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A mitochondrial genome phylogeny of Mytilidae (Bivalvia: Mytilida)
Molecular Phylogenetics and Evolution 139 (2019) 106533
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A mitochondrial genome phylogeny of Mytilidae (Bivalvia: Mytilida) a
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Yucheol Lee , Haena Kwak , Jinkyung Shin , Seung-Chul Kim , Taeho Kim , Joong-Ki Park a b
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Department of Biological Sciences, Sungkyunkwan University, 2066 Seobu-ro, Suwon, Gyeonggi-do 16419, Republic of Korea Division of EcoScience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: Mitochondrial genome Molecular phylogeny Mytilidae Bivalvia Mollusca
The family Mytilidae is a family of bivalve mussels that are distributed worldwide in diverse marine habitats. Within the family, classification systems and phylogenetic relationships among subfamilies remain not yet fully resolved. In this study, we newly determined 9 mitochondrial genome sequences from 7 subfamilies: Bathymodiolus thermophilus (Bathymodiolinae), Modiolus nipponicus (Modiolinae), Lithophaga curta (the first representative of Lithophaginae), Brachidontes mutabilis (Brachidontinae), Mytilisepta virgata (Brachidontinae), Mytilisepta keenae (Brachidontinae), Crenomytilus grayanus (Mytilinae), Gregariella coralliophaga (Crenellinae), and Septifer bilocularis (the first representative of Septiferinae). Phylogenetic trees using maximum likelihood and Bayesian inference methods for 28 mitochondrial genomes (including 19 previously published sequences) showed two major clades with high support values: Clade 1 ((Bathymodiolinae + Modiolinae) + (Lithophaginae + Limnoperninae)) and Clade 2 (((Mytilinae + Crenellinae) + Septiferinae) + Brachidontinae). The position of the genus Lithophaga (representing Lithophaginae) differed from a previously published molecular phylogeny. Divergence time analysis with a molecular clock indicated that lineage splitting among the major subfamilies of Mytilidae (including the habitat transition from marine to freshwater environments by ancestral Limnoperninae) occurred in the Mesozoic period, coinciding with high diversification rates of marine fauna during that time. This is the first mitochondrial genome-based phylogenetic study of the Mytilidae that covers nearly all subfamily members, excluding the subfamily Dacrydiinae.
1. Introduction The family Mytilidae, also known as marine mussels, has received much attention for its economic and environmental importance: some members of this family are well-known as invasive and/or biofouling species (e.g., Limnoperna fortunei) that damage power plant cooling systems and fish farms (Bayne and Bayne, 1976; Jenner et al., 1998), while others (e.g., Mytilus spp., Perna spp.) are edible and have important commercial value (Vakily, 1989; Taylor et al., 1992; Pawiro, 2010). In addition, Mytilus species have long been used as indicator species for assessing environmental pollution (Phillips, 1976; Cossa, 1988; Vázquez-Luis et al., 2016). More recently, they have been the focus of biomaterials research due to the potential commercial importance of the adhesive proteins in its byssal threads (Hwang et al., 2007; Kim et al., 2017). Mytilidae comprises epifaunal or semi-infaunal species that occupy diverse (mostly subfamily-specific) habitats (Stanley, 1970; Distel, 2000) including intertidal, deep sea, and freshwater zones (Soot-Ryen, 1969; Distel, 2000): some species (e.g., Mytilus and Modiolus) secrete byssal threads and attach to hard substrates, while Lithophaginae ⁎
species bore into and inhabit holes in rocks and corals (Bayne, 1976; Owada, 2007). Most species are found in coastal seas, but Bathymodiolinae species inhabit deep sea hydrothermal vents and cold seeps at depths of up to 3000–7000 m, where they have symbiotic associations with chemoautotrophic bacteria (Lutz and Kennish, 1993; Distel et al, 2000; Duperron et al., 2013). Unlike other subfamilies, Limnoperninae is the only member of Mytilidae that has successfully invaded and adapted to brackish and/or freshwater environments (Ricciardi, 1998; Adarraga and Martínez, 2012). Considering these diverse life styles, resolving phylogenetic relationships among major subfamilies within Mytilidae is essential to fully understand their evolutionary history and their diverse ecological specialization pathways. Classification systems of the family Mytilidae based on different morphological features (e.g., shell morphology, internal anatomy, sperm microstructure) in previous studies are very complicated and disagree with each other over the taxonomic arrangement of subordinate subfamily groups (see Supplementary Table 1 for details). Early on, Soot-Ryen (1969) divided the extant Mytilidae (superfamily Mytiloidea) into four subfamilies based on paleontological data, and this system was followed by Newell (1969), Boss (1982) and Bernard
Corresponding author. E-mail address: [email protected] (J.-K. Park).
https://doi.org/10.1016/j.ympev.2019.106533 Received 5 April 2019; Received in revised form 6 June 2019; Accepted 7 June 2019 Available online 08 June 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved.
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(1983): Mytilinae Rafinesque, 1815; Modiolinae Keen, 1958; Crenellinae Gray, 1815; and Lithophaginae H. Adams & A. Adams 1857. Later, the two subfamilies Musculinae Iredale, 1939 and Bathymodiolinae Kenk and B.R. Wilson, 1985 were added to Mytilidae (Habe, 1977). Kafanov and Drozdov (1998) presented a classification system based on sperm microstructures; their phylogeny followed SootRyen (1969)’s system but divided Modiolinae into two subfamilies (Mytilinae, Modiolinae, Crenellinae, Musculinae, and Lithophaginae). Coan et al. (2000) recognized 7 subfamilies (Mytilinae, Modiolinae, Crenellinae, Lithophaginae, Bathymodiolinae, Dacrydiinae, and Septiferinae) by placing Musculinae species into Crenellinae and adding two additional subfamilies: Dacrydiinae Ockelmann, 1983 and Septiferinae Scarlato & Starobogatov, 1979. Recently, Bieler et al. (2010) added Limnoperninae Scarlato & Starobogatov, 1979 into Coan et al. (2000)’s classification system. Unlike many other earlier classifications where Mytilidae was a monotypic family of the superfamily Mytiloidea, there have been some taxonomic authorities that assigned mytilid mussels into three to five separate families (Scarlato and Starobogatov. 1979; Starobogatov, 1992, Bieler and Mikkelsen, 2006; Carter et al., 2011; Morton, 2015). Aside from morphology-based classifications, phylogenetic relationships among Mytilidae species have also been conducted using molecular data. Phylogenetic analyses of 18S rDNA and/or 28S rDNA sequences have indicated that Mytilidae is monophyletic (Distel, 2000; Distel et al., 2000; Steiner and Hammer, 2000; Giribet and Wheeler, 2002; Owada, 2007; Samadi et al., 2007). A recent mitochondrial genome phylogeny of some bivalve taxa with an emphasis on deep sea bivalve species placed Bathymodiolus species as sister to the non-symbiotic, shallow-water mytilid species (Ozawa et al., 2017). More recently, a molecular phylogeny of Mytilidae that included five subfamilies using nuclear (18S, 28S rDNAs and histone H3) and mitochondrial (cox1 and 16S rDNA) genes found two major clades, the Modiolinae + Bathymodiolinae clade and the Mytilinae clade (composed of Septifer, Musculus, and Brachidontes species) with polyphyletic Lithophaginae members (Liu et al., 2018). However, the phylogenetic position of the only freshwater mussel (Limnoperninae) within the family was not clearly resolved in this analysis. Moreover, earlier molecular analyses did not include taxon sampling of subfamily representatives sufficient to fully assess phylogenetic inter-relationships among the subfamilies of Mytilidae. In this study we newly determined the complete mitochondrial genomes of nine Mytilidae species, Bathymodiolus thermophilus Kenk and B.R. Wilson, 1985, Modiolus nipponicus (Oyama, 1950), Lithophaga curta (Lischke, 1874), Brachidontes mutabilis (Gould, 1861), Mytilisepta virgata (Wiegmann, 1837), Mytilisepta keenae (Nomura, 1936), Crenomytilus grayanus (Dunker, 1853), Gregariella coralliophaga (Gmelin, 1791), and Septifer bilocularis (Linnaeus, 1758). These species represent seven Mytilidae subfamilies and include the first sequenced representatives of the subfamilies Septiferinae and Lithophaginae. We used 28 mytilid mitochondrial genomes (including the 9 newly sequenced species) to infer phylogenetic inter-relationships and estimate divergence time among the subfamilies.
2.2. Next generation sequencing and sequence assembly For B. thermophilus and M. nipponicus, genomic DNA was prepared using the Illumina TruSeq DNA library preparation procedure to generate one paired-end library (101 bp read length), which was sequenced on an Illumina HiSeq 4000. Low-quality reads with an error probability of > 50% and Illumina TruSeq adaptor sequences were removed using Trimmomatic v.0.33 (Bolger et al., 2014). The complete genome sequences of B. thermophilus (clean data: 442,593,198,827 bp; reads: 3,034,838,915) and M. nipponicus (clean data: 74,625,809,796 bp; reads: 756,167,692) were assembled using MITObim (Hahn et al., 2013) based on the tutorial “reconstructing mt genomes from mt barcode seeds” on the MITObim website (https://github.com/chrishah/ MITObim). The partial cox1 sequence was used for the barcode seed sequence. 2.3. PCR amplification and assembly For L. curta, B. mutabilis, M. virgata, M. keenae, C. grayanus, G. coralliophaga, and S. bilocularis mitochondrial genomes were obtained using a long-PCR strategy. Five partial fragments from cox1, cox3, cob, rrnS, and rrnL gene regions were amplified using the primer sets LCO1490/HCO2198 (Folmer et al., 1994), cox3F/cox3R (Boore et al., 2005), UCYTB144F/UCYTB272R (Merritt et al., 1998), 12SF/12SR (Machida et al., 2012), and 16SbrL/16SbrH (Palumbi et al., 1991), respectively. PCR reactions were conducted in 50 μL of TaKaRa Ex Taq PCR mixture containing 2 μL of template DNA, 36.75 μL of distilled water, 5 μL of 10x Ex Taq buffer, 1 μL of each primer, 4 μL of dNTP, and 0.25 μL of TaKaRa Ex Taq DNA polymerase. The reaction conditions were 1 cycle of denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 45–60 °C (depending on primer specificity) for 30 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Species-specific primer sets for long PCR amplification were designed (Supplementary Table 2), and long PCR products (size: ∼1.2–6 kb) were obtained in 50 μL reactions containing 1 μL of template DNA, 29.5 μL of distilled water, 5 μL of 10x Taq buffer, 1 μL of each primer, 8 μL of dNTP, 4 μL of MgCl2, and 0.5 μL of LA Taq DNA polymerase, with the following amplification conditions: 1 cycle of denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing using a gradient of 55–65 °C for 30 s, extension at 72 °C for 1 min per kb, and a final extension at 72 °C for 15 min. The amplified PCR products were isolated on 0.6% or 1% agarose gels using a QIAquick gel extraction kit (QIAGEN, Valencia, CA, USA) following standard protocols and sequenced directly using an ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA) with the same primers used for PCR or by primer walking. The PCR amplified fragments were analyzed using Geneious Pro v.8.1.9 (Biomatters, Auckland, New Zealand). 2.4. Mitochondrial genome annotation and gene arrangement The complete mitochondrial genomes were annotated on the MITOS webserver (Bernt et al., 2013), and the protein coding genes (PCGs) were determined using find ORFs (open reading frames) in Geneious and confirmed by comparison with other mytilid species. The two rRNA genes were identified by sequence comparison with previously reported mytilid rRNA genes. The tRNA genes were identified using tRNAscanSE Search Server v.2.0 (Lowe and Chan, 2016) and confirmed by manually inspecting potential cloverleaf secondary structures and anticodon sequences. Alignment of the whole mitochondrial genomes was performed with the progressive Mauve alignment v.2.3.1 (Darling et al., 2004) in Geneious to find and visualize conserved gene order clusters in mitochondrial genomes among mytilid species.
