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.     

Inheritance of two M type mitochondrial DNA from sperm and unfertilized eggs to offspring in Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction-restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.     

Sperm mitochondrial DNA transmission to both male and female offspring in the blue mussel Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type-specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance.     

Sex-Limited Mitochondrial DNA Transmission in the Marine Mussel Mytilus Edulis

Mitochondrial DNA (mtDNA) was thought to be inherited maternally in animals, although paternal leakage has been reported in mice and Drosophila. Recently, direct evidence of extensive paternal inheritance of mtDNA has been found in the marine mussel Mytilus. We give evidence that whereas female mussels are homoplasmic for a genome that is transmitted to eggs, male mussels are heteroplasmic for this genome and for a second genome that is transmitted preferentially to sperm. The results provide support for the existence of separate male and female routes of mtDNA inheritance in mussels. The two genomes show a base sequence divergence exceeding 20% at three protein coding genes, consistent with long term maintenance of the heteroplasmic state. We propose that the two genomes differ in fitness in males and females, possibly as a result of interaction with nuclear genes.     

Male-Dependent Doubly Uniparental Inheritance of Mitochondrial DNA and Female-Dependent Sex-Ratio in the Mussel Mytilus Galloprovincialis

We have investigated sex ratio and mitochondrial DNA inheritance in pair-matings involving five female and five male individuals of the Mediterranean mussel Mytilus galloprovincialis. The percentage of male progeny varied widely among families and was found to be a characteristic of the female parent and independent of the male to which it was mated. Thus sex-ratio in Mytilus appears to be independent of the nuclear genotype of the sperm. With a few exceptions, doubly uniparental inheritance (DUI) of mtDNA was observed in all families fathered by four of the five males: female and male progeny contained the mother's mtDNA (the F genome), but males contained also the father's paternal mtDNA (the M genome). Two hermaphrodite individuals found among the progeny of these crosses contained the F mitochondrial genome in the female gonad and both the F and M genomes in the male gonad. All four families fathered by the fifth male showed the standard maternal inheritance (SMI) of animal mtDNA: both female and male progeny contained only the maternal mtDNA. These observations illustrate the intimate linkage between sex and mtDNA inheritance in species with DUI and suggest different major roles for each gender. We propose a model according to which development of a male gonad requires the presence in the early germ cells of an agent associated with sperm-derived mitochondria, these mitochondria are endowed with a paternally encoded replicative advantage through which they overcome their original minority in the fertilized egg and this advantage (and, therefore, the chance of an early entrance into the germ line) is countered by a maternally encoded egg factor.     

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.     

Doubly uniparental inheritance of mitochondrial DNA: Might it be simpler than we thought?

More than 100 species of bivalve mollusks are currently known to carry two highly diverged mitochondrial DNA (mtDNA) molecules, one of which is transmitted through the egg and the other through the sperm generation after generation, faithfully and uninterruptedly. This mtDNA system, which has become known as doubly uniparental inheritance (DUI), is most likely unique in eukaryotes and constitutes a striking deviation from the strictly maternal inheritance (SMI) of mtDNA that is the rule in the animal kingdom. Here, I present a model of how the paternal mtDNA may escape the mitochondrial destruction that occurs prior to sperm formation and enter the male germ line in the newly formed embryo. In essence, the model treats the sperm-transmitted mtDNA as a molecule that takes a ride with the sperm. The model can be easily tested and, if passed the tests, may open the way for the understanding of DUI at the molecular level and throw light on the mechanisms and evolution of mtDNA transmission in general. In addition, the model shifts attention from nuclear control of paternal mtDNA inheritance, whether systematic (as DUI) or leaky (as the cases reported in a wide variety of animal species), to the mtDNA itself as the protagonist of its own transmission. This possibility has been, so far, ignored in studies of paternal mtDNA transmission in other species including humans.     

Differential segregation patterns of sperm mitochondria in embryos of the blue mussel (Mytilus edulis)

In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.     

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.     

