The complete mitochondrial genome of Sesarmops sinensis reveals gene rearrangements and phylogenetic relationships in Brachyura

Mitochondrial genome (mitogenome) is very important to understand molecular evolution and phylogenetics. Herein, in this study, the complete mitogenome of Sesarmops sinensis was reported. The mitogenome was 15,905 bp in size, and contained 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region (CR). The AT skew and the GC skew are both negative in the mitogenomes of S. sinensis. The nucleotide composition of the S. sinensis mitogenome was also biased toward A + T nucleotides (75.7%). All tRNA genes displayed a typical mitochondrial tRNA cloverleaf structure, except for the trnS1 gene, which lacked a dihydroxyuridine arm. S. sinensis exhibits a novel rearrangement compared with the Pancrustacean ground pattern and other Brachyura species. Based on the 13 PCGs, the phylogenetic analysis showed that S. sinensis and Sesarma neglectum were clustered on one branch with high nodal support values, indicating that S. sinensis and S. neglectum have a sister group relationship. The group (S. sinensis + S. neglectum) was sister to (Parasesarmops tripectinis + Metopaulias depressus), suggesting that S. sinensis belongs to Grapsoidea, Sesarmidae. Phylogenetic trees based on amino acid sequences and nucleotide sequences of mitochondrial 13 PCGs using BI and ML respectively indicate that section Eubrachyura consists of four groups clearly. The resulting phylogeny supports the establishment of a separate subsection Potamoida. These four groups correspond to four subsections of Raninoida, Heterotremata, Potamoida, and Thoracotremata.


Introduction
The Brachyura mostly live in littoral regions of tropical shallow seas and about 7000 species have been described and is the most species rich infraorder within Decapoda [1]. Phylogenetic relationships within the Brachyura are complicated because of the extreme morphological and ecological diversity within the group [2]. Four groups of Brachyura (Dromiacea, Raninoida, Heterotremata and Thoracotremata) were recognized [3][4][5]. Raninoida, Heterotremata and Thoracotremata should be attributed to Eubrachyura, which is a sisiter group to Dromiacea [6,7].
Animal mitochondrial DNA (mtDNA), a double-stranded circular molecule, ranging from 14 to 19 kilobases (kb) in size, containing 37 genes, including 13 PCGs, ATPase subunits 6 and 8 of the ATPase (atp6 and atp8), cytochrome c oxidase subunits 1-3 (cox1-cox3), cytochrome B (cob), NADH dehydrogenase subunits 1-6 and 4 L (nad1-6 and nad4L), 22 tRNA genes, two rRNA genes and CR [8]. The mtDNA can provide important information on rearrangement trends and phylogeny because of its rapid evolutionary rate and lack of genetic recombination [8]. Using complete mitogenomes is becoming increasingly common for phylogenetic reconstruction [9][10][11]. The AT-content, secondary structures of tRNAs, and gene rearrangements can be inferred from animal mitogenomes at the genome level [12]. Partial DNA sequences are often too short to contain sufficient phylogenetic information [13]. Further, the addition of rRNA makes alignment ambiguous [2]. It is becoming increasingly common to use complete animal mitogenomes for phylogenetic reconstruction [9][10][11].
To date, there has been no reports of the complete mitogenome of S. sinensis. Thus, in this paper, the complete mitogenome of S. sinensis was sequenced and compared with other Brachyura mitogenomes. The available complete mitogenomes were used to provide insight into the phylogenetic relationship of S. sinensis and related species. These results will help us to understand features of S. sinensis mitogenome and the evolutionary relationships within Brachyura.

Ethics statement
There are no specific permits for crabs collection in the selected locations. The sampling locations are not privately-owned or natural protected areas. Crabs used for the experiments are not considered endangered or protected species, and its collection is legal in China.