2. Materials and methods 2.1. DNA extraction Genomic DNA from Bathymodiolus thermophilus and Modiolus nipponicus was isolated from frozen tissue following a standard phenol chloroform extraction. For Lithophaga curta, Brachidontes mutabilis, Mytilisepta virgata, Mytilisepta keenae, Crenomytilus grayanus, Gregariella coralliophaga, and Septifer bilocularis DNA was extracted from ethanolfixed tissue (adductor muscle) using an E.Z.N.A. mollusc DNA kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s instructions. 2
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Table 1 Complete mitochondrial genomes used for phylogenetic analysis in this study. Species
Subfamily
Location
bp
GenBank accession no.
Reference
Bathymodiolus japonicus Bathymodiolus platifrons Bathmodiolus septemdierm Bathymodiolus thermophilus Modiolus modiolus Modiolus kurilensis Modiolus nipponicus Modiolus philippinarum Lithophaga curta Limnoperna fortunei Brachidontes exustus Brachidontes mutabilis Mytilisepta virgata Mytilisepta virgata Mytilisepta keenae Mytilus chilensis Mytilus edulis Mytilus galloprovincialls Mytilus trossulus Mytilus coruscus Mytilus californianus Crenomytilus grayanus Perna perna Perna canaliculus Perna viridis Gregariella coralliophaga Musculista senhousia Septifer bilocularis Anadara sativa Crassostrea gigas
Bathymodiolinae Bathymodiolinae Bathymodiolinae Bathymodiolinae Modiolinae Modiolinae Modiolinae Modiolinae Lithophaginae Limnoperninae Brachidontinae Brachidontinae Brachidontinae Brachidontinae Brachidontinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Crenellinae Crenellinae Septiferinae – –
Japan, Sagami Bay Japan, Sagami Bay Japan, Myojin knoll East Pacific Rise 7°N Canada, Nova Scotia China, Qingdao Korea, Jindo China, Hongkong Japan, Umegahama Brazil, Jacui River USA, Madeira Beach Korea, Jejudo Korea, Jindo China, Zhoushan Korea, Jindo Chile, Chiloe – Greece, Heraklion Canada, Nova Scotia Korea, Gageocho – Korea, Yangyang Brazil, Praia Vermelha Australia, Canberra China, Yanghiang Korea, Wando Italy, Venice Lagoon Korea, Jejudo – –
17,510 17,653 17,069 18,819 15,816 16,210 15,638 16,389 16,580 18,145 16,600 16,531 14,703 14,715 15,902 16,765 16,740 16,744 18,652 16,642 16,729 17,582 18,415 16,005 16,014 16,273 21,557 16,253 48,161 18,224
AP014560 AP014561 AP014562 MK721544 KX821782 KY242717 MK721547 KY705073 MK721546 KP756905 KM233636 MK721541 MK721548 KX094521 MK721542 KP100300 AY484747 AY497292 AY823625 KJ577549 JX486124 MK721543 KM655841 MG766134 JQ970425 MK721545 GU001953 MK721549 KF667521 AF177226
Ozawa et al. (2017) Ozawa et al. (2017) Ozawa et al. (2017) This study Robicheau et al. (2017) – This study – This study Uliano-Silva et al. (2016b) Bennett et al. (2016) This study This study – This study Gaitán-Espitia et al. (2016) Hoffmann et al. (1992) Mizi et al. (2005) Breton et al. (2006) Lee and Lee (2016) – This study Uliano-Silva et al. (2016a) – Li et al. (2012) This study Passamonti et al. (2011) This study – –
2.5. Phylogenetic analyses
monophyly of the subfamily Mytilinae, monophyly of Crenellinae, and ((Bathymodiolinae + Modiolinae) + Limnoperninae)) + (((Mytilinae + Crenellinae) + Septiferinae) + Brachidontinae) + Lithophaga) (Supplementary Table 4). Alternative trees were used for the calculation of likelihood under the best-fit substitution model.
We used 28 mitochondrial (27 complete plus 1 partial) genome sequences representing a total of 27 mytilid species (two individuals from Mytilisepta virgata) for phylogenetic analyses (Table 1), with Crassostrea gigas and Anadara sativa as outgroups. Nucleotide sequences of the 12 protein coding genes (PCGs) and two rRNA genes were concatenated, excluding the ATPase 8 gene. The inferred amino acid sequences for each of the 12 PCGs were aligned separately using Clustal W (Thompson et al., 1994) with default options. The two rRNA sequences were individually aligned in Muscle (Edgar, 2004) using default parameters. All alignments were performed in Geneious software. Conserved regions of the aligned sequences were obtained for each of the 12 PCGs and concatenated using Gblocks v.0.91b (Castresana, 2000), with half of the gap positions allowed. The best partition schemes and best-fit substitution model for the dataset were identified using Partition Finder 2 (Lanfear et al., 2016) with the Bayesian information criterion (BIC). Each of the 12 PCGs and rRNA genes was treated as a separate partition (Supplementary Table 3). Phylogenetic relationships were inferred using Maximum Likelihood (ML) with RAxML v.8.2.9 (Stamatakis, 2014) and Bayesian inference (BI) with MrBayes v.3.2.6 (Huelsenbeck and Ronquist, 2001) on the CIPRES portal (Miller et al., 2010). Bootstrap analysis for the ML tree was performed with 10,000 bootstrap replicates. The BI analysis was performed using the Markov chain Monte Carlo (MCMC) method, with two independent runs of 1 × 106 generations with four chains, sampling every 100 generations. Bayesian posterior probability (BPP) values were estimated after discarding the initial 2500 trees (the first 2.5 × 105 generations) as burn-in by confirming the MCMC chains reached full stationarity using Tracer v.1.6 (Rambaut et al., 2013). In order to compare the relationships revealed from the present study with the previous hypotheses, we assessed alternative tree topologies with an approximately unbiased (AU) test using IQ-TREE v.1.6.8 (Nguyen et al., 2014) and 20,000 bootstrap replicates. Specifically, we performed tree topology tests in which the following relationships were constrained:
2.6. Estimation of divergence times Divergence times among subfamilies were estimated using the nucleotide sequences of 12 PCGs and two rRNA genes with a relaxed clock log normal model in BEAST v.2.4.8 (Drummond and Rambaut, 2007). We selected the Calibrated Yule model for the tree prior, and the best partition schemes and the best evolution models were estimated in Partition Finder 2 based on BIC. For estimating divergence time calibration, we used the Modiolinae fossil record data (393–408 MYA), which is similar to the record in the Paleobiology Database (http:// paleodb.org) and Soot-Ryen (1969). The final Markov chain was run twice, for each 1 × 109 generations, sampling every 10,000 generations with the first 25% of generations discarded as burn-in, after confirming convergence of chains with Tracer v.1.6 (Rambaut et al., 2013). The effective sample size of the majority of parameters was > 500. The maximum clade credibility (MCC) tree was visualized in FigTree v.1.4.3 (Rambaut, 2014). 3. Results and discussion 3.1. General features of Mytilidae mitochondrial genomes The complete mitochondrial genomes of the nine new mytilid species are all circular molecules ranging from 14,703 bp (M. virgata) to 18,819 bp (B. thermophilus) in size, which is similar to previously reported bivalve species (Table 1). The size of the control regions is highly variable, as well documented from many other metazoan taxa, 3
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Fig. 1. Linearized mitochondrial gene arrangement patterns of Mytilidae species superimposed on the phylogenetic tree (see Fig. 2 for detailed relationships among mytilid species). Gene and genome size are not scaled. The tRNAs are labelled by single-letter abbreviations of amino acid code.
contributing to the overall genome size variation (Supplementary Tables 5–13). The M. virgata genome is the smallest in size to date (in Mytilidae) and has no control region. Gene order and the number of tRNA genes also vary widely (Fig. 1), as is common in other bivalves (Boore et al., 2004; Breton et al., 2010). Gene arrangement among the species, particularly among different subfamilies, is highly variable, except that all Mytilus species and Crenomytilus grayanus are identical in their gene order. The mitochondrial genomes of B. thermophilus, C. grayanus, G. coralliophaga, and S. bilocularis contain 13 PCGs, 2 rRNA, and 23 tRNA genes (B. thermophilus has three copies of trnL [anticodon: CUG]; C. grayanus, G. coralliophaga, and S. bilocularis have two copies of trnM [AUA]). The M. virgata mtDNA contains 13 PCGs, 22 tRNA and 2 rRNA genes. B. mutabilis contains 13 PCGs, 24 tRNA and 2 rRNA genes (two copies of trnM [AUA] and two copies of trnL [UUU]). M. keenae contains 12 PCGs (lacking atp8), 23 tRNA and 2 rRNA genes (two copies of trnM [AUA]). M. nipponicus and L. curta contain 12 PCGs (lacking atp8), 22 tRNA and 2 rRNA genes. M. nipponicus, L. curta, and M. keenae mtDNAs lack the atp8 gene, which was asserted to occur as a pseudogene in some other Mytilidae species (Uliano-Silva et al., 2016b).
3.2. Phylogenetic relationships and mitochondrial gene arrangements Phylogenetic relationships among Mytilidae subfamilies were inferred for a concatenated dataset (16,550 bp long) of the 12 proteincoding genes and two rRNA genes using ML and BI methods. Tree topology results from both ML and BI analyses were identical in that the family is subdivided into two clades and the overall relationships among their subfamilies (Fig. 2): Clade 1 included the subfamilies Bathymodiolinae, Modiolinae, Limnoperninae, and Lithophaginae, and Clade 2 contained the subfamilies Brachidontinae, Mytilinae, Crenellinae, and Septiferinae, with all branches having high supporting values (100% BP and 1.00 BPP). The Mauve alignment for the mitochondrial genomes indicated that syntenic blocks among the same clade members were much more similar to each other than to species belonging to different clades (Supplementary Fig. S1A and B). Within Clade 1, Bathymodiolinae was sister to Modiolinae, which agrees with some recent studies (Samadi et al., 2007; Ozawa et al., 2017; Liu et al., 2018) but contradicts a previous 18S rDNA phylogeny where Modiolinae occupied a position basal to other mytilid taxa 4
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Fig. 2. Reconstructed phylogenetic tree based on the mitochondrial genomes (12 protein coding genes plus two rRNA genes) of 27 Mytilidae species (two individuals of M. virgata) using maximum likelihood and Bayesian methods. Bayesian posterior probability/maximum likelihood bootstrap support values are shown in the branches. An asterisk denotes mtDNA sequence newly determined in this study.
