Sequencing of the Complete Mitochondrial Genome of the Big Brown Mactra Clam, Mactra grandis (Venerida: Mactridae)

Simple Summary Mitochondrial genomes have become a powerful tool for studying molecular genetics and phylogeny of mollusks. In this study, the complete mitochondrial genome of Mactra grandis was characterized for the first time. The newly sequenced mitochondrial genome fits the typical composition pattern of mollusks with 37 functional genes. Among the Mactridae species with reported mitochondrial genomes, Mactra grandis has the closest relationship with Mactra cygnus. The gene arrangement, genetic distance, and selective pressure of protein-coding genes among Mactra species were also analyzed. This study provides a molecular basis for taxonomy and germplasm research on Mactridae species. Abstract Mitochondrial genomes are playing an increasingly important role in molluscan taxonomy, germplasm, and evolution studies. The first complete mitochondrial genome of the commercial big brown mactra clam, Mactra grandis, was characterized using Illumina next-generation sequencing in this study. The 17,289 bp circular genome has a typical gene organization of 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs, with an obvious (A + T)-bias of 64.54%. All PCGs exhibited a homogeneous bias in nucleotide composition with a (A + T)-bias, a positive GC skew, and a negative AT skew. Results of phylogenetic analysis showed that Mactra grandis was most closely related to Mactra cygnus. The functional gene arrangement of the two species was identical but different from other Mactra species. The congeneric relationships among Mactra species were demonstrated by genetic distance analysis. Additionally, the selective pressure analysis suggested that cox1 was highly efficient for discriminating closely related species in genus Mactra, while nad2 was the most appropriate marker for population genetic analysis.


Introduction
With the continuous decrease in the cost of sequencing high-quality genomes, an increasing number of mitochondrial genomes for mollusks have been reported.These contributions have significantly enriched studies in taxonomy, germplasm research, and investigations into adaptive evolution [1][2][3][4].On one hand, molluscan mitogenomes offer valuable molecular insights for biological taxonomy research, encompassing conserved sequences of functional genes and gene arrangements among closely related species [5][6][7].On the other hand, mitochondrial genomes have a faster evolutionary rate than nuclear genomes and contain appropriate gene markers, mitochondrial single nucleotide polymorphisms, and mutations, which can be used for population genetic diversity and germplasm Animals 2024, 14, 1376 2 of 11 evaluation [8][9][10].Nonetheless, there are still far too few species with sequenced mitogenomes considering the tens of thousands of existing mollusks in the world [11].
The family Mactridae, classified by Lamarck in 1809, encompasses approximately 150 species distributed globally.Commonly characterized by their thin and fragile shells, these bivalves are referred to as surf or trough clams [12,13].Synonyms and taxa rearrangements are quite common in Mactridae species because their shell coloration and forms tend to change with the environment [14].Mitochondrial genomes have been proven effective in recognizing taxonomic and phylogenetic problems in Mactridae species.However, previous phylogenetic data were insufficient to address these issues adequately [15].Mactra grandis (Gmelin, 1791), synonymous with Mactra mera (Reeve, 1854), is an edible large mactrid species distributed in tropical Indo-Pacific regions [14].Known as the big brown mactra clam, Mactra grandis has shells ranging from 6 to 7 cm and is considered the most commonly encountered member of the family Mactridae in Singapore [16].While the type specimen of Mactra mera was collected in China as early as 1854 [17], modern records for this species only began in 1960 [18].Despite its historical and contemporary significance, molecular data for Mactra grandis remain virtually absent.
The mitochondrial genome of Mactra grandis was characterized for the first time in this study.The aims were to (1) provide the first complete mitogenome of the commercial species and (2) verify the classification among Mactra species.

Sample Collection and DNA Extraction
A specimen of Mactra grandis (shell length 6.67 cm, height 4.78 cm, and width 3.06 cm) was collected on 9 August 2023 from the Li'an Bay, Hainan Province (18 • 25.387 ′ N, 110 • 1.039 ′ E).The adductor muscle was exclusively selected for DNA extraction to mitigate potential confounding factors associated with the doubly uniparental inheritance observed in mussels and clams [19].The rest of the specimen was preserved in 95% ethanol and deposited in the State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.Total genomic DNA was extracted using the TIANamp Marine Animals DNA Kit (DP324-03; Tiangen Biotech (Beijing), Beijing, China), following the manufacturer's protocol.

