Mosaic environment-driven evolution of the deep-sea mussel Gigantidas platifrons bacterial endosymbiont.

BACKGROUND: The within-species diversity of symbiotic bacteria represents an important genetic resource for their environmental adaptation, especially for horizontally transmitted endosymbionts. Although strain-level intraspecies variation has recently been detected in many deep-sea endosymbionts, their ecological role in environmental adaptation, their genome evolution pattern under heterogeneous geochemical environments, and the underlying molecular forces remain unclear. RESULTS: Here, we conducted a fine-scale metagenomic analysis of the deep-sea mussel Gigantidas platifrons bacterial endosymbiont collected from distinct habitats: hydrothermal vent and methane seep. Endosymbiont genomes were assembled using a pipeline that distinguishes within-species variation and revealed highly heterogeneous compositions in mussels from different habitats. Phylogenetic analysis separated the assemblies into three distinct environment-linked clades. Their functional differentiation follows a mosaic evolutionary pattern. Core genes, essential for central metabolic function and symbiosis, were conserved across all clades. Clade-specific genes associated with heavy metal resistance, pH homeostasis, and nitrate utilization exhibited signals of accelerated evolution. Notably, transposable elements and plasmids contributed to the genetic reshuffling of the symbiont genomes and likely accelerated adaptive evolution through pseudogenization and the introduction of new genes. CONCLUSIONS: The current study uncovers the environment-driven evolution of deep-sea symbionts mediated by mobile genetic elements. Its findings highlight a potentially common and critical role of within-species diversity in animal-microbiome symbioses. Video Abstract.

Ecological differences among hydrothermal vent symbioses may drive contrasting patterns of symbiont population differentiation.

The intra-host composition of horizontally transmitted microbial symbionts can vary across host populations due to interactive effects of host genetics, environmental, and geographic factors. While adaptation to local habitat conditions can drive geographic subdivision of symbiont strains, it is unknown how differences in ecological characteristics among host-symbiont associations influence the genomic structure of symbiont populations. To address this question, we sequenced metagenomes of different populations of the deep-sea mussel Bathymodiolus septemdierum, which are common at Western Pacific deep-sea hydrothermal vents and show characteristic patterns of niche partitioning with sympatric gastropod symbioses. Bathymodiolus septemdierum lives in close symbiotic relationship with sulfur-oxidizing chemosynthetic bacteria but supplements its symbiotrophic diet through filter-feeding, enabling it to occupy ecological niches with little exposure to geochemical reductants. Our analyses indicate that symbiont populations associated with B. septemdierum show structuring by geographic location, but that the dominant symbiont strain is uncorrelated with vent site. These patterns are in contrast to co-occurring Alviniconcha and Ifremeria gastropod symbioses that exhibit greater symbiont nutritional dependence and occupy habitats with higher spatial variability in environmental conditions. Our results suggest that relative habitat homogeneity combined with sufficient symbiont dispersal and genomic mixing might promote persistence of similar symbiont strains across geographic locations, while mixotrophy might decrease selective pressures on the host to affiliate with locally adapted symbiont strains. Overall, these data contribute to our understanding of the potential mechanisms influencing symbiont population structure across a spectrum of marine microbial symbioses that occupy contrasting ecological niches. IMPORTANCE Beneficial relationships between animals and microbial organisms (symbionts) are ubiquitous in nature. In the ocean, microbial symbionts are typically acquired from the environment and their composition across geographic locations is often shaped by adaptation to local habitat conditions. However, it is currently unknown how generalizable these patterns are across symbiotic systems that have contrasting ecological characteristics. To address this question, we compared symbiont population structure between deep-sea hydrothermal vent mussels and co-occurring but ecologically distinct snail species. Our analyses show that mussel symbiont populations are less partitioned by geography and do not demonstrate evidence for environmental adaptation. We posit that the mussel's mixotrophic feeding mode may lower its need to affiliate with locally adapted symbiont strains, while microhabitat stability and symbiont genomic mixing likely favors persistence of symbiont strains across geographic locations. Altogether, these findings further our understanding of the mechanisms shaping symbiont population structure in marine environmentally transmitted symbioses.