(Bathymodiolinae)+(Lithophaginae+(Mytilinae + Crenellinae))) (Distel et al., 2000). The gene order of these two family members is the same and represented a single syntenic block of 12 PCGs and 2 rRNA genes, excluding tRNA genes, where considerable translocational changes are normally observed (Fig. 1, Supplementary Fig. S2). This result differs from a previous anatomical study (Morton, 2015) that argued Bathymodiolus was more closely related to Musculista (Crenellidae) and Limnoperna (Limnopernidae) than Modiolus (Modiolinae). The freshwater mussel member Limnoperninae was sister to Lithophaginae (100% BP and 1.00 BPP) and they were placed sister to (Bathymodiolinae + Modiolinae) within Clade 1. These relationships are different from an earlier molecular analysis of a combined dataset of mitochondrial and nuclear gene sequences that placed Lithophaga basal
to a grouping of (((Mytilinae + Musculinae) + Septiferinae) + Brachidontinae) of Clade 2, with Leiosolenus (the other Lithophaginae member) positioned basal to Bathymodiolinae + Modiolinae of Clade 1, rendering Lithophaginae polyphyletic (Liu et al., 2018). A tree topology test examining the alternative hypothesis of Lithophaga in a basal position within Clade 2 was statistically rejected in this study (Supplementary Table 4). However, we are not able to test whether Lithophaginae is polyphyletic, since only one Lithophaginae representative (L. curta) is included in this study. The results from the Mauve alignment analysis showed four synteny blocks shared between Lithophaga curta and Bathymodiolinae + Modiolinae; this similarity in mitochondrial genome structure is additional evidence of their close relationship (Supplementary Fig. S3). The four blocks were (1) cox15
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Fig. 3. Estimates of divergence times using BEAST v.2.4.8. Numbers on the nodes indicate the mean divergence times and 95% highest posterior density (HPD) intervals of the divergence time are shown in the parentheses below the horizontal bars. Calibration point is marked by an arrow.
trnL1(CUN), (2) rrnS-trnW, (3) cytochrome b, (4) nad4L-nad1. The only difference in the syntenic arrangements is found in L. curta where the cytochrome b (cob) synteny block is translocated. The results from the synteny block analysis is more complicated when Limnoperna fortunei, the only freshwater mytilid member, is included, as the gene order of this species is idiosyncratic and substantially different from other species belong to Clade 1. In Clade 2, the two Crenellinae species G. coralliophaga and M. senhousia were nested within Mytilinae (i.e., polyphyletic) with a very strong branch support value (100% BP and 1.00 BPP). Tree topology tests of alternative hypotheses in which Mytilinae and Crenellinae were each constrained to be monophyletic were rejected. All Mytilus species (Mytilinae) were clustered with G. coralliophaga, whereas M. senhousia was grouped with another Mytilinae branch of Perna canaliculus and P. viridis with high branch support values (100% BP and 1.00 BPP). Moreover, the result of Mauve alignment between G. coralliophaga and Mytilus + Crenomytilus species showed very strong colinearity in their syntenic structure (Supplementary Fig. S4). The interrelationships of Perna and Musculista still remain unresolved, varying in earlier works according to the characters employed in the analysis: earlier studies based on spermatozoa structure and shell morphology independently placed them in the subfamilies Mytilinae and Crenellinae, respectively (Kafanov and Drozdov, 1998; Coan et al., 2000). On the other hand, based on the anatomical feature of having the pericardial complex located between two posterior byssal retractor muscle blocks, both Perna and Musculista were placed in the same subfamily (Musculinae) separate from Crenellinae (which contained Gregariella) (Morton, 2015). The sister relationship between Perna and Musculista species within
Mytilinae was also supported by a recent molecular phylogeny using mitochondrial and nuclear gene sequences (Liu et al., 2018) and the results of the present study. Nevertheless, there is one unexpected exception found in our mtDNA analysis in regards to the position of Perna perna: surprisingly, it was separated from its congeneric species (P. canaliculus and P. viridis) and instead clustered with Brachidontes species of the subfamily Brachiodontinae (Fig. 2). Both mtDNA gene order comparison and the Mauve alignment analysis showed the highest similarity between P. perna and the two Brachiodontes species (Fig. 1, Supplementary Fig. S1B). To assess whether this unexpected position of P. perna is caused by a representation of paternal M-type sequences, we performed phylogenetic analysis for both maternal F-type and paternal M-type sequences available so far from all Mytilidae species on GenBank. The resulting tree (data not shown) showed that the P. perna mtDNA did not cluster with any M-type sequences from other Mytilidae species. Based on this result, we are not able to postulate that the P. perna mtDNA sequence is of paternal origin, corroborating the interpretation of a previous study (Uliano-Silva et al., 2016a,b). Further sequencing and phylogenetic analysis of the M-type mtDNA sequence from this species will clarify this uncertainty and resolve its enigmatic position. Septiferinae, represented by S. bilocularis, was positioned sister to a grouping of (Mytilinae + Crenellinae), and Brachidontinae was placed as sister to ((Mytilinae + Crenellinae) + Septiferinae), each receiving high branch support values (100% BP and 1.00 BPP). This topology is the same as the relationships recovered in Liu et al. (2018) because they treated Brachidontinae (Hormomya and Brachidontes) and Septiferinae (Septifer) as Mytilinae members. Interestingly, the mitochondrial 6
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genome sequence of M. virgata determined in this study and the partial genome sequence of M. virgata on GenBank (Table 1) showed a 9.1% difference in nucleotide sequence (PCGs showed a difference of 10.4%), although they were recovered as a sister group in our phylogenetic trees and had identical gene arrangement. This nucleotide sequence divergence is not likely to have resulted from the doubly uniparental inheritance (DUI) system, because the difference between M-type and Ftype mtDNAs studied so far ranges over 20% (Breton et al., 2007). It may have originated from historical isolation of populations in different marginal seas during paleoclimate changes (as reviewed by Ni et al., 2014). Similar patterns with cryptic linages (or species) have also been revealed in other species in the northwestern Pacific, including fish (Liu et al., 2007), bivalves (Ni et al., 2012; Wang et al., 2017), and shrimp (Cheng and Sha, 2017).
and Fisheries and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B4011597). Author contributions Study conception and design: JKP. Data collection: YL. Data analysis and interpretation: YL, HK, JS, SCK, TK, and JKP. Manuscript preparation: YL, SCK, TK, and JKP. Declaration of Competing Interest The authors have declared an absence of competing interests. Appendix A. Supplementary material
3.3. Estimates of divergence time among subfamilies
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ympev.2019.106533.
A high ESS (effective sample size) value (> 500) was identified for all parameters in the BEAST analysis to estimate divergence time of Mytilidae species (Fig. 3). The two major Mytilidae clades (Clade 1 and Clade 2) were estimated to have split around 400.40 MYA (95% HPD interval 409.1–391.4 MYA) in the early Devonian period, which is close to the split timing estimated in some previous analyses: Soot-Ryen (1969) and Distel (2000) indicated based on the fossil records that Mytilidae first appeared in the Devonian (with Modiolinae as the ancestral group). A recent molecular analysis (Liu et al., 2018) placed the split of the two major Mytilidae clades at 418.31 MYA, with a 95% highest posterior density (HPD) interval that spans the late Silurian and early Devonian (413.50–423.30 MYA). The Paleobiology Database (https://paleobiodb.org) indicates that the oldest Mytilidae fossil (with Mytilinae as the ancestral group) is from the Silurian. The estimated divergence times of major subfamilies in this study was similar to Liu et al. (2018). Bathymodiolinae and Modiolinae were inferred to have split in the early Jurassic (ca. 200.58 MYA). The divergence time between L. fortunei (a freshwater member of Limnoperninae) and Lithophaga curta (the boring mussel in Lithophaginae) was also estimated in the early Jurassic, dating back to approximately 196.70 MYA. The divergence time of the Limnoperninae lineage from its closest relative, the Lithophaginae group, implies the habitat transition from marine to freshwater environments by the Limnopernae ancestor may have occurred during the early Jurassic period of the Mesozoic Era. The split between (Bathymodiolinae + Modiolinae) and (Lithophaginae + Limnoperninae) was dated to 279.87 MYA in the middle Permian. Within Clade 2, Brachidontinae was the first emerged in the middle Carboniferous (335.46 MYA). Septiferinae was estimated to have branched off 278.90 MYA from the lineage leading to Mytilinae and Crenellinae. The divergence time between the genera Mytilus and Gregarella was estimated to be approximately 163.05 MYA in the late Jurassic. The split time between Musculista and Perna was estimated at 195.08 MYA in the early Jurassic. The divergence between (Mytilus + Gregarella) and (Musculista + Perna) occurred about 253.63 MYA in the early Triassic. The divergence time estimation using molecular clock analysis indicates that lineage splitting among majority of Mytilidae subfamilies occurred in the Mesozoic period (Triassic, Jurassic, and Cretaceous eras). Similarly, during this period many marine fauna also had high family diversification rates (Clarke & Crame, 2003). Since the Mesozoic was a time of warm temperatures, high sea levels, and greater continental shelf area (Hallam 1985; Haq et al., 1987), these conditions may have contributed to diversification rates (Briggs, 2006) and explain why most major bivalve groups expanded in this period (Bieler et al., 2014).
References Adarraga, I., Martínez, J., 2012. First record of the invasive brackish water mytilid Limnoperna securis (Lamarck, 1819) in the Bay of Biscay. Aquat. Invasions 7, 171–180. Bayne, B.L., 1976. Marine Mussels: Their Ecology and Physiology. Cambridge University Press, Cambridge. Bennett, K.F., Bailey, A.W., Brambert, D.J., Ferhati, E.W., Karson, C.A., Nafasat, U., Wadleigh, J.K., Wright, A.H., 2016. The F type mitochondrial genome of the scorched mussel: Brachidontes exustus (Mytiloida, Mytilidae). Mitochondrial DNA Part A 27, 1501–1502. Bernard, F.R., 1983. Catalogue of the living bivalvia of the eastern Pacific Ocean: Bering Strait to Cape Horn. Can. Special Publ. Fish. Aquat. Sci. 61. Bernt, M., Donath, A., Jühling, F., Externbrink, F., Florentz, C., Fritzsch, G., Pütz, J., Middendorf, M., Stadler, P.F., 2013. MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319. Bieler, R., Mikkelsen, P.M., 2006. Bivalvia-a look at the branches. Zool. J. Linn. Soc. 148, 223–235. Bieler, R., Carter, J.G., Coan, E.V., 2010. Classification of bivalve families. In: In: Bouchet, P., Rocroi, J.-.P., Bieler, R., Carter, J.G., Coan, E.V. (Eds.), Nomenclator of bivalve families with a classification of bivalve families, Malacologiapp. 1–184. Bieler, R., Mikkelsen, P.M., Collins, T.M., Glover, E.A., González, V.L., Graf, D.L., Harper, E.M., Healy, J.M., Kawauchi, G.Y., Sharma, P.P., Staubach, S., Strong, E.E., Taylor, J.D., Tëmkin, I., Zardus, J.D., Clark, S., Guzmán, A., McIntyre, E., Sharp, P., Giribet, G., 2014. Investigating the Bivalve Tree of Life - an exemplar-based approach combining molecular and novel morphological characters. Invertebr. Systemat. 28, 32–115. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. Boore, J.L., Macey, J.R., Medina, M., 2005. Sequencing and comparing whole mitochondrial genomes of animals. Methods Enzymol. 395, 311–348. Boore, J.L., Medina, M., Rosenberg, L.A., 2004. Complete sequences of the highly rearranged molluscan mitochondrial genomes of the scaphopod Graptacme eborea and the bivalve Mytilus edulis. Mol. Biol. Evol. 21, 1492–1503. Boss, K.J., 1982. Mollusca. In: Parker, S.P. (Ed.), Synopsis and Classification of Living Organisms. McGraw-Hill Book Company, New York, pp. 945–1166. Breton, S., Burger, G., Stewart, D.T., Blier, P.U., 2006. Comparative analysis of genderassociated complete mitochondrial genomes in marine mussels (Mytilus spp.). Genetics 172, 1107–1119. Breton, S., Doucet-Beaupré, H., Stewart, D.T., Hoeh, W.R., Blier, P.U., 2007. The unusual system of doubly uniparental inheritance of mtDNA: isn't one enough? Trends Genet. 23, 465–474. Breton, S., Stewart, D.T., Hoeh, W.R., 2010. Characterization of a mitochondrial ORF from the gender-associated mtDNAs of Mytilus spp. (Bivalvia: Mytilidae): Identification of the “missing” ATPase 8 gene. Mar. Geonom. 3, 11–18. Briggs, J.C., 2006. Proximate sources of marine biodiversity. J. Biogeogr. 33, 1–10. Carter, J.G., Altaba, C.R., Anderson, L.C., Araujo, R., Biakov, A.S., Bogan, A.E., Campbell, D.C., Campbell, M., Jin-hua, C., Cope, J.C., 2011. A synoptical classification of the Bivalvia (Mollusca). Paleontol. Contrib. 4, 1–47. Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552. Cheng, J., Sha, Z.L., 2017. Cryptic diversity in the Japanese mantis shrimp Oratosquilla oratoria (Crustacea: Squillidae): Allopatric diversification, secondary contact and hybridization. Sci. Rep. 7, 1972. Clarke, A., Crame, J.A., 2003. The importance of historical processes in global patterns of diversity. In: Blackburn, T.M., Gaston, K.J. (Eds.), Macroecology. Blackwell, Oxford, UK, pp. 130–151. Coan, E.V., Scott, P.V., Bernard, F.R., 2000. Bivalve Seashells of Western North America: Marine Bivalve Mollusks from Arctic Alaska to Baja California. Santa Barbara Museum of Natural History.