Sampling and DNA extraction
Adult specimens of S. sinensis were collected from Fujian province in China in June 2014. The total genomic DNA of S. sinensis was isolated from single specimens using the Aidlab Genomic DNA Extraction Kit (Aidlab, China) according to the manufacturer's instructions. DNA from an individual S. sinensis crab was used to amplify the complete mitogenome.

PCR amplification and sequencing
To amplify the entire mitogenome of S. sinensis, specific primers were designed based on the conserved nucleotide sequences of known mitochondrial sequences in the Brachyura [14][15][16][17][18]. The complete mitogenome of S. sinensis was obtained using a combination of conventional PCR and long PCR to amplify overlapping fragments spanning the whole mitogenome. First, six conserved genes (cox1, cox3, 12S, 16S, nad4, cob) were sequencd using the universal primer of crabs. Then the specific primers were designed by conservative sequences. Finally the complete mitogenome was generated by overlapping PCR with specific primers. The fragments were amplified using Aidlab Red Taq (Aidlab) according to the manufacturer's instructions. PCR reactions for the fragments were performed in a 50μL volume with 5μL of 10×Taq plus Buffer (Mg 2+ ), 4μL of dNTPs, 2μL each of the primers, 2μL of DNA, 34.5μL of ddH 2 O, and 0.5μL Red Taq DNA polymerase. The PCR reactions were performed using the following procedures: 94˚C for 3 min; followed by 40 cycles of 30s at 94˚C, annealing for 35s at 48-56˚C (depending on primer combination), elongation for 1-3min (depending on length of the fragments) at 72˚C; and a final extension step of 72˚C for 10 min. The PCR products were separated by agarose gel electrophoresis (1% w/v) and purified using the DNA gel extraction kit (Aidlab, China). The purified PCR products were ligated into the T-vector (SangonBiotech, China) and sequenced at least three times.

Sequence alignment and gene annotation
Thirty-nine complete Brachyura mitogenomes were downloaded from GenBank. In addition, the mitogenomes of Cherax destructor and Cambaroides similis were downloaded from Gen-Bank and used as outgroup taxons. Detailed information is shown in Table 1.
Sequence annotation was performed using NCBI BLAST and the DNAStar package (DNAStar Inc. Madison, WI, USA). Alignments of sequences for each of the available Brachyura mitogenomes were performed using default settings in MAFFT, and then concatenated [19]. The sequences were aligned with those of closely related species. To remove the gaps in sequences and align each gene, poorly aligned positions and divergent regions were removed using Gblocks in our study [20]. The fasta sequences were converted to the nex format and phylip format for BI and ML, respectively. We then used DAMBE to detect the saturated conditions of the sequences [21]; the result of the DAMBE analysis was that ISS was less than ISS.c and p value was extremely significant (0.0000), suggesting that sequences was unsaturated and suit to construct phylogenetic tree. Cloverleaf secondary structure and anticodons of transfer RNAs were identified using the web-server of tRNA-scan SE [22].

Phylogenetic analysis
We estimated the taxonomic status of S. sinensis within the Decapoda by constructing the phylogenetic tree. Two concatenated datasets: amino acid alignments (AA dataset) and nucleotide alignments (NT dataset) from 42 mitogenomes PCGs were combined. Each dataset was processed using two inference methods: Bayesian inference (BI) and Maximum likelihood (ML). BI was performed using MrBayes v 3.2.1 [23]. ML was performed using raxmlGUI [24]. Nucleotide substitution models were selected using Akaike information criterion implemented in Mrmodeltest v 2.3 [25]. The GTR+I+G model was chosen as the best model of nucleotide phylogenetic analysis and molecular evolution. MtArt + I+ G +F was chosen as the best model for the AA dataset, according to the results of Prottest version 1.4 [26]. There was no MtArt + I+ G +F model in MrBayes; therefore, MtREV + I+ G +F was selected as the second best model. ML analyses were performed on 1000 bootstrapped replicates [27]. The Bayesian analysis ran as 4 simultaneous MCMC chains for 10,000,000 generations, sampled every 100 generations, and a burn-in of 2,500,000 generations was used. Convergence was deduced for the Bayesian analysis on the following basis: the average standard deviation of split frequencies was less than 0.01. Additionally, we observe sufficient parameter sampling by using software Tracer v1.6. The value of ESS is more than 200. The above two points show that our data is convergent [28]. BI was performed under the GTRCAT model with the NT dataset. ML was performed with ML+rapid bootstrap under the GTRCAT model with the NT dataset. BI and ML were performed under the MtREV + I+ G +F model with the AA dataset.