Cytogenetic characterization of the golden mussel (Limnoperna fortunei) reveals the absence of sex heteromorphic chromosomes.

The golden mussel (Limnoperna fortunei) is an aggressive invasive species in South America, where it endangers native species and freshwater ecosystems, in addition to causing extensive economic losses, mainly to the hydroelectric sector. Currently, there's no efficient control method available and the invasion has progressed across the continent. Its high reproduction rate is one of the key factors of the golden mussel's high invasive potential and, recently, efforts have been done in order to understand the reproduction and the sexual features of this species. However, its cytogenetics characterization is incipient and the possible occurrence of sex-specific cytogenetic features was never investigated. In this study, we aimed to characterize the chromosomal morphometry, the distribution profile of heterochromatin, and to detect possible sex-related epigenetic marks in the golden mussel. Results revealed that the karyotypic structure is similar in both sexes and no chromosome heteromorphism was observed between males and females specimens. The data increment the cytogenetic characterization of Limnoperna fortunei and contribute for future studies that aim to further investigate its reproduction and underlying sex determination processes.

The Complete Mitochondrial Genome of Mytilisepta virgata (Mollusca: Bivalvia), Novel Gene Rearrangements, and the Phylogenetic Relationships of Mytilidae.

The circular mitochondrial genome of Mytilisepta virgata spans 14,713 bp, which contains 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. Analysis of the 13 PCGs reveals that the mitochondrial gene arrangement of Mytilisepta is relatively conserved at the genus level. The location of the atp8 gene in Mytilisepta keenae differs from that of other species. However, compared with the putative molluscan ancestral gene order, M. virgata exhibits a high level of rearrangement. We constructed phylogenetic trees based on concatenated 12 PCGs from Mytilidae. As a result, we found that M. virgata is in the same clade as other Mytilisepta spp. The result of estimated divergence times revealed that M. virgata and M. keenae diverged around the early Paleogene period, although the oldest Mytilisepta fossil was from the late or upper Eocene period. Our results provide robust statistical evidence for a sister-group relationship within Mytilida. The findings not only confirm previous results, but also provide valuable insights into the evolutionary history of Mytilidae.

Insights into symbiotic interactions from metatranscriptome analysis of deep-sea mussel Gigantidas platifrons under long-term laboratory maintenance.

Symbioses between invertebrates and chemosynthetic bacteria are of fundamental importance in deep-sea ecosystems, but the mechanisms that enable their symbiont associations are still largely undescribed, owing to the culturable difficulties of deep-sea lives. Bathymodiolinae mussels are remarkable in their ability to overcome decompression and can be maintained successfully for an extended period under atmospheric pressure, thus providing a model for investigating the molecular basis of symbiotic interactions. Herein, we conducted metatranscriptome sequencing and gene co-expression network analysis of Gigantidas platifrons under laboratory maintenance with gradual loss of symbionts. The results revealed that one-day short-term maintenance triggered global transcriptional perturbation in symbionts, but little gene expression changes in mussel hosts, which were mainly involved in responses to environmental changes. Long-term maintenance with depleted symbionts induced a metabolic shift in the mussel host. The most notable changes were the suppression of sterol biosynthesis and the complementary activation of terpenoid backbone synthesis in response to the reduction of bacteria-derived terpenoid sources. In addition, we detected the upregulation of host proteasomes responsible for amino acid deprivation caused by symbiont depletion. Additionally, a significant correlation between host microtubule motor activity and symbiont abundance was revealed, suggesting the possible function of microtubule-based intracellular trafficking in the nutritional interaction of symbiosis. Overall, by analyzing the dynamic transcriptomic changes during the loss of symbionts, our study highlights the nutritional importance of symbionts in supplementing terpenoid compounds and essential amino acids and provides insight into the molecular mechanisms and strategies underlying the symbiotic interactions in deep-sea ecosystems.

Transcriptome analysis of gene expression profiling from the deep sea in situ to the laboratory for the cold seep mussel Gigantidas haimaensis.