Funding This research was supported by a grant from the Marine Biotechnology Program (20170431) funded by the Ministry of Oceans 7
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Y. Lee, et al.
from the pericardial–posterior byssal retractor musculature complex. Molluscan Res. 35, 227–245. Newell, N.D., 1969. Classification of Bivalvia. In: Moore, R.C. (Ed.), Treatise on Invertebrate Paleontology. Geological Society of America and University of Kansas Press, pp. 205–224. Ni, G., Li, Q., Kong, L., Yu, H., 2014. Comparative phylogeography in marginal seas of the northwestern Pacific. Mol. Ecol. 23, 534–548. Ni, G., Li, Q., Kong, L.F., Zheng, X.D., 2012. Phylogeography of bivalve Cyclina sinensis: testing the historical glaciations and Changjiang River outflow hypotheses in northwestern Pacific. PLoS ONE 7, e49487. Owada, M., 2007. Functional morphology and phylogeny of the rock-boring bivalves Leiosolenus and Lithophaga (Bivalvia: Mytilidae): a third functional clade. Mar. Biol. 150, 853–860. Ozawa, G., Shimamura, S., Takaki, Y., Yokobori, S.I., Ohara, Y., Takishita, K., Maruyama, T., Fujikura, K., Yoshida, T., 2017. Updated mitochondrial phylogeny of Pteriomorph and Heterodont Bivalvia, including deep-sea chemosymbiotic Bathymodiolus mussels, vesicomyid clams and the thyasirid clam Conchocele cf. bisecta. Mar. Geonom. 31, 43–52. Palumbi, S., Martin, A., Romano, S., Mcmillan, W.O., Stice, L., Grabowski, G., 1991. The Simple Fool’s Guide to PCR. Version 2. Department of Zoology and Kewalo Marine Laboratory. University of Hawaii, Honolulu, USA. Passamonti, M., Ricci, A., Milani, L., Ghiselli, F., 2011. Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae). BMC Genom. 12, 442. Pawiro, S., 2010. Bivalves: global production and trade trends. In: Rees, G., Pond, K., Kay, D., Bartram, J., Santo Domingo, J. (Eds.), Safe Management of Shellfish and Harvest Waters. IWA Publishing, London, UK, pp. 11–19. Phillips, D.J.H., 1976. The common mussel Mytilus edulis as an indicator of pollution by zinc, cadmium, lead and copper. I. Effects of environmental variables on uptake of metals. Mar. Biol. 38, 59–69. Rambaut, A., 2014. FigTree v1.4.3. Available from: http://tree.bio.ed.ac.uk/software/ figtree/. Rambaut, A., Suchard, M.A., Xie, W., Drummond, A., 2013. Tracer v1.6. Available from: http://tree.bio.ed.ac.uk/software/tracer/. Ricciardi, A., 1998. Global range expansion of the Asian mussel Limnoperna fortunei (Mytilidae): another fouling threat to freshwater systems. Biofouling 13, 97–106. Robicheau, B.M., Powell, A.E., Del Bel, L., Breton, S., Stewart, D.T., 2017. Evidence for extreme sequence divergence between the male-and female-transmitted mitochondrial genomes in the bivalve mollusc, Modiolus modiolus (Mytilidae). J. Zool. Syst. Evol. Res. 55, 89–97. Samadi, S., Quéméré, E., Lorion, J., Tillier, A., von Cosel, R., Lopez, P., Cruaud, C., Couloux, A., Boisselier-Dubayle, M.C., 2007. Molecular phylogeny in mytilids supports the wooden steps to deep-sea vents hypothesis. C.R. Biol. 330, 446–456. Scarlato, O.A., Starobogatov, Y.I., 1979. The system of the suborder Mytileina (Bivalvia). In: Molljuski. Osnoynye rezultalty ikh izuchenija. 6. pp. 22–25. Soot-Ryen, T., 1969. Superfamily Mytilacea Rafinesque, 1815. In: Moore, R.C. (Ed.), Treatise on Invertebrate Paleontology, Mollusca. Geological Society of America Inc. and University of Kansas Press, Lawrence, Kansas, pp. 271–280. Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies. Bioinformatics 30, 1312–1313. Stanley, S.M., 1970. Relation of Shell form to Life Habits of the Bivalvia (Mollusca). Geological Society of America. Starobogatov, Y.I., 1992. Morphological basis for phylogeny and classification of Bivalvia. Ruthenica 2, 1–25. Steiner, G., Hammer, S., 2000. Molecular phylogeny of the Bivalvia inferred from 18S rDNA sequences with particular reference to the Pteriomorphia. In: Harpre, E.M., Taylor, J.D., Crame, J.A. (Eds.), The Evolutionary Biology of the Bivalvia. Geological Society, London, Special Publications, pp. 11–29. Taylor, B.E., Jamieson, G., Carefoot, T.H., 1992. Mussel culture in British Columbia: the influence of salmon farms on growth of Mytilus edulis. Aquaculture 108, 51–66. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Uliano-Silva, M., Americo, J., Bastos, A.S., Furtado, C., Rebelo, M.D.F., Prosdocimi, F., 2016a. Complete mitochondrial genome of the brown mussel Perna perna (Bivalve, Mytilidae). Mitochondrial DNA Part A 27, 3955–3956. Uliano-Silva, M., Americo, J.A., Costa, I., Schomaker-Bastos, A., de Freitas Rebelo, M., Prosdocimi, F., 2016b. The complete mitochondrial genome of the golden mussel Limnoperna fortunei and comparative mitogenomics of Mytilidae. Gene 577, 202–208. Vakily, J.M., 1989. The Biology and Culture of Mussels of the Genus Perna. ICLARM Studies and Reviews 17. International Centre for Living Aquatic Resources Management, Manila, Philippines, and Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ) GmbH, Eschborn, Germany, pp. 1–63. Vázquez-Luis, M., Morató, M., Campillo, J.A., Guitart, C., Deudero, S., 2016. High metal contents in the fan mussel Pinna nobilis in the Balearic Archipelago (western Mediterranean Sea) and a review of concentrations in marine bivalves (Pinnidae). Scientia Marina. 80, 111–122. Wang, X., Kong, L., Chen, J., Matsukuma, A., Li, Q., 2017. Phylogeography of bivalve Meretrix petechialis in the Northwestern Pacific indicated by mitochondrial and nuclear DNA data. PLoS ONE 12, e0183221.
Cossa, D., 1988. Cadmium in Mytilus spp.: worldwide survey and relationship between seawater and mussel content. Mar. Environ. Res. 26, 265–284. Darling, A.E., Mau, B., Blattner, F.R., Perna, N.T., 2004. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14, 1394–1403. Distel, D.L., 2000. Phylogenetic relationships among Mytilidae (Bivalvia): 18S rRNA data suggest convergence in mytilid body plans. Mol. Phylogenet. Evol. 15, 25–33. Distel, D.L., Baco, A.R., Chuang, E., Morrill, W., Cavanaugh, C.M., Smith, C., 2000. Do mussels take wooden steps to deep-sea vents? Nature 403, 725–726. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Duperron, S., Gaudron, S.M., Rodrigues, C.F., Cunha, M.R., Decker, C., Olu, K., 2013. An overview of chemosynthetic symbioses in bivalves from the North Atlantic and Mediterranean Sea. Biogeosciences. 10, 3241–3267. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech, 3, 294–299. Gaitán-Espitia, J.D., Quintero-Galvis, J.F., Mesas, A., D’Elía, G., 2016. Mitogenomics of southern hemisphere blue mussels (Bivalvia: Pteriomorphia): Insights into the evolutionary characteristics of the Mytilus edulis complex. Sci. Rep. 6, 26853. Giribet, G., Wheeler, W., 2002. On bivalve phylogeny: a high-level analysis of the Bivalvia (Mollusca) based on combined morphology and DNA sequence data. Invertebr. Biol. 121, 271–324. Habe, T., 1977. Systematics of Mollusca in Japan: Bivalvia and Scaphopoda. Hokuryukan, Tokyo. Hahn, C., Bachmann, L., Chevreux, B., 2013. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Res. 41 e129-e129. Hallam, A., 1985. A review of Mesozoic climates. J. Geol. Soc. 142, 433–445. Haq, B.U., Hardenbol, J.A.N., Vail, P.R., 1987. Chronology of fluctuating sea levels since the Triassic. Science 235, 1156–1167. Hoffmann, R.J., Boore, J.L., Brown, W.M., 1992. A novel mitochondrial genome organization for the blue mussel, Mytilus edulis. Genetics 131, 397–412. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Hwang, D.S., Gim, Y., Yoo, H.J., Cha, H.J., 2007. Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials 28, 3560–3568. Jenner, H.A., Whitehouse, J.W., Taylor, C.J.L., Khalanski, M., 1998. Cooling water management in European power stations: biology and control of fouling. Hydroécologie Appliquée 10, 1–225. Kafanov, A.I., Drozdov, A.L., 1998. Comparative sperm morphology and phylogenetic classification of Recent Myiloidea (Bivalvia). Malacologia 39, 129–139. Kenk, V.C., Wilson, B.R., 1985. A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos rift zone. Malacologia 26, 253–271. Kim, H.J., Yang, B., Park, T.Y., Lim, S., Cha, H.J., 2017. Complex coacervates based on recombinant mussel adhesive proteins: their characterization and applications. Soft Matter 13, 7704–7716. Nguyen, L.T., Schmidt, H.A., von Haeseler, A., Minh, B.Q., 2014. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274. Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B., 2016. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. 34, 772–773. Lee, Y.C., Lee, Y.H., 2016. The F type mitochondrial genome of hard-shelled mussel: Mytilus coruscus (Mytiloida, Mytilidae). Mitochondrial DNA Part A 27, 624–625. Li, X., Wu, X., Yu, Z., 2012. Complete mitochondrial genome of the Asian green mussel Perna viridis (Bivalvia, Mytilidae). Mitochondrial DNA 23, 358–360. Liu, J., Liu, H., Zhang, H., 2018. Phylogeny and evolutionary radiation of the marine mussels (Bivalvia: Mytilidae) based on mitochondrial and nuclear genes. Mol. Phylogenet. Evol. 126, 233–240. Liu, J.X., Gao, T.X., Wu, S.F., Zhang, Y.P., 2007. Pleistocene isolation in the Northwestern Pacific marginal seas and limited dispersal in a marine fish, Chelon haematocheilus (Temminck & Schlegel, 1845). Mol. Ecol. 16, 275–288. Lowe, T.M., Chan, P.P., 2016. tRNAscan-SE On-line: integrating search and contextual for analysis of transfer RNA genes. Nucleic Acids Res. 44, 54–57. Lutz, R.A., Kennish, M.J., 1993. Ecology of deep-sea hydrothermal vent communities: a review. Rev. Geophys. 31, 211–242. Machida, R.J., Kweskin, M., Knowlton, N., 2012. PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences. PLoS ONE 7, e35887. Merritt, T.J.S., Shi, L., Chase, M.C., Rex, M.A., Etter, R.J., Quattro, J.M., 1998. Universal cytochrome b primers facilitate intraspecific studies in molluscan taxa. Mol. Mar. Biol. Biotech. 7, 7–11. Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: 2010 Gateway Computing Environments Workshop (GCE). IEEE, New Orleans, pp. 1–8. Mizi, A., Zouros, E., Moschonas, N., Rodakis, G.C., 2005. The complete maternal and paternal mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis: implications for the doubly uniparental inheritance mode of mtDNA. Mol. Biol. Evol. 22, 952–967. Morton, B., 2015. Evolution and adaptive radiation in the Mytiloidea (Bivalvia): clues
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A mitochondrial genome phylogeny of Mytilidae (Bivalvia: Mytilida) a
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Yucheol Lee , Haena Kwak , Jinkyung Shin , Seung-Chul Kim , Taeho Kim , Joong-Ki Park a b
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Department of Biological Sciences, Sungkyunkwan University, 2066 Seobu-ro, Suwon, Gyeonggi-do 16419, Republic of Korea Division of EcoScience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: Mitochondrial genome Molecular phylogeny Mytilidae Bivalvia Mollusca
The family Mytilidae is a family of bivalve mussels that are distributed worldwide in diverse marine habitats. Within the family, classification systems and phylogenetic relationships among subfamilies remain not yet fully resolved. In this study, we newly determined 9 mitochondrial genome sequences from 7 subfamilies: Bathymodiolus thermophilus (Bathymodiolinae), Modiolus nipponicus (Modiolinae), Lithophaga curta (the first representative of Lithophaginae), Brachidontes mutabilis (Brachidontinae), Mytilisepta virgata (Brachidontinae), Mytilisepta keenae (Brachidontinae), Crenomytilus grayanus (Mytilinae), Gregariella coralliophaga (Crenellinae), and Septifer bilocularis (the first representative of Septiferinae). Phylogenetic trees using maximum likelihood and Bayesian inference methods for 28 mitochondrial genomes (including 19 previously published sequences) showed two major clades with high support values: Clade 1 ((Bathymodiolinae + Modiolinae) + (Lithophaginae + Limnoperninae)) and Clade 2 (((Mytilinae + Crenellinae) + Septiferinae) + Brachidontinae). The position of the genus Lithophaga (representing Lithophaginae) differed from a previously published molecular phylogeny. Divergence time analysis with a molecular clock indicated that lineage splitting among the major subfamilies of Mytilidae (including the habitat transition from marine to freshwater environments by ancestral Limnoperninae) occurred in the Mesozoic period, coinciding with high diversification rates of marine fauna during that time. This is the first mitochondrial genome-based phylogenetic study of the Mytilidae that covers nearly all subfamily members, excluding the subfamily Dacrydiinae.
