Biodiversity Data Journal 11: e101333 OO) doi: 10.3897/BDJ.11.e101333 open access Research Article The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) indicates the genetic diversity within Gryphaeidae Fengping Li*§, Hongyue Liu!, Xin Heng?, Yu Zhang", Mingfu Fan?, Shunshun Wang*, Chunsheng Liuf, Zhifeng Gut, Aimin Wang§, Yi Yang$+# + College of Marine Science, Hainan University, Haikou, China § State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China | Institute of Marine Science and Technology, Shandong University, Qingdao, China |] Sanya Oceanographic Institution, Ocean University of China, Sanya, China # Sanya Nanfan Research Institute, Hainan University, Sanya, China Corresponding author: Yi Yang (yiyangouc@outlook.com) Academic editor: Graham Oliver Received: 01 Feb 2023 | Accepted: 12 Mar 2023 | Published: 20 Mar 2023 Citation: Li F, Liu H, Heng X, Zhang Y, Fan M, Wang §S, Liu C, Gu Z, Wang A, Yang Y (2023) The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) indicates the genetic diversity within Gryphaeidae. Biodiversity Data Journal 11: €101333. https://doi.org/10.3897/BDJ.11.e101333 Abstract Different from the true oyster (family Ostreidae), the molecular diversity of the gryphaeid oyster (family Gryphaeidae) has never been sufficiently investigated. In the present study, the complete mitochondrial (mt) genome of Hyotissa sinensis was sequenced and compared with those of other ostreoids. The total length of H. sinensis mtDNA is 30,385 bp, encoding 12 protein-coding-genes (PCGs), 26 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. The nucleotide composition and codon usage preference of H. sinensis mtDNA is similar to that of H. hyotis within the same genus. On the other hand, the presence of three trnM and three trnL genes of H. sinensis was not detected neither in H. hyotis nor other ostroid species. Another unique character of H. sinensis mtDNA is that both rrnS and rmL have a nearly identical duplication. The PCG order of H. sinensis is identical to H. hyotis and the two congener species also share an identical block of 12 tRNA genes. The tRNA rearrangements mostly happen in the region from Cox7 to Nad3, the same area where the duplicated genes are located. The rearrangements within © LiF et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Li F et al Gryphaeidae could be explained by a "repeat-random loss model". Phylogenetic analyses revealed Gryphaeidae formed by H. sinensis + H. hyotis as sister to Ostreidae, whereas the phylogenetic relationship within the latter group remains unresolved. The present study indicated the mitogenomic diversity within Gryphaeidae and could also provide important data for future better understanding the gene order rearrangements within superfamily Ostreoidea. Keywords Mitochondrial genome, gryphaeid oyster, gene order rearrangement, phylogeny Introduction Oysters belong to superfamily Ostreoidea, which is comprised of Gryphaeidae and Ostreidae (Bouchet et al. 2010). Distributed worldwide, oysters are important fishery and aquaculture species (Guo et al. 2018). As the leading molluscan species by production, oysters have one of the longest cultured histories and remains cultured on all continents, except Antarctica (Botta et al. 2020). However, the oyster populations have declined throughout the world due to the influence of overfishing, habitat loss and degradation, disease and parasitic outbreaks (Wilberg et al. 2011). The protection and management of oyster resources depend on comprehensive information of genetic diversity at both species and population levels. With the development of molecular biology technologies, DNA sequences have been integrated into oyster identification to eliminate the influence from plasticity of shell shape and provide better understanding of oyster genetic diversity (Liu et al. 2011). Previous studies have implied the effectiveness of mitochondrial DNA (mtDNA) as the molecular marker to reveal genetic diversity (Hebert et al. 2003). The mtDNA, especially the cytochrome c oxidase subunit 1 (COI) and the large ribosomal subunit (16S rDNA), has been applied in the species delimitation (Salvi et al. 2022), population genetics (Li et al. 2015) and phylogeographic analyses (Lazoski et al. 2011) of oyster resources. The complete mitochondrial genome which includes both the sequence and gene order information, has been widely used in oyster phylogenetic analyses (Salvi and Mariottini 2021). These previous studies revealed several mitogenomic characteristics within Ostreidae. Above all, the ostroid mitochondrial genome contains a split of the rmL gene and a duplication of trnM (Danic-Tchaleu et al. 2011), compared with the typical metazoan mtDNA containing 13 protein coding genes (PCGs), two rRNA and 22 tRNA genes (@oore 1999). In the mtDNA of some Asian oysters, the duplications of the rrnS gene and trnK and trnQ genes have been disclosed (Wu et al. 2010). Compared with the tRNAs, the gene order of PCGs is more conserved amongst four genera (Ostrea, Saccostrea, Magallana and Crassostrea), despite some translocations and/or transversions happening between genera (Danic-Tchaleu et al. 2011). The gryphaeid oysters (family Gryphaedae) differ from the true oysters (family Ostreidae) in the morphology of larval shell and soft tissues (Bayne 2017). In addition, the vesicular microstructure of shell is uniquely found amongst The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 3 Gryphaeidae species (Checa et al. 2020). Some gryphaeid species are also commercially important; however, their genetic diversity has seldom been well studied. Li et al. (2022) reported