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The complete mitochondrial genome of Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) and comparison with other Brachyuran crabs
Genomics xxx (xxxx) xxx–xxx
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Original Article
The complete mitochondrial genome of Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) and comparison with other Brachyuran crabs ⁎
Zhengfei Wang , Xuejia Shi, Yitao Tao, Qiong Wu, Yuze Bai, Huayun Guo, Dan Tang Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, School of Ocean and Biological Engineering, Yancheng Teachers University, Yancheng 224001, Jiangsu Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sesarmidae Parasesarma pictum Mitogenome Gene order Phylogentic
Mitochondrial DNA (mtDNA) is an extrachromosomal genome which can provide important information for evolution and phylogenetic analysis. In this study, we assembled a complete mitogenome of a crab Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) from next generation sequencing reads at the first time. P. pictum is a mudflat crab, belonging to the Sesarmidae family (subfamily Sesarminae), which is perched on East Asia. The 15,716 bp mitogenome covers 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and one control region (CR). The control region spanns 420 bp. The genome composition was highly A+T biased 75.60% and showed negative AT-skew (−0.03) and negative GC-skew (−0.47). Compared with the ancestor of Brachyura, the gene order of Sesarmidae has several differences and the gene order of P. pictum is typical for mitogenomes of Sesarmidae. Phylogenetic tree based on nucleotide sequences of mitochondrial 13 PCGs using BI and ML determined that P. pictum has a sister group relationship with Parasesarma tripectinis and belongs to Sesarmidae.
1. Introduction Decapoda is an economically important order of crustaceans comprising 18,000 living and extinct species, including freshwater crayfish, lobsters, hermit crabs, shrimps, and “true” crab [1,2]. Among these species, the Sesarmidae (Decapoda; Brachyura; Grapsoidea) is the most speciose family of crabs occurring in the mangroves [3]. Even though they are not important in commercial fishery, previous researches have demonstrated that Sesarmidae crabs play an important ecological role in mangrove ecosystems [4,5]. The taxonomy of the Sesarminae is one of the most challenging problems in the Sesarminae [6] and there are many new species have been reported gradually recent years [7,8]. Parasesarma pictum is a mudflat crab, belonging to the Sesarmidae family (subfamily Sesarminae), which is endemic to East Asia [9,10]. This crab typically inhabits mangrove swamps, preferring the upper intertidal region of estuaries, and living in small crevices and abandoned holes made by other species [10]. Mitochondrial DNA (mtDNA) is an extrachromosomal genome which has been widely used as an informative molecular marker for diverse evolutionary studies among species, including molecular evolution, phylogenetics, population genetics, and comparative and evolutionary genomics [11,12]. In the majority of animals, mitogenomes
⁎
are typically closed circular molecule approximately ranging in size from 14 to 20 kilobases (Kb) [13]. It contains 37 genes, including 13 protein coding genes (PCGs) [subunits 6 and 8 of the ATPase (atp6 and atp8), cytochrome coxidase subunits 1–3 (cox1–cox3), cytochrome B (cob), NADH dehydrogenase subunits 1–6 and 4 l (nad1–6 and nad4l)], two ribosomal RNA genes encoding the small and large subunit rRNAs (rrnS and rrnL), 22 transfer RNA (tRNA) genes and a control region (CR) of variable length, known as the A+T-rich region [11,14,15]. To date, the complete mitogenome of P. pictum has not been reported. Therefore, in this study, the complete mitogenome of P. pictum was initially sequenced and compared with other Brachyura mitogenomes. The available complete mitogenomes were used to provide insight into the phylogenetic relationship of P. pictum and related species. These results will perform an important role in understanding features of P. pictum mitogenome and the evolutionary relationships within Brachyura. 2. Materials and methods 2.1. Samples and DNA extraction The specimen used in this study was collected in Shanghai, China.
Corresponding author. E-mail address: [email protected] (Z. Wang).
https://doi.org/10.1016/j.ygeno.2018.05.002 Received 10 March 2018; Received in revised form 29 April 2018; Accepted 4 May 2018 0888-7543/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Wang, Z., Genomics (2018), https://doi.org/10.1016/j.ygeno.2018.05.002
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2.3. Sequence analysis and gene annotation
Table 1 List of Brachyuran species analyzed in this study with their GenBank accession numbers. Species
Family
Size (bp)
Accession no.
Parasesarma pictum Pyrhila pisum Scylla olivacea Scylla serrata Scylla paramamosain Scylla tranquebarica Charybdis japonica Charybdis feriata Callinectes sapidus Portunus sanguinolentus Portunus pelagicus Portunus trituberculatus Maja squinado Maja crispata Maguimithrax spinosissimus Leptodius sanguineus Pseudocarcinus gigas Segonzacia mesatlantica Gandalfus puia Gandalfus yunohana Austinograea alayseae Austinograea rodriguezensis Macrophthalmus japonicus Mictyris longicarpus Hemigrapsus sanguineus Eriocheir japonica japonica Eriocheir japonica sinensis Eriocheir japonica hepuensis Helicana wuana Helice tientsinensis Ilyoplax deschampsi Xenograpsus testudinatus Parasesarma tripectinis Metopaulias depressus Clistocoeloma sinense Sesarma neglectum Sesarmops sinensis Grapsus tenuicrustatus Pachygrapsus crassipes Ocypode cordimanus Somanniathelphusa boyangensis Huananpotamon lichuanense Sinopotamon xiushuiense Geothelphusa dehaani Umalia orientalis Lyreidus brevifrons Homologenus malayensis Moloha majora Clibanarius infraspinatus
Sesarmidae Leucosiidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Majidae Majidae Mithracidae Xanthidae Menippidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Macrophthalmidae Mictyridae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Dotillidae Xenograpsidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Grapsidae Grapsidae Ocypodidae Parathelphusidae Potamidae Potamidae Potamidae Raninidae Raninidae Homolidae Homolidae Diogenidae
15,613 15,516 15,723 15,775 15,825 15,833 15,738 15,660 16,263 16,024 16,157 16,026 16,598 16,592 15,817 15,480 15,515 15,521 15,548 15,567 15,620 15,611 16,170 15,548 16,275 16,352 16,354 16,335 16,359 16,212 15,460 15,798 15,612 15,765 15,706 15,920 15,905 15,858 15,652 15,604 17,032 15,380 18,460 18,197 15,466 16,112 15,793 15,903 16,504
This study NC_030047.1 NC_012569.1 NC_012565.1 NC_012572.1 NC_012567.1 NC_013246.1 NC_024632.1 NC_006281.1 NC_028225.1 NC_026209.1 NC_005037.1 NC_035425.1 NC_035424.1 NC_025518.1 NC_029726.1 NC_006891.1 NC_035300.1 NC_027414.1 NC_013713.1 NC_020314.1 NC_020312.1 NC_030048.1 NC_025325.1 NC_035307.1 NC_011597.1 NC_006992.1 NC_011598.1 NC_034995.1 NC_030197.1 NC_020040.1 NC_013480.1 NC_030046.2 NC_030535.1 NC_033866.1 NC_031851.1 NC_030196.1 NC_029724.1 NC_021754.1 NC_029725.1 NC_032044.1 NC_031406.1 NC_029226.1 NC_007379.1 NC_026688.1 NC_026721.1 NC_026080.1 NC_029361.1 NC_025776.1
The assembled mitochondrial genes were compared with Parasesarma tripectinis (NC_030046) and identified by BLAST searches in the NCBI database [17] to ensure that the sequences were correct. The locations of the PCGs were identified using ORF Finder via NCBI with the selection of invertebrate mitochondrial genetic code. Abnormal start codons and stop codons were determined based on comparisons with other crabs. Codon usage was calculated using MEGA 7.0 [18]. Identification of tRNA genes was verified using the MITOS WebServer (http://mitos2.bioinf.uni-leipzig.de/index.py). The rRNA genes were determined based on the locations of adjacent tRNA genes and by comparisons with other crabs. Strand asymmetry was calculated using the formulae: AT-skew = (A − T)/(A + T); GC-skew = (G − C)/ (G + C) [19]. The graphical map of the mitochondrial genome was drawn using the online mitochondrial visualization tool Organellar Genome DRAW [20,21]. 2.4. Phylogenetic analysis The complete nucleotide sequences of other 47 Brachyura (Table 1) were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/ genbank/). The mitogenome of Clibanarius infraspinatus was used as outgroup. We estimated the taxonomic status of P. pictum within Brachyura by reconstructing phylogenetic trees. Nucleotide sequences of each gene and their deduced amino acid sequences were aligned separately using MUSCLE 3.8 [22] and MEGA 7.0 [18]. The amino acid sequences for each of the 13 mitochondrial PCGs from 49 mitogenomes were aligned using default settings and concatenated. The concatenated set of nucleotide sequences were used for phylogenetic analysis, which was performed with the Bayesian inference (BI) and Maximum Likelihood (ML) methods using MrBayes v3.2.6 [23] and RAxML [24], respectively. The best model selected with jModeltest [25] and the model of SYM+I+G was optimal for analysis with nucleotide alignment. In BI analysis, two simultaneous runs of 10,000,000 generations conducted for the matrix. We made two simultaneous runs, sampling trees every 1000 generations, with three heated and one cold chain to encourage swapping among the Markov-chain Monte Carlo (MCMC) chains. Convergence of sampled parameters and potential autocorrelation (effective sampling size/ESS for all parameters > 200) was investigated in Tracer 1.6 (http://tree.bio.ed.ac.uk/software/tracer/). Additionally, the average standard deviation of split frequencies between both runs was checked (< 0.01). The Bayesian posterior probabilities were obtained from the 50% majority rule consensus of the post-burn-in trees sampled at stationarity, after removing the first 25% of trees as a “burnin” stage. The resulting phylogenetic trees were visualized in FigTree v1.4.2.
