Confirmation of the first intronic sequence in the bivalvian mitochondrial genome of Macoma balthica (Linnaeus, 1758).

In 2020, the first male-type mitochondrial genome from the clam Macoma balthica was published. Apart from the unusual doubly uniparental inheritance of mtDNA, scientists observed a unique (over 4k bp long) extension in the middle of the cox2 gene. We have attempted to replicate these data by NGS DNA sequencing and explore further the expression of the long cox2 gene. In our study, we report an even longer cox2 gene (over 5.5 kbp) with no stop codon separating conserved cox2 domains, as well as, based on the rtPCR, a lower relative gene expression pattern of the middle part of the gene (5' = 1; mid = 0.46; 3' = 0.89). Lastly, we sequenced the cox2 gene transcript proving the excision of the intronic sequence.

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.

A study of aanat and asmt expression in the three-spined stickleback eye and skin: Not only "on the way to melatonin".

Melatonin synthesis is controlled by aralkylamine N-acetyltransferase (AANAT: EC 2.3.1.87) acetylating serotonin (5-hydroxytryptamine; 5-HT) to N-acetylserotonin (NAS), and N-acetylserotonin O-methyltransferase (ASMT: EC 2.1.1.4) methylating NAS to melatonin (Mel; N-acetyl-5-methoxytryptamine). We examined the levels of expression of the aanat and asmt genes, Mel concentrations as well as AANAT isozyme activity in the eyeball (with retina) and skin of the three-spined stickleback (Gasterosteus aculeatus), at noon and midnight. We found mRNA of four genes (aanat1a, snat, asmt and asmt2) in the eyeball, and two (aanat1a and asmt2) in the skin. The presence of two transcripts of genes encoding AANAT and two of ASMT in the eyeball at noon and midnight, suggests activity of AANAT and ASMT isozymes in metabolic pathways besides "the way to melatonin", all the more so because day/night changes in Mel concentration do not follow the changes in either the expression of genes or the activity of AANAT. The high effectiveness of noon NAS synthesis in the eyeball at low substrate concentrations, which is not reflected in high Mel production, suggests the function of eye NAS beyond that of a precursor to the biosynthesis of Mel. The inhibition of AANAT isozyme activity by product observed in the eyeball may be one of the mechanisms of 5-HT husbanding in the eye (retina). The presence of transcripts of genes encoding both AANAT and ASMT and the activity of AANAT, at noon and midnight, supports a local Mel synthesis in the sticklebacks' skin.

The complete maternal and paternal mitochondrial genomes of Unio crassus: Mitochondrial molecular clock and the overconfidence of molecular dating.

The availability of a rapidly growing number of complete mitochondrial genome sequences provokes high confidence dating approaches. However, even if the congruence between mitochondrial and nuclear markers is reasonable, the resulting topologies are frequently questionable. The unique opportunity to study the evolutionary history of two independent mitochondrial genomes in one phylogenetic context exists in the freshwater mussels family Unionidae. The two lineages function under doubly uniparental inheritance since well before the emergence of the family. Despite the relatively high number of available complete sequences of maternally inherited genomes, comparative analyses are limited by the small number of sequences of counterpart paternally inherited genomes. We have sequenced for the first time the representative set of five sequences (two maternal and three paternal) from the species Unio crassus. Comparative analysis of the phylogenies reconstructed using relevant mitogenomic data available in GenBank (13 species in total) reveal that single - genome inferences are congruent only if the relaxed clock is assumed.

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression.

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large-scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time-calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.

Mitogenomics of recombinant mitochondrial genomes of Baltic Sea Mytilus mussels.