the mtDNA of Hyotissa hyotis which represents the first complete mitochondrial genome within family Gryphaeidae. Different from the mitogenomic organisation of Ostreidae, neither the split of the rrnL nor the duplication of trnM was detected in that of H. hyotis. Furthermore, the PCG order of H. hyotis showed little shared gene blocks with ostroids, indicating that extensive rearrangements happened within superfamily Ostreoidea. Despite the existence of one complete mitochondrial genome within Gryphaeidae, it is still necessary to include more data to conclude the mitogenomic features of this family. In the present study, the complete mitochondrial genome of H. sinensis was sequenced. Our aims are: 1. to characterise the mitogenomic features of H. sinensis and compare with H. hyotis; 2. to explore the gene order rearrangements within Gryphaeidae. Materials and methods Sample collection and DNA extraction The specimen of H. sinensis was collected by scuba diving on the artificial fish reef in the marine ranching area of Wuzhizhou Island (18°18'55"N; 109°46'3"E). The adductor muscle of the specimen was deposited in 95% alcohol in the Laboratory of Economic Shellfish Genetic Breeding and Culture Technology (LESGBCT), Hainan University. Whole genomic DNA was extracted from the adductor muscle of one individual using TIANamp Marine Animals DNA Kit (Tiangen, Beijing, China) in accordance with the manufacturer’s instructions. The genomic DNA was visualised on 1% agarose gel for quality inspection. DNA Sequencing and mitogenome assembly Genomic DNA of H. sinensis was sent to Novogene (Beijing, China) for library construction and next-generation sequencing. The DNA library, with insert size of approximately 300 bp, was generated using NEB Next Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s instructions. It was then sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end reads and 34,429,854 clean reads of each direction were finally generated. Clean data were imported in Geneious Prime 2021.0.1 for mitogenome assembly, with the strategy following Li et al. (2022). NOVOPlasty 4.2 (Dierckxsens et al. 2017) was also employed to avoid incorrect assembly. Due to the existence of a duplicated region, which is more than 2,000 bp, this mitogenome is not able to be completely assembled only with the Illumina short reads. Therefore, a long PCR amplification was intended to fill the assembled gap using the 1F forward (5'- 4 Li F et al GGGGGTAAGATATTTTGTGCAGCGA-3') and 1R_ reverse (5'-TCGACAGGTGG GCTAGACTTAACGC-3’) specific primers designed in the present study. The long PCR reactions contained 2.5 ul of 10x buffer (Mg2* plus), 3 yl of dNTPs (2.5 mM), 0.5 ul of each primer (10 uM), 0.8 ul of template DNA (25-40 ng/ul), 0.2 yl of TaKaRa LA Taq DNA polymerase (5 U/ul) and DEPC (Diethypyrocarbonate) water up to 25 ul. Long PCR reactions were conducted by initial denaturation step at 94°C for 60 s, followed by 35 cycles of: 10 s at 98°C, 30 s at 57°C and 5 min at 68°C, then a final extension step at 68°C for 10 min. The PCR products were purified by ethanol precipitation and sequenced at Beijing Liuhe BGI (Beijing, China). The PCR primers were used as sequence primers. Mitogenomic annotation and sequence analysis The mitogenome of H. sinensis was annotated using Geneious Prime. The PCGs were determined by ORF Finder (http:/Awww.ncbi.nim.nih.gov/orffinder) and MITOS Webserver (Bernt et al. 2013) with the invertebrate mitochondrial genetic code and their boundaries were modified by comparing them with those of congener species H. hyotis (GenBank Accession Number OP 151093). The secondary structure of tRNA genes was predicted by MITOS and ARWEN (Laslett and Canback 2008), while the boundaries of rRNA genes were obtained using MITOS and modified according to those of other ostreoids. The nucleotide composition of the whole complete mitogenome, PCGs, rRNA and tRNA genes was computed using MEGA X (Kumar et al. 2018). The base skew values for a given strand were determined as: AT skew = (A - T)/(A + T) and GC skew = (G - C/)((G + C), where A, T, G and C are the occurrences of the four nucleotides (Perna and Kocher 1995). Codon usage of PCGs was estimated using MEGA X. The mitochondrial genome map was generated using CGView (Grant and Stothard 2008). Phylogenetic analysis A total of 21 ostreoid species was included for phylogenetic reconstruction (Table 1), with two pearl oysters Pinctata maxima and P margaritifera as outgroup following Plazzi and Passamonti (2010). The dataset concatenating the nucleotide sequences of the 12 PCGs (Atp8 was not included) and two rRNA genes were constructed. The PCGs were aligned separately as codons using ClustalW integrated in MEGA X. The rRNA genes were aligned separately with MAFFT v.7 (Katoh and Standley 2013) and the ambiguously aligned positions were removed using Gblocks v.0.91b (Castresana 2000) with default parameters. The 14 separated alignments were finally concatenated into a single dataset using Geneious Prime and DAMBE5 (Xia 2018) was employed to generate different formats for further phylogenetic analyses. The best fit partition schemes and corresponding substitution models were identified using PartitionFinder 2 (Lanfear et al. 2017) under the Bayesian Information Criterion (BIC). The partitions tested in the present study referred to Li et al. (2022). The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... Table 1. List of mitochondrial genomes used in this study. New mt genomes Family Gryphaeidae Species Length (bp) Hyotissa sinensis 30,385 GenBank mt genome Family Species Length (bp) Accession No. Gryphaeidae