The complete mitochondrial genome of Hyotissasinensis (Bivalvia, Ostreoidea) indicates the genetic diversity within Gryphaeidae

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 Hyotissasinensis 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 rrnL 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 Cox1 to Nad3, the same area where the duplicated genes are located. The rearrangements within 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.


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 rrnL 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 (Boore 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 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.

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.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′-TCGACAGGTGG GCTAGACTTAACGC-3′) specific primers designed in the present study.The long PCR reactions contained 2.5 μl of 10× buffer (Mg 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.

Mitogenomic annotation and sequence analysis
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).

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)  List of mitochondrial genomes used in this study.
The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 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.

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 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).
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.

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 (  (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).Table 2.
Gene annotations of the complete mt genome of Hyotissa sinensis.
The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 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).

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 ( Cox1, Nad1, Cox2, Nad6, Nad4 and Atp6) and ATA 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.Mitochondrial genome map of Hyotissa sinensis.
List of AT content, AT skew and GC skew of Hyotissa sinensis mtDNA.
Codon and relative synonymous codon usage (RSCU) of 12 protein-coding genes (PCGs) in the mtDNA of Hyotissa sinensis.
The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 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).
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 Relative synonymous codon usage (RSCU) of mitochondrial genome for Hyotissa sinensis.
The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 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.

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 (−lnL = 154,942.703)and BI (−lnL = 150,197.79for run 1; −lnL = 150,198.99for 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.
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

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 The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) ... 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.Linearised PCG order of Ostreoidea, based on the phylogenetic tree.
(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 posttranscriptional polyadenylation (Ojala et al. 1981).The relative synonymous codon usage (RSCU) values of H. sinensis are shown in Table Figure 2.

Figure 5 .
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.

Figure 6 .
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.
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).

Table 1 .
under the Bayesian Information Criterion (BIC).The partitions tested in the present study referred to Li et al. (2022).
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 (Wu et al. 2010e 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.