Recombination in the control region (CR) of Mytilus mitochondrial DNA (mtDNA) was originally reported based on the relatively short, sequenced fragments of mitochondrial genomes. Recombination outside the CR has been reported recently with the suggestion that such processes are common in Mytilus. We have fully sequenced a set of 11 different mitochondrial haplotypes representing the high diversity of paternally inherited mitochondrial genomes of Baltic Sea Mytilus mussels, including the haplotype close to the native Mytilus trossulus mitochondrial genome, which was thought to have been entirely eliminated from this population. Phylogenetic and comparative analysis showed that the recombination is limited to the vicinity of the CR in all sequenced genomes. Coding sequence comparison indicated that all paternally inherited genomes showed increased accumulation of nonsynonymous substitutions, including the genomes which switched their transmission route very recently. The acquisition of certain CR sequences through recombination with highly divergent paternally inherited genomes seems to precede and favor the switch, but it is not a prerequisite for this process. Interspecies hybridization in the Baltic Sea during the recent 10,000 years created conditions for both structural and evolutionary mitochondrial instability which resulted in the observed variation and dynamics of mtDNA in Baltic Sea Mytilus mussels. In conclusion, the data shows that the effects of mitochondrial recombination are limited to the CR of few phylogenetic lineages.

Co-expressed mitochondrial genomes: recently masculinized, recombinant mitochondrial genome is co-expressed with the female-transmitted mtDNA genome in a male Mytilus trossulus mussel from the Baltic Sea.

BACKGROUND: Few exceptions have been described from strict maternal inheritance of mitochondrial DNA in animals, including sea mussels (Mytilidae), clams (Donacidae, Veneridae and Solenidae) and freshwater mussels (Unionoidae) order. In these bivalves mitochondria and their DNA are transferred through two separate routes. The females inherit only the maternal mitochondrial DNA whereas the males inherit maternal as well as paternal mitochondrial DNA, which is usually present only in gonads and sperm. The mechanism controlling this phenomenon is unclear but leads to the existence of two separate mitochondrial DNA lineages in a single species. The lineages are usually well differentiated: up to 20-50% divergence in nucleotide sequence. Occasionally, a maternal mitochondrial DNA can invade the paternal transmission route, eventually replacing the diverged M-type and lowering the divergence. Such role reversal (masculinization) event has happened recently in the Mytilus population of the Baltic Sea which consists of M. edulis × M. trossulus hybrids, but the functional status of the resulting mitochondrial genome was unknown. RESULTS: In this paper we sequenced transcripts from one specimen that was identified as male carrying both the female mitochondrial genome and a recently masculinized mitochondrial genome. Additionally, the analysis of the control region has showed that the recently masculinized, recombinant genome, not only has an M-type control region and all coding regions derived from the F-type, but also is transcriptionally active along side the maternally inherited F-type genome. In the comparative analysis, the two genomes exhibit different substitution patterns, typical for the M vs. F genome comparisons. The genetic distances and ratios of non-synonymous substitutions also suggest that one of the genomes is transitioning from the maternal to the paternal inheritance mode, consistent with its recent masculinization. CONCLUSION: We have shown, for the first time, that the recently masculinized mitochondrial genome is active and that it accumulates excess of non-synonymous substitutions across its coding sequence. This suggests, that, under certain cytonuclear incompatibility conditions, masculinization may serve to restore the endangered functionality of the paternally inherited genome. This is also another example of a mitochondrial genome in which the recombination in the control region predated its transition from paternal to maternal transmission route.

The absence of canonical respiratory complex I subunits in male-type mitogenomes of three Donax species.

Bivalves are an extraordinary class of animals in which species with a doubly uniparental inheritance (DUI) of mitochondrial DNA have been described. DUI is characterized as a mitochondrial homoplasmy of females and heteroplasmy of male individuals where F-type mitogenomes are passed to the progeny with mother egg cells and divergent M-type mitogenomes are inherited with fathers sperm cells. However, in most cases only male individuals retain divergent mitogenome inherited with spermatozoa. Additionally, in many of bivalves, unique mitochondrial features, like additional genes, gene duplication, gene extensions, mitochondrial introns, and recombination, were observed. In this study, we sequenced and assembled male-type mitogenomes of three Donax species. Comparative analysis of mitochondrial sequences revealed a lack of all seven NADH dehydrogenase subunits as well as the presence of three long additional open reading frames lacking identifiable homology to any of the existing genes.

Plant mitochondrial genome map (PMGmap): A software tool for the comprehensive visualization of coding, noncoding and genome features of plant mitochondrial genomes.