Genome structure and organization
The S. sinensis mitogenome is a closed circular molecule of 15,905 bp in length. It has been deposited in GenBank under the accession number KR336554 and contains typical animal mitochondrial genes, including 13 PCGs, 22 tRNA genes, a large ribosomal RNA (lrRNA) gene and a small ribosomal RNA (srRNA) genes, and CR (  [29]. The AT skew and GC skew were calculated for the selected complete mitogenomes ( Table 3). The A+T skew value is in the range from -0.080 (Pachygrapsus crassipes) to 0.040 (Homologenus malayensis). The GC skew values were negative in all sequenced Brachyura Although the AT skew and GC skew of S. sinensis are all negative, GC skew is far lower than that of AT indicates an obvious bias toward the use of As and Cs.

Protein-coding genes
The mitogenome of S. sinensis contains 13 PCGs, starting with the typical ATN codons ( (S1 Table). The RSCU (relative synonymous codon usage) values for S. sinensis for the third positions is shown in Fig 2A and Table 4. The codon usage is biased: there is a high frequency of AT compared with GC in the third codon position, which is consistent with other crabs. The most common amino acids in mitochondrial proteins are Leu (UUR), Ile and Phe ( Fig  2B). Transfer RNAs, ribosomal RNAs, and control region The S. sinensis mitogenome contains 22 tRNA genes, as do most Brachyura mtDNAs. The tRNA genes range from 64 to 73 bp. Fourteen tRNA genes are encoded on the majority strand and eight are encoded on then minority strand ( Table 2). All the tRNA genes have a typical cloverleaf structure, except for the trnS1 gene, whose dihydroxyuridine (DHU) arm had been simplified down to a loop (Fig 3). The loss of the DHU arm is common in animal mitogenomes and has been considered a typical feature of metazoan mitogenomes [30][31][32][33][34]. The cloverleaf secondary structures of 19 transfer RNAs were identified using the web-server of tRNAscan SE. The three tRNAs not detected by tRNAscan-SE were determined in the unannotated regions by sequence similarity to tRNAs of other crabs. The average AT content of the tRNA genes is 74.6%; their AT skew and GCskew are all negative (S1 Table), showing an obvious bias toward the use of Ts and Cs. Two rRNA genes were identified on the minority strand in the S. sinensis, with the lrRNA gene located between trnL (CUN) and trnV, and the srRNA gene located between trnV and CR, respectively. The lrRNA gene is 999 bp and the srRNA gene is 822 bp long. The AT-skew of rRNAs (0.001), the GC-skew of rRNAs (-0.296) shows clearly that more As and more Cs than Ts and Gs in rRNAs (S1 Table). CR is located between rrns and trnQ. The average AT content of the CR is 83.2%. The overall AT-skew and GC-skew in the CR of S. sinensis are 0.107 and −0.111, respectively (S1 Table), indicating an obvious bias toward the use of As and Cs.