Total DNA was extracted from the muscle tissues and using the Aidlab Genomic DNA Extraction Kit (Aidlab Biotech, Beijing, China). Specimen was incubated at 55 °C for 5 h to lyse completely and the total genomic DNA was eluted in 50 mL double-distill water (ddH2O), while the remaining steps were conducted in accordance with the manufacturer's protocol. Genomic DNA was stored at −20 °C.
3. Results and discussion 3.1. Genome organization and nucleotide composition In the present study, we determined the complete mitochondrial genome sequence of the P. pictum by next-generation sequencing methods. The results of the assembled mitogenomes from two lanes were consistent. We finally achieved an average sequence depth of ~55× per base. The mitogenome of P. pictum is a closed circular molecule of 15,613 bp in length. The mitogenome contained 37 typical mitochondrial genes [13 PCGs (cox1-3, nad1-6, nad4l, cob, atp6 and atp8), 22 tRNAs (one for each amino acid, two for leucine and serine), two rRNAs (rrnS and rrnL) and a major non-coding region known as the CR] and all the genes were identified (Table 2 and Fig. 1). The mitogenome of P. pictum has been deposited in GenBank under accession number MG580780. The nucleotide compositions of the mitogenome of P. pictum are as follows: (A) 36.60%; (T) 39.00%; (G) 9.83%; and (C) 14.57% (Table 3). The whole mitogenome of P. pictum was biased
2.2. Mitogenome sequencing The mitogenome of P. pictum was sequenced by next-generation sequencing. Two lanes for P. pictum were sequenced as 150 bp reads using Illumina HisSeq 4000 (5 GB raw and 4 GB raw; Origin gene, Shanghai, China). Raw sequence data were deposited into Short Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra/) with the accession no. SRR6315542. Clean data without sequencing adapters were de novo assembled using the NOVOPlasty software [16]. To evaluate the single-base accuracy of the assembled mitogenome, we compared the assembled genome with three confirmed sequences by PCR and Sanger sequencing methods. 2
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Table 2 Summary of Parasesarma pictum mitogenome. Gene
Direction
Location
Size
Anticodon
Start codon
Stop codon
Intergenic nucletide
cob trnS2 nad1 trnL1 rrnL trnV rrnS CR trnQ trnI trnM nad2 trnW trnC trnY cox1 trnL2 cox2 trnK trnD atp8 atp6 cox3 trnG nad3 trnA trnR1 trnN trnR2 trnE trnH trnF nad5 nad4 nad4l trnT trnP nad6
+ + − − − − −
484–1618 1619–1685 1703–2641 2677–2743 2721–4074 4077–4149 4151–4986 4987–5616 5617–5684 5702–5768 5789–5859 5860–6867 6866–6934 6943–7007 7008–7074 7077–8610 8612–8680 8689–9376 9377–9446 9447–9513 9514–9672 9666–10,340 10,340–11,134 11,134–11,198 11,199–11,549 11,552–11,617 11,623–11,687 11,690–11,755 11,757–11,823 11,824–11,890 11,894–11,957 11,959–12,024 12,029–13,759 13,806–15,155 15,149–15,451 15,458–15,524 15,525–15,589 15,592–484
1135 67 939 67 1354 73 836 630 68 67 71 1008 69 65 67 1534 69 688 70 67 159 675 795 65 351 66 65 66 67 67 64 66 1731 1350 303 67 65 504
− TGA − TAG − TAC − − TTG GAT CAT − TCA GCA GTA − TAA − TTT GTC − − − TCC − TGC TCG GTT TCT TTC GTG GAA − − − TGT TGG −
ATG − ATA − − − − − − − − ATG − − − ATG − ATG − − ATG ATT ATG − ATC − − − − − − − ATG ATG ATG − − ATT
T − TAA − − − − − − − − TAG − − − T − T − − TAA TAA TAA − TAG − − − − − − − TAA TAA TAA − − TAA
0 17 35 −23 2 1 0 0 17 20 0 −2 8 0 2 1 8 0 0 0 −7 −1 −1 0 2 5 2 1 0 3 1 4 46 −7 6 0 2 −1
− + + + + − − + + + + + + + + + + + + + + + − − − − − + − +
atp8, nad1, nad4, nad5, nad6 and nad4l), and the nad2 and nad3 used TAG as a stop codon whereas the cox1, cox2 and cob terminated by a single T (Table 2). These features are somewhat alike to other invertebrate mitochondrial genomes, and the incomplete stop codon was presumably completed as TAA via post-transcriptional polyadenylation [12,27,28]. Relative synonymous codon usage (RSCU) values for the 13 PCGs are summarised in Table 4 and Fig. 2. Excluding start and stop codons, the mitogenome of P. pictum consists of 3719 codons. In the 13 PCGs of P. pictum, the most frequently used amino acids were Leu (15.70%), followed by Phe (9.38%), Ser (6.13%), and Met (6.05%). A common feature in most metazoan mitochondrial genomes is a bias toward a higher representation of nucleotides A and T that leads to a subsequent bias in the corresponding encoded amino acids [29,30]. In the mitogenome of P. pictum, the overall A+T content of 13 PCGs was 74.88% and AT-skew was negative, indicating a higher occurrence of T than A (Table 3).
toward AT nucleotides (75.60%) in a similar manner to Sesarma neglectum (75.50%) and Sesarmops sinensis (75.55%), both of which have the same gene arrangements with P. pictum [26]. Most genes (24 of 37) were encoded on the heavy (+) strand while the remaining 13 genes were located on the light (−) strand (Table 2). There is a 42 bp overlap between genes in seven locations, with the longest 23 bp overlap located in between nad1 (CUN) and trnL1 in mitogenome of P. pictum (Table 2). The mitogenome of P. pictum contains 280 of intergenic spacer sequence spread over 20 regions, ranging in size from 1 to 46 bp (Table 2). The whole mitogenome of P. pictum was biased toward AT nucleotides (75.60%). The AT-skew and GC-skew for the whole mitogenome were both negative, respectively, indicating a higher occurrence of T than A and C than G (Table 3). As a result of the gene arrangement and compactness of mitogenome, the P. pictum mitogenome has a total of 42 bp overlap between genes in 7 locations; seven pairs of overlapping genes are present: trnL1/nad1, nad2/trnM, atp8/trnD, atp6/ atp8, cox3/atp6, nad4/nad5 and nad6/trnP, with the longest 23 bp overlap located between trnL1 and rrnL.