1. Introduction The family Mytilidae, also known as marine mussels, has received much attention for its economic and environmental importance: some members of this family are well-known as invasive and/or biofouling species (e.g., Limnoperna fortunei) that damage power plant cooling systems and fish farms (Bayne and Bayne, 1976; Jenner et al., 1998), while others (e.g., Mytilus spp., Perna spp.) are edible and have important commercial value (Vakily, 1989; Taylor et al., 1992; Pawiro, 2010). In addition, Mytilus species have long been used as indicator species for assessing environmental pollution (Phillips, 1976; Cossa, 1988; Vázquez-Luis et al., 2016). More recently, they have been the focus of biomaterials research due to the potential commercial importance of the adhesive proteins in its byssal threads (Hwang et al., 2007; Kim et al., 2017). Mytilidae comprises epifaunal or semi-infaunal species that occupy diverse (mostly subfamily-specific) habitats (Stanley, 1970; Distel, 2000) including intertidal, deep sea, and freshwater zones (Soot-Ryen, 1969; Distel, 2000): some species (e.g., Mytilus and Modiolus) secrete byssal threads and attach to hard substrates, while Lithophaginae ⁎
species bore into and inhabit holes in rocks and corals (Bayne, 1976; Owada, 2007). Most species are found in coastal seas, but Bathymodiolinae species inhabit deep sea hydrothermal vents and cold seeps at depths of up to 3000–7000 m, where they have symbiotic associations with chemoautotrophic bacteria (Lutz and Kennish, 1993; Distel et al, 2000; Duperron et al., 2013). Unlike other subfamilies, Limnoperninae is the only member of Mytilidae that has successfully invaded and adapted to brackish and/or freshwater environments (Ricciardi, 1998; Adarraga and Martínez, 2012). Considering these diverse life styles, resolving phylogenetic relationships among major subfamilies within Mytilidae is essential to fully understand their evolutionary history and their diverse ecological specialization pathways. Classification systems of the family Mytilidae based on different morphological features (e.g., shell morphology, internal anatomy, sperm microstructure) in previous studies are very complicated and disagree with each other over the taxonomic arrangement of subordinate subfamily groups (see Supplementary Table 1 for details). Early on, Soot-Ryen (1969) divided the extant Mytilidae (superfamily Mytiloidea) into four subfamilies based on paleontological data, and this system was followed by Newell (1969), Boss (1982) and Bernard
Corresponding author. E-mail address: [email protected] (J.-K. Park).
https://doi.org/10.1016/j.ympev.2019.106533 Received 5 April 2019; Received in revised form 6 June 2019; Accepted 7 June 2019 Available online 08 June 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved.
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(1983): Mytilinae Rafinesque, 1815; Modiolinae Keen, 1958; Crenellinae Gray, 1815; and Lithophaginae H. Adams & A. Adams 1857. Later, the two subfamilies Musculinae Iredale, 1939 and Bathymodiolinae Kenk and B.R. Wilson, 1985 were added to Mytilidae (Habe, 1977). Kafanov and Drozdov (1998) presented a classification system based on sperm microstructures; their phylogeny followed SootRyen (1969)’s system but divided Modiolinae into two subfamilies (Mytilinae, Modiolinae, Crenellinae, Musculinae, and Lithophaginae). Coan et al. (2000) recognized 7 subfamilies (Mytilinae, Modiolinae, Crenellinae, Lithophaginae, Bathymodiolinae, Dacrydiinae, and Septiferinae) by placing Musculinae species into Crenellinae and adding two additional subfamilies: Dacrydiinae Ockelmann, 1983 and Septiferinae Scarlato & Starobogatov, 1979. Recently, Bieler et al. (2010) added Limnoperninae Scarlato & Starobogatov, 1979 into Coan et al. (2000)’s classification system. Unlike many other earlier classifications where Mytilidae was a monotypic family of the superfamily Mytiloidea, there have been some taxonomic authorities that assigned mytilid mussels into three to five separate families (Scarlato and Starobogatov. 1979; Starobogatov, 1992, Bieler and Mikkelsen, 2006; Carter et al., 2011; Morton, 2015). Aside from morphology-based classifications, phylogenetic relationships among Mytilidae species have also been conducted using molecular data. Phylogenetic analyses of 18S rDNA and/or 28S rDNA sequences have indicated that Mytilidae is monophyletic (Distel, 2000; Distel et al., 2000; Steiner and Hammer, 2000; Giribet and Wheeler, 2002; Owada, 2007; Samadi et al., 2007). A recent mitochondrial genome phylogeny of some bivalve taxa with an emphasis on deep sea bivalve species placed Bathymodiolus species as sister to the non-symbiotic, shallow-water mytilid species (Ozawa et al., 2017). More recently, a molecular phylogeny of Mytilidae that included five subfamilies using nuclear (18S, 28S rDNAs and histone H3) and mitochondrial (cox1 and 16S rDNA) genes found two major clades, the Modiolinae + Bathymodiolinae clade and the Mytilinae clade (composed of Septifer, Musculus, and Brachidontes species) with polyphyletic Lithophaginae members (Liu et al., 2018). However, the phylogenetic position of the only freshwater mussel (Limnoperninae) within the family was not clearly resolved in this analysis. Moreover, earlier molecular analyses did not include taxon sampling of subfamily representatives sufficient to fully assess phylogenetic inter-relationships among the subfamilies of Mytilidae. In this study we newly determined the complete mitochondrial genomes of nine Mytilidae species, Bathymodiolus thermophilus Kenk and B.R. Wilson, 1985, Modiolus nipponicus (Oyama, 1950), Lithophaga curta (Lischke, 1874), Brachidontes mutabilis (Gould, 1861), Mytilisepta virgata (Wiegmann, 1837), Mytilisepta keenae (Nomura, 1936), Crenomytilus grayanus (Dunker, 1853), Gregariella coralliophaga (Gmelin, 1791), and Septifer bilocularis (Linnaeus, 1758). These species represent seven Mytilidae subfamilies and include the first sequenced representatives of the subfamilies Septiferinae and Lithophaginae. We used 28 mytilid mitochondrial genomes (including the 9 newly sequenced species) to infer phylogenetic inter-relationships and estimate divergence time among the subfamilies.
2.2. Next generation sequencing and sequence assembly For B. thermophilus and M. nipponicus, genomic DNA was prepared using the Illumina TruSeq DNA library preparation procedure to generate one paired-end library (101 bp read length), which was sequenced on an Illumina HiSeq 4000. Low-quality reads with an error probability of > 50% and Illumina TruSeq adaptor sequences were removed using Trimmomatic v.0.33 (Bolger et al., 2014). The complete genome sequences of B. thermophilus (clean data: 442,593,198,827 bp; reads: 3,034,838,915) and M. nipponicus (clean data: 74,625,809,796 bp; reads: 756,167,692) were assembled using MITObim (Hahn et al., 2013) based on the tutorial “reconstructing mt genomes from mt barcode seeds” on the MITObim website (https://github.com/chrishah/ MITObim). The partial cox1 sequence was used for the barcode seed sequence. 2.3. PCR amplification and assembly For L. curta, B. mutabilis, M. virgata, M. keenae, C. grayanus, G. coralliophaga, and S. bilocularis mitochondrial genomes were obtained using a long-PCR strategy. Five partial fragments from cox1, cox3, cob, rrnS, and rrnL gene regions were amplified using the primer sets LCO1490/HCO2198 (Folmer et al., 1994), cox3F/cox3R (Boore et al., 2005), UCYTB144F/UCYTB272R (Merritt et al., 1998), 12SF/12SR (Machida et al., 2012), and 16SbrL/16SbrH (Palumbi et al., 1991), respectively. PCR reactions were conducted in 50 μL of TaKaRa Ex Taq PCR mixture containing 2 μL of template DNA, 36.75 μL of distilled water, 5 μL of 10x Ex Taq buffer, 1 μL of each primer, 4 μL of dNTP, and 0.25 μL of TaKaRa Ex Taq DNA polymerase. The reaction conditions were 1 cycle of denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 45–60 °C (depending on primer specificity) for 30 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Species-specific primer sets for long PCR amplification were designed (Supplementary Table 2), and long PCR products (size: ∼1.2–6 kb) were obtained in 50 μL reactions containing 1 μL of template DNA, 29.5 μL of distilled water, 5 μL of 10x Taq buffer, 1 μL of each primer, 8 μL of dNTP, 4 μL of MgCl2, and 0.5 μL of LA Taq DNA polymerase, with the following amplification conditions: 1 cycle of denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing using a gradient of 55–65 °C for 30 s, extension at 72 °C for 1 min per kb, and a final extension at 72 °C for 15 min. The amplified PCR products were isolated on 0.6% or 1% agarose gels using a QIAquick gel extraction kit (QIAGEN, Valencia, CA, USA) following standard protocols and sequenced directly using an ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA) with the same primers used for PCR or by primer walking. The PCR amplified fragments were analyzed using Geneious Pro v.8.1.9 (Biomatters, Auckland, New Zealand). 2.4. Mitochondrial genome annotation and gene arrangement The complete mitochondrial genomes were annotated on the MITOS webserver (Bernt et al., 2013), and the protein coding genes (PCGs) were determined using find ORFs (open reading frames) in Geneious and confirmed by comparison with other mytilid species. The two rRNA genes were identified by sequence comparison with previously reported mytilid rRNA genes. The tRNA genes were identified using tRNAscanSE Search Server v.2.0 (Lowe and Chan, 2016) and confirmed by manually inspecting potential cloverleaf secondary structures and anticodon sequences. Alignment of the whole mitochondrial genomes was performed with the progressive Mauve alignment v.2.3.1 (Darling et al., 2004) in Geneious to find and visualize conserved gene order clusters in mitochondrial genomes among mytilid species.