Hyotissa hyotis 22,185 OP151093 Ostreidae Dendostrea sandvichensis 16,338 MT635133 Ostreidae Magallana gigas 18,225 MW143047 Ostreidae Magallana gigas 18,225 EU672831 Ostreidae Magallana hongkongensis 18,617 MZ337404 Ostreidae Magallana bilineata 22,420 MT985154 Ostreidae Magallana belcheri 21,020 MH051332 Ostreidae Magallana nippona 20,030 HM015198 Ostreidae Magallana iredalei 22,446 FJ841967 Ostreidae Magallana ariakensis 18,414 EU672835 Ostreidae Magallana sikamea 18,243 EU672833 Ostreidae Magallana angulata 18,225 EU672832 Ostreidae Crassostrea gasar 17,685 KR856227 Ostreidae Crassostrea virginica 17,244 AY905542 Ostreidae Ostrea denselameliosa 16,277 HM015199 Ostreidae Ostrea edulis 16,320 JF274008 Ostreidae Ostrea lurida 16,344 KC768038 Ostreidae Saccostrea mordax 16,532 FJ841968 Ostreidae Saccostrea cucullata 16,396 KP967577 Ostreidae Saccostrea kegaki 16,260 KX065089 Margaritidae Pinctada margaritifera 15,680 HM467838 Margaritidae Pinctada maxima 16,994 GQ452847 Sampling time Accession No. The Maximum Likelihood method (ML) and Bayesian Inference method (BI) were used for phylogenetic reconstruction. ML trees were constructed by lQtree 1.6.12 (Nguyen et al. 2015), which allows different partitions to have different evolutionary rates (-spp option) and with 10,000 ultrafast bootstrap replicates (-bb option). BI trees were constructed using MrBayes v.3.2.6 (Ronquist et al. 2012), running four simultaneous Monte Carlo Markov Chains (MCMC) for 10,000,000 generations, sampling every 1000 generations and discarding the first 25% generations as burn-in. Two independent runs were performed to increase the chance of adequate mixing of the Markov chains and to increase the chance 6 Li F et al of detecting failure to converge, as determined using Tracer v.1.6. The effective sample size (ESS) of all parameters was above 200. The generated phylogenetic trees were visualised in Fig Tree v.1.4.2. Results and discussion Species identification and mitogenome assembly Misidentifications are quite frequent in oyster mitogenomics. This is the case for the example of the recently-published mitogenome of Alectryonella plicatula (with GenBank Accession Number M\VV143047) that, in fact, was found to be a misidentified Magallana gigas as reported by Salvi et al. (2021). The identification of H. sinensis was conducted, based on both morphological and molecular evidence. The specimen in the present study possesses an oval shell with a length of about 14 cm (Fig. 1). The shell surface irregularly folds with radial ribs on both valves, which are weaker than those of H. hyotis. The margin of interior shell is dark purple, while the central part is white. The adductor muscle scar is large and located at the posterior side of the centre of the shell. Molecular identification was following Salvi et al. (2021), based on the rmL fragment, which shows identity values from 99.19% to 99.79% to the previously published sequences on GenBank (KC847135 and MT332230). Figure 1. EESl The image of Hyotissa sinensis. This mitogenome was firstly assembled. based on the next-generation data using two different types of software, resulting in almost identical results. However, two repetitive The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... if sequences that corresponded to the partial rrnL and rrnS genes were discovered on both sides of the draft mitogenome, indicating the incomplete assembly derived from short Illunima sequencing reads. The long PCR amplification which generated a product with 3,068 bp in length finally covered the assembly gap and completed the duplicated rmnL and rns genes. No additional gene was discovered within this Sanger-sequencing fragment. Mitochondrial genome composition The total length of H. sinensis mtDNA is 30,385 bp, encoding 40 genes including 12 PCGs, 26 tRNA genes and two rRNA genes (Table 2). The size of H. sinensis mitogenome is obviously longer than the other species from superfamily Ostreoidea (Wu et al. 2010, Li et al. 2022). All mitochondrial genes of H. sinensis are encoded on the same strand (Fig. 2), as previously indicated in other marine bivalves (Ghiselli et al. 2021). Different from the typical metazoan mtDNA, the Atp8 gene is not detected in H. sinensis. Although Atop8 was found in H. hyotis (Li et al. 2022), the identification of this short sequence is laborious due to its high substitution rate that led to the low homology even to its congener species. Although the absence of the Afp8 gene in family Ostreidae was reported by Ren et al. (2010), subsequent studies discovered this ATP gene in oyster mitogenome where it was thought to be absent (Vu et al. 2012). Amongst the 26 tRNAs of H. sinensis, three trnM were discovered (Fig. 3). In addition, H. sinensis consists of one extra copy of trnL-UUR and one of trnW. Another unique character is that both rnS and rmL have a nearly identical duplication (Fig. 2). The largest non-coding region located between trnL and Nad3 is 3,901 bp in length (Table 2). Table 2. Gene annotations of the complete mt genome of Hyotissa sinensis. Gene Strand _ Location Size (bp) StartCodon Stopcodon _ Intergenic nucleotides Cox1 H 1-1986 1986 ATG TAA 1936 tRNA-Met? H 3923-3989 67 842 tRNA-Lys H 4832-4898 67 81 tRNA-Gin H 4944-5006 63 0 rm H 5007-6335 1329 212 rms H 6548-7491 944 74 tRNA-/le H 7566-7632 67 97 tRNA-Ser7 H 7730-7799 70 105 Nad 1 H 7905-8978 1074 ATG TAG 185 tRNA-Ala H 9164-9230 67 220 tRNA-Met2 H 9451-9539 89 54 tRNA-Met3 =H 9594-9682 89 203 tRNA-Tyr H 9886-9948 63 37 tRNA-Glu H 9986-10051 66 9 Gene tRNA-Phe rnc rms tRNA-Thr tRNA-Pro tRNA-Leu1 tRNA-Asp tRNA-Leu2 Nad3 tRNA-Asn tRNA-Gly