Gene rearrangement
In Pancrustaceans, the tRNA gene order between CR and trnM is trnI-trnQ [35,36] (Fig 4A). The arrangement of the tRNA genes between CR and trnM is trnQ-trnI in S. sinensis (Fig 4G). The tRNA rearrangements are generally considered to be a consequence of tandem duplication of part of the mitogenome [37,38]. The arrangement of the tRNA gene between trnE and trnF is trnH in S. sinensis, which is different from the tRNA genes arrangement of the Pancrustacean ground pattern. In most arthropods, trnH is between nad4 and nad5 [39], whereas it was found between trnE and trnF in S. sinensis. The phenomenon of gene rearrangements in the mitochondrial genome is a relatively common event in crustacean species [40]. The gene order of S. sinensis is identical to that of S. neglectum [41], M. depressus and P. tripectinis ( Fig  4G), which supports the view that S. sinensis belongs to the Grapsoidea, Sesarmidae. The above results suggested that S. sinensis, S. neglectum, M. depressus and P. tripectinis are sister groups. As shown in Fig 4B, the gene orders of these species are identical. The order of the genes in the mitogenome of S. sinensis is different from that in these Brachyura mitogenomes sequences because of the rearrangement of two tRNA genes between CR and trnM. The arrangement of the tRNA genes is trnQ-trnI between CR and trnM in S. sinensis, which is different from the trnI-trnQ of the these Brachyura species. In this case, the tandem duplication of the gene regions that include trnI, trnQ, and trnM, followed by losses of the supernumerary genes might represent the most ideal mechanism for mitochondrial gene rearrangement [42][43][44]. It is believed that slipped-strand mispairing takes place first, followed by gene deletion [45]. The gene orders of Eriocheir japonica sinensis [46], E. j. hepuensis, E. j. japonica, Helice latimera,

Phylogenetic analysis
Phylogenetic analyses were based on two datasets: the AA datasets and the NT datasets using two methods (BI and ML) and alignment method of MAFFT, the topologies of phylogenetic analysis in BI and ML were roughly the same, with some slight differences.  [51]. BI and ML trees of the nucleotide sequences and amino acid sequences of mitochondrial 13 PCGs (Figs 5 and 6) with C. destructor and C. similis as outgroups generated identical tree topologies. The section Eybrachyura crabs consist of four groups, one comprising famlies of Heterotremata, the second Thoracotremata famlies, the third Potamoidea crabs, and the fourth Raninoida species. All the bootstrap values for the branches separating these groups are high. The resulting phylogeny supports the establishment of a separate subsection Potamoida corresponding to Group 3. The present molecular study gives additional evidence for the Potamoida status in these taxa. In all trees Potamoida does closely cluster with subsection Thoracotremata. The relationship between Potamoida and Thoracotremata is much closer than it between the former and Heterotremata, which was proposed by Bowman and Abele [4]. These four subsections (groups) constitute a monophyletic sister group to Section Dromiacea (Group 5) in all phylogenetic trees.

Conclusion
This study presents one mitogenome of S. sinensis. The mitogenome contains 13 PCGs, 22 tRNA genes, two rRNA genes and CR. The AT skew and the GC skew are both negative in the mitogenomes of S. sinensis, indicating an obvious bias toward the use of Ts and Cs, which is consistent with most sequenced brachyuran crabs. The gene arrangement of S. sinensis is identical to that of S. neglectum, P. tripectinis and M. depressus. In comparison to Pancrustacean ground pattern and common arrangement for brachyuran crabs, S. sinensis exhibits a novel rearrangement. Tandem duplication followed by random deletion is widely considered to explain generation of gene rearrangement of mitogenome in S. sinensis. The phylogenetic analyses indicate that S. sinensis and S. neglectum have sister group relationships, and the clade (S. sinensis + S. neglectum) is well supported to be the sister group to (P. tripectinis + M. depressus), suggesting that S. sinensis should belong to Grapoidea, Sesarmidae. The topology of BI and ML trees of Brachyura species (Fig 5) inferred from nucleotide sequences of mitochondrial 13 PCGs sequences are similar to those of Fig 6 constructed from amino acid sequences of whole mitogenomes. The resulting phylogeny strongly supports the establishment of a separate subsection Potamoida, so section Eubrachyura consists of four subsections which are Raninoida, Heterotremata, Potamoida, and Thoracotremata. However, the four subsections and two sections are monophyletic, respectively, whereas the relationships within families of each subsection were not resolved absolutely in the present study.
Supporting information S1