BACKGROUND: The deep-sea mussel Gigantidas haimaensis is a representative species from the Haima cold seep ecosystem in the South China Sea that establishes endosymbiosis with chemotrophic bacteria. During long-term evolution, G. haimaensis has adapted well to the local environment of cold seeps. Until now, adaptive mechanisms responding to environmental stresses have remained poorly understood. RESULTS: In this study, transcriptomic analysis was performed for muscle tissue of G. haimaensis in the in situ environment (MH) and laboratory environment for 0 h (M0), 3 h (M3) and 9 h (M9), and 187,368 transcript sequences and 22,924 annotated differentially expressed genes (DEGs) were generated. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, these DEGs were enriched with a broad spectrum of biological processes and pathways, including those associated with antioxidants, apoptosis, chaperones, immunity and metabolism. Among these significantly enriched pathways, protein processing in the endoplasmic reticulum and metabolism were the most affected metabolic pathways. These results may imply that G. haimaensis struggles to support the life response to environmental change by changing gene expression profiles. CONCLUSION: The present study provides a better understanding of the biological responses and survival strategies of the mussel G. haimaensis from deep sea in situ to the laboratory environment.

A regulatory hydrogenase gene cluster observed in the thioautotrophic symbiont of Bathymodiolus mussel in the East Pacific Rise.

The mytilid mussel Bathymodiolus thermophilus lives in the deep-sea hydrothermal vent regions due to its relationship with chemosynthetic symbiotic bacteria. It is well established that symbionts reside in the gill bacteriocytes of the mussel and can utilize hydrogen sulfide, methane, and hydrogen from the surrounding environment. However, it is observed that some mussel symbionts either possess or lack genes for hydrogen metabolism within the single-ribotype population and host mussel species level. Here, we found a hydrogenase cluster consisting of additional H2-sensing hydrogenase subunits in a complete genome of B. thermophilus symbiont sampled from an individual mussel from the East Pacific Rise (EPR9N). Also, we found methylated regions sparsely distributed throughout the EPR9N genome, mainly in the transposase regions and densely present in the rRNA gene regions. CRISPR diversity analysis confirmed that this genome originated from a single symbiont strain. Furthermore, from the comparative analysis, we observed variation in genome size, gene content, and genome re-arrangements across individual hosts suggesting multiple symbiont strains can associate with B. thermophilus. The ability to acquire locally adaptive various symbiotic strains may serve as an effective mechanism for successfully colonizing different chemosynthetic environments across the global oceans by host mussels.

Mitochondrial genomic analyses provide new insights into the "missing" atp8 and adaptive evolution of Mytilidae.

BACKGROUND: Mytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length. RESULTS: In this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater. CONCLUSIONS: Our analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments.

Comparative mitogenomic analyses and gene rearrangements reject the alleged polyphyly of a bivalve genus.

Background: The order and orientation of genes encoded by animal mitogenomes are typically conserved, although there is increasing evidence of multiple rearrangements among mollusks. The mitogenome from a Brazilian brown mussel (hereafter named B1) classified as Perna perna Linnaeus, 1758 and assembled from Illumina short-length reads revealed an unusual gene order very different from other congeneric species. Previous mitogenomic analyses based on the Brazilian specimen and other Mytilidae suggested the polyphyly of the genus Perna. Methods: To confirm the proposed gene rearrangements, we sequenced a second Brazilian P. perna specimen using the "primer-walking" method and performed the assembly using as reference Perna canaliculus. This time-consuming sequencing method is highly effective when assessing gene order because it relies on sequentially-determined, overlapping fragments. We also sequenced the mitogenomes of eastern and southwestern South African P. perna lineages to analyze the existence of putative intraspecific gene order changes as the two lineages show overlapping distributions but do not exhibit a sister relationship. Results: The three P. perna mitogenomes sequenced in this study exhibit the same gene order as the reference. CREx, a software that heuristically determines rearrangement scenarios, identified numerous gene order changes between B1 and our P. perna mitogenomes, rejecting the previously proposed gene order for the species. Our results validate the monophyly of the genus Perna and indicate a misidentification of B1.

Pangenome Evolution in Environmentally Transmitted Symbionts of Deep-Sea Mussels Is Governed by Vertical Inheritance.

Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbionts inhabiting Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each symbiont species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared with core genes, accessory genes are enriched in genome plasticity functions. We found no evidence for recent HGT between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting that the MOX association with B. brooksi is more recent in comparison to that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that genome evolution of environmentally transmitted symbionts that associate with individual hosts over their lifetime is affected by a narrow symbiosis where the frequency of HGT is constrained.

Genetic diversity and connectivity of chemosynthetic cold seep mussels from the U.S. Atlantic margin.

BACKGROUND: Deep-sea mussels in the subfamily Bathymodiolinae have unique adaptations to colonize hydrothermal-vent and cold-seep environments throughout the world ocean. These invertebrates function as important ecosystem engineers, creating heterogeneous habitat and promoting biodiversity in the deep sea. Despite their ecological significance, efforts to assess the diversity and connectivity of this group are extremely limited. Here, we present the first genomic-scale diversity assessments of the recently discovered bathymodioline cold-seep communities along the U.S. Atlantic margin, dominated by Gigantidas childressi and Bathymodiolus heckerae. RESULTS: A Restriction-site Associated DNA Sequencing (RADSeq) approach was used on 177 bathymodiolines to examine genetic diversity and population structure within and between seep sites. Assessments of genetic differentiation using single-nucleotide polymorphism (SNP) data revealed high gene flow among sites, with the shallower and more northern sites serving as source populations for deeper occurring G. childressi. No evidence was found for genetic diversification across depth in G. childressi, likely due to their high dispersal capabilities. Kinship analyses indicated a high degree of relatedness among individuals, and at least 10-20% of local recruits within a particular site. We also discovered candidate adaptive loci in G. childressi and B. heckerae that suggest differences in developmental processes and depth-related and metabolic adaptations to chemosynthetic environments. CONCLUSIONS: These results highlight putative source communities for an important ecosystem engineer in the deep sea that may be considered in future conservation efforts. Our results also provide clues into species-specific adaptations that enable survival and potential speciation within chemosynthetic ecosystems.

Evidence of multiple genome duplication events in Mytilus evolution.

BACKGROUND: Molluscs remain one significantly under-represented taxa amongst available genomic resources, despite being the second-largest animal phylum and the recent advances in genomes sequencing technologies and genome assembly techniques. With the present work, we want to contribute to the growing efforts by filling this gap, presenting a new high-quality reference genome for Mytilus edulis and investigating the evolutionary history within the Mytilidae family, in relation to other species in the class Bivalvia. RESULTS: Here we present, for the first time, the discovery of multiple whole genome duplication events in the Mytilidae family and, more generally, in the class Bivalvia. In addition, the calculation of evolution rates for three species of the Mytilinae subfamily sheds new light onto the taxa evolution and highlights key orthologs of interest for the study of Mytilus species divergences. CONCLUSIONS: The reference genome presented here will enable the correct identification of molecular markers for evolutionary, population genetics, and conservation studies. Mytilidae have the capability to become a model shellfish for climate change adaptation using genome-enabled systems biology and multi-disciplinary studies of interactions between abiotic stressors, pathogen attacks, and aquaculture practises.

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.

A chromosome-level assembly supports genome-wide investigation of the DMRT gene family in the golden mussel (Limnoperna fortunei).

BACKGROUND: The golden mussel (Limnoperna fortunei) is a highly invasive species that causes environmental and socioeconomic losses in invaded areas. Reference genomes have proven to be a valuable resource for studying the biology of invasive species. While the current golden mussel genome has been useful for identifying new genes, its high fragmentation hinders some applications. FINDINGS: In this study, we provide the first chromosome-level reference genome for the golden mussel. The genome was built using PacBio HiFi, 10X, and Hi-C sequencing data. The final assembly contains 99.4% of its total length assembled to the 15 chromosomes of the species and a scaffold N50 of 97.05 Mb. A total of 34,862 protein-coding genes were predicted, of which 84.7% were functionally annotated. A significant (6.48%) proportion of the genome was found to be in a hemizygous state. Using the new genome, we have performed a genome-wide characterization of the Doublesex and Mab-3 related transcription factor gene family, which has been proposed as a target for population control strategies in other species. CONCLUSIONS: From the applied research perspective, a higher-quality genome will support genome editing with the aim of developing biotechnology-based solutions to control invasion. From the basic research perspective, the new genome is a high-quality reference for molecular evolutionary studies of Mytilida and other Lophotrochozoa, and it may be used as a reference for future resequencing studies to assess genomic variation among different golden mussel populations, unveiling potential routes of dispersion and helping to establish better control policies.

