution. Male-transmitted mtDNA (M type) evolves faster than female-transmitted (F type) mtDNA. Relatively relaxed selection on the M type has been proposed as an explanation for this phenomenon. To further evaluate the selective forces acting upon the M-type genome, we used RT-PCR to determine where it is expressed. M-type mtDNA expression was detected in all gonad samples and in 50% of somatic tissues of males, and in a single female tissue. F-type mtDNA expression was detected in all female tissues, all male somatic tissues, and all but one male gonad sample. We argue that the expression of M-type mtDNA in male somatic and male gonad tissues has implications for the strength of selection acting upon it.     

Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm

A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.     

Presence of two mitochondrial genomes in the mytilid Perumytilus purpuratus: Phylogenetic evidence for doubly uniparental inheritance

This study presents evidence, using sequences of ribosomal 16S and COI mtDNA, for the presence of two mitochondrial genomes in Perumytilus purpuratus. This may be considered evidence of doubly uniparental mtDNA inheritance. The presence of the two types of mitochondrial genomes differentiates females from males. The F genome was found in the somatic and gonadal tissues of females and in the somatic tissues of males; the M genome was found in the gonads and mantle of males only. For the mitochondrial 16S region, ten haplotypes were found for the F genome (nucleotide diversity 0.004), and 7 haplotypes for the M genome (nucleotide diversity 0.001), with a distance Dxy of 0.125 and divergence Kxy of 60.33%. For the COI gene 17 haplotypes were found for the F genome (nucleotide diversity 0.009), and 10 haplotypes for the M genome (nucleotide diversity 0.010), with a genetic distance Dxy of 0.184 and divergence Kxy of 99.97%. Our results report the presence of two well-differentiated, sex-specific types of mitochondrial genome (one present in the male gonad, the other in the female gonad), implying the presence of DUI in P. purpuratus. These results indicate that care must be taken in phylogenetic comparisons using mtDNA sequences of P. purpuratus without considering the sex of the individuals.     

Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.)

Marine mussels of the genus Mytilus have an unusual mode of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Female mussels are homoplasmic for the F mitotype, which is inherited maternally, while males are usually heteroplasmic, carrying a mixture of the maternal F mitotype and the paternally inherited M genome. Two classes of M genomes have been observed: "standard" M genomes and "recently masculinized" M genomes. The latter are more similar to F genomes at the sequence level but are transmitted paternally like standard M genomes. In this study we report the complete sequences of two standard male M. edulis and one recently masculinized male M. trossulus mitochondrial genome. A comparative analysis, including the previously sequenced M. edulis F and M. galloprovincialis F and M mtDNAs, reveals that these genomes are identical in gene order, but highly divergent in nucleotide and amino acid sequence. The large amount (>20%) of nucleotide substitutions that fall in coding regions implies that there are several amino acid replacements between the F and M genomes, which likely have an impact on the structural and functional properties of the mitochondrial proteome. Correlation of the divergence rate of different protein-coding genes indicates that mtDNA-encoded proteins of the M genome are still under selective constraints, although less highly than genes of the F genome. The mosaic F/M control region of the masculinized F genome provides evidence for lineage-specific sequences that may be responsible for the different mode of transmission genetics. This analysis shows the value of comparative genomics to better understand the mechanisms of maintenance and segregation of mtDNA sequence variants in mytilid mussels.     

An unusual case of gender-associated mitochondrial DNA heteroplasmy: the mytilid <Emphasis Type="Italic">Musculista senhousia</Emphasis> (Mollusca Bivalvia)

Background

Doubly Uniparental Inheritance (DUI) represents the most outstanding exception to matrilinear inheritance of mitochondrial DNA (mtDNA), typical of Metazoa. In a few bivalve mollusks, two sex-linked mtDNAs (the so-called M and F) are inherited in a peculiar way: both daughters and sons receive their F from the mother, whereas sons inherit M from the father (males do not transmit F to their progeny). This realizes a double mechanism of transmission, in which M and F mtDNAs are inherited uniparentally.DUI systems represent a unique experimental model for testing the evolutionary mechanisms that apply to mitochondrial genomes and their transmission patterns as well as to mtDNA recombination.

Results

A new case of DUI is described in Musculista senhousia (Mollusca: Bivalvia: Mytilidae). Its heteroplasmy pattern is in line with standard DUI. Sequence variability analysis evidenced two main results: F haplotypes sequence variability is higher than that of M haplotypes, and F mitochondrial haplotypes experience a higher mutation rate in males' somatic tissues than in females' ones. Phylogenetic analysis revealed also that M. senhousia M and F haplotypes cluster separately from that of the other mytilids.

Conclusion

Sequence variability analysis evidenced some unexpected traits. The inverted variability pattern (the F being more variable than M) was new and it challenges most of the rationales proposed to account for sex-linked mtDNA evolution. We tentatively related this to the history of the Northern Adriatic populations analyzed. Moreover, F sequences evidenced a higher mutation level in male's soma, this variability being produced de novo each generation. This suggests that mechanisms evolved to protect mtDNA in females (f.i. antioxidant gene complexes) might be under relaxed selection in males. Phylogenetic analysis of sex-linked haplotypes confirmed that they have switched their roles during the evolutionary history of mytilids, at variance to what has been observed in unionids. Consequently, reciprocal monophyly of M and F lineages got easily lost because of role-reversals and consequent losses of M lineages, as already observed in Mytilus.