Female‐dependent transmission of paternal mtDNA is a shared feature of bivalve species with doubly uniparental inheritance (DUI) of mitochondrial DNA

Several species from a number of bivalve molluscan families are known to have a paternally transmitted mitochondrial genome, along with the standard maternally transmitted one. The main characteristic of the phenomenon, known as doubly uniparental inheritance (DUI), is the coupling of sex and mtDNA inheritance: males receive both genomes but transmit only the paternal to their progeny; females either do not have the paternal genome or, if they do, they do not transmit it to their progeny. In the families Mytilidae and Veneridae, both of which have DUI, a female individual is either female‐biased (it produces only, or nearly so, female progeny), male‐biased (it produces mainly male progeny) or non‐biased (it produces both genders in intermediate frequencies). Here we present evidence for a same pattern in the freshwater mussel, Unio delphinus (Unionidae). These results suggest that the maternal control of whether a fertilized egg will develop into a male or a female individual (and the associated feature of whether it will inherited or not inherit the paternal mtDNA) is a general characteristic of species with DUI.     

Sperm motility in Mytilus edulis in relation to mitochondrial DNA polymorphisms: implications for the evolution of doubly uniparental inheritance in bivalves

Bivalves of the families Mytilidae, Unionidae, and Veneridae have an unusual mode of mitochondrial DNA (mtDNA) transmission called doubly uniparental inheritance (DUI). A characteristic feature of DUI is the presence of two gender-associated mtDNA genomes that are transmitted through males (M-type mtDNA) and females (F-type mtDNA), respectively. Female mussels are predominantly homoplasmic with only the F-type expressed in both somatic and gonadal tissue; males are heteroplasmic with the M-type expressed in the gonad and F-type in somatic tissue for the most part. An unusual evolutionary feature of this system is that an mt genome with F-coding sequences occasionally invades the male route of inheritance (i.e., a "role reversal" event), and is thereafter transmitted as a new M-type. Phylogenetic studies have demonstrated that the new or "recently masculinized" M-types may eventually replace the older or "standard" M-types over time. To investigate whether this replacement process could be due to an advantage in sperm swimming behavior, we measured differences in motility parameters and found that sperm with the recently masculinized M-type had significantly faster curvilinear velocity and average path velocity when compared to sperm with standard M-type. This increase in sperm swimming speed could explain the multiple evolutionary replacements of standard M-types by masculinized M-types that have been hypothesized for the mytilid lineage. However, our observations do not support the hypothesis that DUI originated because it permits the evolution of mitochondrial adaptations specific to sperm performance, otherwise, the evolutionarily older, standard M genome should perform better.     

Maternal inheritance of mitochondrial DNA (mtDNA) in the Pacific oyster (Crassostrea gigas): a preliminary study using mtDNA sequence analysis with evidence of random distribution of MitoTracker-stained sperm mitochondria in fertilized eggs

In many bivalve species, paternal and maternal mitochondrial DNA (mtDNA) from sperm and eggs is transmitted to the offspring. This phenomenon is known as doubly uniparental inheritance (DUI). In these species, sperm mtDNA (M type) is inherited by the male gonad of the offspring. Egg mtDNA (F type) is inherited by both male and female somatic cells and female gonadal cells. In Mytilidae, sperm mitochondria are distributed in the cytoplasm of differentiating male germ cells because they are transmitted to the male gonad. In the present study, we investigated maternal inheritance of mtDNA in the Pacific oyster, Crassostrea gigas. Sequence analysis of two mitochondrial non-coding regions revealed an identical sequence pattern in the gametes and adductor muscle samples taken from six males and five females. To observe whether sperm mitochondria were specifically located in the cytoplasm of differentiating germ cells, their distribution was recorded in C. gigas fertilized eggs by vital staining with MitoTracker Green. Although the 1D blastomere was identified in the cytoplasm of differentiating germ cells, sperm mitochondria were located at the 1D blastomere in only 32% of eggs during the 8-cell stage. Thus, in C. gigas, sperm mitochondria do not specifically locate in the germ cell region at the 1D blastomere. We suggest that the distribution of sperm mitochondria is not associated with germ cell formation in C. gigas. Furthermore, as evidenced by the mtDNA sequences of two non-coding regions, we conclude that mitochondrial DNA is maternally inherited in this species.     

Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex

A novel form of mitochondrial DNA (mtDNA) inheritance has previously been documented for the blue mussel (Mytilus edulis). Female mussels inherit their mtDNA solely from their mother while males inherit mtDNA from both their mother and their father. In males, the paternal mtDNA is preferentially amplified so that the male gonad is highly enriched for the paternal mtDNA that is then transmitted from fathers to sons. We demonstrate that this mode of mtDNA inheritance also operates in the closely related species M. galloprovincialis and M. trossulus. The evolutionary relationship between the male and female mtDNA lineages is estimated by phylogenetic analysis of 455 nucleotides from the large subunit ribosomal RNA gene. We have found that the male and female lineages are highly divergent; the divergence of these lineages began prior to the speciation of the three species of blue mussels. Further, the separation between the male and female lineages is estimated to have occurred between 5.3 and 5.7 MYA.      