Sequencing, Assembly, and Genome Annotation
The genomic library was constructed with the whole-genome shotgun strategy and sequenced on the Illumina NovaSeq platform (Illumina, San Diego, CA, USA) at Shanghai Personal Biotechnology Co., Ltd.(Shanghai, China) by using the 2 × 150 bp paired-end sequencing mode and with an insert size of 400 bp.The software NGS QC Toolkit v2.3.3 [20] was used for quality control of raw data.Then, GetOrganelle v1.7.7.0 [21] and SPAdes v3.9.0 [22] were employed for the de novo assembly to construct contig and scaffold sequences.BLASTN was conducted in the NCBI nucleotide database using Blast v2.2.31+.And finally, Mummer v3.1 [23] and Pilon v1.18 [24] were used to fill gaps between contigs.
The complete mitogenome sequence was uploaded to the MITOS2 Web Server (http:// mitos2.bioinf.uni-leipzig.de/index.py,accessed on 15 October 2023) for functional annotation [25].The genetic code was selected as "5 Invertebrate Mitochondrial".The boundaries of PCGs were determined by an ORF finder (https://www.ncbi.nlm.nih.gov/orffinder,accessed on 15 October 2023) and corrected manually by comparison with genes from the same family [15].The mitochondrial genome circular map was drawn using the Proksee [26].

Genome Composition and Codon Usage
MEGA 7.0 software [27] was applied to calculate the nucleotide base composition of the newly sequenced mitogenome.GC skew was determined using the following formulae: GC skew= (G − C)/ (G + C) and AT skew = (A − T)/ (A + T), where G, C, A, and T represent the frequency of each nucleotide base.PhyloSuite v1.1.16[28] was used to analyze the relative synonymous codon usage (RSCU) of the mitogenome.

Phylogenetic Analysis and Gene Arrangement
Phylogenetic relationships within the family Mactridae were determined based on the datasets of 13 PCGs and 2 rRNAs by PhyloSuite [28], with Donax trunculus from the family Donacidae and Mercenaria mercenaria from the family Veneridae being the outgroup.MAFFT was employed to align the PCGs under codon mode and rRNAs under normal mode independently [29].Then, all PCG and rRNA alignments files were concatenated into a data matrix.The best partitioning scheme and evolutionary models for 15 predefined partitions were selected using PartitionFinder2 [30], with greedy algorithm and AICc criterion.
Phylogenetic trees were reconstructed using Bayesian inference (BI) and maximum likelihood (ML) analyses.Bayesian inference phylogenies were inferred using MrBayes 3.2.6 [31] under partition model (2 parallel runs, 200,000 generations), in which the initial 25% of sampled data were discarded as burn-in.Maximum likelihood phylogenies were inferred using IQ-TREE [32] under an edge-linked partition model for 5000 ultrafast bootstraps [33], as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test [34].Phylogenetic trees and gene arrangements were visualized using the Interactive Tree of Life [35].The branch support values of Bayesian posterior probabilities (PP) and the maximum likelihood bootstrap support values (BS) were shown on the trees.The CREx algorithm was employed to reconstruct the putative gene order rearrangement events that might have transpired within the genus Mactra [36].

Selective Pressure and Genetic Distance Analysis
Based on the phylogenetic results, the selective pressure on PCGs in the two main Mactra clades was analyzed, respectively.Software PhyloSuite [28] was employed to perform the preparation of the mitochondrial PCG sequences.The sequences were aligned in batches with MAFFT [29] using '-auto' strategy and codon alignment mode.The alignments were refined using the codon-aware program MACSE v. 2.03 [37], which preserved the reading frame and allowed incorporation of sequencing errors or sequences with frameshifts.Ambiguously aligned fragments of 13 alignments were removed in batches using Gblocks [38].DnaSP6 software [39] was then used to calculate the nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) of each PCG.Given their widespread utilization as genetic markers in population, phylogeny, and evolution studies of bivalves, cox1 and 16S were chosen alongside PCG nad2, which experienced the least selective pressure in this investigation, to assess the genetic distances between the two primary clades within the genus Mactra [8,40,41].The genetic distances were analyzed using the Kimura 2-parameter model in MEGA 7.0 [29] to elucidate their taxonomic relationships, with only one sequence utilized per species.