Tools for visualizing genomes are essential for investigating genomic features and their interactions. Currently, tools designed originally for animal mitogenomes and plant plastomes are used to visualize the mitogens of plants but cannot accurately display features specific to plant mitogenomes, such as nonlinear exon arrangement for genes, the prevalence of functional noncoding features and complex chromosomal architecture. To address these problems, a software package, plant mitochondrial genome map (PMGmap), was developed using the Python programming language. PMGmap can draw genes at exon levels; draw cis- and trans-splicing gene maps, noncoding features and repetitive sequences; and scale genic regions by using the scaling of the genic regions on the mitogenome (SAGM) algorithm. It can also draw multiple chromosomes simultaneously. Compared with other state-of-the-art tools, PMGmap showed better performance in visualizing 405 plant mitogenomes, showing potential as an invaluable tool for plant mitogenome research. The web and container versions and the source code of PMGmap can be accessed through the following link: http://www.1kmpg.cn/pmgmap.

No evidence of DUI in the Mediterranean alien species Brachidontes pharaonis (P. Fisher, 1870) despite mitochondrial heteroplasmy.

Two genetically different mitochondrial haplogroups of Brachidontes pharaonis (p-distance 6.8%) have been identified in the Mediterranean Sea. This hinted at a possible presence of doubly uniparental inheritance in this species. To ascertain this possibility, we sequenced two complete mitogenomes of Brachidontes pharaonis mussels and performed a qPCR analysis to measure the relative mitogenome copy numbers of both mtDNAs. Despite the presence of two very similar regions composed entirely of repetitive sequences in the two haplogroups, no recombination between mitogenomes was detected. In heteroplasmic individuals, both mitogenomes were present in the generative tissues of both sexes, which argues against the presence of doubly uniparental inheritance in this species.

First Glimpse at the Diverse Aquaporins of Amphipod Crustaceans.

The importance of aquaporins (AQPs) in the transport of water and solutes through cell membranes is well recognized despite being relatively new. To date, despite their abundance, diversity, and presence in disparate environments, amphipods have only been mentioned in studies about the AQPs of other animals and have never been further investigated. In this work, we aimed to recover from public data available AQPs of these crustaceans and reconstruct phylogenetic affinities. We first performed BLAST searches with several queries of diverse taxa against different NCBI databases. Then, we selected the clades of AQPs retrieving the amphipod superfamily Gammaroidea as monophyletic and ran phylogenetic analyses to assess their performances. Our results show how most of the AQPs of amphipods are similar to those of other crustaceans, despite the Prip-like displayed different paralogs, and report for the first time a putative Aqp8-like for arthropods. We also found that the candidate genes of Prip-like, Bib-like, Aqp12-like, and Glp-like help solve deeper relationships in phylogenies of amphipods while leaving uncertainties in shallower parts. With our findings, we hope to increase attention to the study of amphipods as models for AQP functioning and evolution.

Is interlineage recombination responsible for low divergence of mitochondrial nad3 genes in Mytilus galloprovincialis?

The existence of mtDNA recombination in animals has been confirmed by several case studies. Still, for Mytilus mussels possessing two divergent mitochondrial genomes (M and F), which can recombine, no recombination between coding sequences of highly diverged M and F genomes has been shown. Based on the full sequences of both genomes, it has been suggested that particularly low divergence observed within the mitochondrial nad3 gene of the Mytilus galloprovincialis mussel may be caused by its exceptionally low evolutionary rate. Here, we contribute a new pair of mitochondrial genomes typical for M. galloprovincialis and show that this low divergence is not a sign of evolutionary conservation but is rather caused by the acquisition of an F-related sequence by the published M genome of M. galloprovincialis. The most likely scenario for this apparent mtDNA-coding region recombination case is an assembly artifact.

Comparative genomics of marine mussels (Mytilus spp.) gender associated mtDNA: rapidly evolving atp8.