2. Materials and methods 2.1. DNA extraction Genomic DNA from Bathymodiolus thermophilus and Modiolus nipponicus was isolated from frozen tissue following a standard phenol chloroform extraction. For Lithophaga curta, Brachidontes mutabilis, Mytilisepta virgata, Mytilisepta keenae, Crenomytilus grayanus, Gregariella coralliophaga, and Septifer bilocularis DNA was extracted from ethanolfixed tissue (adductor muscle) using an E.Z.N.A. mollusc DNA kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s instructions. 2
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Table 1 Complete mitochondrial genomes used for phylogenetic analysis in this study. Species
Subfamily
Location
bp
GenBank accession no.
Reference
Bathymodiolus japonicus Bathymodiolus platifrons Bathmodiolus septemdierm Bathymodiolus thermophilus Modiolus modiolus Modiolus kurilensis Modiolus nipponicus Modiolus philippinarum Lithophaga curta Limnoperna fortunei Brachidontes exustus Brachidontes mutabilis Mytilisepta virgata Mytilisepta virgata Mytilisepta keenae Mytilus chilensis Mytilus edulis Mytilus galloprovincialls Mytilus trossulus Mytilus coruscus Mytilus californianus Crenomytilus grayanus Perna perna Perna canaliculus Perna viridis Gregariella coralliophaga Musculista senhousia Septifer bilocularis Anadara sativa Crassostrea gigas
Bathymodiolinae Bathymodiolinae Bathymodiolinae Bathymodiolinae Modiolinae Modiolinae Modiolinae Modiolinae Lithophaginae Limnoperninae Brachidontinae Brachidontinae Brachidontinae Brachidontinae Brachidontinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Mytilinae Crenellinae Crenellinae Septiferinae – –
Japan, Sagami Bay Japan, Sagami Bay Japan, Myojin knoll East Pacific Rise 7°N Canada, Nova Scotia China, Qingdao Korea, Jindo China, Hongkong Japan, Umegahama Brazil, Jacui River USA, Madeira Beach Korea, Jejudo Korea, Jindo China, Zhoushan Korea, Jindo Chile, Chiloe – Greece, Heraklion Canada, Nova Scotia Korea, Gageocho – Korea, Yangyang Brazil, Praia Vermelha Australia, Canberra China, Yanghiang Korea, Wando Italy, Venice Lagoon Korea, Jejudo – –
17,510 17,653 17,069 18,819 15,816 16,210 15,638 16,389 16,580 18,145 16,600 16,531 14,703 14,715 15,902 16,765 16,740 16,744 18,652 16,642 16,729 17,582 18,415 16,005 16,014 16,273 21,557 16,253 48,161 18,224
AP014560 AP014561 AP014562 MK721544 KX821782 KY242717 MK721547 KY705073 MK721546 KP756905 KM233636 MK721541 MK721548 KX094521 MK721542 KP100300 AY484747 AY497292 AY823625 KJ577549 JX486124 MK721543 KM655841 MG766134 JQ970425 MK721545 GU001953 MK721549 KF667521 AF177226
Ozawa et al. (2017) Ozawa et al. (2017) Ozawa et al. (2017) This study Robicheau et al. (2017) – This study – This study Uliano-Silva et al. (2016b) Bennett et al. (2016) This study This study – This study Gaitán-Espitia et al. (2016) Hoffmann et al. (1992) Mizi et al. (2005) Breton et al. (2006) Lee and Lee (2016) – This study Uliano-Silva et al. (2016a) – Li et al. (2012) This study Passamonti et al. (2011) This study – –
2.5. Phylogenetic analyses
monophyly of the subfamily Mytilinae, monophyly of Crenellinae, and ((Bathymodiolinae + Modiolinae) + Limnoperninae)) + (((Mytilinae + Crenellinae) + Septiferinae) + Brachidontinae) + Lithophaga) (Supplementary Table 4). Alternative trees were used for the calculation of likelihood under the best-fit substitution model.
We used 28 mitochondrial (27 complete plus 1 partial) genome sequences representing a total of 27 mytilid species (two individuals from Mytilisepta virgata) for phylogenetic analyses (Table 1), with Crassostrea gigas and Anadara sativa as outgroups. Nucleotide sequences of the 12 protein coding genes (PCGs) and two rRNA genes were concatenated, excluding the ATPase 8 gene. The inferred amino acid sequences for each of the 12 PCGs were aligned separately using Clustal W (Thompson et al., 1994) with default options. The two rRNA sequences were individually aligned in Muscle (Edgar, 2004) using default parameters. All alignments were performed in Geneious software. Conserved regions of the aligned sequences were obtained for each of the 12 PCGs and concatenated using Gblocks v.0.91b (Castresana, 2000), with half of the gap positions allowed. The best partition schemes and best-fit substitution model for the dataset were identified using Partition Finder 2 (Lanfear et al., 2016) with the Bayesian information criterion (BIC). Each of the 12 PCGs and rRNA genes was treated as a separate partition (Supplementary Table 3). Phylogenetic relationships were inferred using Maximum Likelihood (ML) with RAxML v.8.2.9 (Stamatakis, 2014) and Bayesian inference (BI) with MrBayes v.3.2.6 (Huelsenbeck and Ronquist, 2001) on the CIPRES portal (Miller et al., 2010). Bootstrap analysis for the ML tree was performed with 10,000 bootstrap replicates. The BI analysis was performed using the Markov chain Monte Carlo (MCMC) method, with two independent runs of 1 × 106 generations with four chains, sampling every 100 generations. Bayesian posterior probability (BPP) values were estimated after discarding the initial 2500 trees (the first 2.5 × 105 generations) as burn-in by confirming the MCMC chains reached full stationarity using Tracer v.1.6 (Rambaut et al., 2013). In order to compare the relationships revealed from the present study with the previous hypotheses, we assessed alternative tree topologies with an approximately unbiased (AU) test using IQ-TREE v.1.6.8 (Nguyen et al., 2014) and 20,000 bootstrap replicates. Specifically, we performed tree topology tests in which the following relationships were constrained:
2.6. Estimation of divergence times Divergence times among subfamilies were estimated using the nucleotide sequences of 12 PCGs and two rRNA genes with a relaxed clock log normal model in BEAST v.2.4.8 (Drummond and Rambaut, 2007). We selected the Calibrated Yule model for the tree prior, and the best partition schemes and the best evolution models were estimated in Partition Finder 2 based on BIC. For estimating divergence time calibration, we used the Modiolinae fossil record data (393–408 MYA), which is similar to the record in the Paleobiology Database (http:// paleodb.org) and Soot-Ryen (1969). The final Markov chain was run twice, for each 1 × 109 generations, sampling every 10,000 generations with the first 25% of generations discarded as burn-in, after confirming convergence of chains with Tracer v.1.6 (Rambaut et al., 2013). The effective sample size of the majority of parameters was > 500. The maximum clade credibility (MCC) tree was visualized in FigTree v.1.4.3 (Rambaut, 2014). 3. Results and discussion 3.1. General features of Mytilidae mitochondrial genomes The complete mitochondrial genomes of the nine new mytilid species are all circular molecules ranging from 14,703 bp (M. virgata) to 18,819 bp (B. thermophilus) in size, which is similar to previously reported bivalve species (Table 1). The size of the control regions is highly variable, as well documented from many other metazoan taxa, 3
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Fig. 1. Linearized mitochondrial gene arrangement patterns of Mytilidae species superimposed on the phylogenetic tree (see Fig. 2 for detailed relationships among mytilid species). Gene and genome size are not scaled. The tRNAs are labelled by single-letter abbreviations of amino acid code.
contributing to the overall genome size variation (Supplementary Tables 5–13). The M. virgata genome is the smallest in size to date (in Mytilidae) and has no control region. Gene order and the number of tRNA genes also vary widely (Fig. 1), as is common in other bivalves (Boore et al., 2004; Breton et al., 2010). Gene arrangement among the species, particularly among different subfamilies, is highly variable, except that all Mytilus species and Crenomytilus grayanus are identical in their gene order. The mitochondrial genomes of B. thermophilus, C. grayanus, G. coralliophaga, and S. bilocularis contain 13 PCGs, 2 rRNA, and 23 tRNA genes (B. thermophilus has three copies of trnL [anticodon: CUG]; C. grayanus, G. coralliophaga, and S. bilocularis have two copies of trnM [AUA]). The M. virgata mtDNA contains 13 PCGs, 22 tRNA and 2 rRNA genes. B. mutabilis contains 13 PCGs, 24 tRNA and 2 rRNA genes (two copies of trnM [AUA] and two copies of trnL [UUU]). M. keenae contains 12 PCGs (lacking atp8), 23 tRNA and 2 rRNA genes (two copies of trnM [AUA]). M. nipponicus and L. curta contain 12 PCGs (lacking atp8), 22 tRNA and 2 rRNA genes. M. nipponicus, L. curta, and M. keenae mtDNAs lack the atp8 gene, which was asserted to occur as a pseudogene in some other Mytilidae species (Uliano-Silva et al., 2016b).
3.2. Phylogenetic relationships and mitochondrial gene arrangements Phylogenetic relationships among Mytilidae subfamilies were inferred for a concatenated dataset (16,550 bp long) of the 12 proteincoding genes and two rRNA genes using ML and BI methods. Tree topology results from both ML and BI analyses were identical in that the family is subdivided into two clades and the overall relationships among their subfamilies (Fig. 2): Clade 1 included the subfamilies Bathymodiolinae, Modiolinae, Limnoperninae, and Lithophaginae, and Clade 2 contained the subfamilies Brachidontinae, Mytilinae, Crenellinae, and Septiferinae, with all branches having high supporting values (100% BP and 1.00 BPP). The Mauve alignment for the mitochondrial genomes indicated that syntenic blocks among the same clade members were much more similar to each other than to species belonging to different clades (Supplementary Fig. S1A and B). Within Clade 1, Bathymodiolinae was sister to Modiolinae, which agrees with some recent studies (Samadi et al., 2007; Ozawa et al., 2017; Liu et al., 2018) but contradicts a previous 18S rDNA phylogeny where Modiolinae occupied a position basal to other mytilid taxa 4
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Fig. 2. Reconstructed phylogenetic tree based on the mitochondrial genomes (12 protein coding genes plus two rRNA genes) of 27 Mytilidae species (two individuals of M. virgata) using maximum likelihood and Bayesian methods. Bayesian posterior probability/maximum likelihood bootstrap support values are shown in the branches. An asterisk denotes mtDNA sequence newly determined in this study.
