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 [3-5]. 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 [9-11]. 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 [9-11].

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.

Materials and methods

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 [14-18]. 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 (Mg2+), 4μL of dNTPs, 2μL each of the primers, 2μL of DNA, 34.5μL of ddH2O, 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 GenBank and used as outgroup taxons. Detailed information is shown in Table 1.

Table 1

List of 40 Brachyura species analysed in this paper with their GenBank accession numbers.

Species	Family	Size (bp)	Accession No.
Sesarmops sinensis	Sesarmidae	15,905	KR336554
Sesarma neglectum	Sesarmidae	15,920	KX156954
Metopaulias depressus	Sesarmidae	15,765	KX118277
Parasesarmops tripectinis	Sesarmidae	15,612	KU343209
Helice latimera	Varunidae	16,246	KU589291
Eriocheir japonica sinensis	Varunidae	16,378	KM516908
Eriocheir japonica hepuensis	Varunidae	16,335	FJ455506
Eriocheir japonica japonica	Varunidae	16,352	FJ455505
Cyclograpsus granulosus	Varunidae	16,300	LN624373
Gandalfus yunohana	Bythograeidae	15,567	EU647222
Gandalfus puia	Bythograeidae	15,548	KR002727
Austinograea alayseae	Bythograeidae	15,620	JQ035660
Austinograea rodriguezensis	Bythograeidae	15,611	JQ035658
Ocypode cordimanus	Ocypodidae	15,604	KT896743
Ocypode ceratophthalmus	Ocypodidae	15,564	LN611669
Umalia orientalis	Raninidae	15,466	KM365084
Lyreidus brevifrons	Raninidae	16,112	KM983394
Homologenus malayensis	Homolidae	15,793	KJ612407
Moloha majora	Homolidae	15,903	KT182069
Pseudocarcinus gigas	Menippidae	15,515	AY562127
Myomenippe fornasinii	Menippidae	15,658	LK391943
Geothelphusa dehaani	Potamidae	18,197	AB187570
Huananpotamon lichuanense	Potamidae	15,380	KX639824
Portunus pelagicus	Portunidae	16,157	KM977882
Callinectes sapidus	Portunidae	16,263	AY363392
Portunus trituberculatus	Portunidae	16,026	AB093006
Portunus sanguinolentus	Portunidae	16,024	KT438509
Charybdis japonica	Portunidae	15,738	FJ460517
Scylla paramamosain	Portunidae	15,824	JX457150
Scylla olivacea	Portunidae	15,723	FJ827760
Scylla tranquebarica	Portunidae	15,833	FJ827759
Scylla serrata	Portunidae	15,775	FJ827758
Charybdis feriata	Portunidae	15,660	KF386147
Pachygrapsus crassipes	Grapsidae	15,652	KC878511
Ilyoplax deschampsi	Dotillidae	15,460	JF909979
Damithrax spinosissimus	Mithracidae	15,817	KM405516
Macrophthalmus japonicus	Macrophthalmidae	16,170	KU343211
Dynomene pilumnoides	Dynomenidae	16,475	KT182070
Xenograpsus testudinatus	Xenograpsidae	15,798	EU727203
Mictyris longicarpus	Mictyridae	15,548	LN611670

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.

Results and discussion

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 (Table 2 and Fig 1). Twenty-three genes are coded on the majority strand and the remaining fourteen genes are transcribed on the minority strand. The S. sinensis nucleotide composition is (A) 37.4%, (T) 38.3%, (G) 9.4%, and (C) 14.9%. It shows a high A+T bias: the A+T nucleotide content is 75.7%. In addition, the A+T skew value ([A–T]/[A+T]) is –0.012, and the G+C skew value ([G–C]/[G+C]) is –0.228 [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 mitogenomes, ranging from –0.349 (Macrophthalmus japonicus) to –0.215 (P. tripectinis). 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.

Table 2

Summary of mitogenome of S. sinensis.