3.3. Skewness 3.2. PCGs and codon usage In the mitogenome of P. pictum, the skew of AT was negative and the skew of GC was consistently negative, indicating an obvious bias toward the use of T and C (Table 3). The AT-skew and GC-skew of the selected complete Brachyura mitogenomes were also calculated. From the Table 5, the GC-skew for the complete mitogenomes was negative (from −0.35 to −0.19) and the AT-skew was slightly negative (from −0.09 to 0) except Leptodius sanguineus (0.04) [31], Gandalfus puia (0.01) [32], Austinograea alayseae (0.03) [33], Austinograea rodriguezensis (0.02) [34], Macrophthalmus japonicas (0.01), Homologenus
The mitogenome of P. pictum has 13 typical PCGs contain seven NADH genes (nad1-6 and nad4L), two ATP genes (atp6 and atp8) and three cytochrome genes (cox1-3 and cob) found in invertebrate animals (Fig. 1). The region of PCGs is 11,172 bp in size. In the mitogenome of P. pictum, nad1, nad4, nad4l and nad5 were encoded on the light strand, while the remaining nine PCGs (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cob) were encoded on the heavy strand. All the PCGs started with ATN and eight PCGs were terminated by TAA (cox3, atp6, 3
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Fig. 1. Gene map of the mitogenome of P. pictum. Protein-coding genes are color-coded (cox: purple; nad: yellow; atp: green; cob: kelly); rRNA genes are in red; tRNA genes are in blue. Abbreviations of protein-coding genes and ribosomal genes are as follows: atp6 and atp8 for ATP synthase subunits 6 and 8, cox1–3 for cytochrome oxidase subunits 1–3, cob for cytochrome b, nad1–6 and nad4L for NADH dehydrogenase subunits 1–6 and 4 L, rrnL and rrnS for large and small rRNA subunits. In addition, trnS1, trnS2, trnL1, and trnL2 denote codons tRNA-Ser (AGN), tRNA-Ser (UCN), tRNA-Leu (CUN), and tRNA-Leu (UUR), respectively. CR = Control Region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 3 Composition and skewness of Parasesarma pictum mitogenome. P. pictum
Size(bp)
A%
T%
G%
C%
A + T%
AT-skew
GC-skew
Mitogenome cob nad1 nad2 cox1 cox2 atp8 atp6 cox3 nad3 nad5 nad4 nad4l nad6 PCGs tRNAs rRNAs CR
15,613 1135 939 1008 1534 688 159 675 795 351 1731 1350 303 504 11,172 1478 2190 420
36.60 29.07 29.71 29.46 29.60 32.77 30.19 30.22 29.06 32.76 33.33 32.22 29.37 29.17 30.53 38.31 40.31 41.19
39.00 42.11 45.90 47.82 39.57 38.98 50.94 41.78 41.13 43.30 43.33 44.00 47.19 50.40 44.34 38.18 39.09 38.10
9.83 12.95 15.55 8.63 15.65 12.85 5.66 10.81 14.09 9.69 15.02 15.93 16.83 6.35 12.31 13.48 13.35 8.33
14.57 15.86 8.84 14.09 15.19 15.40 13.21 17.19 15.72 14.25 8.32 7.85 6.60 14.09 12.81 10.03 7.25 12.38
75.60 71.19 75.61 77.28 69.17 71.75 81.13 72.00 70.19 76.07 76.66 76.22 76.57 79.56 74.88 76.49 79.40 79.29
−0.032 −0.183 −0.214 −0.237 −0.144 −0.087 −0.256 −0.160 −0.172 −0.139 −0.130 −0.155 −0.233 −0.267 −0.184 0.002 0.015 0.039
−0.194 −0.101 0.275 −0.240 0.015 −0.090 −0.400 −0.228 −0.055 −0.190 0.287 0.340 0.437 −0.379 −0.020 0.147 0.296 −0.196
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Table 4 The codon number and relative synonymous codon usage in Parasesarma pictum mitochondrial protein coding genes. Codon
Count
RSCU
Codon
Count
RSCU
Codon
Count
RSCU
Codon
Count
RSCU
UUU(F) UUC(F) UUA(L) UUG(L) CUU(L) CUC(L) CUA(L) CUG(L) AUU(I) AUC(I) AUA(M) AUG(M) GUU(V) GUC(V) GUA(V) GUG(V)
314 35 415 33 77 4 50 5 331 23 189 36 104 3 99 13
1.80 0.20 4.26 0.34 0.79 0.04 0.51 0.05 1.87 0.13 1.68 0.32 1.90 0.05 1.81 0.24
UCU(S) UCC(S) UCA(S) UCG(S) CCU(P) CCC(P) CCA(P) CCG(P) ACU(T) ACC(T) ACA(T) ACG(T) GCU(A) GCC(A) GCA(A) GCG(A)
120 13 87 8 80 12 47 4 85 7 77 4 112 13 60 5
2.61 0.28 1.89 0.17 2.24 0.34 1.31 0.11 1.97 0.16 1.78 0.09 2.36 0.27 1.26 0.11
UAU(Y) UAC(Y) UAA(*) UAG(*) CAU(H) CAC(H) CAA(Q) CAG(Q) AAU(N) AAC(N) AAA(K) AAG(K) GAU(D) GAC(D) GAA(E) GAG(E)
146 15 8 2 68 9 61 13 124 26 89 6 57 12 68 9
1.81 0.19 1.60 0.40 1.77 0.23 1.65 0.35 1.65 0.35 1.87 0.13 1.65 0.35 1.77 0.23
UGU(C) UGC(C) UGA(W) UGG(W) CGU(R) CGC(R) CGA(R) CGG(R) AGU(S) AGC(S) AGA(S) AGG(S) GGU(G) GGC(G) GGA(G) GGG(G)
33 0 94 7 21 0 30 3 39 3 88 10 82 7 124 10
2.00 0.00 1.86 0.14 1.56 0.00 2.22 0.22 0.85 0.07 1.91 0.22 1.47 0.13 2.22 0.18
Fig. 2. Relative synonymous codon usage in Parasesarma pictum mitogenome.
malayensis (0.04) [35], Moloha majora (0.04) [36] and Clibanarius infraspinatus (0.04) [30].
79.29% AT nucleotides, with a positive AT skew (0.039) and negative GC skew (−0.196) (Table 3).
