Doubly Uniparental Inheritance of mtDNA: An Unappreciated Defiance of a General Rule.

We recount the basic observations about doubly uniparental inheritance (DUI) of mtDNA in bivalvian mollusks with an emphasis on those that were obtained from work in Mytilus and appeared after the review by Zouros (Evol Biol 40:1-31, 2013). Using this information, we present a new model about DUI that is a revised version of previously suggested models. The model can be summarized as follows. A Mytilus female either provides its eggs with the "masculinizing" factor S and the "sperm mitochondria binding" factor Z, or it does not. This property of the female is determined by two nuclear genes, S and Z, that are always in the on/on or the off/off phase. In fertilized eggs without factors S and Z the embryo develops into a female and the sperm mitochondria are randomly dispersed among cells following development. In fertilized eggs with factors S and Z, the first factor causes the cell to become eventually sperm and the second causes the sperm mitochondria to aggregate and anchor to the nuclear membrane by binding to a specific motif of the sperm-derived mtDNA. Factors S and Z are continuously co-synthesized and co-localized in the cell line from the egg to the sperm. The sperm mitochondria of the aggregate escape the mechanism that eliminates the cell's mitochondria before the formation of the sperm. The rescued mitochondria are subsequently packed into five mega-mitochondria in the sperm and are delivered in the egg.

Proteomic analysis of eggs from Mytilus edulis females differing in mitochondrial DNA transmission mode.

Many bivalves have an unusual mechanism of mitochondrial DNA (mtDNA) inheritance called doubly uniparental inheritance (DUI) in which distinctly different genomes are inherited through the female (F genome) and male (M genome) lineages. In fertilized eggs that will develop into male embryos, the sperm mitochondria remain in an aggregation, which is believed to be delivered to the primordial germ cells and passed to the next generation through the sperm. In fertilized eggs that will develop into female embryos, the sperm mitochondria are dispersed throughout the developing embryo and make little if any contribution to the next generation. The frequency of embryos with the aggregated or dispersed mitochondrial type varies among females. Previous models of DUI have predicted that maternal nuclear factors cause molecular differences among unfertilized eggs from females producing embryos with predominantly dispersed or aggregated mitochondria. We test this hypothesis using females of each of the two types from a natural population. We have found small, yet detectable, differences of the predicted type at the proteome level. We also provide evidence that eggs of females giving the dispersed pattern have consistently lower expression for different proteasome subunits than eggs of females giving the aggregated pattern. These results, combined with those of an earlier study in which we used hatchery lines of Mytilus, and with a transcriptomic study in a clam that has the DUI system of mtDNA transmission, reinforce the hypothesis that the ubiquitin-proteasome system plays a key role in the mechanism of DUI and sex determination in bivalves. We also report that eggs of females giving the dispersed pattern have higher expression for arginine kinase and enolase, enzymes involved in energy production, whereas ferritin, which is involved in iron homeostasis, has lower expression. We discuss these results in the context of genetic models for DUI and suggest experimental methods for further understanding the role of these proteins in DUI.

Homologous recombination between highly diverged mitochondrial sequences: examples from maternally and paternally transmitted genomes.

Homologous recombination is restricted to sequences of low divergence. This is attributed to the mismatch repairing system (MMR), which does not allow recombination between sequences that are highly divergent. This acts as a safeguard against recombination between nonhomologous sequences that could result in genome imbalance. Here, we report recombination between maternal and paternal mitochondrial genomes of the sea mussel, whose sequences differ by >20%. We propose that the strict maternal inheritance of the animal mitochondrial DNA and the ensuing homoplasmy has relieved the MMR system of the animal mitochondrion from the pressure to tolerate recombination only among sequences with a high degree of similarity.

Genetic variation underlying protein expression in eggs of the marine mussel Mytilus edulis.

Study of the genetic basis of gene expression variation is central to attempts to understand the causes of evolutionary change. Although there are many transcriptomics studies estimating genetic variance and heritability in model organisms such as humans there is a lack of equivalent proteomics studies. In the present study, the heritability underlying egg protein expression was estimated in the marine mussel Mytilus. We believe this to be the first such measurement of genetic variation for gene expression in eggs of any organism. The study of eggs is important in evolutionary theory and life history analysis because maternal effects might have profound effects on the rate of evolution of offspring traits. Evidence is presented that the egg proteome varies significantly between individual females and that heritability of protein expression in mussel eggs is moderate to high suggesting abundant genetic variation on which natural selection might act. The study of the mussel egg proteome is also important because of the unusual system of mitochondrial DNA inheritance in mussels whereby different mitochondrial genomes are transmitted independently through female and male lineages (doubly uniparental inheritance). It is likely that the mechanism underlying this system involves the interaction of specific egg factors with sperm mitochondria following fertilization, and its elucidation might be advanced by study of the proteome in females having different progeny sex ratios. Putative identifications are presented here for egg proteins using MS/MS in Mytilus lines differing in sex ratio. Ontology terms relating to stress response and protein folding occur more frequently for proteins showing large expression differences between the lines. The distribution of ontology terms in mussel eggs was compared with those for previous mussel proteomics studies (using other tissues) and with mammal eggs. Significant differences were observed between mussel eggs and mussel tissues but not between the two types of eggs.