Redescription of Bathymodiolus septemdierum Hashimoto and Okutani, 1994 (Bivalvia, Mytilida, Mytilidae), a mussel broadly distributed across hydrothermal vent locations in the western Pacific and Indian Oceans.

Mussels of the genus Bathymodiolus Kenk & Wilson belong to the foundation fauna at hydrothermal vents in the global deep sea. In the western Pacific and Indian oceans, the three nominal taxa B. septemdierum Hashimoto and Okutani, B. brevior Cosel, Métivier & Okutani and B. marisindicus Hashimoto are currently recognized as separate species despite morphological and genetic evidence for their conspecificity. All three are listed with the International Union for Conservation of Nature Red List based on highly restricted ranges. We compile and supplement existing morphometric and molecular data to revise the Bathymodiolus septemdierum species group. We redescribe B. septemdierum as a single species with B. brevior and B. marisindicus recognized as junior synonyms. Given the exceptionally broad range of B. septemdierum, we propose removal of these three taxa from the IUCN Red List.

microRNAs facilitate comprehensive responses of Bathymodiolinae mussel against symbiotic and nonsymbiotic bacteria stimulation.

Bathymodiolinae mussels are dominant species in cold seeps and hydrothermal vents and could harbor endosymbionts in gill bacteriocytes. However, mechanisms underlying the symbiosis have remained largely undisclosed for years. In the present study, the global expression pattern of immune-related genes and miRNAs were surveyed in Gigantidas platifrons during bacterial challenges using enriched symbiotic methane oxidation bacteria MOBs or nonsymbiotic Vibrio. As a result, multiple pattern recognition receptors were found differentially expressed at 12 h and 24 h post bacteria challenges and distinctly clustered between stimulations. Dozens of immune effectors along with signal transducers were also modulated simultaneously during MOB or Vibrio challenge. A total of 459 miRNAs were identified in the gill while some were differentially expressed post MOB or nonsymbiotic bacteria challenge. A variety of immune-related genes were annotated as target genes of aforesaid differentially expressed miRNAs. As a result, biological processes including the immune recognition, lysosome activity and bacteria engulfment were suggested to be dynamically modulated by miRNAs in either symbiotic or nonsymbiotic bacteria challenge. It was suggested that G. platifrons mussels could maintain a robust immune response against invading pathogens while establishing symbiosis with chemosynthetic bacteria with the orchestra of immune-related genes and miRNAs.

Deep-sea mussels from a hybrid zone on the Mid-Atlantic Ridge host genetically indistinguishable symbionts.

The composition and diversity of animal microbiomes is shaped by a variety of factors, many of them interacting, such as host traits, the environment, and biogeography. Hybrid zones, in which the ranges of two host species meet and hybrids are found, provide natural experiments for determining the drivers of microbiome communities, but have not been well studied in marine environments. Here, we analysed the composition of the symbiont community in two deep-sea, Bathymodiolus mussel species along their known distribution range at hydrothermal vents on the Mid-Atlantic Ridge, with a focus on the hybrid zone where they interbreed. In-depth metagenomic analyses of the sulphur-oxidising symbionts of 30 mussels from the hybrid zone, at a resolution of single nucleotide polymorphism analyses of ~2500 orthologous genes, revealed that parental and hybrid mussels (F2-F4 generation) have genetically indistinguishable symbionts. While host genetics does not appear to affect symbiont composition in these mussels, redundancy analyses showed that geographic location of the mussels on the Mid-Atlantic Ridge explained most of the symbiont genetic variability compared to the other factors. We hypothesise that geographic structuring of the free-living symbiont population plays a major role in driving the composition of the microbiome in these deep-sea mussels.

First transcriptome assembly of a newly discovered vent mussel, Gigantidas vrijenhoeki, at Onnuri Vent Field on the northern Central Indian Ridge.