3.4. Transfer RNAs, ribosomal RNAs, and A+T-rich region
3.5. Gene order
Like most Brachyura mtDNA, the P. pictum mitogenome contains a set of 22 tRNAs genes (Fig. 3), although this feature is not very well conserved in animal mtDNA. The tRNAs ranged in size from 64 to 73 bp and showed a strong A + T bias, as these bases accounted for 76.49% of the DNA. Further, they exhibited a postive AT-skew (0.002) and a positive GC-skew (0.147) (Table 3). Fifteen tRNA genes were present on the heavy strand and seven were on the light strand. All the tRNA genes had the typical cloverleaf structure. These features are common in most Brachyura mitogenomes. The secondary cloverleaf structure of 22 tRNAs was examined using MITOS WebServer (http://mitos2.bioinf. uni-leipzig.de/index.py). The tRNAs were predicted to fold into the expected cloverleaf secondary structures with normal base pairing and G–U (e.g. tRNAArg, Met). However, there were other base pair arrangements, such as U–U (e.g. tRNAAla). The rrnL is 1354 bp and rrnS is 836 bp, one locating between trnL1 and trnV and another locating between trnV and CR (Table 2). The A+T content of two rRNA genes was totally 79.40% and they had positive AT skew (0.015) (Table 3). The CR located between rrnS and trnQ spans 630 bp. This region contains
The complete gene order in the P. pictum mitogenome is shown in Fig. 4. Compared with the gene arrangement in ancestral Brachyura [37], the gene order of P. pictum only had two differences of ancestral Brachyura. In short, only two pairs of genes appeared to be rearranged, along with trnH and trnQ. As usual, trnH located between nad5 and nad4 which, however, was shifted into trnE and trnF junction. Another rearrangement was the transversion of trnI and trnQ. The gene order of P. pictum mitogenome was also compared with P. tripectinis (Sesarmidae: Brachyura) which went to have the same arrangement (Accession: NC_030046.2). Futhermore, the gene orders of P. pictum and P. tripectinis are the same as that ancestral Sesarmidae [38]. The same gene order of P. pictum and P. tripectinis identifies that the two species have a close relationship in evolution. 4. Phylogentic analysis The phylogenetic relationships were analyzed based on the concatenated nucleotide sequences of 13 PCGs from 48 Brachyura species
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Table 5 Composition and skewness of mitogenome in 49 Brachyura species. Species
A%
T%
A + T%
G%
C%
G + C%
AT-skew
GC-skew
Parasesarma pictum Pyrhila pisum Scylla olivacea Scylla serrata Scylla paramamosain Scylla tranquebarica Charybdis feriata Charybdis japonica Callinectes sapidus Portunus pelagicus Portunus sanguinolentus Portunus trituberculatus Maja crispata Maja squinado Maguimithrax spinosissimus Leptodius sanguineus Pseudocarcinus gigas Segonzacia mesatlantica Gandalfus puia Gandalfus yunohana Austinograea alayseae Austinograea rodriguezensis Macrophthalmus japonicus Mictyris longicarpus Hemigrapsus sanguineus Eriocheir japonica hepuensis Eriocheir japonica japonica Eriocheir japonica sinensis Helicana wuana Helice tientsinensis Ilyoplax deschampsi Xenograpsus testudinatus Geothelphusa dehaani Grapsus tenuicrustatus Huananpotamon lichuanense Parasesarma tripectinis Metopaulias depressus Clistocoeloma sinense Sesarma neglectum Sesarmops sinensis Pachygrapsus crassipes Ocypode cordimanus Sinopotamon xiushuiense Somanniathelphusa boyangensis Lyreidus brevifrons Umalia orientalis Homologenus malayensis Moloha majora Clibanarius infraspinatus
36.60 37.28 33.45 34.55 34.79 35.04 34.09 33.79 34.17 33.71 31.59 33.33 33.59 33.75 33.30 33.62 35.02 33.83 35.15 34.29 34.37 35.26 33.65 32.43 34.27 35.10 35.16 35.28 32.95 33.93 34.15 36.70 36.92 31.92 35.76 36.23 37.94 37.11 37.42 37.37 30.49 31.81 36.03 35.13 34.21 33.09 37.31 38.36 35.39
39.00 37.20 35.91 37.95 38.19 38.72 36.05 35.45 34.93 35.03 34.01 36.89 36.71 37.06 36.77 31.23 35.46 33.99 34.75 35.65 32.43 33.55 32.75 36.58 35.50 36.41 36.45 36.35 35.47 35.13 35.47 37.21 38.00 33.11 37.44 37.98 39.38 38.62 38.21 38.31 35.79 34.53 38.51 37.25 36.38 34.94 34.42 35.50 32.55
75.60 74.48 69.36 72.51 72.98 73.76 70.15 69.24 69.10 68.74 65.60 70.22 70.30 70.80 70.07 64.85 70.47 67.82 69.90 69.94 66.79 68.80 66.39 69.01 69.76 71.51 71.62 71.63 68.42 69.06 69.62 73.91 74.92 65.03 73.21 74.22 77.32 75.72 75.63 75.68 66.28 66.34 74.54 72.38 70.59 68.03 71.73 73.86 67.94
9.83 10.22 11.23 10.42 10.17 9.74 11.25 11.88 11.15 12.21 12.91 11.30 11.13 11.02 10.56 11.16 10.80 10.53 10.34 10.80 11.36 10.28 10.95 11.83 11.18 10.76 10.72 10.67 11.55 11.00 10.72 9.32 8.26 12.13 9.31 10.11 8.71 9.36 9.51 9.39 12.73 11.89 8.61 9.25 11.31 11.79 10.01 9.82 12.84
14.57 15.31 19.40 17.07 16.85 16.50 18.60 18.88 19.76 19.06 21.50 18.48 18.57 18.18 19.37 23.99 18.72 21.65 19.76 19.25 21.85 20.91 22.66 19.15 19.05 17.73 17.66 17.70 20.03 19.94 19.66 16.77 16.82 22.85 17.48 15.67 13.97 14.92 14.86 14.93 20.99 21.76 16.85 18.38 18.10 20.17 18.25 16.32 19.23
24.40 25.52 30.64 27.49 27.02 26.24 29.85 30.76 30.90 31.26 34.40 29.78 29.70 29.20 29.93 35.15 29.53 32.18 30.10 30.06 33.21 31.20 33.61 30.99 30.24 28.49 28.38 28.37 31.58 30.94 30.38 26.09 25.08 34.97 26.79 25.78 22.68 24.28 24.37 24.32 33.72 33.66 25.46 27.62 29.41 31.97 28.27 26.14 32.06
−0.03 0.00 −0.04 −0.05 −0.05 −0.05 −0.03 −0.02 −0.01 −0.02 −0.04 −0.05 −0.05 −0.05 −0.05 0.04 −0.01 0.00 0.01 −0.02 0.03 0.02 0.01 −0.06 −0.02 −0.02 −0.02 −0.02 −0.04 −0.02 −0.02 −0.01 −0.01 −0.02 −0.02 −0.02 −0.02 −0.02 −0.01 −0.01 −0.09 −0.04 −0.03 −0.03 −0.03 −0.03 0.04 0.04 0.04
−0.47 −0.50 −0.47 −0.46 −0.46 −0.45 −0.47 −0.48 −0.49 −0.48 −0.46 −0.45 −0.46 −0.46 −0.45 −0.54 −0.49 −0.50 −0.51 −0.48 −0.53 −0.53 −0.51 −0.44 −0.48 −0.48 −0.48 −0.49 −0.46 −0.48 −0.48 −0.49 −0.49 −0.48 −0.48 −0.48 −0.48 −0.48 −0.49 −0.49 −0.43 −0.46 −0.47 −0.47 −0.47 −0.47 −0.54 −0.54 −0.54
Potamidae, Bythograeidae, and Portunidae was also identified and relationship on these families is also consistent with previous researches [38–40].
and one outgroup. The results of the BI and ML analyses generated consistent tree topologies (Fig. 5). In this study, both the BI and ML analyses showed that each superfamily in the tree formed a monophyletic clade. It is obvious that P. pictum and P. tripectinis clustered in one branch in the phylogenetic tree with high nodal support values (BI posterior probabilities [PP] = 1; ML bootstrap [BP] = 100) (Fig. 5), indicating that P. pictum and P. tripectinis have a sister group relationship. Additionally, the results of the phylogenetic analyses revealed Metopaulias depressus and Clistocoeloma sinense were grouped into one clade with high nodal support value. These two pairs of Sesarmide apparently have a stable sister relationship which is consistent with previous study [38]. This result supported that P. pictum belongs to Grapsoidea, Sesarmidae. Further, Xenograpsus testudinatus, which was originally placed in Varunidae, has been transferred to its own family (Xenograpsidae) [39,40]. From the phylogenetic tree, we also found that X. testudinatus and Sesarmidae species formed a group and showed close relationships. Additionally, the relationship on Sesarmidae, Xenograpsidae, Grapsidae, Varunidae,
5. Conclusion In this study, we firstly sequenced the complete 15,613 bp mitogenome of P. pictum, in which 37 genes (13 PCGs, 22 tRNA genes and 2 rRNA genes) and one control region are located as a typical of Brachyura mitogenome. With all PCGs are initiated by ATN codon, the cox1, cox2 and cob genes have incomplete stop codons consisting of just a T, and the other 10 PCGs stop with the canonical TAA or TAG. The AT-skew and the GC-skew are both negative in the mitogenomes of P. pictum which are consistent with most sequenced brachyuran crabs. The gene order of P. pictum is the same as P. tripectinis which is consistent of Sesarmidae, indicating the relationship between P. pictum and P. tripectinis. Futhermore, the phylogenetic analyses support that P. pictum belongs to Grapsoidea, Sesarmidae.
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Fig. 3. Secondary structures of the 22 transfer RNA genes of P. pictum. tRNAs are labeled with the abbreviations of their corresponding amino acids. Dashes (−) indicate Watson-Crick pairing.
Fig. 4. Linear representation of gene rearrangements of Ancestor of Brachyura, Sesarmidae, Parasesarma tripectinis and Parasesarma pictum. All genes are transcribed from left to right. tRNA genes are represented by the corresponding single-letter amino acid code. S1 (AGN), S2 (UCN), L1 (CUN), L2 (UUR), CR (control region). rrnL and rrnS are the large and small ribosomal RNA subunits.
Fig. 5. Phylogeny of P.pictum based on nucleotide sequences. The phylogenetic tree was inferred from the nucleotide sequences of 13 mitogenome PCGs using BI and 8 ML methods. Numbers on branches indicate posterior probability (BI) and bootstrap (ML).