Gene	Direction	Location	Size	Anticodon	Start codon	Stop codon	Intergenic nucleotides
cox1	F	1–1559	1559	—	ATG	TAA	-24
trnL2	F	1536–1601	66	TAA	—	—	6
cox2	F	1608–2315	708	—	ATG	TAA	-20
trnK	F	2296–2365	70	TTT	—	—	-1
trnD	F	2365–2428	64	GTC	—	—	0
atp8	F	2429–2587	159	—	ATG	TAA	-7
atp6	F	2581–3254	674	—	ATG	TA	0
cox3	F	3255–4045	791	—	ATG	TA	0
trnG	F	4046–4110	65	TCC	—	—	0
nad3	F	4111–4461	351	—	ATT	TAA	4
trnA	F	4466–4529	64	TGC	—	—	10
trnR	F	4540–4605	66	TCG	—	—	2
trnN	F	4608–4674	67	GTT	—	—	3
trnS1	F	4678–4744	67	TCT	—	—	0
trnE	F	4745–4810	66	TTC	—	—	4
trnH	R	4815–4878	64	TAC	—	—	2
trnF	R	4881–4945	65	GAA	—	—	7
nad5	R	4953–6680	1728	—	ATG	TAA	19
nad4	R	6700–8061	1392	—	ATG	TAA	-7
nad4L	R	8055–8357	303	—	ATG	TAA	9
trnT	F	8367–8432	66	TGT	—	—	0
trnP	R	8433–8499	67	TGG	—	—	2
nad6	F	8502–9004	503	—	ATT	TA	0
cob	F	9005–10,159	1155	—	ATG	TAA	-20
trnS2	F	10,140–10206	67	TGA	—	—	19
nad1	R	10,226–11164	939	—	ATA	TAA	40
trnL1	R	11,205–11,268	64	TAG	—	—	0
rrnL	R	11,269–12,267	999	—	—	—	340
trnV	R	12,608–12680	73	TAC	—	—	0
rrnS	R	12,681–13,502	822	—	—	—	0
CR	—	13,503–14,253	751	—	—	—	0
trnQ	R	14,254–14,322	69	TTG	—	—	192
trnI	F	14,515–14,581	67	GAT	—	—	47
trnM	F	14,629–14,698	70	CAT	—	—	0
nad2	F	14,699–15,706	1008	—	ATG	TAG	2
trnW	F	15,709–15,778	70	TCA	—	—	-3
trnC	R	15,776–15,839	64	GCA	—	—	0
trnY	R	15,840–15,905	66	GTA	—	—	—

Fig 1

Circular map of the mitogenome of S. sinensis.

Protein coding and ribosomal genes are shown with standard abbreviations. Genes for tRNAs are abbreviated by a single letter, with S1 = AGN, S2 = UCN, L1 = CUN, and L2 = UUR. 13 Protein coding genes are yellow colored. tRNAs are purple colored. rRNAs are red colored.

Table 3

The A+T skew value and the G+C skew value of 40 Brachyura species.