tRNA-Ser2 tRNA-Trp1 tRNA-Leu3 tRNA-Val tRNA-His Cox2 Cytb Nad2 tRNA-Trp2 Nad5 Nad6 tRNA-Cys Nad4 Atp6 Nad4L tRNA-Arg Cox3 The overall AT content of the H. sinensis mtDNA is 57.2%, similar to that of H. hyotis (59.2%; Li et al. (2022)). The AT skew and GC skew are -0.15 and 0.27, respectively (Table 3), indicating that the nucleotide composition is skewed from A in favour of T and from C to G. The negative AT skew and positive GC skew have also been reported in other ostreoid Strand ps fee Col Qe Vs | ec CO Ve ies Ut Qe CO Qe TOO) te em Cd Veen a (eC) er KO Vs P| ees a eC eC es Pen (en esd ee em et Qe Om Location 10061-10125 10142-11507 11700-12643 12808-12870 13050-13115 13207-13269 13807-13874 14987-15049 18951-19290 19735-19801 19804-19869 19877-19946 19972-20039 20053-20115 20145-20211 20242-20305 20333-21028 21154-22785 22864-23883 23898-23965 23975-25828 25925-26527 26558-26620 26622-27965 28040-28786 28855-29145 29181-29243 29453-30331 mitogenomes (VVu et al. 2010). Size (bp) 65 1366 944 63 66 63 68 63 340 67 66 70 68 63 67 64 696 1632 1020 68 1857 603 63 1344 747 291 63 879 Li F et al Start Codon ATT ATG ATA ATT TTG ATG ATG ATG TTG TTT Stop codon TAG TAG TAA TAG TAG TAG TAG TAG TAA Intergenic nucleotides 16 125 74 35 209 54 The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... FS) PCGs, tRNA and rRNA genes The AT content of the concatenated PCGs is 57.0% (Table 3). Amongst the individual PCGs, the AT content values range from 54.7% (Nad4L) to 60.3% (Atp6). The AT and GC skews of PCGs also show the same tendency of asymmetry as the mitogenome. The PCG start/stop codon usage preference of H. sinensis is different from that of H. hyotis. Amongst the 12 PCGs, seven genes start with the conventional initiation codons ATG (Cox7, Nad1, Cox2, Nad6, Nad4 and Atp6) and ATA (Cytb). The alternative start codons ATT (Nad2 and Nad3), TTG (Nad4L and Nad5) and TTT (Cox3) are detected in the remaining five genes. All PCGs employ the conventional stop codons TAA (Cox7, Cox3 and Nad2) and TAG, except for Nad3 which use the truncated stop codon T. The incomplete stop codons (TA and T) could be presumably modified to TAA through post- transcriptional polyadenylation (Ojala et al. 1981). The relative synonymous codon usage (RSCU) values of H. sinensis are shown in Table 4. Amongst all the amino acids, the frequency of leucine is the highest, as suggested in H. hyotis as well as in other invertebrate groups (Sun et al. 2020, Yang et al. 2020). Significant synonymous codon usage bias is also observed in the PCGs of H. sinensis, similar to that of H. hyotis (Fig. 4). Most of the preferred codons (e.g. TTT and TTG) are composed of T and G, which could explain the negative AT skew and positive GC skew of the PCGs to some extent. tRNA-Ile Hyotissa sinensis 30,385 bp ~l- ~ ~ ~, _ A da a gt te tRNA-Al a a a >/ Seniattet2 Cox2 QZ Ge ma. rae ak / S_ tRNA-Met3 wip cy. a tRNA-Glu a . tRNA-Phe eBMACTrpl 7 oe : y rrnL tRNA-Tyr Sig: 2 tRNA-Val“ tRNAmAsn Nad3 4 tRNA-Gly s tRNA-Leu3 L. \\ ies \ tRNA-Pro tRNA-As * tRNA-Leu2 4 tRNA-Thr tRNA-Leul Figure 2. EE Mitochondrial genome map of Hyotissa sinensis. 10 Li F et al yi Glutanine sotcucine dysK) (Giln-Q) (Heel) Alanine Methionine Tyrosine Glotarene (Ala-A) (Met-M2) (Ty) (Ghe-B) Aspartic i Threonine Proline Leucine yo (The-T) {Pro-P) (LewL I) (AD) Gilyeme (Gly) (Trp-WI) cucene Tryptophan \ Cysteine U Valine Histdine (Trp-W2) (Cys-C) (Lou-l3) (ValeV) (Hist) ote oe Discriminator nucleotide Arginine (Arg-R) Amnino ackd aecepeer (AA) arm TyC arn Variable {V) boop Anticodoe (AC) arm Dihydrouridene (DIU) arm Figure 3. EES] Inferred secondary structures of 26 transfer RNAs from Hyotissa sinensis. The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 11 Table 3. List of AT content, AT skew and GC skew of Hyotissa sinensis mtDNA. Feature (A+T)% AT skew GC skew Whole genome 57.2 -0.15 0.27 PCGs 57.0 -0.23 0.29 PCGs1 55.7 -0.24 0.40 PCGs2 56.4 -0.13 0.28 PCGs3 58.7 -0.31 0.19 Atp6 60.3 -0.26 0.39 Cox1 58.4 -0.16 0.24 Cox2 57.8 -0.25 0.29 Cox3 56.2 -0.30 0.20 Cytb 57.1 -0.16 0.23 Nad1 55.8 -0.18 0.21 Nad2 5725 -0.29 0.26 Nad3 55.8 -0.35 0.44 Nad4 55:5 -0.30 0.34 Nad4L 54.7 -0.17 0.45 Nad5 56.3 -0.25 0.37 Nad6 56.2 -0.15 0.39 tRNAs 56.3 -0.14 0.19 rel 57.6 -0.03 0.20 rst 52.8 0.09 0.13 rmnL2 58.4 -0.02 0.20 mns2 52.6 0.10 0.13 Table 4. Codon and relative synonymous codon usage (RSCU) of 12 protein-coding genes (PCGs) in the mtDNA of Hyotissa sinensis. Amino Acid Codon Count (RSCU) Amino Acid Codon Count (RSCU) Phe UUU 254.0(1.69) Ala GCU 101.0(1.54) UUC 47.0(0.31) GCC 47.0(0.71) Leu UUA 83.0(1.06) GCA 47.0(0.71) UUG 189.0(2.42) GCG 68.0(1.03) CUU 66.0(0.84) Gly GGU 73.0(0.83) CUC 21.0(0.27) GGC 47.0(0.53) 12 Li F et al Amino Acid Codon Count (RSCU) Amino Acid Codon Count (RSCU) CUA 39.0(0.50) GGA 82.0(0.93) CUG 71.0(0.91) GGG 151.0(1.71) lle AUU 191.0(1.65) Arg CGU 35.0(1.26) AUC 40.0(0.35) CGC 20.0(0.72) Met AUA 63.0(0.57) CGA 30.0(1.08) AUG 159.0(1.43) CGG 26.0(0.94) Val GUU 151.0(1.52) Tyr UAU 136.0(1.50) GUC 48.0(0.48) UAC 45.0(0.50) GUA 69.0(0.69) His CAU 47.0(1.08) GUG 130.0(1.31) CAC 40.0(0.92) Ser UCU 90.0(1.97) Gln CAA 22.0(0.80) UCC 21.0(0.46) CAG 33.0(1.20) UCA 29.0(0.63) Asn AAU 77.0(1.43) UCG 43.0(0.94) AAC 31.0(0.57) AGU 27.0(0.59) Lys AAA 86.0(0.98) AGC 20.0(0.44) AAG 89.0(1.02) AGA 73.0(1.60) Asp GAU 75.0(1.61) AGG 63.0(1.38) GAC 18.0(0.39) Pro CCU 65.0(1.70) Glu GAA 57.0(0.77) CCC 26.0(0.68) GAG 92.0(1.23) CCA 28.0(0.73) Cys UGU 64.0(1.28) CCG 34.0(0.89) UGC 36.0(0.72) Thr ACU 70.0(1.74) Trp UGA 62.0(0.73) ACC 27.0(0.67) UGG 107.0(1.27) ACA 28.0(0.70) - UAA 3.0(0.55) ACG 36.0(0.89) UAG 8.0(1.45) The AT content of the