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Acknowledgements
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This study was funded by the National Natural Science Foundation of China (grant number 31702014), and Doctoral Scientific Research Foundation of Yancheng Teachers University to ZFW, and Open Foundation of Jiangsu Key Laboratory for Bioresources of Saline Soils (grant number JKLBS2016007), and Yancheng Teachers University Undergraduate Training Programs for Innovation and Entrepreneurship to YTT. Conflict of interest The authors declare there are no competing interests. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Authors' contributions HYG, XJS, QW, ZFW and YTT designed and conceived the experiment. YTT, ZFW, YZB and DT performed the data analysis and draft the manuscript. All authors read and approved the final manuscript. References [1] J. Krieger, A. Sombke, F. Seefluth, et al., Comparative brain architecture of the European shore crab Carcinus maenas (Brachyura) and the common hermit crab Pagurus bernhardus (Anomura) with notes on other marine hermit crabs, Cell Tissue Res. 348 (1) (2012) 47–69. [2] H. Shen, A. Braband, G. Scholtz, Mitogenomic analysis of decapod crustacean phylogeny corroborates traditional views on their relationships, Mol. Phylogenet. Evol. 66 (3) (2013) 776–789. [3] S. Cannicci, C.D. Schubart, G. Innocenti, et al., A new species of the genus Parasesarma De Man 1895 from East African mangroves and evidence for mitochondrial introgression in sesarmid crabs, Zool. Anz. 269 (2017) 89–99. [4] A.A. Flores, J. Saraiv, J. Paula, Sexual, maturity, reproductive cycles, and juvenile recruitment of Perisesarma guttatum (brachyura, sesarmidae) at ponta rasa mangrove swamp, inhaca island, mozambique, J. Crustac. Biol. 22 (1) (2002) 143–156. [5] S.A. Khan, S.M. Raffi, P.S. Lyla, Brachyuran crab diversity in natural (Pichavaram) and artificially developed mangroves (Vellar estuary), Curr. Sci. 88 (2005) 1316–1324. [6] D.L. Rahayu, P.K.L. Ng, Revision of the Parasesarma plicatum (Latreille, 1803) species-group (Crustacea: Decapoda: Brachyura: Sesarmidae), Zootaxa 2327 (2010) 1–22. [7] A. Shahdadi, P.J.F. Davie, C.D. Schubart, Perisesarma tuerkayi, a new species of mangrove crab from Vietnam (Decapoda;Brachyura;Sesarmidae),with an assessment of its phylogenetic relationships, Crustaceana 90 (7–10) (2017) 1155–1175. [8] T. Naruse, P.K.L. Ng, A new species of Chiromantes s. str. (Decapoda: Brachyura: Sesarmidae) from the Ryukyu Islands, Japan, with a note on the identity of Holometopus serenei Soh, 1978, Crustacean Res. 37 (2008) 1–13. [9] G. Guerao, K. Anger, U.W.E. Nettelmann, et al., Complete larval and early juvenile development of the mangrove crab Perisesarma fasciatum (Crustacea: Brachyura: Sesarmidae) from Singapore, with a larval comparison of Parasesarma and Perisesarma, J. Plankton Res. 26 (12) (2004) 1389–1408. [10] L.M. Surhone, M.T. Tennoe, S.F. Henssonow, Parasesarma Pictum, Betascript Publishing, 2010. [11] J.L. Boore, Animal mitochondrial genomes, Nucleic Acids Res. 27 (8) (1999) 1767–1780. [12] Q.N. Liu, B.J. Zhu, L.S. Dai, et al., The complete mitochondrial genome of the wild silkworm moth, Actias selene, Gene 505 (2) (2012) 291–299. [13] L.S. Dai, B.J. Zhu, Q.N. Liu, et al., Characterization of the complete mitochondrial genome of Bombyx mori strain H9 (Lepidoptera: Bombycidae), Gene 519 (2) (2013) 326–334.
9
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Genomics journal homepage: www.elsevier.com/locate/ygeno
Original Article
The complete mitochondrial genome of Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) and comparison with other Brachyuran crabs ⁎
Zhengfei Wang , Xuejia Shi, Yitao Tao, Qiong Wu, Yuze Bai, Huayun Guo, Dan Tang Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, School of Ocean and Biological Engineering, Yancheng Teachers University, Yancheng 224001, Jiangsu Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sesarmidae Parasesarma pictum Mitogenome Gene order Phylogentic
Mitochondrial DNA (mtDNA) is an extrachromosomal genome which can provide important information for evolution and phylogenetic analysis. In this study, we assembled a complete mitogenome of a crab Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) from next generation sequencing reads at the first time. P. pictum is a mudflat crab, belonging to the Sesarmidae family (subfamily Sesarminae), which is perched on East Asia. The 15,716 bp mitogenome covers 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and one control region (CR). The control region spanns 420 bp. The genome composition was highly A+T biased 75.60% and showed negative AT-skew (−0.03) and negative GC-skew (−0.47). Compared with the ancestor of Brachyura, the gene order of Sesarmidae has several differences and the gene order of P. pictum is typical for mitogenomes of Sesarmidae. Phylogenetic tree based on nucleotide sequences of mitochondrial 13 PCGs using BI and ML determined that P. pictum has a sister group relationship with Parasesarma tripectinis and belongs to Sesarmidae.
1. Introduction Decapoda is an economically important order of crustaceans comprising 18,000 living and extinct species, including freshwater crayfish, lobsters, hermit crabs, shrimps, and “true” crab [1,2]. Among these species, the Sesarmidae (Decapoda; Brachyura; Grapsoidea) is the most speciose family of crabs occurring in the mangroves [3]. Even though they are not important in commercial fishery, previous researches have demonstrated that Sesarmidae crabs play an important ecological role in mangrove ecosystems [4,5]. The taxonomy of the Sesarminae is one of the most challenging problems in the Sesarminae [6] and there are many new species have been reported gradually recent years [7,8]. Parasesarma pictum is a mudflat crab, belonging to the Sesarmidae family (subfamily Sesarminae), which is endemic to East Asia [9,10]. This crab typically inhabits mangrove swamps, preferring the upper intertidal region of estuaries, and living in small crevices and abandoned holes made by other species [10]. Mitochondrial DNA (mtDNA) is an extrachromosomal genome which has been widely used as an informative molecular marker for diverse evolutionary studies among species, including molecular evolution, phylogenetics, population genetics, and comparative and evolutionary genomics [11,12]. In the majority of animals, mitogenomes
⁎
are typically closed circular molecule approximately ranging in size from 14 to 20 kilobases (Kb) [13]. It contains 37 genes, including 13 protein coding genes (PCGs) [subunits 6 and 8 of the ATPase (atp6 and atp8), cytochrome coxidase subunits 1–3 (cox1–cox3), cytochrome B (cob), NADH dehydrogenase subunits 1–6 and 4 l (nad1–6 and nad4l)], two ribosomal RNA genes encoding the small and large subunit rRNAs (rrnS and rrnL), 22 transfer RNA (tRNA) genes and a control region (CR) of variable length, known as the A+T-rich region [11,14,15]. To date, the complete mitogenome of P. pictum has not been reported. Therefore, in this study, the complete mitogenome of P. pictum was initially sequenced and compared with other Brachyura mitogenomes. The available complete mitogenomes were used to provide insight into the phylogenetic relationship of P. pictum and related species. These results will perform an important role in understanding features of P. pictum mitogenome and the evolutionary relationships within Brachyura. 2. Materials and methods 2.1. Samples and DNA extraction The specimen used in this study was collected in Shanghai, China.
Corresponding author. E-mail address: [email protected] (Z. Wang).
https://doi.org/10.1016/j.ygeno.2018.05.002 Received 10 March 2018; Received in revised form 29 April 2018; Accepted 4 May 2018 0888-7543/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Wang, Z., Genomics (2018), https://doi.org/10.1016/j.ygeno.2018.05.002
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2.3. Sequence analysis and gene annotation
Table 1 List of Brachyuran species analyzed in this study with their GenBank accession numbers. Species
Family
Size (bp)
Accession no.