The control region of maternally and paternally inherited mitochondrial genomes of three species of the sea mussel genus Mytilus.

Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial genomes. In the interbreeding taxa Mytilus edulis and M. galloprovincialis, several genomes of both types have been fully sequenced. The genome consists of the coding part (which, in addition to protein and RNA genes, contains several small noncoding sequences) and the main control region (CR), which in turn consists of three distinct parts: the first variable (VD1), the conserved (CD), and the second variable (VD2) domain. The maternal and paternal genomes are very similar in gene content and organization, even though they differ by >20% in primary sequence. They differ even more at VD1 and VD2, yet they are remarkably similar at CD. The complete sequence of a genome from the closely related species M. trossulus was previously reported and found to consist of a maternal-like coding part and a paternal-like and a maternal-like CR. From this and from the fact that it was extracted from a male individual, it was inferred that this is a genome that switched from maternal to paternal transmission. Here we provide clear evidence that this genome is the maternal genome of M. trossulus. We have found that in this genome the tRNA(Gln) in the coding region is apparently defective and that an intact copy of this tRNA occurs in the CR, that one of the two conserved domains is missing essential motifs, and that one of the two first variable domains has a high rate of divergence. These features may explain the large size and mosaic structure of the CR of the maternal genome of M. trossulus. We have also obtained CR sequences of the maternal and paternal genomes of M. californianus, a more distantly related species. We compare the control regions from all three species, focusing on the divergence among genomes of different species origin and among genomes of different transmission routes.

Promoting evolution: the brand new Hellenic Evolutionary Society (HEVOS).

Herein we present the recently founded Hellenic Evolutionary Society (HEVOS) that has been recently instituted to promote evolution and scientific thinking among the Greek-speaking public. HEVOS is a timely initiative, given the low levels of acceptance of evolution by Greek society and the almost complete lack of evolution teaching in primary and secondary education in Greece. Herein, the main aims of the Society are presented.

Increasing genomic information in bivalves through new EST collections in four species: development of new genetic markers for environmental studies and genome evolution.

The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.

Doubly uniparental inheritance (DUI) of mitochondrial DNA in Donax trunculus (Bivalvia: Donacidae) and the problem of its sporadic detection in Bivalvia.

Mitochondrial DNA is transmitted maternally in metazoan species. This rule does not hold in several species of bivalves that have two mtDNA types, one that is transmitted maternally and the other paternally. This system of mitochondrial DNA transmission is known as doubly uniparental inheritance (DUI). Here we present evidence of DUI in the clam Donax trunculus making Donacidae the sixth bivalve family in which the phenomenon has been found. In addition, we present the taxonomic affiliation of all species in which DUI is currently known to occur and construct a phylogeny of the maternal and paternal genomes of these species. We use this information to address the question of a single or multiple origins of DUI and to discuss whether failed attempts to demonstrate the presence of DUI in several bivalve species might be due to problems of detection or to genuine absence of the phenomenon.

No evidence for absence of paternal mtDNA in male progeny from pair matings of the mussel Mytilus galloprovincialis.

The claim that a Mytilus galloprovincialis male failed to transmit mtDNA to its sons in controlled crosses is shown to be false. At present there is no evidence for mussel males lacking a paternal mtDNA. This makes unlikely the hypothesis that maternal genomes may become paternally transmitted by invading the germ line of males that lack a paternal genome.

A mitochondrial genome with a reversed transmission route in the Mediterranean mussel Mytilus galloprovincialis.

Species of the marine mussel genus Mytilus are known to contain two mitochondrial genomes, one transmitted maternally (the F genome) and the other paternally (the M genome). The two genomes have diverged by more than 20% in DNA sequence. Here we present the complete sequence of a third genome, genome C, which we found in the sperm of a Mytilus galloprovincialis male. The coding part of the new genome resembles in sequence the F genome, from which it differs by about 2% on average, but differs from the M genome by as much as the F from the M. Its major control region (CR) is more than three times larger than that of the F or the M genome and consists of repeated sequence domains of the CR of the M genome flanked by domains of the CR of the F genome. We present a sequence of events that reconstruct most parsimoniously the derivation of the C genome from the F and M genomes. The sequence consists of a duplication of CR elements of the M genome and subsequent insertion of these tandemly repeated elements in the F genome by recombination. The fact that the C genome was found as the only mitochondrial genome in the sperm of the male from which it was extracted suggests that it is transmitted paternally.

