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.

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′-GGGGGTAAGATATTTTGTGCAGCGA-3′) and 1R reverse (5′-TCGACAGGTGGGCTAGACTTAACGC-3′) specific primers designed in the present study. The long PCR reactions contained 2.5 μl of 10× buffer (Mg²⁺ plus), 3 μl of dNTPs (2.5 mM), 0.5 μl of each primer (10 μM), 0.8 μl of template DNA (25–40 ng/μl), 0.2 μl of TaKaRa LA Taq DNA polymerase (5 U/μl) and DEPC (Diethypyrocarbonate) water up to 25 μl. 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.

The mitogenome of H. sinensis was annotated using Geneious Prime. The PCGs were determined by ORF Finder (http://www.ncbi.nlm.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 OP151093). The secondary structure of tRNA genes was predicted by MITOS and ARWEN (Laslett and Canbäck 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).

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 Maximum Likelihood method (ML) and Bayesian Inference method (BI) were used for phylogenetic reconstruction. ML trees were constructed by IQtree 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 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 FigTree v.1.4.2.

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 MW143047) 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 rrnL fragment, which shows identity values from 99.19% to 99.79% to the previously published sequences on GenBank (KC847135 and MT332230).

Figure 1.  

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 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 rrnL and rrnS genes. No additional gene was discovered within this Sanger-sequencing fragment.

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 Atp8 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 Atp8 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 (Wu 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 rrnS and rrnL 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).

Figure 2.  

Mitochondrial genome map of Hyotissa sinensis.

Figure 3.  

Inferred secondary structures of 26 transfer RNAs from Hyotissa sinensis.

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 mitogenomes (Wu et al. 2010).

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 (Cox1, 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 (Cox1, 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.

Figure 4.  

Relative synonymous codon usage (RSCU) of mitochondrial genome for Hyotissa sinensis.

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 trnM in most bivalve mitogenomes (Lee et al. 2019, Wang et al. 2021). The presence of three trnM in H. sinensis has never been reported in Ostreoidea before. All three trnM genes recognise codon AUG, but trnM2 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 trnW genes that occur only in Gryphaeidae should be considered as an occasional event within Ostreoidea (Wu et al. 2012).

Figure 5.  

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.

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 rrnS is considered as a common feature of the Asian genus Magallana. Similarly, it is assumed that the duplication of rrnL and rrnS 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 rrnS 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.

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 (−lnL = 154,942.703) and BI (−lnL = 150,197.79 for run 1; −lnL = 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 (Dietl 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.

Figure 6.  

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 (BI) 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 Likelihood (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.

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 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).

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.

Figure 7.  

Linearised PCG order of Ostreoidea, based on the phylogenetic tree.

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 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.