Parasesarma pictum Pyrhila pisum Scylla olivacea Scylla serrata Scylla paramamosain Scylla tranquebarica Charybdis japonica Charybdis feriata Callinectes sapidus Portunus sanguinolentus Portunus pelagicus Portunus trituberculatus Maja squinado Maja crispata Maguimithrax spinosissimus Leptodius sanguineus Pseudocarcinus gigas Segonzacia mesatlantica Gandalfus puia Gandalfus yunohana Austinograea alayseae Austinograea rodriguezensis Macrophthalmus japonicus Mictyris longicarpus Hemigrapsus sanguineus Eriocheir japonica japonica Eriocheir japonica sinensis Eriocheir japonica hepuensis Helicana wuana Helice tientsinensis Ilyoplax deschampsi Xenograpsus testudinatus Parasesarma tripectinis Metopaulias depressus Clistocoeloma sinense Sesarma neglectum Sesarmops sinensis Grapsus tenuicrustatus Pachygrapsus crassipes Ocypode cordimanus Somanniathelphusa boyangensis Huananpotamon lichuanense Sinopotamon xiushuiense Geothelphusa dehaani Umalia orientalis Lyreidus brevifrons Homologenus malayensis Moloha majora Clibanarius infraspinatus
Sesarmidae Leucosiidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Majidae Majidae Mithracidae Xanthidae Menippidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Macrophthalmidae Mictyridae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Dotillidae Xenograpsidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Grapsidae Grapsidae Ocypodidae Parathelphusidae Potamidae Potamidae Potamidae Raninidae Raninidae Homolidae Homolidae Diogenidae
15,613 15,516 15,723 15,775 15,825 15,833 15,738 15,660 16,263 16,024 16,157 16,026 16,598 16,592 15,817 15,480 15,515 15,521 15,548 15,567 15,620 15,611 16,170 15,548 16,275 16,352 16,354 16,335 16,359 16,212 15,460 15,798 15,612 15,765 15,706 15,920 15,905 15,858 15,652 15,604 17,032 15,380 18,460 18,197 15,466 16,112 15,793 15,903 16,504
This study NC_030047.1 NC_012569.1 NC_012565.1 NC_012572.1 NC_012567.1 NC_013246.1 NC_024632.1 NC_006281.1 NC_028225.1 NC_026209.1 NC_005037.1 NC_035425.1 NC_035424.1 NC_025518.1 NC_029726.1 NC_006891.1 NC_035300.1 NC_027414.1 NC_013713.1 NC_020314.1 NC_020312.1 NC_030048.1 NC_025325.1 NC_035307.1 NC_011597.1 NC_006992.1 NC_011598.1 NC_034995.1 NC_030197.1 NC_020040.1 NC_013480.1 NC_030046.2 NC_030535.1 NC_033866.1 NC_031851.1 NC_030196.1 NC_029724.1 NC_021754.1 NC_029725.1 NC_032044.1 NC_031406.1 NC_029226.1 NC_007379.1 NC_026688.1 NC_026721.1 NC_026080.1 NC_029361.1 NC_025776.1
The assembled mitochondrial genes were compared with Parasesarma tripectinis (NC_030046) and identified by BLAST searches in the NCBI database [17] to ensure that the sequences were correct. The locations of the PCGs were identified using ORF Finder via NCBI with the selection of invertebrate mitochondrial genetic code. Abnormal start codons and stop codons were determined based on comparisons with other crabs. Codon usage was calculated using MEGA 7.0 [18]. Identification of tRNA genes was verified using the MITOS WebServer (http://mitos2.bioinf.uni-leipzig.de/index.py). The rRNA genes were determined based on the locations of adjacent tRNA genes and by comparisons with other crabs. Strand asymmetry was calculated using the formulae: AT-skew = (A − T)/(A + T); GC-skew = (G − C)/ (G + C) [19]. The graphical map of the mitochondrial genome was drawn using the online mitochondrial visualization tool Organellar Genome DRAW [20,21]. 2.4. Phylogenetic analysis The complete nucleotide sequences of other 47 Brachyura (Table 1) were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/ genbank/). The mitogenome of Clibanarius infraspinatus was used as outgroup. We estimated the taxonomic status of P. pictum within Brachyura by reconstructing phylogenetic trees. Nucleotide sequences of each gene and their deduced amino acid sequences were aligned separately using MUSCLE 3.8 [22] and MEGA 7.0 [18]. The amino acid sequences for each of the 13 mitochondrial PCGs from 49 mitogenomes were aligned using default settings and concatenated. The concatenated set of nucleotide sequences were used for phylogenetic analysis, which was performed with the Bayesian inference (BI) and Maximum Likelihood (ML) methods using MrBayes v3.2.6 [23] and RAxML [24], respectively. The best model selected with jModeltest [25] and the model of SYM+I+G was optimal for analysis with nucleotide alignment. In BI analysis, two simultaneous runs of 10,000,000 generations conducted for the matrix. We made two simultaneous runs, sampling trees every 1000 generations, with three heated and one cold chain to encourage swapping among the Markov-chain Monte Carlo (MCMC) chains. Convergence of sampled parameters and potential autocorrelation (effective sampling size/ESS for all parameters > 200) was investigated in Tracer 1.6 (http://tree.bio.ed.ac.uk/software/tracer/). Additionally, the average standard deviation of split frequencies between both runs was checked (< 0.01). The Bayesian posterior probabilities were obtained from the 50% majority rule consensus of the post-burn-in trees sampled at stationarity, after removing the first 25% of trees as a “burnin” stage. The resulting phylogenetic trees were visualized in FigTree v1.4.2.
Total DNA was extracted from the muscle tissues and using the Aidlab Genomic DNA Extraction Kit (Aidlab Biotech, Beijing, China). Specimen was incubated at 55 °C for 5 h to lyse completely and the total genomic DNA was eluted in 50 mL double-distill water (ddH2O), while the remaining steps were conducted in accordance with the manufacturer's protocol. Genomic DNA was stored at −20 °C.
3. Results and discussion 3.1. Genome organization and nucleotide composition In the present study, we determined the complete mitochondrial genome sequence of the P. pictum by next-generation sequencing methods. The results of the assembled mitogenomes from two lanes were consistent. We finally achieved an average sequence depth of ~55× per base. The mitogenome of P. pictum is a closed circular molecule of 15,613 bp in length. The mitogenome contained 37 typical mitochondrial genes [13 PCGs (cox1-3, nad1-6, nad4l, cob, atp6 and atp8), 22 tRNAs (one for each amino acid, two for leucine and serine), two rRNAs (rrnS and rrnL) and a major non-coding region known as the CR] and all the genes were identified (Table 2 and Fig. 1). The mitogenome of P. pictum has been deposited in GenBank under accession number MG580780. The nucleotide compositions of the mitogenome of P. pictum are as follows: (A) 36.60%; (T) 39.00%; (G) 9.83%; and (C) 14.57% (Table 3). The whole mitogenome of P. pictum was biased
2.2. Mitogenome sequencing The mitogenome of P. pictum was sequenced by next-generation sequencing. Two lanes for P. pictum were sequenced as 150 bp reads using Illumina HisSeq 4000 (5 GB raw and 4 GB raw; Origin gene, Shanghai, China). Raw sequence data were deposited into Short Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra/) with the accession no. SRR6315542. Clean data without sequencing adapters were de novo assembled using the NOVOPlasty software [16]. To evaluate the single-base accuracy of the assembled mitogenome, we compared the assembled genome with three confirmed sequences by PCR and Sanger sequencing methods. 2
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Table 2 Summary of Parasesarma pictum mitogenome. Gene
Direction
Location
Size
Anticodon
Start codon
Stop codon
Intergenic nucletide
cob trnS2 nad1 trnL1 rrnL trnV rrnS CR trnQ trnI trnM nad2 trnW trnC trnY cox1 trnL2 cox2 trnK trnD atp8 atp6 cox3 trnG nad3 trnA trnR1 trnN trnR2 trnE trnH trnF nad5 nad4 nad4l trnT trnP nad6
+ + − − − − −
484–1618 1619–1685 1703–2641 2677–2743 2721–4074 4077–4149 4151–4986 4987–5616 5617–5684 5702–5768 5789–5859 5860–6867 6866–6934 6943–7007 7008–7074 7077–8610 8612–8680 8689–9376 9377–9446 9447–9513 9514–9672 9666–10,340 10,340–11,134 11,134–11,198 11,199–11,549 11,552–11,617 11,623–11,687 11,690–11,755 11,757–11,823 11,824–11,890 11,894–11,957 11,959–12,024 12,029–13,759 13,806–15,155 15,149–15,451 15,458–15,524 15,525–15,589 15,592–484
1135 67 939 67 1354 73 836 630 68 67 71 1008 69 65 67 1534 69 688 70 67 159 675 795 65 351 66 65 66 67 67 64 66 1731 1350 303 67 65 504
− TGA − TAG − TAC − − TTG GAT CAT − TCA GCA GTA − TAA − TTT GTC − − − TCC − TGC TCG GTT TCT TTC GTG GAA − − − TGT TGG −
ATG − ATA − − − − − − − − ATG − − − ATG − ATG − − ATG ATT ATG − ATC − − − − − − − ATG ATG ATG − − ATT
T − TAA − − − − − − − − TAG − − − T − T − − TAA TAA TAA − TAG − − − − − − − TAA TAA TAA − − TAA
0 17 35 −23 2 1 0 0 17 20 0 −2 8 0 2 1 8 0 0 0 −7 −1 −1 0 2 5 2 1 0 3 1 4 46 −7 6 0 2 −1
− + + + + − − + + + + + + + + + + + + + + + − − − − − + − +
atp8, nad1, nad4, nad5, nad6 and nad4l), and the nad2 and nad3 used TAG as a stop codon whereas the cox1, cox2 and cob terminated by a single T (Table 2). These features are somewhat alike to other invertebrate mitochondrial genomes, and the incomplete stop codon was presumably completed as TAA via post-transcriptional polyadenylation [12,27,28]. Relative synonymous codon usage (RSCU) values for the 13 PCGs are summarised in Table 4 and Fig. 2. Excluding start and stop codons, the mitogenome of P. pictum consists of 3719 codons. In the 13 PCGs of P. pictum, the most frequently used amino acids were Leu (15.70%), followed by Phe (9.38%), Ser (6.13%), and Met (6.05%). A common feature in most metazoan mitochondrial genomes is a bias toward a higher representation of nucleotides A and T that leads to a subsequent bias in the corresponding encoded amino acids [29,30]. In the mitogenome of P. pictum, the overall A+T content of 13 PCGs was 74.88% and AT-skew was negative, indicating a higher occurrence of T than A (Table 3).