Multiple events are responsible for an insertion in a paternally inherited mitochondrial genome of the mussel Mytilus galloprovincialis.

In a sperm-transmitted mtDNA of Mytilus galloprovincialis we found an insertion that is not present in the typical genome and whose origin can be explained by a sequence of three events: a tandem duplication, a nonhomologous recombination, and a deletion. Unless such events are extremely rare in this species, the identical gene arrangement of the two gender-specific genomes should imply strong selection for same gene order and size.

Evolution and inheritance of animal mitochondrial DNA: rules and exceptions.

Mitochondrial DNA (mtDNA) has been studied intensely for "its own" merit. Its role for the function of the cell and the organism remains a fertile field, its origin and evolution is an indispensable part of the evolution of life and its interaction with the nuclear DNA is among the most important cases of genome synergism and co-evolution. Also, mtDNA was proven one of the most useful tools in population genetics and molecular phylogenetics. In this article we focus on animal mtDNA and discuss briefly how our views about its structure, function and transmission have changed, how these changes affect the information we have accumulated through its use in the fields of phylogeny and population structure and what are the most important questions that remain open for future research.

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.

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.

Evidence that the large noncoding sequence is the main control region of maternally and paternally transmitted mitochondrial genomes of the marine mussel (Mytilus spp.).

Both the maternal (F-type) and paternal (M-type) mitochondrial genomes of the Mytilus species complex M. edulis/galloprovincialis contain a noncoding sequence between the l-rRNA and the tRNA(Tyr) genes, here called the large unassigned region (LUR). The LUR, which is shorter in M genomes, is capable of forming secondary structures and contains motifs of significant sequence similarity with elements known to have specific functions in the sea urchin and the mammalian control region. Such features are not present in other noncoding regions of the F or M Mytilus mtDNA. The LUR can be divided on the basis of indels and nucleotide variation in three domains, which is reminiscent of the tripartite structure of the mammalian control region. These features suggest that the LUR is the main control region of the Mytilus mitochondrial genome. The middle domain has diverged by only 1.5% between F and M genomes, while the average divergence over the whole molecule is approximately 20%. In contrast, the first domain is among the most divergent parts of the genome. This suggests that different parts of the LUR are under different selection constraints that are also different from those acting on the coding parts of the molecule.

Genetics of mother-dependent sex ratio in blue mussels (Mytilus spp.) and implications for doubly uniparental inheritance of mitochondrial DNA.

Previous studies have shown that in most pair matings of Mytilus edulis, M. trossulus, and M. galloprovincialis there is a large sex-ratio bias in favor of either males or females. The degree of bias is a characteristic property of the female parent, as matings of the same female with different males produce the same sex ratio, but matings of the same male with different females produce different sex ratios. All three species possess the unusual feature of doubly uniparental inheritance of mitochondrial DNA (mtDNA); i.e., they contain two distinct types of mtDNA, one that is transmitted matrilinearly and one that is transmitted patrilinearly. This coupling of sex and mtDNA transmission raises the possibility that the mechanism of sex-ratio determination in mussels might be under the control of the mtDNA of the female parent. Here we present data from pedigreed crosses that confirm the previous observations that in mussel matings there is a strong sex-ratio bias and that the bias is under the control of the female parent. In addition, these data strongly suggest that this control is exercised by the mother's nuclear rather than mitochondrial genotype. Making use of these findings we develop a model of mother-dependent sex determination and use data from crosses involving wild females to test the model's predictions at the population level.

A protein binding site in the M mitochondrial genome of Mytilus galloprovincialis may be responsible for its paternal transmission.

Sea mussels (genus Mytilus) have two mitochondrial genomes in obligatory co-existence, one that is transmitted through the egg and the other through the sperm. The phenomenon, known as Doubly Uniparental Inheritance (DUI) of mitochondrial DNA (mtDNA), is presently known to occur in more than 40 molluscan bivalve species. Females and the somatic tissues of males contain mainly the maternal (F) genome. In contrast, the sperm contains only the paternal (M) genome. Through electrophoretic mobility shift assay (EMSA) experiments we have identified a sequence element in the control region (CR) of the M genome that acts as a binding site for the formation of a complex with a protein factor that occurs in the male gonad. An adenine tract upstream to the element is also essential for the formation of the complex. The reaction is highly specific. It does not occur with protein extracts from the female gonad or from a male or female somatic tissue. Further experiments showed that the interaction takes place in mitochondria surrounding the nucleus of the cells of male gonads, suggesting a distinct role of perinuclear mitochondria. We propose that at a certain point during spermatogenesis mitochondria are subject to degradation and that perinuclear mitochondria with the M mtDNA-protein complex are protected from this degradation with the result that mature spermatozoa contain only the paternal mitochondrial genome.