The unusual mode of mitochondrial DNA inheritance, with two separate: maternal (F) and paternal (M) lineages, gives unique opportunities to study the evolution of the mitochondrial genome. This system was first discovered in the marine mussels Mytilus. The three related species: Mytilus edulis, Mytilus galloprovincialis and Mytilus trossulus form a complex in which the divergence of M and F lineages pre-dates the speciation. The complete mitochondrial genomes of both lineages were known for all species except Pacific M. trossulus. Here we report, for the first time, the complete sequences of both mitochondrial genomes of Pacific M. trossulus, filling the gap. While the reported M and F genomes are highly diverged (26%), they have similar organisation. The only difference is the translocation of one tRNA gene into the long, mosaic control region of the F genome. Consistent presence of an ORF which most likely represents the atp8 gene was confirmed in both genomes. The predicted protein has characteristics expected of the functional atp8 even though the M and F versions are markedly different in length. Comparative analysis involving all three species led to the conclusion that the cause of a faster evolution of atp8 and Mytilus mtDNA in general is most likely the Compensation-Draft Feedback process coupled with relatively relaxed selection in the M lineage. Thus, we postulate that the adaptive changes may have played a role in the emergence of highly diverged, barely recognizable atp8 in Mytilus mussels.

Semimytilus algosus: first known hermaphroditic mussel with doubly uniparental inheritance of mitochondrial DNA.

Doubly uniparental inheritance (DUI) of mitochondrial DNA is a rare phenomenon occurring in some freshwater and marine bivalves and is usually characterized by the mitochondrial heteroplasmy of male individuals. Previous research on freshwater Unionida mussels showed that hermaphroditic species do not have DUI even if their closest gonochoristic counterparts do. No records showing DUI in a hermaphrodite have ever been reported. Here we show for the first time that the hermaphroditic mussel Semimytilus algosus (Mytilida), very likely has DUI, based on the complete sequences of both mitochondrial DNAs and the distribution of mtDNA types between male and female gonads. The two mitogenomes show considerable divergence (34.7%). The presumably paternal M type mitogenome dominated the male gonads of most studied mussels, while remaining at very low or undetectable levels in the female gonads of the same individuals. If indeed DUI can function in the context of simultaneous hermaphroditism, a change of paradigm regarding its involvement in sex determination is needed. It is apparently associated with gonadal differentiation rather than with sex determination in bivalves.

The ORF in the control region of the female-transmitted Mytilus mtDNA codes for a protein.

Bivalve species with doubly uniparental inheritance of mitochondria have been shown to contain additional mtDNA-encoded proteins suspected to be involved in sex-specific transmission of the female (F) and male (M) mitochondrial genomes. This is true for freshwater mussels and marine clams but was still unclear for marine mussel Mytilus spp. Here we present evidence that a F mtDNA-specific open reading frame (ORF) identified in the control region of M. edulis codes for a protein. The protein was detected, using western blots, in both female and male mantle tissues, which contain the gonads. The protein was also localized, using immunochemistry, in sperm mitochondria.

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

Frequent nonhomologous recombination has been previously postulated to explain the 1045-bp insertion in one mitochondrial sperm-transmitted haplotype of Mytilus galloprovincialis. Such recombination would lead to the disruption of gene order and so the existence of a specific mechanism for maintaining the same gene order in both mitochondrial genomes of Mytilus has been proposed. Here the simpler explanation of the observed structure, involving a tandem duplication and a deletion, is presented. Their occasional occurrence in Mytilus mtDNA proves the similarity, not the difference, between animals with and without DUI.

Complete paternally inherited mitogenomes of two freshwater mussels Unio pictorum and Sinanodonta woodiana (Bivalvia: Unionidae).

Freshwater bivalves from the family Unionidae usually have two very divergent mitogenomes, inherited according to the doubly uniparental model. The early divergence of these two mitogenomic lineages gives a unique opportunity to use two mitogenomic data sets in a single phylogenetic context. However, the number of complete sequences of the maternally inherited mitogenomes of these animals available in GenBank greatly exceeds that of the paternally inherited mitogenomes. This is a problem for phylogenetic reconstruction because it limits the use of both mitogenomic data sets. Moreover, since long branch attraction phenomenon can bias reconstructions if only a few but highly divergent taxa are considered, the shortage of the faster evolving paternally inherited mitogenome sequences is a real problem. Here we provide, for the first time, complete sequences of the M mitogenomes sampled from Polish populations of two species: native Unio pictorum and invasive Sinanodonta woodiana. It increases the available set of mitogenomic pairs to 18 species per family, and allows unambiguous reconstruction of phylogenetic relationships among them. The reconstructions based on M and F mitogenomes which were separated for many millions of years, and subject to differing evolutionary dynamics, are fully congruent.