Species	Size (bp)	A %	G %	T %	C %	A+T %	A+T skew	G+C skew
S. sinensis	15,905	37.4	9.4	38.3	14.9	75.7	-0.012	-0.228
H. latimera	16,246	34.0	11.0	35.1	19.9	69.1	-0.017	-0.290
G. puia	15,548	35.1	10.3	34.8	19.8	69.9	0.006	-0.313
P. sanguinolentus	16,024	31.6	12.9	34.0	21.5	65.6	-0.037	-0.243
E. j. sinensis	16,378	35.2	10.8	36.4	17.6	71.6	-0.016	-0.243
E. j. hepuensis	16,335	35.1	10.8	36.4	17.7	71.5	-0.018	-0.245
E. j. japonica	16,352	35.2	10.7	36.5	17.7	71.7	-0.018	-0.245
X. testudinatus	15,798	36.7	9.3	37.2	16.8	73.9	-0.007	-0.297
P. gigas	15,515	35.0	10.8	35.5	18.7	70.5	-0.006	-0.268
G. dehaani	18,197	36.9	8.3	38.0	16.8	74.9	-0.014	-0.341
L. brevifrons	16,112	34.2	11.3	36.4	18.1	70.6	-0.031	-0.231
C. sapidus	16,263	34.2	11.1	34.9	19.8	69.1	-0.011	-0.279
P. trituberculatus	16,026	33.3	11.3	36.9	18.5	70.2	-0.051	-0.241
H. malayensis	15,793	37.3	10.0	34.4	18.3	71.7	0.040	-0.292
C. japonica	15,738	33.8	11.9	35.4	18.9	69.2	-0.024	-0.228
S. paramamosain	15,824	34.9	10.1	38.2	16.8	73.1	-0.045	-0.247
U. orientalis	15,466	33.1	11.8	34.9	20.2	68.0	-0.027	-0.262
S. olivacea	15,723	33.5	11.2	35.9	19.4	69.4	-0.035	-0.267
S. tranquebarica	15,833	35.0	9.8	38.7	16.5	73.7	-0.050	-0.258
S. serrata	15,775	34.5	10.4	38.0	17.1	72.5	-0.047	-0.242
D. spinosissimus	15,817	33.3	10.5	36.8	19.4	70.1	-0.050	-0.294
C. feriata	15,660	34.1	11.2	36.1	18.6	70.2	-0.028	-0.246
G. yunohana	15,567	34.3	10.8	35.6	19.3	69.9	-0.019	-0.281
P. pelagicus	16,157	33.7	12.2	35.0	19.1	68.8	-0.019	-0.219
A. alayseae	15,620	34.4	11.4	32.4	21.8	66.8	0.029	-0.316
A. rodriguezensis	15,611	35.3	10.3	33.5	20.9	68.8	0.025	-0.341
P. crassipes	15,652	30.5	12.7	35.8	21.0	66.3	-0.080	-0.245
I. deschampsi	15,460	34.1	10.7	35.5	19.7	69.6	-0.019	-0.294
O. cordimanus	15,604	31.8	11.9	34.5	21.8	66.3	-0.043	-0.293
P. tripectinis	15,612	36.2	10.1	38.0	15.7	74.2	-0.023	-0.215
M. japonicus	16,170	33.6	10.9	32.8	22.7	66.4	0.014	-0.349
S. neglectum	15,920	37.4	9.5	38.2	14.9	75.6	-0.010	-0.219
M. depressus	15765	37.9	8.7	39.4	14.0	77.3	-0.0038	-0.231
O. ceratophthalmus	15,564	33.7	11.1	35.8	19.4	69.5	-0.029	-0.269
D. pilumnoides	16,475	37.5	9.5	34.7	18.3	72.2	0.037	-0.316
M. majora	15,903	38.4	9.8	35.5	16.3	73.9	0.039	-0.248
H. lichuanense	15,380	35.8	9.3	37.4	17.5	73.2	-0.023	-0.305
M. fornasinii	15,658	35.5	9.9	36.1	18.5	71.6	-0.0087	-0.303
C. granulosus	16,300	33.2	11.2	36.1	19.5	69.3	-0.043	-0.272
M. longicarpus	15,548	32.4	11.8	36.6	19.2	69.0	-0.060	-0.236

Protein-coding genes

The mitogenome of S. sinensis contains 13 PCGs, starting with the typical ATN codons (Table 2). One (nad1) starts with ATA, two (nad3, nad6) with ATT, and ten (cox1, cox2, atp8, atp6, cox3, cob, nad2, nad5, nad4, and nad4l) with ATG. Nine PCGs (cox1, cox2, atp8, nad3, nad5, nad4, nad4l, cob, and nad1) have a complete TAA stop codon, while the remaining four terminate with either TA (atp6, cox3, and nad6) or TAG (nad2). Nine PCGs (cox1, cox2, atp8, atp6, cox3, nad3, nad6, cob, and nad2) are encoded on the majority strand, while the rest are encoded on the minority strand. The A+T content was 74.0% and AT skew was –0.163 (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).

Fig 2

Relative synonymous codon usage (RSCU) in S. sinensis mitogenome (A). Amino acid composition in the mitogenome of S. sinensis (B).

Table 4

RSCU (relative synonymous codon usage) of S. sinensis.