(Bathymodiolinae)+(Lithophaginae+(Mytilinae + Crenellinae))) (Distel et al., 2000). The gene order of these two family members is the same and represented a single syntenic block of 12 PCGs and 2 rRNA genes, excluding tRNA genes, where considerable translocational changes are normally observed (Fig. 1, Supplementary Fig. S2). This result differs from a previous anatomical study (Morton, 2015) that argued Bathymodiolus was more closely related to Musculista (Crenellidae) and Limnoperna (Limnopernidae) than Modiolus (Modiolinae). The freshwater mussel member Limnoperninae was sister to Lithophaginae (100% BP and 1.00 BPP) and they were placed sister to (Bathymodiolinae + Modiolinae) within Clade 1. These relationships are different from an earlier molecular analysis of a combined dataset of mitochondrial and nuclear gene sequences that placed Lithophaga basal
to a grouping of (((Mytilinae + Musculinae) + Septiferinae) + Brachidontinae) of Clade 2, with Leiosolenus (the other Lithophaginae member) positioned basal to Bathymodiolinae + Modiolinae of Clade 1, rendering Lithophaginae polyphyletic (Liu et al., 2018). A tree topology test examining the alternative hypothesis of Lithophaga in a basal position within Clade 2 was statistically rejected in this study (Supplementary Table 4). However, we are not able to test whether Lithophaginae is polyphyletic, since only one Lithophaginae representative (L. curta) is included in this study. The results from the Mauve alignment analysis showed four synteny blocks shared between Lithophaga curta and Bathymodiolinae + Modiolinae; this similarity in mitochondrial genome structure is additional evidence of their close relationship (Supplementary Fig. S3). The four blocks were (1) cox15
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Fig. 3. Estimates of divergence times using BEAST v.2.4.8. Numbers on the nodes indicate the mean divergence times and 95% highest posterior density (HPD) intervals of the divergence time are shown in the parentheses below the horizontal bars. Calibration point is marked by an arrow.
trnL1(CUN), (2) rrnS-trnW, (3) cytochrome b, (4) nad4L-nad1. The only difference in the syntenic arrangements is found in L. curta where the cytochrome b (cob) synteny block is translocated. The results from the synteny block analysis is more complicated when Limnoperna fortunei, the only freshwater mytilid member, is included, as the gene order of this species is idiosyncratic and substantially different from other species belong to Clade 1. In Clade 2, the two Crenellinae species G. coralliophaga and M. senhousia were nested within Mytilinae (i.e., polyphyletic) with a very strong branch support value (100% BP and 1.00 BPP). Tree topology tests of alternative hypotheses in which Mytilinae and Crenellinae were each constrained to be monophyletic were rejected. All Mytilus species (Mytilinae) were clustered with G. coralliophaga, whereas M. senhousia was grouped with another Mytilinae branch of Perna canaliculus and P. viridis with high branch support values (100% BP and 1.00 BPP). Moreover, the result of Mauve alignment between G. coralliophaga and Mytilus + Crenomytilus species showed very strong colinearity in their syntenic structure (Supplementary Fig. S4). The interrelationships of Perna and Musculista still remain unresolved, varying in earlier works according to the characters employed in the analysis: earlier studies based on spermatozoa structure and shell morphology independently placed them in the subfamilies Mytilinae and Crenellinae, respectively (Kafanov and Drozdov, 1998; Coan et al., 2000). On the other hand, based on the anatomical feature of having the pericardial complex located between two posterior byssal retractor muscle blocks, both Perna and Musculista were placed in the same subfamily (Musculinae) separate from Crenellinae (which contained Gregariella) (Morton, 2015). The sister relationship between Perna and Musculista species within
Mytilinae was also supported by a recent molecular phylogeny using mitochondrial and nuclear gene sequences (Liu et al., 2018) and the results of the present study. Nevertheless, there is one unexpected exception found in our mtDNA analysis in regards to the position of Perna perna: surprisingly, it was separated from its congeneric species (P. canaliculus and P. viridis) and instead clustered with Brachidontes species of the subfamily Brachiodontinae (Fig. 2). Both mtDNA gene order comparison and the Mauve alignment analysis showed the highest similarity between P. perna and the two Brachiodontes species (Fig. 1, Supplementary Fig. S1B). To assess whether this unexpected position of P. perna is caused by a representation of paternal M-type sequences, we performed phylogenetic analysis for both maternal F-type and paternal M-type sequences available so far from all Mytilidae species on GenBank. The resulting tree (data not shown) showed that the P. perna mtDNA did not cluster with any M-type sequences from other Mytilidae species. Based on this result, we are not able to postulate that the P. perna mtDNA sequence is of paternal origin, corroborating the interpretation of a previous study (Uliano-Silva et al., 2016a,b). Further sequencing and phylogenetic analysis of the M-type mtDNA sequence from this species will clarify this uncertainty and resolve its enigmatic position. Septiferinae, represented by S. bilocularis, was positioned sister to a grouping of (Mytilinae + Crenellinae), and Brachidontinae was placed as sister to ((Mytilinae + Crenellinae) + Septiferinae), each receiving high branch support values (100% BP and 1.00 BPP). This topology is the same as the relationships recovered in Liu et al. (2018) because they treated Brachidontinae (Hormomya and Brachidontes) and Septiferinae (Septifer) as Mytilinae members. Interestingly, the mitochondrial 6
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genome sequence of M. virgata determined in this study and the partial genome sequence of M. virgata on GenBank (Table 1) showed a 9.1% difference in nucleotide sequence (PCGs showed a difference of 10.4%), although they were recovered as a sister group in our phylogenetic trees and had identical gene arrangement. This nucleotide sequence divergence is not likely to have resulted from the doubly uniparental inheritance (DUI) system, because the difference between M-type and Ftype mtDNAs studied so far ranges over 20% (Breton et al., 2007). It may have originated from historical isolation of populations in different marginal seas during paleoclimate changes (as reviewed by Ni et al., 2014). Similar patterns with cryptic linages (or species) have also been revealed in other species in the northwestern Pacific, including fish (Liu et al., 2007), bivalves (Ni et al., 2012; Wang et al., 2017), and shrimp (Cheng and Sha, 2017).
and Fisheries and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B4011597). Author contributions Study conception and design: JKP. Data collection: YL. Data analysis and interpretation: YL, HK, JS, SCK, TK, and JKP. Manuscript preparation: YL, SCK, TK, and JKP. Declaration of Competing Interest The authors have declared an absence of competing interests. Appendix A. Supplementary material
3.3. Estimates of divergence time among subfamilies
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ympev.2019.106533.
A high ESS (effective sample size) value (> 500) was identified for all parameters in the BEAST analysis to estimate divergence time of Mytilidae species (Fig. 3). The two major Mytilidae clades (Clade 1 and Clade 2) were estimated to have split around 400.40 MYA (95% HPD interval 409.1–391.4 MYA) in the early Devonian period, which is close to the split timing estimated in some previous analyses: Soot-Ryen (1969) and Distel (2000) indicated based on the fossil records that Mytilidae first appeared in the Devonian (with Modiolinae as the ancestral group). A recent molecular analysis (Liu et al., 2018) placed the split of the two major Mytilidae clades at 418.31 MYA, with a 95% highest posterior density (HPD) interval that spans the late Silurian and early Devonian (413.50–423.30 MYA). The Paleobiology Database (https://paleobiodb.org) indicates that the oldest Mytilidae fossil (with Mytilinae as the ancestral group) is from the Silurian. The estimated divergence times of major subfamilies in this study was similar to Liu et al. (2018). Bathymodiolinae and Modiolinae were inferred to have split in the early Jurassic (ca. 200.58 MYA). The divergence time between L. fortunei (a freshwater member of Limnoperninae) and Lithophaga curta (the boring mussel in Lithophaginae) was also estimated in the early Jurassic, dating back to approximately 196.70 MYA. The divergence time of the Limnoperninae lineage from its closest relative, the Lithophaginae group, implies the habitat transition from marine to freshwater environments by the Limnopernae ancestor may have occurred during the early Jurassic period of the Mesozoic Era. The split between (Bathymodiolinae + Modiolinae) and (Lithophaginae + Limnoperninae) was dated to 279.87 MYA in the middle Permian. Within Clade 2, Brachidontinae was the first emerged in the middle Carboniferous (335.46 MYA). Septiferinae was estimated to have branched off 278.90 MYA from the lineage leading to Mytilinae and Crenellinae. The divergence time between the genera Mytilus and Gregarella was estimated to be approximately 163.05 MYA in the late Jurassic. The split time between Musculista and Perna was estimated at 195.08 MYA in the early Jurassic. The divergence between (Mytilus + Gregarella) and (Musculista + Perna) occurred about 253.63 MYA in the early Triassic. The divergence time estimation using molecular clock analysis indicates that lineage splitting among majority of Mytilidae subfamilies occurred in the Mesozoic period (Triassic, Jurassic, and Cretaceous eras). Similarly, during this period many marine fauna also had high family diversification rates (Clarke & Crame, 2003). Since the Mesozoic was a time of warm temperatures, high sea levels, and greater continental shelf area (Hallam 1985; Haq et al., 1987), these conditions may have contributed to diversification rates (Briggs, 2006) and explain why most major bivalve groups expanded in this period (Bieler et al., 2014).
References Adarraga, I., Martínez, J., 2012. First record of the invasive brackish water mytilid Limnoperna securis (Lamarck, 1819) in the Bay of Biscay. Aquat. Invasions 7, 171–180. Bayne, B.L., 1976. Marine Mussels: Their Ecology and Physiology. Cambridge University Press, Cambridge. Bennett, K.F., Bailey, A.W., Brambert, D.J., Ferhati, E.W., Karson, C.A., Nafasat, U., Wadleigh, J.K., Wright, A.H., 2016. The F type mitochondrial genome of the scorched mussel: Brachidontes exustus (Mytiloida, Mytilidae). Mitochondrial DNA Part A 27, 1501–1502. Bernard, F.R., 1983. Catalogue of the living bivalvia of the eastern Pacific Ocean: Bering Strait to Cape Horn. Can. Special Publ. Fish. Aquat. Sci. 61. Bernt, M., Donath, A., Jühling, F., Externbrink, F., Florentz, C., Fritzsch, G., Pütz, J., Middendorf, M., Stadler, P.F., 2013. MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319. Bieler, R., Mikkelsen, P.M., 2006. Bivalvia-a look at the branches. Zool. J. Linn. Soc. 148, 223–235. Bieler, R., Carter, J.G., Coan, E.V., 2010. Classification of bivalve families. In: In: Bouchet, P., Rocroi, J.-.P., Bieler, R., Carter, J.G., Coan, E.V. (Eds.), Nomenclator of bivalve families with a classification of bivalve families, Malacologiapp. 1–184. Bieler, R., Mikkelsen, P.M., Collins, T.M., Glover, E.A., González, V.L., Graf, D.L., Harper, E.M., Healy, J.M., Kawauchi, G.Y., Sharma, P.P., Staubach, S., Strong, E.E., Taylor, J.D., Tëmkin, I., Zardus, J.D., Clark, S., Guzmán, A., McIntyre, E., Sharp, P., Giribet, G., 2014. Investigating the Bivalve Tree of Life - an exemplar-based approach combining molecular and novel morphological characters. Invertebr. Systemat. 28, 32–115. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. Boore, J.L., Macey, J.R., Medina, M., 2005. Sequencing and comparing whole mitochondrial genomes of animals. Methods Enzymol. 395, 311–348. Boore, J.L., Medina, M., Rosenberg, L.A., 2004. Complete sequences of the highly rearranged molluscan mitochondrial genomes of the scaphopod Graptacme eborea and the bivalve Mytilus edulis. Mol. Biol. Evol. 21, 1492–1503. Boss, K.J., 1982. Mollusca. In: Parker, S.P. (Ed.), Synopsis and Classification of Living Organisms. McGraw-Hill Book Company, New York, pp. 945–1166. Breton, S., Burger, G., Stewart, D.T., Blier, P.U., 2006. Comparative analysis of genderassociated complete mitochondrial genomes in marine mussels (Mytilus spp.). Genetics 172, 1107–1119. Breton, S., Doucet-Beaupré, H., Stewart, D.T., Hoeh, W.R., Blier, P.U., 2007. The unusual system of doubly uniparental inheritance of mtDNA: isn't one enough? Trends Genet. 23, 465–474. Breton, S., Stewart, D.T., Hoeh, W.R., 2010. Characterization of a mitochondrial ORF from the gender-associated mtDNAs of Mytilus spp. (Bivalvia: Mytilidae): Identification of the “missing” ATPase 8 gene. Mar. Geonom. 3, 11–18. Briggs, J.C., 2006. Proximate sources of marine biodiversity. J. Biogeogr. 33, 1–10. Carter, J.G., Altaba, C.R., Anderson, L.C., Araujo, R., Biakov, A.S., Bogan, A.E., Campbell, D.C., Campbell, M., Jin-hua, C., Cope, J.C., 2011. A synoptical classification of the Bivalvia (Mollusca). Paleontol. Contrib. 4, 1–47. Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552. Cheng, J., Sha, Z.L., 2017. Cryptic diversity in the Japanese mantis shrimp Oratosquilla oratoria (Crustacea: Squillidae): Allopatric diversification, secondary contact and hybridization. Sci. Rep. 7, 1972. Clarke, A., Crame, J.A., 2003. The importance of historical processes in global patterns of diversity. In: Blackburn, T.M., Gaston, K.J. (Eds.), Macroecology. Blackwell, Oxford, UK, pp. 130–151. Coan, E.V., Scott, P.V., Bernard, F.R., 2000. Bivalve Seashells of Western North America: Marine Bivalve Mollusks from Arctic Alaska to Baja California. Santa Barbara Museum of Natural History.
