Biodiversity Data Journal : Research Article
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Research Article
The complete mitochondrial genome of Hyotissa sinensis (Bivalvia, Ostreoidea) indicates the genetic diversity within Gryphaeidae
expand article infoFengping Li‡,§, Hongyue Liu|, Xin Heng, Yu Zhang, Mingfu Fan, Shunshun Wang, Chunsheng Liu, Zhifeng Gu, 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
Open Access

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 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.

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 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.

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′-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.

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

Table 1.

List of mitochondrial genomes used in this study.

New mt genomes

Family

Species

Length (bp)

Sampling time

Accession No.

Gryphaeidae

Hyotissa sinensis

30,385

June 2022

OQ333008

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 denselamellosa

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

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.

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 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.

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 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).

Table 2.

Gene annotations of the complete mt genome of Hyotissa sinensis.

Gene

Strand

Location

Size (bp)

Start Codon

Stop codon

Intergenic nucleotides

Cox1

H

1-1986

1986

ATG

TAA

1936

tRNA-Met1

H

3923-3989

67

842

tRNA-Lys

H

4832-4898

67

81

tRNA-Gln

H

4944-5006

63

0

rrnL

H

5007-6335

1329

212

rrnS

H

6548-7491

944

74

tRNA-Ile

H

7566-7632

67

97

tRNA-Ser1

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

tRNA-Phe

H

10061-10125

65

16

rrnL

H

10142-11507

1366

192

rrnS

H

11700-12643

944

164

tRNA-Thr

H

12808-12870

63

179

tRNA-Pro

H

13050-13115

66

91

tRNA-Leu1

H

13207-13269

63

537

tRNA-Asp

H

13807-13874

68

1112

tRNA-Leu2

H

14987-15049

63

3901

Nad3

H

18951-19290

340

ATT

T

444

tRNA-Asn

H

19735-19801

67

2

tRNA-Gly

H

19804-19869

66

7

tRNA-Ser2

H

19877-19946

70

25

tRNA-Trp1

H

19972-20039

68

13

tRNA-Leu3

H

20053-20115

63

29

tRNA-Val

H

20145-20211

67

30

tRNA-His

H

20242-20305

64

27

Cox2

H

20333-21028

696

ATG

TAG

125

Cytb

H

21154-22785

1632

ATA

TAG

78

Nad2

H

22864-23883

1020

ATT

TAA

14

tRNA-Trp2

H

23898-23965

68

6

Nad5

H

23975-25828

1857

TTG

TAG

96

Nad6

H

25925-26527

603

ATG

TAG

30

tRNA-Cys

H

26558-26620

63

1

Nad4

H

26622-27965

1344

ATG

TAG

74

Atp6

H

28040-28786

747

ATG

TAG

Nad4L

H

28855-29145

291

TTG

TAG

35

tRNA-Arg

H

29181-29243

63

209

Cox3

H

29453-30331

879

TTT

TAA

54

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).

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

57.5

-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

rrnL1

57.6

-0.03

0.20

rrnS1

52.8

0.09

0.13

rrnL2

58.4

-0.02

0.20

rrnS2

52.6

0.10

0.13

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 (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.

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)

CUA

39.0(0.50)

GGA

82.0(0.93)

CUG

71.0(0.91)

GGG

151.0(1.71)

Ile

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)

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.

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 = 150,197.79 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.

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 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.

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).

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

Supplementary material

Suppl. material 1: Best fit partitions and substitution models  
Authors:  Fengping Li, Yi Yang
Data type:  Table
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