Protein-Coding Genes
Variation and heterogeneity of DNA base compositions among species or gene fragments were the results of evolutionary adaptation to the environment [42].Interestingly, all 13 PCGs of Mactra grandis mitogenome exhibited a homogeneous bias in nucleotide composition with a (A + T)-bias from 61.45% (nad5) to 72.81% (atp8), a positive GC skew from 0.2070 (cytb) to 0.5484 (atp8), and a negative AT skew from −0.4171 (nad4l) to −0.2028 (cox2).Cox1, nad3, and nad4 had GTG and nad1 had ATA at the sequence start, while the other 9 PCGs had ATG at the sequence start.All the PCGs except nad4l, which was truncated with nucleotide T, had TAA or TAG at the sequence end.
The relative synonymous codon usage (RSCU) of Mactra grandis mitogenome indicated Phe, Val, and Leu were the three most frequently used amino acids (417, 405, and 370 counts, respectively) (Figure 2).Consistent with other mactrid mitogenomes, NNU and NNA were dominant in most codons, indicating a preferred sequence ending with A or T. UUA-Leu2, UCU-Ser2, CCU-Pro, ACU-Thr, and GCU-Ala were the five most frequently used codons.All five codons had RSCU values over 2.

Ribosomal RNAs and Transfer RNAs
The lengths of 12S and 16S ribosomal RNAs of Mactra grandis were 895 bp and 1193 bp, respectively.The 12S rRNA was located between trnP and trnY, while the 16S rRNA was located between cytb and atp8.The AT base contents for 12S and 16S were 64.43% and 67.79%, respectively, indicating AT biases.Both rRNAs showed positive GC skew (0.2366 and 0.2727 for 12S and 16S, respectively).However, the 12S showed a negative AT skew (−0.0138) while the 16S showed a positive AT skew (0.0050).
The 22 mitochondrial tRNAs of Mactra grandis ranged from 61 bp (trnH and trnS2) to 70 bp (trnC).All the tRNAs showed positive GC skews, ranging from 0.0526 in trnE to 0.5000 in trnD.Except for trnL1, for which the AT skew was positive (0.0667), all tRNAs showed negative AT skews, ranging from −0.2973 in trnS2 to −0.0233 in trnW.All tRNAs showed a typical cloverleaf model.The origin for the heavy strand replication (OH), 416 bp, was located between trnH and trnR.

Ribosomal RNAs and Transfer RNAs
The lengths of 12S and 16S ribosomal RNAs of Mactra grandis were 895 bp and 1,193 bp, respectively.The 12S rRNA was located between trnP and trnY, while the 16S rRNA was located between cytb and atp8.The AT base contents for 12S and 16S were 64.43% and 67.79%, respectively, indicating AT biases.Both rRNAs showed positive GC skew (0.2366 and 0.2727 for 12S and 16S, respectively).However, the 12S showed a negative AT skew (−0.0138) while the 16S showed a positive AT skew (0.0050).
The 22 mitochondrial tRNAs of Mactra grandis ranged from 61 bp (trnH and trnS2) to 70 bp (trnC).All the tRNAs showed positive GC skews, ranging from 0.0526 in trnE to 0.5000 in trnD.Except for trnL1, for which the AT skew was positive (0.0667), all tRNAs showed negative AT skews, ranging from −0.2973 in trnS2 to −0.0233 in trnW.All tRNAs showed a typical cloverleaf model.The origin for the heavy strand replication (OH), 416 bp, was located between trnH and trnR.

Phylogeny and Gene Arrangement Analysis
The best partitioning scheme in this study had 164 params, 18,324 sites, and 10 subsets, with InL and AICc being −221,561.8095703125and 443,454.599481,respectively.The best evolutionary models were listed in Table 2. Phylograms derived from ML and BI analyses had identical topologies, suggesting that the family Mactridae was subdivided into two main clades (Figure 3, PP = 1, BS = 100).One clade covered only Mactra species, including eighteen mitochondrial genome sequences from 7 species and a cryptic species