Funding This research was supported by a grant from the Marine Biotechnology Program (20170431) funded by the Ministry of Oceans 7
Molecular Phylogenetics and Evolution 139 (2019) 106533
Y. Lee, et al.
from the pericardial–posterior byssal retractor musculature complex. Molluscan Res. 35, 227–245. Newell, N.D., 1969. Classification of Bivalvia. In: Moore, R.C. (Ed.), Treatise on Invertebrate Paleontology. Geological Society of America and University of Kansas Press, pp. 205–224. Ni, G., Li, Q., Kong, L., Yu, H., 2014. Comparative phylogeography in marginal seas of the northwestern Pacific. Mol. Ecol. 23, 534–548. Ni, G., Li, Q., Kong, L.F., Zheng, X.D., 2012. Phylogeography of bivalve Cyclina sinensis: testing the historical glaciations and Changjiang River outflow hypotheses in northwestern Pacific. PLoS ONE 7, e49487. Owada, M., 2007. Functional morphology and phylogeny of the rock-boring bivalves Leiosolenus and Lithophaga (Bivalvia: Mytilidae): a third functional clade. Mar. Biol. 150, 853–860. Ozawa, G., Shimamura, S., Takaki, Y., Yokobori, S.I., Ohara, Y., Takishita, K., Maruyama, T., Fujikura, K., Yoshida, T., 2017. Updated mitochondrial phylogeny of Pteriomorph and Heterodont Bivalvia, including deep-sea chemosymbiotic Bathymodiolus mussels, vesicomyid clams and the thyasirid clam Conchocele cf. bisecta. Mar. Geonom. 31, 43–52. Palumbi, S., Martin, A., Romano, S., Mcmillan, W.O., Stice, L., Grabowski, G., 1991. The Simple Fool’s Guide to PCR. Version 2. Department of Zoology and Kewalo Marine Laboratory. University of Hawaii, Honolulu, USA. Passamonti, M., Ricci, A., Milani, L., Ghiselli, F., 2011. Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae). BMC Genom. 12, 442. Pawiro, S., 2010. Bivalves: global production and trade trends. In: Rees, G., Pond, K., Kay, D., Bartram, J., Santo Domingo, J. (Eds.), Safe Management of Shellfish and Harvest Waters. IWA Publishing, London, UK, pp. 11–19. Phillips, D.J.H., 1976. The common mussel Mytilus edulis as an indicator of pollution by zinc, cadmium, lead and copper. I. Effects of environmental variables on uptake of metals. Mar. Biol. 38, 59–69. Rambaut, A., 2014. FigTree v1.4.3. Available from: http://tree.bio.ed.ac.uk/software/ figtree/. Rambaut, A., Suchard, M.A., Xie, W., Drummond, A., 2013. Tracer v1.6. Available from: http://tree.bio.ed.ac.uk/software/tracer/. Ricciardi, A., 1998. Global range expansion of the Asian mussel Limnoperna fortunei (Mytilidae): another fouling threat to freshwater systems. Biofouling 13, 97–106. Robicheau, B.M., Powell, A.E., Del Bel, L., Breton, S., Stewart, D.T., 2017. Evidence for extreme sequence divergence between the male-and female-transmitted mitochondrial genomes in the bivalve mollusc, Modiolus modiolus (Mytilidae). J. Zool. Syst. Evol. Res. 55, 89–97. Samadi, S., Quéméré, E., Lorion, J., Tillier, A., von Cosel, R., Lopez, P., Cruaud, C., Couloux, A., Boisselier-Dubayle, M.C., 2007. Molecular phylogeny in mytilids supports the wooden steps to deep-sea vents hypothesis. C.R. Biol. 330, 446–456. Scarlato, O.A., Starobogatov, Y.I., 1979. The system of the suborder Mytileina (Bivalvia). In: Molljuski. Osnoynye rezultalty ikh izuchenija. 6. pp. 22–25. Soot-Ryen, T., 1969. Superfamily Mytilacea Rafinesque, 1815. In: Moore, R.C. (Ed.), Treatise on Invertebrate Paleontology, Mollusca. Geological Society of America Inc. and University of Kansas Press, Lawrence, Kansas, pp. 271–280. Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies. Bioinformatics 30, 1312–1313. Stanley, S.M., 1970. Relation of Shell form to Life Habits of the Bivalvia (Mollusca). Geological Society of America. Starobogatov, Y.I., 1992. Morphological basis for phylogeny and classification of Bivalvia. Ruthenica 2, 1–25. Steiner, G., Hammer, S., 2000. Molecular phylogeny of the Bivalvia inferred from 18S rDNA sequences with particular reference to the Pteriomorphia. In: Harpre, E.M., Taylor, J.D., Crame, J.A. (Eds.), The Evolutionary Biology of the Bivalvia. Geological Society, London, Special Publications, pp. 11–29. Taylor, B.E., Jamieson, G., Carefoot, T.H., 1992. Mussel culture in British Columbia: the influence of salmon farms on growth of Mytilus edulis. Aquaculture 108, 51–66. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Uliano-Silva, M., Americo, J., Bastos, A.S., Furtado, C., Rebelo, M.D.F., Prosdocimi, F., 2016a. Complete mitochondrial genome of the brown mussel Perna perna (Bivalve, Mytilidae). Mitochondrial DNA Part A 27, 3955–3956. Uliano-Silva, M., Americo, J.A., Costa, I., Schomaker-Bastos, A., de Freitas Rebelo, M., Prosdocimi, F., 2016b. The complete mitochondrial genome of the golden mussel Limnoperna fortunei and comparative mitogenomics of Mytilidae. Gene 577, 202–208. Vakily, J.M., 1989. The Biology and Culture of Mussels of the Genus Perna. ICLARM Studies and Reviews 17. International Centre for Living Aquatic Resources Management, Manila, Philippines, and Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ) GmbH, Eschborn, Germany, pp. 1–63. Vázquez-Luis, M., Morató, M., Campillo, J.A., Guitart, C., Deudero, S., 2016. High metal contents in the fan mussel Pinna nobilis in the Balearic Archipelago (western Mediterranean Sea) and a review of concentrations in marine bivalves (Pinnidae). Scientia Marina. 80, 111–122. Wang, X., Kong, L., Chen, J., Matsukuma, A., Li, Q., 2017. Phylogeography of bivalve Meretrix petechialis in the Northwestern Pacific indicated by mitochondrial and nuclear DNA data. PLoS ONE 12, e0183221.
Cossa, D., 1988. Cadmium in Mytilus spp.: worldwide survey and relationship between seawater and mussel content. Mar. Environ. Res. 26, 265–284. Darling, A.E., Mau, B., Blattner, F.R., Perna, N.T., 2004. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14, 1394–1403. Distel, D.L., 2000. Phylogenetic relationships among Mytilidae (Bivalvia): 18S rRNA data suggest convergence in mytilid body plans. Mol. Phylogenet. Evol. 15, 25–33. Distel, D.L., Baco, A.R., Chuang, E., Morrill, W., Cavanaugh, C.M., Smith, C., 2000. Do mussels take wooden steps to deep-sea vents? Nature 403, 725–726. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Duperron, S., Gaudron, S.M., Rodrigues, C.F., Cunha, M.R., Decker, C., Olu, K., 2013. An overview of chemosynthetic symbioses in bivalves from the North Atlantic and Mediterranean Sea. Biogeosciences. 10, 3241–3267. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech, 3, 294–299. Gaitán-Espitia, J.D., Quintero-Galvis, J.F., Mesas, A., D’Elía, G., 2016. Mitogenomics of southern hemisphere blue mussels (Bivalvia: Pteriomorphia): Insights into the evolutionary characteristics of the Mytilus edulis complex. Sci. Rep. 6, 26853. Giribet, G., Wheeler, W., 2002. On bivalve phylogeny: a high-level analysis of the Bivalvia (Mollusca) based on combined morphology and DNA sequence data. Invertebr. Biol. 121, 271–324. Habe, T., 1977. Systematics of Mollusca in Japan: Bivalvia and Scaphopoda. Hokuryukan, Tokyo. Hahn, C., Bachmann, L., Chevreux, B., 2013. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Res. 41 e129-e129. Hallam, A., 1985. A review of Mesozoic climates. J. Geol. Soc. 142, 433–445. Haq, B.U., Hardenbol, J.A.N., Vail, P.R., 1987. Chronology of fluctuating sea levels since the Triassic. Science 235, 1156–1167. Hoffmann, R.J., Boore, J.L., Brown, W.M., 1992. A novel mitochondrial genome organization for the blue mussel, Mytilus edulis. Genetics 131, 397–412. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Hwang, D.S., Gim, Y., Yoo, H.J., Cha, H.J., 2007. Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials 28, 3560–3568. Jenner, H.A., Whitehouse, J.W., Taylor, C.J.L., Khalanski, M., 1998. Cooling water management in European power stations: biology and control of fouling. Hydroécologie Appliquée 10, 1–225. Kafanov, A.I., Drozdov, A.L., 1998. Comparative sperm morphology and phylogenetic classification of Recent Myiloidea (Bivalvia). Malacologia 39, 129–139. Kenk, V.C., Wilson, B.R., 1985. A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos rift zone. Malacologia 26, 253–271. Kim, H.J., Yang, B., Park, T.Y., Lim, S., Cha, H.J., 2017. Complex coacervates based on recombinant mussel adhesive proteins: their characterization and applications. Soft Matter 13, 7704–7716. Nguyen, L.T., Schmidt, H.A., von Haeseler, A., Minh, B.Q., 2014. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274. Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B., 2016. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. 34, 772–773. Lee, Y.C., Lee, Y.H., 2016. The F type mitochondrial genome of hard-shelled mussel: Mytilus coruscus (Mytiloida, Mytilidae). Mitochondrial DNA Part A 27, 624–625. Li, X., Wu, X., Yu, Z., 2012. Complete mitochondrial genome of the Asian green mussel Perna viridis (Bivalvia, Mytilidae). Mitochondrial DNA 23, 358–360. Liu, J., Liu, H., Zhang, H., 2018. Phylogeny and evolutionary radiation of the marine mussels (Bivalvia: Mytilidae) based on mitochondrial and nuclear genes. Mol. Phylogenet. Evol. 126, 233–240. Liu, J.X., Gao, T.X., Wu, S.F., Zhang, Y.P., 2007. Pleistocene isolation in the Northwestern Pacific marginal seas and limited dispersal in a marine fish, Chelon haematocheilus (Temminck & Schlegel, 1845). Mol. Ecol. 16, 275–288. Lowe, T.M., Chan, P.P., 2016. tRNAscan-SE On-line: integrating search and contextual for analysis of transfer RNA genes. Nucleic Acids Res. 44, 54–57. Lutz, R.A., Kennish, M.J., 1993. Ecology of deep-sea hydrothermal vent communities: a review. Rev. Geophys. 31, 211–242. Machida, R.J., Kweskin, M., Knowlton, N., 2012. PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences. PLoS ONE 7, e35887. Merritt, T.J.S., Shi, L., Chase, M.C., Rex, M.A., Etter, R.J., Quattro, J.M., 1998. Universal cytochrome b primers facilitate intraspecific studies in molluscan taxa. Mol. Mar. Biol. Biotech. 7, 7–11. Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: 2010 Gateway Computing Environments Workshop (GCE). IEEE, New Orleans, pp. 1–8. Mizi, A., Zouros, E., Moschonas, N., Rodakis, G.C., 2005. The complete maternal and paternal mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis: implications for the doubly uniparental inheritance mode of mtDNA. Mol. Biol. Evol. 22, 952–967. Morton, B., 2015. Evolution and adaptive radiation in the Mytiloidea (Bivalvia): clues
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