toward AT nucleotides (75.60%) in a similar manner to Sesarma neglectum (75.50%) and Sesarmops sinensis (75.55%), both of which have the same gene arrangements with P. pictum [26]. Most genes (24 of 37) were encoded on the heavy (+) strand while the remaining 13 genes were located on the light (−) strand (Table 2). There is a 42 bp overlap between genes in seven locations, with the longest 23 bp overlap located in between nad1 (CUN) and trnL1 in mitogenome of P. pictum (Table 2). The mitogenome of P. pictum contains 280 of intergenic spacer sequence spread over 20 regions, ranging in size from 1 to 46 bp (Table 2). The whole mitogenome of P. pictum was biased toward AT nucleotides (75.60%). The AT-skew and GC-skew for the whole mitogenome were both negative, respectively, indicating a higher occurrence of T than A and C than G (Table 3). As a result of the gene arrangement and compactness of mitogenome, the P. pictum mitogenome has a total of 42 bp overlap between genes in 7 locations; seven pairs of overlapping genes are present: trnL1/nad1, nad2/trnM, atp8/trnD, atp6/ atp8, cox3/atp6, nad4/nad5 and nad6/trnP, with the longest 23 bp overlap located between trnL1 and rrnL.
3.3. Skewness 3.2. PCGs and codon usage In the mitogenome of P. pictum, the skew of AT was negative and the skew of GC was consistently negative, indicating an obvious bias toward the use of T and C (Table 3). The AT-skew and GC-skew of the selected complete Brachyura mitogenomes were also calculated. From the Table 5, the GC-skew for the complete mitogenomes was negative (from −0.35 to −0.19) and the AT-skew was slightly negative (from −0.09 to 0) except Leptodius sanguineus (0.04) [31], Gandalfus puia (0.01) [32], Austinograea alayseae (0.03) [33], Austinograea rodriguezensis (0.02) [34], Macrophthalmus japonicas (0.01), Homologenus
The mitogenome of P. pictum has 13 typical PCGs contain seven NADH genes (nad1-6 and nad4L), two ATP genes (atp6 and atp8) and three cytochrome genes (cox1-3 and cob) found in invertebrate animals (Fig. 1). The region of PCGs is 11,172 bp in size. In the mitogenome of P. pictum, nad1, nad4, nad4l and nad5 were encoded on the light strand, while the remaining nine PCGs (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cob) were encoded on the heavy strand. All the PCGs started with ATN and eight PCGs were terminated by TAA (cox3, atp6, 3
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Fig. 1. Gene map of the mitogenome of P. pictum. Protein-coding genes are color-coded (cox: purple; nad: yellow; atp: green; cob: kelly); rRNA genes are in red; tRNA genes are in blue. Abbreviations of protein-coding genes and ribosomal genes are as follows: atp6 and atp8 for ATP synthase subunits 6 and 8, cox1–3 for cytochrome oxidase subunits 1–3, cob for cytochrome b, nad1–6 and nad4L for NADH dehydrogenase subunits 1–6 and 4 L, rrnL and rrnS for large and small rRNA subunits. In addition, trnS1, trnS2, trnL1, and trnL2 denote codons tRNA-Ser (AGN), tRNA-Ser (UCN), tRNA-Leu (CUN), and tRNA-Leu (UUR), respectively. CR = Control Region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 3 Composition and skewness of Parasesarma pictum mitogenome. P. pictum
Size(bp)
A%
T%
G%
C%
A + T%
AT-skew
GC-skew
Mitogenome cob nad1 nad2 cox1 cox2 atp8 atp6 cox3 nad3 nad5 nad4 nad4l nad6 PCGs tRNAs rRNAs CR
15,613 1135 939 1008 1534 688 159 675 795 351 1731 1350 303 504 11,172 1478 2190 420
36.60 29.07 29.71 29.46 29.60 32.77 30.19 30.22 29.06 32.76 33.33 32.22 29.37 29.17 30.53 38.31 40.31 41.19
39.00 42.11 45.90 47.82 39.57 38.98 50.94 41.78 41.13 43.30 43.33 44.00 47.19 50.40 44.34 38.18 39.09 38.10
9.83 12.95 15.55 8.63 15.65 12.85 5.66 10.81 14.09 9.69 15.02 15.93 16.83 6.35 12.31 13.48 13.35 8.33
14.57 15.86 8.84 14.09 15.19 15.40 13.21 17.19 15.72 14.25 8.32 7.85 6.60 14.09 12.81 10.03 7.25 12.38
75.60 71.19 75.61 77.28 69.17 71.75 81.13 72.00 70.19 76.07 76.66 76.22 76.57 79.56 74.88 76.49 79.40 79.29
−0.032 −0.183 −0.214 −0.237 −0.144 −0.087 −0.256 −0.160 −0.172 −0.139 −0.130 −0.155 −0.233 −0.267 −0.184 0.002 0.015 0.039
−0.194 −0.101 0.275 −0.240 0.015 −0.090 −0.400 −0.228 −0.055 −0.190 0.287 0.340 0.437 −0.379 −0.020 0.147 0.296 −0.196
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Table 4 The codon number and relative synonymous codon usage in Parasesarma pictum mitochondrial protein coding genes. Codon
Count
RSCU
Codon
Count
RSCU
Codon
Count
RSCU
Codon
Count
RSCU
UUU(F) UUC(F) UUA(L) UUG(L) CUU(L) CUC(L) CUA(L) CUG(L) AUU(I) AUC(I) AUA(M) AUG(M) GUU(V) GUC(V) GUA(V) GUG(V)
314 35 415 33 77 4 50 5 331 23 189 36 104 3 99 13
1.80 0.20 4.26 0.34 0.79 0.04 0.51 0.05 1.87 0.13 1.68 0.32 1.90 0.05 1.81 0.24
UCU(S) UCC(S) UCA(S) UCG(S) CCU(P) CCC(P) CCA(P) CCG(P) ACU(T) ACC(T) ACA(T) ACG(T) GCU(A) GCC(A) GCA(A) GCG(A)
120 13 87 8 80 12 47 4 85 7 77 4 112 13 60 5
2.61 0.28 1.89 0.17 2.24 0.34 1.31 0.11 1.97 0.16 1.78 0.09 2.36 0.27 1.26 0.11
UAU(Y) UAC(Y) UAA(*) UAG(*) CAU(H) CAC(H) CAA(Q) CAG(Q) AAU(N) AAC(N) AAA(K) AAG(K) GAU(D) GAC(D) GAA(E) GAG(E)
146 15 8 2 68 9 61 13 124 26 89 6 57 12 68 9
1.81 0.19 1.60 0.40 1.77 0.23 1.65 0.35 1.65 0.35 1.87 0.13 1.65 0.35 1.77 0.23
UGU(C) UGC(C) UGA(W) UGG(W) CGU(R) CGC(R) CGA(R) CGG(R) AGU(S) AGC(S) AGA(S) AGG(S) GGU(G) GGC(G) GGA(G) GGG(G)
33 0 94 7 21 0 30 3 39 3 88 10 82 7 124 10
2.00 0.00 1.86 0.14 1.56 0.00 2.22 0.22 0.85 0.07 1.91 0.22 1.47 0.13 2.22 0.18
Fig. 2. Relative synonymous codon usage in Parasesarma pictum mitogenome.