     

Evolution of a Unique Mitotype-Specific Protein-Coding Extension of the Cytochrome c Oxidase II Gene in Freshwater Mussels (Bivalvia: Unionoida)

A unique mode of mitochondrial DNA inheritance, designated doubly-uniparental inheritance (DUI), occurs in three bivalve subclasses (Pteriomorpha: Mytiloida, Palaeoheterodonta: Unionoida, Heterodonta: Veneroida), indicating that DUI may be a widespread phenomenon among bivalves. In mytiloids, breakdown of this pattern of inheritance (gender-switching) is observed in natural populations and in a phylogenetic context. In contrast, gender-switching has not occurred during the evolutionary history of unionoids. Here we present sequences for the male (M) and female (F) mitotypes from an additional 8 species of Unionoida. Consistent with previous observations, the M and F mitotypes of all species form reciprocally monophyletic clades supporting the hypothesis of taxon-specific rates of gender-switching. Coinciding with the absence of gender-switching is an ≈185 codon extension of the cytochrome c oxidase II (MTCO2) locus in the male genome. The extension is present in all 12 unionoid species examined, including a representative of the family Margaritiferidae, indicating that this protein-coding polymorphism originated ≥ 200 MYBP. Although the extension is well conserved in length among 11 of the 12 species, one taxon has a significantly shortened extension. Lastly, examination of the rates and patterns of substitution indicate that the extension is evolving under relaxed purging selection, a pattern inconsistent with the conserved nature of MTCO2 or any cytochrome c oxidase locus.[Reviewing Editor: Dr. J. William Ballard]     

Nonhomologous Recombination Between the Large Unassigned Region of the Male and Female Mitochondrial Genomes in the Mussel, Mytilus trossulus

Doubly uniparental inheritance of mtDNA (DUI) is commonly observed in several genera of bivalves. Under DUI, female offspring inherit mtDNA from their mothers, while male offspring inherit mtDNA from both parents but preferentially transmit the paternally inherited mtDNA to their sons. Several studies have shown that the female- and male-specific mtDNA lineages in blue mussels, Mytilus spp., vary by upward of 20% at the nucleotide level. In addition to high levels of nucleotide substitution, the present study observed substantial gender-based length polymorphism in the presumptive mitochondrial control region (=large unassigned region; LUR) of North American M. trossulus. In this species, female lineage LUR haplotypes are over 2 kb larger than male lineage LUR haplotypes. Analysis of sequence data for these length variants indicates that the F LUR haplotypes of North American M. trossulus contain sequences similar to the F lineage control region in the congeners M. edulis and M. galloprovincialis. Relative to the F LUR in the latter two species, however, the F lineage LUR haplotypes in M. trossulus contain two large sequence insertions, each nearly 1 kb in size. One of these insertions has high sequence similarity to the male lineage LUR of M. trossulus. The tandem arrangement of F and M control region sequences in the F lineage LUR of M. trossulus is most likely the result of nonhomologous recombination between the male and the female mitochondrial genomes in M. trossulus, a finding that has important implications regarding the transmission and evolution of blue mussel mitochondrial genomes. [Reviewing Editor: Dr. Martin Kreitman]     

Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae)

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16S RNA gene that indicates the DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, M. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as "masculinization" of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.      

Mitochondrial DNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the doubly uniparental inheritance of mitochondrial DNA system in the blue mussel Mytilus galloprovincialis

Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real‐time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male‐biased crosses, but decreased significantly in female‐biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.     

Doubly uniparental inheritance of mitochondrial DNA in freshwater mussels: History and status of the European species

Doubly uniparental inheritance (DUI) is a mode of inheriting mitochondrial DNA that is distinct from strictly maternal inheritance. It has been described in nine and three families of marine and freshwater mussels, respectively, including the European margaritiferids and unionids. Among the 16 freshwater species of Unionida inhabiting Europe, DUI has been described in 9 species of dioecious mussels and was absent from a single hermaphroditic species and from secondary hermaphroditic specimens. The DUI freshwater mussels include two vastly genetically different mitochondrial genomes: maternal (F genome) and paternal (M genome), which coexist within the same specimen but in different tissues. The F genome is present in all female tissues and somatic male tissues. It is inherited in the typical, maternal, manner. Conversely, the M genome is located primarily in the male gonads and generative cells, and is inherited paternally. Dioecious Unionidae display unique characteristics that have been interrelated for over 200 million years: a high fidelity of the transmission of the F and M genomes in DUI and two paths of spermatogenesis–the typical path that produces sperm cells containing mitochondria with the F genome and the atypical path that produces sperm cells with the M genome. The mitogenomes of freshwater mussels display unique features that are not present in any other animal, that is, an additional, gender-specific gene and an elongated cox2 gene occurring exclusively in the M genome. These features mean that the mitochondria, in addition to their basic function of producing energy, also may take part in determining sex in these dioecious organisms.