Extensive mitochondrial heteroplasmy in hybrid water frog (Pelophylax spp.) populations from Southeast Europe.

Water frogs of the genus Pelophylax (previous Rana) species have been much studied in Europe for their outstanding reproductive mechanism in which sympatric hybridization between genetically distinct parental species produces diverse genetic forms of viable hybrid animals. The most common hybrid is P. esculentus that carries the genomes of both parental species, P. ridibundus and P. lessonae, but usually transfers the whole genome of only one parent to its offsprings (hybridogenesis). The evolutionary cost of transfer of the intact genome and hence the hemiclonal reproduction is the depletion of heterozygosity in the hybrid populations. Pelophylax esculentus presents an excellent example of the long-term sustained hybridization and hemiclonal reproduction in which the effects of the low genetic diversity are balanced through the novel mutations and periodic recombinations. In this study, we analyzed the mitochondrial (mt) and microsatellites DNA variations in hybrid Pelophylax populations from southern parts of the Pannonian Basin and a north-south transect of the Balkan Peninsula, which are home for a variety of Pelophylax genetic lineages. The mtDNA haplotypes found in this study corresponded to P. ridibundus and P. epeiroticus of the Balkan - Anatolian lineage (ridibundus-bedriagae) and to P. lessonae and a divergent lessonae haplotype of the lessonae lineage. The mtDNA genomes showed considerable intraspecific variation and geographic differentiation. The Balkan wide distributed P. ridibundus was found in all studied populations and its nuclear genome, along with either the lessonae or the endemic epeiroticus genome, in all hybrids. An unexpected finding was that the hybrid populations were invariably heteroplasmic, that is, they contained the mtDNA of both parental species. We discussed the possibility that such extensive heteroplasmy is a result of hybridization and it comes from regular leakage of the paternal mtDNA from a sperm of one species that fertilizes eggs of another. In this case, the mechanisms that protect the egg from heterospecific fertilization and further from the presence of sperm mtDNA could become compromised due to their differences and divergence at both, mitochondrial and nuclear DNA. The heteroplasmy once retained in the fertilized egg could be transmitted by hybrid backcrossing to the progeny and maintained in a population over generations. The role of interspecies and heteroplasmic hybrid animals due to their genomic diversity and better fitness compare to the parental species might be of the special importance in adaptations to miscellaneous and isolated environments at the Balkan Peninsula.

The rRNA and tRNA transcripts of maternally and paternally inherited mitochondrial DNAs of Mytilus galloprovincialis suggest presence of a "degradosome" in mussel mitochondria and necessitate the re-annotation of the l-rRNA/CR boundary.

Species of the genus Mytilus carry two mitochondrial genomes in obligatory coexistence; one transmitted though the eggs (the F type) and one through the sperm (the M type). We have studied the 3' and 5' ends of rRNA and tRNA transcripts using RT-PCR and RNA circularization techniques in both the F and M genomes of Mytilus galloprovincialis. We have found polyadenylated and non-adenylated transcripts for both ribosomal and transfer RNAs. In all these genes the 5' ends of the transcripts coincided with the first nucleotide of the annotated genes, but the 3' ends were heterogeneous. The l-rRNA 3' end is 47 or 48 nucleotides upstream from the one assigned by a previous annotation, which makes the adjacent first domain (variable domain one, VD1) of the main control region (CR) correspondingly longer. We have observed s-rRNA and l-rRNA transcripts with truncated 3' end and polyadenylated tRNA transcripts carrying the CCA trinucleotide. We have also detected polyadenylated RNA remnants carrying the sequences of the control region, which strongly suggests RNA degradation activity and thus presence of degradosomes in Mytilus mitochondria.

Does the ORF in the control region of Mytilus mtDNA code for a protein product?

The control region of the mtDNA of Mytilus is known to contain sequences that determine whether the genome will be paternally or maternally transmitted. An open reading frame (ORF) in this region raised suspicion that it may code for a protein involved in this mechanism. An analysis of the mtDNA transcriptome failed to produce evidence for this hypothesis.