concatenated tRNAs is 56.3%, while the AT skew and GC skew are -0.14 and 0.19, respectively (Table 3). The length of tRNA genes ranges from 63 to 89 bp (Table 2). All the 26 tRNA genes could be folded into typical clover-leaf secondary structures, except for trnS-UCN and trnS-AGN which lack the dihydrouracil (DHU) arm, but are simplified down to a loop (Fig. 3). The missing DHU arm in the secondary structure of trnS-AGN is quite common in metazoan mitogenomes (Wolstenholme 1992). However, lack of the DHU arm in trnS-UCN is not a common feature observed in invertebrate mitogenomes, though it has been found in some arthropod taxa (Wang et al. 2016). The typical metazoan mtDNA possesses a total of 22 tRNA genes, including two copies of trnL and two of trnS. However, the bivalve mtDNA usually shows deviations especially in the number of tRNAs. A typical example is the existence of one extra tml in most bivalve mitogenomes (Lee et al. 2019, Wang et al. 2021). The presence of three trnM in H. The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 13 sinensis has never been reported in Ostreoidea before. All three trnM/ genes recognise codon AUG, but tmM2 and trnM3 share almost identical sequences, indicating the evidence of the tRNA duplication event which happens quite commonly in molluscan mitogenomes (Ghiselli et al. 2021). Sequence comparison suggests that trnM2 and trnM3 in H. sinensis are homologous to the single trnM in H. hyotis (Fig. 5). Amongst the three trnL, two copies that recognise the codon UUA indicate another case of tRNA duplication (Fig. 3). The two trnW genes in H. sinensis could also be traced in H. hyotis (Fig. 5). The appearance of two tmW genes that occur only in Gryphaeidae should be considered as an occasional event within Ostreoidea (Wu et al. 2012). to Phe Leu Ile Met Val Ser Pro Thr Ala Tyr His Gln Asn Lys Asp Glu Cys Trp Arg Gly cus | a hava || ucA cca. [GGA] CULE UNG TAYE TAU GUE LCE COE AEE | GEE CAC CAE CAG AAG ANG GAC GAG NOC UGG COCK Coan) [Uta |! aun [AAS Seut |fucu [ecu | “ACU |/aCU. WAU [CAU || CAA | “AAT | AAA || GAUL [GAA YoU.|| UGA | Cau, | Gsu | Figure 4. ETSI Relative synonymous codon usage (RSCU) of mitochondrial genome for Hyotissa sinensis. The H. sinensis contains two almost identical copies of rrnL and two of rrnS, which were not detected in H. hyotis (Li et al. 2022). The duplication of rmS is considered as a common feature of the Asian genus Magallana. Similarly, it is assumed that the duplication 14 Li F etal of rrnL and rmS in H. sinensis is a derived character, but still needs to be further determined by the inclusion of more data within Gryphaeidae. The two copies of rmS are 944 bp in length, while the two rrnL copies are 1,329 bp and 1,366 bp, respectively. The AT content, AT skew and GC skew values of rRNA genes are shown in Table 3. Amino Acid arm DHU arm & loop Anticodon arm & loop TwC arm & loop Amino Acid arm a is tnM-H. hyotis (GGCGGAGTAAGTTATT AGACTATCGGGCICATECCCCGGAAGTGCA CTGAATAAAATAGGGCTGTCCCTAAAGTCGGTGCCTCCGTCT T--ATAGTG -AAAGTAATTTCCACGGTAGGTAC TGCTT trnM2-H. sinensis SQCARCTERSISERESED GRE O™ CICATCACCCGGAGGTGT 5 =. TCGGGCTC CCCGGAGGTGTGCCGCT- -ATGATG RAAGCAATTTCCACGGTAGGTACTCCTGCTT B Amino Acid arm =DHU arm & loop Anticodon arm & loop TwC arm & loop Amino Acid arm had Ea traW1-H. hyotis AAGACTTTAGGTTATGACAAACCTCGATGTT CARGCATTGCAAAATCCTG- ~TGAAGGTTAGGTCTTT traW1-H. sinensis AGGGCTTTAGGTTATG~ TAAACCTCGATGTTITCARACATTGCAAAATCCTAATTATAGGTTAAGCCTIT Amino Acid arm DHU arm & loop Anticodon arm & loop TwC arm & loop Amino Acid arm ——_z = trnW2-H. hyotis AGGGC CTTTAGGTTATGGAGACCAACGCATICC trnW2-H. sinensis GGAACTCTAGGTTATGAAGACCACCGTATICCA Figure 5. EES] Alignment of trnM/ sequences (A) and trnW (B) in mitochondrial genomes of Hyotissa sinensis and H. hyotis. tRNA secondary structure is displayed above the alignment and the position of the anticodon is highlighted within the rectangular frame. GTGCGTAAGAAGTGATGTAATC GCTAAGCTTTT ATACGGCAGAAGCATTATTGTTITGCTGGGTTTTT Phylogenetic analyses According to the BIC, the best partition scheme is the one combining genes by subunits, but analysing each codon position separately (Suppl. material 1). ML (-InL = 154,942.703) and BI (-InL = 150,197.79 for run 1; -InL = 150,198.99 for run 2) analyses arrived at almost identical topologies (Fig. 6). Within Ostreoidea, Gryphaeidae formed by H. hyotis and H. sinensis was recovered as sister to Ostreidae. Different from the extinct gryphaeids which have been widely researched (Diet! et al. 2000, Hautmann et al. 2017, Kosenko 2019), only a few studies focused on the living gryphaeids. Based on several short gene fragments, Li et al. (2021) reconstructed the phylogenetic relationships of Ostreoidea, within which the monophyletic Hyotissa (including both H. hyotis and H. sinensis) was sister to Pycnodonte + Neopycnodonte despite the poor support values at some points. However, future studies with the inclusion of broader mitogenomic data are still needed to solve the phylogenetic relationships of Gryphaeidae. The phylogenetic relationships within Ostreidae generated from ML and BI methods arrived at different topologies (Fig. 6). The BI tree in the present study is consistent with Li et al. (2022), in which the rRNA genes were not included, while the ML tree here suggests Crassostreinae as sister to (Ostreinae + Saccostreinae), which is supported by previous phylogenies (Danic-Tchaleu et al. 2011, Salvi et al. 2014). This controversy has been discussed by Li et al. (2022). In addition to the inclusion of rRNA genes for phylogenetic reconstruction, this study also included the mitogenomic data of genus Dendostrea