Phylogeny and Gene Arrangement Analysis
The best partitioning scheme in this study had 164 params, 18,324 sites, and 10 subsets, with InL and AICc being −221,561.8095703125and 443,454.599481,respectively.The best evolutionary models were listed in Table 2. Phylograms derived from ML and BI analyses had identical topologies, suggesting that the family Mactridae was subdivided into two main clades (Figure 3, PP = 1, BS = 100).One clade covered only Mactra species, including eighteen mitochondrial genome sequences from 7 species and a cryptic species in Mactra antiquata.The newly sequenced species, Mactra grandis, clustered with Mactra cygnus, demonstrating a sister relationship (Clade B).Other Mactra species clustered to Clade A, including Mactra quadrangularis, Mactra sp., Mactra chinensis, Mactra antiquata, the cryptic species in Mactra antiquata, and Mactra cumingii, with the first five species having two or more sequences.Although markedly morphologically different from Mactra antiquata, Mactra cumingii occupied the phylogenetic position to divide the Mactra antiquata mitogenomes into two types, which supported the existence of a cryptic species in Mactra antiquata [2,43].The other ten Mactridae species from 6 genera constituted another main clade, including four genera from subfamily Mactrinae (Mactrinula, Rangia, Mulinia, and Spisula), one genus from subfamily Lutrariinae (Lutraria), and one family from subfamily Anatinellidae (Raeta).Our results provided support for removing Mactrinula, Rangia, Mulinia, and Spisula out from subfamily Mactrinae [15].Two Raeta species first clustered with Mactrinula dolabrata, prompting the attribution of Raeta to family Mactridae instead of Anatinellidae [44].Results also showed that all congeneric species in Mactridae clustered together, indicating closer relationships.Anatinellidae (Raeta).Our results provided support for removing Mactrinula, Rangia, Mulinia, and Spisula out from subfamily Mactrinae [15].Two Raeta species first clustered with Mactrinula dolabrata, prompting the attribution of Raeta to family Mactridae instead of Anatinellidae [44].Results also showed that all congeneric species in Mactridae clustered together, indicating closer relationships.Mitochondrial gene arrangements among closely related mollusks were usually highly conserved, although gene rearrangements could be relatively frequent in Mollusca [45][46][47].In this study, most Mactridae species had 22 tRNAs, besides Spisula sibyllae (MG431821) and Mactra antiquata (KC503290, KC503291, and JQ423460), which had a duplication of trnM (Figure 4).Although the genus Mactra was shown as a monophyletic group, two types of gene arrangement were observed within the genus.Clade A and Clade B had independent gene orders.The differences involved the translocations of two long gene chains: -trnT-nad1-trnG-nad2-trnD-and -trnP-12S-trnY-trnS1-cox3-cytb-16S-atp8-nad4-trnH-trnR-trnL2-trnE-trnS2-atp6-nad3-trnK.CREx analysis indicated the occurrence of a tandem duplication random loss (TDRL) event from Mactra grandis to Mactra quadrangularis.This difference put forward the question of whether the two clades belonged to one genus.Additionally, three species in genus Spisula also showed differences in not only generic makeup but also gene orders.However, in genera Lutraria and Raeta, gene arrangements were highly conserved.
atp8-nad4-trnH-trnR-trnL2-trnE-trnS2-atp6-nad3-trnK.CREx analysis indicated the occurrence of a tandem duplication random loss (TDRL) event from Mactra grandis to Mactra quadrangularis.This difference put forward the question of whether the two clades belonged to one genus.Additionally, three species in genus Spisula also showed differences in not only generic makeup but also gene orders.However, in genera Lutraria and Raeta, gene arrangements were highly conserved.

Genetic Comparison within Genus Mactra
To further clarify the relationships between Clade A and Clade B, selective pressure and genetic distance analyses were conducted in this study.The results of selective pressure analysis within the two clades in Mactra showed that all PCGs had Ka/Ks < 1 (Table 3), indicating purifying selection in Mactra species.The minimum Ka/Ks value of Cox1 was 0.02989 in Clade A and 0.07025 in clade B, indicating that mitochondrial cox1 would be highly efficient for discriminating closely related species in genus Mactra [48][49][50].Except for the atp8 in Clade A, nad2 showed the highest Ka/Ks values in both clades, suggesting that nad2 was subject to the least selective pressure among the 12 PCGs and had the highest variation.This makes nad2 the most suitable marker for population genetic analysis [8] and may lead to gene chain translocation between the two clades.The genetic distances within Clade A were determined to be 0.17266, 0.29388, and 0.13860 based on the cox1, nad2, and 16S rRNA, respectively.However, the genetic distances between the two clades were comparable with that within Clade B based on cox1 (0.21858 < 0.22756), nad2 (0.49023 < 0.49587), and 16S rRNA (0.26458 < 0.28222).The results were consistent