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.      

The Effects of Natural Hybridization on the Regulation of Doubly Uniparental Mtdna Inheritance in Blue Mussels (Mytilus Spp.)

Blue mussels in the Mytilus edulis species complex have a doubly uniparental mode of mtDNA inheritance with separate maternal and paternal mtDNA lineages. Female mussels inherit their mtDNA solely from their mother, while males inherit mtDNA from both parents. In the male gonad the paternal mtDNA is preferentially replicated so that only paternal mtDNA is transmitted from fathers to sons. Hybridization is common among differentiated blue mussel taxa; whenever it involves M. trossulus, doubly uniparental mtDNA inheritance is disrupted. We have found high frequencies of males without and females with paternal mtDNA among hybrid mussels produced by interspecific matings between M. galloprovincialis and M. trossulus. In contrast, hybridization between M. galloprovincialis and M. edulis does not affect doubly uniparental inheritance, indicating a difference in the divergence of the mechanisms regulating mtDNA inheritance among the three blue mussel taxa. Our data indicate a high frequency of disrupted mtDNA transmission in F(1) hybrids and suggest that two separate mechanisms, one regulating the transmission of paternal mtDNA to males and another inhibiting the establishment of paternal mtDNA in females, act to regulate doubly uniparental inheritance. We propose a model for the regulation of doubly uniparental inheritance that is consistent with these observations.     

No evidence for presence of maternal mitochondrial DNA in the sperm of Mytilus galloprovincialis males

Species of the mussel family Mytilidae have a special mitochondrial DNA (mtDNA) transmission system, known as doubly uniparental inheritance (DUI), which consists of a maternally inherited (F) and a paternally inherited (M) mitochondrial genome. Females are normally homoplasmic for the F genome and males are heteroplasmic mosaics, with their somatic tissues dominated by the maternal and their gonads dominated by the paternal genome. Several studies have indicated that the maternal genome may often be present in the male germ line. Here we report the results from the examination of mtDNA in pure sperm from more than 30 males of Mytilus galloprovincialis. In all cases, except one, we detected only the M genome. In the sperm of one male, we detected a paternal genome with an F-like primary sequence that was different from the sequence of the maternal genome in the animal's somatic tissues. We conclude that the male germ line is protected against invasion by the maternal genome. This is important because fidelity of gamete-specific transmission of the two mitochondrial genomes is a basic requirement for the stability of DUI.     

Hybrid male sterility is caused by mitochondrial DNA deletion

Although it is known that the hybrid male mouse is sterile just like any other animal’s heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.     

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.     

Strict sex-specific mtDNA segregation in the germ line of the DUI species Venerupis philippinarum (Bivalvia: Veneridae)

Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of standard maternal inheritance of metazoan mitochondria. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-type) and the other through sperm (M-type). In this paper, we report results from a multiplex real-time quantitative polymerase chain reaction analysis on the Manila clam Venerupis philippinarum (formerly Tapes philippinarum). We quantified M- and F-types in somatic tissues, gonads, and gametes. Nuclear and external reference sequences were used, and the whole experimental process was designed to avoid any possible cross-contamination. In most male somatic tissues, the M-type is largely predominant: This suggests that the processes separating sex-linked mitochondrial DNAs (mtDNAs) in somatic tissues are less precise than in other DUI species. In the germ line, we evidenced a strict sex-specific mtDNA segregation because both sperm and eggs do carry exclusively M- and F-types, respectively, an observation that is in contrast with a previous analysis on Mytilus galloprovincialis. More precisely, whereas two mtDNAs are present in the whole gonad, only the sex-specific one is detected in gametes. Because of this, we propose that the mtDNA transmission is achieved through a three-checkpoint process in V. philippinarum. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the embryo development (here named Checkpoint #1 and Checkpoint #2) are already well known for DUI species; a Checkpoint #3 would act when primordial germ cells (PGCs) are first formed and would work in both males and females. We believe that Checkpoint #3 is a mere variation of the "mitochondrial bottleneck" in species with standard maternal inheritance, established when their PGCs separate during embryo cleavage.