compared with Li et al. (2022). Firstly, Dendostrea sandvichensis and its sister group The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 15 Ostrea constitute subfamily Ostreinae, which is in accordance with the current Classification (MolluscaBase 2023). Morphologically, Dendostrea species could be distinguished from its radiating ribs on the surface of the right valve (Hu et al. 2019). Magallana gigas MW143047 Magallana gigas EU672831 Magallana angulata EU672832 Magallana sikamea EU672833 ee _ me Magallana ariakensis EU672835 a Magallana hongkongensis MZ337404 Magallana nippona HMO015198 Crassostreinae Magallana belcheri MHO051332 Magallana iredalei FJ841967 Magallana bilineata MT985154 Crassostrea gasar KR856227 Crassostrea virginica AY905542 Hyotissa hyotis OP151093 Gryphaeidae Hyotissa sinensis 0Q333008 2.00 Figure 6. EES] Phylogenetic relationships of Ostreoidea, based on the concatenated nucleotide sequences of 12 mitochondrial protein-coding genes and two ribosomal RNA genes. The reconstructed Bayesian Inference (Bl) phylogram is shown. The first number at each node is Bayesian posterior probability (PP) and the second number is the bootstrap proportion (BP) of Maximum Likelinood (ML) analyses. The nodal with maximum statistical supports (PP = 1; BP = 100) is marked with a solid red circle. BP values under 80 and PP values under 0.90 are marked as a dash line. Within Crassostreinae, two separated clades corresponding to the Asia-Pacific and Atlantic Regions are clearly presented (Figs 6, 7). Recently, the Pacific cupped oysters which were previously included in Crassostrea along with the Atlantic cupped oysters, were re- assigned to genus Magallana (Salvi and Mariottini 2017). Subsequent studies have demonstrated that Magallana was well-founded, based on a scientific basis and its validity has been thoroughly discussed (Willan 2021, Salvi and Mariottini 2021, Spencer et al. 2022). The present phylogeny also provides support for this classification. Gene rearrangement Within Ostreidae, the gene rearrangement events are most common in tRNA genes (Ren et al. 2010). Although some shared PCG blocks could be detected amongst the four ostreid 16 Li F et al genera, Magallana, Crassostrea, Ostrea and Saccostrea, it is still not possible to assume a pleisomorphic gene order in Ostreidae, based on available data as discussed in Salvi and Mariottini (2021). Within Ostreoidea, one shared gene block (Nad5-Nad6-Nad4-Atp6), plus one inverted gene block (Nad1-Nad3-Cox2-Cytb) were detected between H. hyotis (Gryphaeidae) and Saccostrea (Ostreidae). The newly-sequenced mitogenome of H. sinensis further confirms this feature of Gryphaeidae. Above all, the PCG order of H. sinensis (excluding the ATP8 gene since it is missing in H. sinensis) is identical to H. hyotis (Fig. 7), in agreement with the pattern that PCG order is conserved within the genus as mentioned in Ostreidae. Furthermore, H. sinensis also shares an identical block of 12 tRNAs with H. hyotis (Fig. 7). The tRNA rearrangements mostly happen in the region from Cox1 to Nad3, the same area where the duplicated genes are located. As a result, the rearrangements within Gryphaeidae could be explained by a "repeat-random loss model" ( San Mauro et al. 2005). To understand how the PCG orders evolved within superfamily Ostreoidea, more mitogenomes belonging to Gryphaeidae (including genera Pycnodonte and Neopycnodonte), as well as a robust phylogenetic framework, are still needed. Scat aa ma TE Cytb |D) Cox2 MiS; LiMzS:[P Ki C QiimL|N rmS|¥ JABS) G] V [RAS] R | RRR RB Nac]. | F | 5 Ew “cox mt mire Cytb [Dj Cox MiSiLiM:S: P|rmSK|C|QLmmill] N rmS|y [ARGO] G) V SR] RET SRS ac]. RR F | Rw GBRG +e] Co 1D Coxd MSM PSK Cll my A | RS. || BR 1] Cb | Cox? MiSs LMS. P mS KOEN ry A GV RR I So Rw nL rec 0 Com MS, LMS ms CORRE Ns YA VR A I A BY | TE Cot [D| Cond MSL NGS, Plesk aN rs AV A RR | © | Rw Crassostreinae 1/T)E Cytb |D] Cox? MiSi Li MJS.|P rmS)K. Cm] G)N rm] [AGS v (SG! r+ RATS 6) © ER | F | Rw 1 T]E| Cy [| Coxd [Mss CN my JA |v | | | Rw 1/T]E Cu (D|Cox® M/S: MSS) Poms mi Noms || SR RR | | Rw TE) Cyt |b) Cox? Mi Si LiMzS: P rm /K|C|mmil|N rrns|y [AGS | v NIG | ARR 6] © RR | | RRS 1] [E|Coab [Cox SL [P1G ims Mi vO RNS. ¥ [SR | | Rw See RHA] e cv Coa | sir Gms kc v!OAENS.¥ PAN | T rms G BR 1 Cytb | Cox? M{Si Mi|S:j|Plimi| RV IRR Lo F | [RRR on. NS) Jv S| Nc wR, Hc +6 Rc [ox SNS | | RS i i wa Saccostreinae SH rs\ CB iw [ca coo Maso OR A A A UE Cox2 |MiSiM:SiL1 P| rm RRB) | Y JARBS)N RV HR ems K/L F Aa wb RRB 68 | Coa oi a A sR A Ostrea den — nrellos. TE Cyt Cox? | MjSi MSL, P]rmi RI) Cy JAISG)N| RV 1 RRR ce |e, RG «| FA Dendostrea sandvic ne ae Cyt, _Cox2 [My Ss BBE: S| P rent JR@5 C|V ARDS) | |v, H IRR re. RR | I KF Rw Hyotissa hyotis x : i ALQ|KM YE F rma |ems) 1] |S, RARE! P| BBN | Gs.) Ls Vi) Cox2 | Cyt (SRW RRS] Nad c [RRR GRAY SERS Gryphaeidae Hyotissa sinensis MjK) Q|rmit rms| 1S, RMBIBJA MoM, YE F Jim rms T| P | DL RBBB N/G |S. Wi)L VH| Cox2 | Cyt (RISRBW RIS Nd) c (SR ASSIA ES Figure 7. EESI Linearised PCG order of Ostreoidea, based on the phylogenetic tree. Magallana belcheri Magallana iredalei Magallana bilineata Saceo Ostrea edulis Ostreinae Funding program This research was funded by the Hainan Provincial Natural Science Foundation of China (322QN260), the Key Research and Development Project of Hainan Province (ZDYF2021SHFZ269), the National Key Research and Development Program of China (2019YFD0901301) and the Hainan Province Graduate’ Innovation Project (Qhys2022-124). The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 17 Author contributions Fengping Li and Hongyue Liu contributed