This is the first report of a transcriptome assembly of a newly discovered hydrothermal vent mussel, Gigantidas vrijenhoeki (Bivalvia: Mytilidae), on the Central Indian Ridge. Gigantidas vrijenhoeki was identified from material collected at the newly discovered Onnuri Vent Field (OVF) on the Central Indian Ridge in 2018, and was reported as a new species, distinct from another dominant hydrothermal vent mussel, Bathymodiolus marisindicus, in 2020. We sequenced the transcriptome of G. vrijenhoeki using the Illumina HiSeq X System. De novo assembly and analysis of the coding regions predicted 25,405 genes, 84.76% of which was annotated by public databases. The transcriptome of G. vrijenhoeki will be a valuable resource in studying the ecological and biological characteristics of this new species, which is distinct from other deep-sea mussels. These data should also support the investigation of the relationship between the environmental conditions of hydrothermal vents and the unique distribution of G. vrijenhoeki in the OVF of the Central Indian Ridge.

Phylogeography of Mytilisepta virgata (Mytilidae: Bivalvia) in the northwestern Pacific: Cryptic mitochondrial lineages and mito-nuclear discordance.

The purplish bifurcate mussel Mytilisepta virgata is widely distributed and represents one of the major components of the intertidal community in the northwestern Pacific (NWP). Here, we characterized population genetic structure of NWP populations throughout nearly their whole distribution range using both mitochondrial (mtDNA cox1) and nuclear (ITS1) markers. Population genetic analyses for mtDNA cox 1 sequences revealed two monophyletic lineages (i.e., southern and northern lineages) geographically distributed according to the two different surface water temperature zones in the NWP. The timing of the lineage split is estimated at the Pliocene- mid-Pleistocene (5.49-1.61 Mya), which is consistent with the timing of the historical isolation of the East Sea/Sea of Japan from the South and East China Seas due to sea level decline during glacial cycles. Historical sea level fluctuation during the Pliocene-Pleistocene and subsequent adaptation of mussels to different surface water temperature zones may have contributed to shaping the contemporary genetic diversity and deep divergence of the two mitochondrial lineages. In contrast to mtDNA sequences, a clear lineage split between the two mitochondrial lineages was not found in ITS1 sequences, which showed a star-like structure composed of a mixture of southern and northern mitochondrial lineages. Possible reasons for this type of mito-nuclear discordance include stochastic divergence in the coalescent processes of the two molecular markers, or balancing selection under different marine environments. Cryptic speciation cannot be ruled out from these results, and future work using genomic analyses is required to address whether the thermal physiology of these mussels corresponds to the deep divergence of their mitochondrial genes and to test for the existence of morphologically indistinguishable but genetically separate cryptic species.

Molecular Identification, Characterization, and Expression Analysis of a Metallothionein Gene from Septifer virgatus.

This study provides a preliminary characterization of a metallothionein (MT) gene in Septifer virgatus and highlights its potential use in biomonitoring. The full-length SvMT cDNA and the complete sequence of the SvMT gene were identified using reverse transcriptase PCR coupled with the rapid amplification of cDNA ends and the primer walking method. The SvMT cDNA encodes a protein of 72 amino acids having nine classical Cys-X-Cys motifs. Moreover, the deduced amino acids contained the conserved motif (Cys-x-Cys-x(3)-Cys-Thr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys) of MT family 2. Its molecular mass and isoelectric point were estimated to be 7.01 kDa and 7.00, respectively. BLAST-based searching indicated that SvMT shared 81.0% amino acid sequence identity with Mytilus edulis MT-20-II. The SvMT gene has three coding exons and two introns. After exposure to 1 mg/L cadmium chloride, the expression of SvMT increased 15-fold by 3 days (d), with a maximum expression of 27-fold by 5 d compared with the pre-exposure level. After exposure to 2 mg/L zinc chloride, the expression of SvMT increased 2.5-fold by 3 d and 4.7-fold by 5 d compared with the pre-exposure level. A significant increase in the expression level of SvMT mRNA was observed after the exposure of S. virgatus to the combination of 0.003 mg/L cadmium chloride and 0.2 mg/L zinc chloride compared with the pre-exposure level. Our work indicates that the SvMT gene is associated with stress responses and could be a potential biomarker for marine pollution.