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	314	1.76	UCU(S)	106	2.41	UAU(Y)	132	1.69	UGU(C)	32	1.83
UUC(F)	42	0.24	UCC(S)	14	0.32	UAC(Y)	24	0.31	UGC(C)	3	0.17
UUA(L)	408	4.2	UCA(S)	102	2.32	UAA(*)	11	1.83	UGA(W)	98	1.9
UUG(L)	35	0.36	UCG(S)	3	0.07	UAG(*)	1	0.17	UGG(W)	5	0.1
CUU(L)	70	0.72	CCU(P)	77	2.15	CAU(H)	59	1.55	CGU(R)	19	1.41
CUC(L)	9	0.09	CCC(P)	14	0.39	CAC(H)	17	0.45	CGC(R)	0	0
CUA(L)	55	0.57	CCA(P)	50	1.4	CAA(Q)	68	1.81	CGA(R)	30	2.22
CUG(L)	6	0.06	CCG(P)	2	0.06	CAG(Q)	7	0.19	CGG(R)	5	0.37
AUU(I)	325	1.83	ACU(T)	88	2.11	AAU(N)	140	1.74	AGU(S)	40	0.91
AUC(I)	31	0.17	ACC(T)	10	0.24	AAC(N)	21	0.26	AGC(S)	1	0.02
AUA(M)	209	1.81	ACA(T)	67	1.6	AAA(K)	86	1.74	AGA(S)	74	1.68
AUG(M)	22	0.19	ACG(T)	2	0.05	AAG(K)	13	0.26	AGG(S)	12	0.27
GUU(V)	92	1.69	GCU(A)	109	2.26	GAU(D)	63	1.77	GGU(G)	82	1.48
GUC(V)	7	0.13	GCC(A)	19	0.39	GAC(D)	8	0.23	GGC(G)	6	0.11
GUA(V)	99	1.82	GCA(A)	60	1.24	GAA(E)	70	1.82	GGA(G)	119	2.15
GUG(V)	20	0.37	GCG(A)	5	0.1	GAG(E)	7	0.18	GGG(G)	14	0.25

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 [30-34]. The cloverleaf secondary structures of 19 transfer RNAs were identified using the web-server of tRNA-scan 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.

Fig 3

Secondary structures of 22 transfer tRNA genes of S. sinensis.

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.

Fig 4

Linearized represantation of gene rearrangements of Brachyura circle mitogeomes.

All genes are transcribed from left to right. tRNA genes are exhibited by the corresponding single-letter amino acid code with S1 = AGN, S2 = UCN, L1 = CUN, and L2 = UUR. CR represents control region. rrnL, rrnS, large and small subunit ribosomal RNA.

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 [42-44]. 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, Cyclograpsus granulosus and M. japonicus are same (Fig 4C). H. latimera, C. granulosus, E. j. sinensis, E. j. hepuensis, and E. j. japonica belong to the Grapoidea, Varunidae [47].

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. S. sinensis and S. neglectum were clustered in one branch in the phylogenetic tree with high nodal support values in BI and ML trees. (S. sinensis + S. neglectum) clade is well supported to be the sister group to the (P. tripectinis + M. depressus) clade. This supported the view that S. sinensis belongs to the Grapoidea, Sesarmidae, which is consistent with the results of the gene rearrangement analysis. H. latimera, C. granulosus, E. j. sinensis, E. j. hepuensis, and E. j. japonica clustered together with high statistical support (Figs 5 and 6), showing that these species have sister group relationships and belong to Grapsoidea, Varunidae. P. crassipes belong to the Grapoidea, Grapsidae [48]. Previous studies noted an ambiguous classification for Ilyoplax deschampsi. I. deschampsi is part of the Dotillidae in this ambiguous classification. The phylogenetic position of I. deschampsi is within the Grapsoidea [2,49,50]. The real phylogenetic position of I. deschampsi should be closer to the Grapsoidea species that shown in Figs 5 and 6. Xenograpsus testudinatus, originally placed in the Varunidae, has been transferred to its own family (Xenograpsidae) [51].

Fig 5

Inferred phylogenetic relationship among Brachyura based on nucleotide sequences of mitochondrial 13PCGs using BI and ML.

C. destructor and C. similis were used as outgroups.

Fig 6

Inferred phylogenetic relationship among Brachyura based on amino acid sequences of mitochondrial 13PCGs using BI and ML.

C. destructor and C. similis were used as outgroups.

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.

Composition and skewness in the S. sinensis mitogenome.

(DOCX)

Click here for additional data file.