equally to this work and should be considered co-first authors. AW and YY designed the study; XH, YZ, MF, SW, ZG and CL collected the data; FL and HL performed the analyses; FL, HL and YY wrote the first draft of the manuscript; and all authors contributed intellectually to the manuscript. Conflicts of interest The authors report no conflicts of interest. References ° Bayne B (2017) Biology of oysters. Academic Press, London, 860 pp. [ISBN 9780128034729] ° Bernt M, Donath A, Juhling F, Externbrink F, Florentz C, Fritzsch G, Putz J, Middendorf M, Stadler P (2013) MITOS: Improved de novo metazoan mitochondrial genome annotation. Molecular Phylogenetics and Evolution 69 (2): 313-319. https://doi.org/ 10.1016/j.ympev.2012.08.023 ° Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Research 27 (8): 1767-1780. https://doi.org/10.1093/nar/27.8.1767 ° Botta R, Asche F, Borsum JS, Camp E (2020) A review of global oyster aquaculture production and consumption. Marine Policy 117: 103952. https://doi.org/10.1016/ j.marpol.2020.103952 ° Bouchet P, Rocroi J, Bieler R, Carter J, Coan E (2010) Nomencliator of bivalve families with a classification of bivalve families. Malacologia 52 (2): 1-184. https://doi.org/ 10.4002/040.052.0201 ° Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17 (4): 540-552. https:// doi.org/10.1093/oxfordjournals.molbev.a026334 ° Checa AG, Linares F, Maldonado-Valderrama J, Harper EM (2020) Foamy oysters: vesicular microstructure production in the Gryphaeidae via emulsification. Journal of the Royal Society Interface 17 (170): 20200505. https://doi.org/10.1098/rsif.2020.0505 ° Danic-Tchaleu G, Heurtebise S, Morga B, Lapegue S (2011) Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification. BMC Research Notes 4 (1): 400. https://doi.org/10.1186/1756-0500-4-400 ° Dierckxsens N, Mardulyn P, Smits G (2017) NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Research 45 (4): e18. https://doi.org/10.1093/nar/qkw955 ° Dietl GP, Alexander RR, Bien WF (2000) Escalation in Late Cretaceous-early Paleocene oysters (Gryphaeidae) from the Atlantic Coastal Plain. Paleobiology 26 (2): 215-237. https://doi.org/10.1666/0094-8373(2000)026<0215:EILCEP>2.0.CO;2 ° Ghiselli F, Gomes-dos-Santos A, Adema CM, Lopes-Lima M, Sharbrough J, Boore JL (2021) Molluscan mitochondrial genomes break the rules. Philosophical Transactions of the Royal Society B 376 (1825): 20200159. https://doi.org/10.1098/rstb.2020.0159 18 Li F et al Grant JR, Stothard P (2008) The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Research 36: 181-184. https://doi.org/10.1093/nar/ gkn179 Guo X, Li C, Wang H, Xu Z (2018) Diversity and evolution of living oysters. Journal of Shellfish Research 37 (4): 755-771. https://doi.org/10.2983/035.037.0407 Hautmann M, Ware D, Bucher H (2017) Geologically oldest oysters were epizoans on Early Triassic ammonoids. Journal of Molluscan Studies 83 (3): 253-260. https://doi.org/ 10.1093/mollus/eyx018 Hebert PD, Ratnasingham S, De Waard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London. Series B: Biological Sciences 270 (Suppl_1): 96-99. https://doi.org/ 10.1098/rsbl.2003.0025 Hu L, Wang H, Zhang Z, Li C, Guo X (2019) Classification of small flat oysters of Ostrea stentina species complex and a new species Ostrea neostentina sp. nov. Bivalvia: Ostreidae). Journal of Shellfish Research 38 (2): 295-308. https://doi.org/ 10.2983/035.038.0210 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30 (4): 772-780. https://doi.org/10.1093/molbev/mst010 Kosenko IN (2019) On the Upper Maastrichtian oysters of the genus Rhynchostreon Bayle (Bivalvia, Gryphaeidae) from the Mountainous Crimea. Paleontological Journal 53 (6): 583-592. https://doi.org/10.1134/S0031030119060042 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35 (6): 1547-1549. https://doi.org/10.1093/molbev/msy096 Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34 (3): 772-773. https://doi.org/ 10.1093/molbev/msw260 Laslett D, Canback B (2008) ARWEN: A program to detect tRNA genes in metazoan mitochondrial nucleotide sequences. Bioinformatics 24 (2): 172-175. https://doi.org/ 10.1093/bioinformatics/btm573 Lazoski C, Gusmao J, Boudry P, Solé-Cava A (2011) Phylogeny and phylogeography of Atlantic oyster species: Evolutionary history, limited genetic connectivity and isolation by distance. Marine Ecology Progress Series 426: 197-212. https://doi.org/10.3354/ meps09035 Lee Y, Kwak H, Shin J, Kim SC, Kim T, Park JK (2019) A mitochondrial genome phylogeny of Mytilidae (Bivalvia: Mytilida). Molecular Phylogenetics and Evolution 139: 106533. https://doi.org/10.1016/j.ympev.2019.106533 Li C, Kou Q, Zhang Z, Hu L, Huang W, Cui Z, Liu Y, Ma P, Wang H (2021) Reconstruction of the evolutionary biogeography reveal the origins and diversification of oysters (Bivalvia: Ostreidae). Molecular Phylogenetics and Evolution 164: 107268. https://doi.org/10.1016/j.ympev.2021.107268 LiF, Fan M, Wang S, Gu Z, Wang A, Liu C, Yang Y, Liu S (2022) The complete mitochondrial genome of Hyotissa hyotis (Bivalvia: Gryphaeidae) reveals a unique gene order within Ostreoidea. Fishes 7 (6): 317. https://doi.org/10.3390/fishes 7060317 The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 19 Li S, LiQ, Yu H, Kong L, Liu S (2015) Genetic variation and population structure of the Pacific oyster Crassostrea gigas in the northwestern Pacific inferred from mitochondrial COI sequences. Fisheries Science 81 (6): 1071-1082. https://doi.org/10.1007/ $12562-015-0928-x Liu J, LiQ, Kong L, Yu H, Zheng X (2011) Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding. Molecular Ecology Resources 11 (5): 820-830. https://doi.org/10.1111/j.1755-0998.2011.03025.x MolluscaBase (Ed.) (2023) Dendostrea Swainson, 1835. https:// www.marinespecies.org/aphia.php?p=taxdetails&id=4 15280. Accessed on: 2023-1-20. Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelinood phylogenies. Molecular Biology and Evolution 32 (1): 268-274. https://doi.org/10.1093/molbev/msu300 Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290: 470-474. https://doi.org/10.1038/290470a0 Perna NT, Kocher TD (1995) Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. Journal of Molecular Evolution 41: 353-358. https://doi.org/10.1007/BF00186547 Plazzi F, Passamonti M (2010) Towards a molecular phylogeny of Mollusks: Bivalves’ early evolution as revealed by mitochondrial genes. Molecular Phylogenetics and Evolution 57 (2): 641-657. https://doi.org/10.1016/j.ympev.2010.08.032 Ren J, Liu X, Jiang F, Guo X, Liu B (2010) Unusual conservation of mitochondrial gene order in Crassostrea oysters: evidence for recent speciation in Asia. BMC Evolutionary Biology 10: 394. https://doi.org/10.1186/1471-2148-10-394 Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, H6hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61 (3): 539-542. https://doi.org/10.1093/sysbio/sys029 Salvi D, Macali A, Mariottini P (2014) Molecular phylogenetics and systematics of the bivalve family Ostreidae based on rRNA sequence-structure models and multilocus species tree. PLOS One 9 (9): 108696. https://doi.org/10.1371/journal.pone.0108696 Salvi D, Mariottini P (2017) Molecular taxonomy in 2D: a novel ITS2 rRNA sequence- structure approach guides the description of the oysters' subfamily Saccostreinae and the genus Magallana (Bivalvia: Ostreidae). Zoological Journal of the Linnean Society 179 (2): 263-276. https://doi.org/10.1111/Z0j.12455 Salvi D, Mariottini P (2021) Revision shock in Pacific oysters taxonomy: the genus Magallana (formerly Crassostrea in part) is well-founded and necessary. Zoological Journal of the Linnean Society 192 (1): 43-58. https://doi.org/10.1093/zoolinnean/ zlaai12 Salvi D, Berrilli E, Garzia M, Mariottini P (2021) Yet Another Mitochondrial Genome of the Pacific Cupped Oyster: The Published Mitogenome of Alectryonella plicatula (Ostreinae) Is Based on a Misidentified Magallana gigas (Crassostreinae). Frontiers in Marine Science 8: 741455. https://doi.org/10.3389/fmars.2021.741455 Salvi D, Al-Kandari M, Oliver PG, Berrilli E, Garzia M (2022) Cryptic Marine Diversity in the Northern Arabian Gulf: An Integrative Approach Uncovers a New Species of Oyster (Bivalvia: Ostreidae), Ostrea oleomargarita. Journal of Zoological Systematics and Evolutionary Research 2022: 7058975. https://doi.org/10.1155/2022/7058975 20 Li F et al San Mauro D, Gower D, Zardoya R, Wilkinson M (2005) A hotspot of gene order rearrangement by tandem duplication and random loss in the vertebrate mitochondrial genome. Molecular Biology and Evolution 23 (1): 227-234. https://doi.org/10.1093/ molbev/msj025 Spencer H, Willan R, Mariottini P, Salvi D (2022) Taxonomic consistency and nomenclatural rules within oysters: Comment on Li et al. (2021). Molecular Phylogenetics and Evolution 170: 107437. https://doi.org/10.1016/j.ympev.2022.107437 Sun S, Cheng J, Sun S, Sha Z (2020) Complete mitochondrial genomes of two deep- sea pandalid shrimps, Heterocarpus ensifer and Bitias brevis: insights into the phylogenetic position of Pandalidae (Decapoda: Caridea). Journal of Oceanology and Limnology 38 (3): 816-825. https://doi.org/10.1007/s00343-019-9040-x Wang Y, Yang Y, Liu H, Kong L, Yu H, Liu S, Li Q (2021) Phylogeny of Veneridae (Bivalvia) based on mitochondrial genomes. Zoologica Scripta 50 (1): 58-70. https:// doi.org/10.1111/zsc.12454 Wang Z, Li C, Fang W, Yu X (2016) The complete mitochondrial genome of two Tetragnatha spiders (Araneae: Tetragnathidae): severe truncation of tRNAs and novel gene rearrangements in Araneae. International Journal of Biological Sciences 12 (1): 109-119. https://doi.org/10.7150/ijbs. 12358 Wilberg MJ, Livings ME, Barkman JS, Morris BT, Robinson JM (2011) Overfishing, disease, habitat loss, and potential extirpation of oysters in upper Chesapeake Bay. Marine Ecology Progress Series 436: 131-144. htips://doi.org/10.3354/meps09161 Willan R (2021) Magallana or mayhem? Molluscan Research 41 (1): 75-79. https:// doi.org/10.1080/13235818.2020.1865514 Wolstenholme DR (1992) Animal mitochondrial DNA: structure and evolution. International Review of Cytology 141: 173-216. https://doi.org/10.1016/ S0074-7696(08)62066-5 Wu X, Xu X, Yu Z, Wei Z, Xia J (2010) Comparison of seven Crassostrea mitogenomes and phylogenetic analyses. Molecular Phylogenetics and Evolution 57 (1): 448-454. https://doi.org/10.1016/j.ympev.2010.05.029 Wu X, Li X, LiL, Xu X, Xia J, Yu Z (2012) New features of Asian Crassostrea oyster mitochondrial genomes: a novel alloacceptor tRNA gene recruitment and two novel ORFs. Gene 507 (2): 112-118. https://doi.org/10.1016/j.gene.2012.07.032 Xia X (2018) DAMBE7: New and improved tools for data analysis in molecular biology and evolution. Molecular Biology and Evolution 35: 1550-1552. https://doi.org/10.1093/ molbev/msy073 Yang Y, Liu H, Qi L, Kong L, Li Q (2020) Complete mitochondrial genomes of two toxin- accumulated nassariids (Neogastropoda: Nassariidae: Nassarius) and their implication for phylogeny. International Journal of Molecular Sciences 21 (10): 3545. https://doi.org/ 10.3390/ijms21103545 The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... Supplementary material Suppl. material 1: Best fit partitions and substitution models EJ Authors: Fengping Li, Yi Yang Data type: Table Download file (15.00 kb) 21