Genetic Comparison within Genus Mactra
To further clarify the relationships between Clade A and Clade B, selective pressure and genetic distance analyses were conducted in this study.The results of selective pressure analysis within the two clades in Mactra showed that all PCGs had Ka/Ks < 1 (Table 3), indicating purifying selection in Mactra species.The minimum Ka/Ks value of Cox1 was 0.02989 in Clade A and 0.07025 in clade B, indicating that mitochondrial cox1 would be highly efficient for discriminating closely related species in genus Mactra [48][49][50].Except for the atp8 in Clade A, nad2 showed the highest Ka/Ks values in both clades, suggesting that nad2 was subject to the least selective pressure among the 12 PCGs and had the highest variation.This makes nad2 the most suitable marker for population genetic analysis [8] and may lead to gene chain translocation between the two clades.The genetic distances within Clade A were determined to be 0.17266, 0.29388, and 0.13860 based on the cox1, nad2, and 16S rRNA, respectively.However, the genetic distances between the two clades were comparable with that within Clade B based on cox1 (0.21858 < 0.22756), nad2 (0.49023 < 0.49587), and 16S rRNA (0.26458 < 0.28222).The results were consistent with the interspecific genetic distances within Mactra, based on either cox1 (0.085 ~0.284) or 16S (0.014 ~0.271) [48].As a result, species from both Clade A and Clade B should belong to the same genus.

Conclusions
The complete mitochondrial genome of Mactra grandis is a circular molecule of 17,289 bp, with 13 protein-coding genes, 2 rRNAs, and 22 tRNAs.All protein-coding genes in Mactra grandis mitogenome exhibited a homogeneous bias in nucleotide composition with a (A + T)-bias, a positive GC skew, and a negative AT skew.Among the Mactridae species with mitogenomes reported, Mactra grandis is the most closely related to Mactra cygnus in terms of both the mitochondrial molecular dendrogram and the functional gene arrangement.By contrast, other Mactra species share another gene arrangement, with differences in the translocations of two long gene chains: -trnT-nad1-trnG-nad2-trnD-and -trnP-12S-trnY-trnS1-cox3-cytb-16S-atp8-nad4-trnH-trnR-trnL2-trnE-trnS2-atp6-nad3-trnK.Still, the congeneric relationships among Mactra species are determined.The selective pressure analysis of mitochondrial protein-coding genes further suggests that cox1 is highly efficient for discriminating closely related species in genus Mactra and that nad2 is the most appropriate marker for population genetic analysis.Institutional Review Board Statement: Ethical review and approval were waived for this study because the mactrid in this study is an invertebrate with no sense or subjective experience.
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.The mitochondrial genome map of Mactra grandis, collected in Li'an Bay, Hainan, China.

Figure 3 .Figure 3 .
Figure 3. Phylogenetic tree of Mactridae species based on 13 protein-coding genes and 2 rRNAs, with Mercenaria mercenaria and Donax trunculus being the outgroup.Numbers near the nodes areFigure 3. Phylogenetic tree of Mactridae species based on 13 protein-coding genes and 2 rRNAs, with Mercenaria mercenaria and Donax trunculus being the outgroup.Numbers near the nodes are branch support values of Bayesian posterior probabilities, followed by maximum likelihood bootstrap support values.Mitogenome sequence obtained in this study was marked in red.

Figure 4 .
Figure 4. Gene arrangements of mitogenomes in Mactridae.Mitogenome sequence obtained in this study was marked in red.

Figure 4 .
Figure 4. Gene arrangements of mitogenomes in Mactridae.Mitogenome sequence obtained in this study was marked in red.

Author
Contributions: X.W. and B.W. designed the experiment.X.W. collected the sample and interpreted the data.P.M. conducted the experiment, wrote the first draft of the manuscript, and was accountable for all aspects of the work.Z.L. (Zhuanzhuan Li) and Z.L. (Zhihong Liu) provided technical assistance on data analysis.X.S. and L.Z. revised the manuscript critically.B.W. was responsible for the funding acquisition and final approval of the version to be published.All authors commented on the previous version of the manuscript and edited the language.All authors have read and agreed to the published version of the manuscript.Funding: This work was partly supported by the National Marine Genetic Resource Center, the Project of Yellow River Fisheries Resources and Environment Investigation from the MARA, P. R. China, the National Natural Science Foundation of China (No. 42006080), and the Central Publicinterest Scientific Institution Basal Research Fund, CAFS (No. 2023TD30).

Table 1 .
Organization of the mitochondrial genome of Mactra grandis.
Figure 1.The mitochondrial genome map of Mactra grandis, collected in Li'an Bay, Hainan, China.

Table 1 .
Organization of the mitochondrial genome of Mactra grandis.

Table 2 .
Partitions and evolutionary models selected by PartitionFinder2 for phylogenetic analyses.

Table 2 .
Partitions and evolutionary models selected by PartitionFinder2 for phylogenetic analyses.

Table 3 .
The evolutionary constraint (Ka/Ks) analyses of 13 mitochondrial protein-coding genes in two clades of genus Mactra.Ka: nonsynonymous substitution rate; Ks: synonymous substitution rate calculations.