Actively transcribed and expressed atp8 gene in Mytilus edulis mussels.

BACKGROUND: Animal mitochondrial genomes typically encode 37 genes: 13 proteins, 22 tRNAs and two rRNAs. However, many species represent exceptions to that rule. Bivalvia along with Nematoda and Platyhelminthes are often suspected to fully or partially lack the ATP synthase subunit 8 (atp8) gene. This raises the question as to whether they are really lacking this gene or is this maybe an annotation problem? Among bivalves, Mytilus edulis has been inferred to lack an ATP8 gene since the characterization of its mitochondrial genome in 1992. Even though recent bioinformatic analyses suggested that atp8 is present in Mytilus spp., due to high divergence in predicted amino acid sequences, the existence of a functional atp8 gene in this group remains controversial. RESULTS: Here we demonstrate that M. edulis mitochondrial open reading frames suggested to be atp8 (in male and female mtDNAs) are actively translated proteins. We also provide evidence that both proteins are an integral part of the ATP synthase complex based on in-gel detection of ATP synthase activity and two-dimensional Blue-Native and SDS polyacrylamide electrophoresis. CONCLUSION: Many organisms (e.g., Bivalvia along with Nematoda and Platyhelminthes) are considered to be lacking certain mitochondrial genes often only based on poor similarity between protein coding gene sequences in genetically closed species. In some situations, this may lead to the inference that the ATP8 gene is absent, when it is in fact present, but highly divergent. This shows how important complementary role protein-based approaches, such as those in the present study, can provide to bioinformatic, genomic studies (i.e., ability to confirm the presence of a gene).

Mitogenomics of Perumytilus purpuratus (Bivalvia: Mytilidae) and its implications for doubly uniparental inheritance of mitochondria.

Animal mitochondria are usually inherited through the maternal lineage. The exceptional system allowing fathers to transmit their mitochondria to the offspring exists in some bivalves. Its taxonomic spread is poorly understood and new mitogenomic data are needed to fill the gap. Here, we present for the first time the two divergent mitogenomes from Chilean mussel Perumytilus purpuratus. The existence of these sex-specific mitogenomes confirms that this species has the doubly uniparental inheritance (DUI) of mitochondria. The genetic distance between the two mitochondrial lineages in P. purpuratus is not only much bigger than in the Mytilus edulis species complex but also greater than the distance observed in Musculista senhousia, the only other DUI-positive member of the Mytilidae family for which both complete mitochondrial genomes were published to date. One additional, long ORF (open reading frame) is present exclusively in the maternal mitogenome of P. purpuratus. This ORF evolves under purifying selection, and will likely be a target for future DUI research.

Family of Tc1-like elements from fish genomes and horizontal transfer.

The involvement of horizontal transfer (HT) in the evolution of vertebrate transposable elements (TEs) is a matter of an ongoing debate. The phylogenetic relationships between Tc1 TEs, based on limited dataset have been previously used to infer a case of Tc1 HT between the genomes of fish and frogs. Here this hypothesis has been critically evaluated by the experimental approach including comparative data on the range of fish species available today. The distribution of a Tc1 subfamily of TE in selected fish species was investigated by PCR with a single primer complementary to ITRs and showed that they are widespread in the studied 17 fish species. They belong to five different subfamilies of Tc1 TEs, as revealed by the comparison with current genomic data for fish and amphibians. The original hypothesis would get much weaker support from the current data, although at least one novel potential and more convincing case of HT was identified between genomes of Perciformes fish. An interesting case of recombination-driven mobilisation of a degenerated TE by distantly related TE from different subfamily was discovered in the genome of pike. The occurrence of such cases widens the range of TE elements identifiable with the employed experimental approach. Further similar studies would help to explain the evolution of the multiple Tc1 lineages including species for which full genome sequences will not be available soon.