malayensis (0.04) [35], Moloha majora (0.04) [36] and Clibanarius infraspinatus (0.04) [30].
79.29% AT nucleotides, with a positive AT skew (0.039) and negative GC skew (−0.196) (Table 3).
3.4. Transfer RNAs, ribosomal RNAs, and A+T-rich region
3.5. Gene order
Like most Brachyura mtDNA, the P. pictum mitogenome contains a set of 22 tRNAs genes (Fig. 3), although this feature is not very well conserved in animal mtDNA. The tRNAs ranged in size from 64 to 73 bp and showed a strong A + T bias, as these bases accounted for 76.49% of the DNA. Further, they exhibited a postive AT-skew (0.002) and a positive GC-skew (0.147) (Table 3). Fifteen tRNA genes were present on the heavy strand and seven were on the light strand. All the tRNA genes had the typical cloverleaf structure. These features are common in most Brachyura mitogenomes. The secondary cloverleaf structure of 22 tRNAs was examined using MITOS WebServer (http://mitos2.bioinf. uni-leipzig.de/index.py). The tRNAs were predicted to fold into the expected cloverleaf secondary structures with normal base pairing and G–U (e.g. tRNAArg, Met). However, there were other base pair arrangements, such as U–U (e.g. tRNAAla). The rrnL is 1354 bp and rrnS is 836 bp, one locating between trnL1 and trnV and another locating between trnV and CR (Table 2). The A+T content of two rRNA genes was totally 79.40% and they had positive AT skew (0.015) (Table 3). The CR located between rrnS and trnQ spans 630 bp. This region contains
The complete gene order in the P. pictum mitogenome is shown in Fig. 4. Compared with the gene arrangement in ancestral Brachyura [37], the gene order of P. pictum only had two differences of ancestral Brachyura. In short, only two pairs of genes appeared to be rearranged, along with trnH and trnQ. As usual, trnH located between nad5 and nad4 which, however, was shifted into trnE and trnF junction. Another rearrangement was the transversion of trnI and trnQ. The gene order of P. pictum mitogenome was also compared with P. tripectinis (Sesarmidae: Brachyura) which went to have the same arrangement (Accession: NC_030046.2). Futhermore, the gene orders of P. pictum and P. tripectinis are the same as that ancestral Sesarmidae [38]. The same gene order of P. pictum and P. tripectinis identifies that the two species have a close relationship in evolution. 4. Phylogentic analysis The phylogenetic relationships were analyzed based on the concatenated nucleotide sequences of 13 PCGs from 48 Brachyura species
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Table 5 Composition and skewness of mitogenome in 49 Brachyura species. Species
A%
T%
A + T%
G%
C%
G + C%
AT-skew
GC-skew
Parasesarma pictum Pyrhila pisum Scylla olivacea Scylla serrata Scylla paramamosain Scylla tranquebarica Charybdis feriata Charybdis japonica Callinectes sapidus Portunus pelagicus Portunus sanguinolentus Portunus trituberculatus Maja crispata Maja squinado Maguimithrax spinosissimus Leptodius sanguineus Pseudocarcinus gigas Segonzacia mesatlantica Gandalfus puia Gandalfus yunohana Austinograea alayseae Austinograea rodriguezensis Macrophthalmus japonicus Mictyris longicarpus Hemigrapsus sanguineus Eriocheir japonica hepuensis Eriocheir japonica japonica Eriocheir japonica sinensis Helicana wuana Helice tientsinensis Ilyoplax deschampsi Xenograpsus testudinatus Geothelphusa dehaani Grapsus tenuicrustatus Huananpotamon lichuanense Parasesarma tripectinis Metopaulias depressus Clistocoeloma sinense Sesarma neglectum Sesarmops sinensis Pachygrapsus crassipes Ocypode cordimanus Sinopotamon xiushuiense Somanniathelphusa boyangensis Lyreidus brevifrons Umalia orientalis Homologenus malayensis Moloha majora Clibanarius infraspinatus
36.60 37.28 33.45 34.55 34.79 35.04 34.09 33.79 34.17 33.71 31.59 33.33 33.59 33.75 33.30 33.62 35.02 33.83 35.15 34.29 34.37 35.26 33.65 32.43 34.27 35.10 35.16 35.28 32.95 33.93 34.15 36.70 36.92 31.92 35.76 36.23 37.94 37.11 37.42 37.37 30.49 31.81 36.03 35.13 34.21 33.09 37.31 38.36 35.39
39.00 37.20 35.91 37.95 38.19 38.72 36.05 35.45 34.93 35.03 34.01 36.89 36.71 37.06 36.77 31.23 35.46 33.99 34.75 35.65 32.43 33.55 32.75 36.58 35.50 36.41 36.45 36.35 35.47 35.13 35.47 37.21 38.00 33.11 37.44 37.98 39.38 38.62 38.21 38.31 35.79 34.53 38.51 37.25 36.38 34.94 34.42 35.50 32.55
75.60 74.48 69.36 72.51 72.98 73.76 70.15 69.24 69.10 68.74 65.60 70.22 70.30 70.80 70.07 64.85 70.47 67.82 69.90 69.94 66.79 68.80 66.39 69.01 69.76 71.51 71.62 71.63 68.42 69.06 69.62 73.91 74.92 65.03 73.21 74.22 77.32 75.72 75.63 75.68 66.28 66.34 74.54 72.38 70.59 68.03 71.73 73.86 67.94
9.83 10.22 11.23 10.42 10.17 9.74 11.25 11.88 11.15 12.21 12.91 11.30 11.13 11.02 10.56 11.16 10.80 10.53 10.34 10.80 11.36 10.28 10.95 11.83 11.18 10.76 10.72 10.67 11.55 11.00 10.72 9.32 8.26 12.13 9.31 10.11 8.71 9.36 9.51 9.39 12.73 11.89 8.61 9.25 11.31 11.79 10.01 9.82 12.84
14.57 15.31 19.40 17.07 16.85 16.50 18.60 18.88 19.76 19.06 21.50 18.48 18.57 18.18 19.37 23.99 18.72 21.65 19.76 19.25 21.85 20.91 22.66 19.15 19.05 17.73 17.66 17.70 20.03 19.94 19.66 16.77 16.82 22.85 17.48 15.67 13.97 14.92 14.86 14.93 20.99 21.76 16.85 18.38 18.10 20.17 18.25 16.32 19.23
24.40 25.52 30.64 27.49 27.02 26.24 29.85 30.76 30.90 31.26 34.40 29.78 29.70 29.20 29.93 35.15 29.53 32.18 30.10 30.06 33.21 31.20 33.61 30.99 30.24 28.49 28.38 28.37 31.58 30.94 30.38 26.09 25.08 34.97 26.79 25.78 22.68 24.28 24.37 24.32 33.72 33.66 25.46 27.62 29.41 31.97 28.27 26.14 32.06
−0.03 0.00 −0.04 −0.05 −0.05 −0.05 −0.03 −0.02 −0.01 −0.02 −0.04 −0.05 −0.05 −0.05 −0.05 0.04 −0.01 0.00 0.01 −0.02 0.03 0.02 0.01 −0.06 −0.02 −0.02 −0.02 −0.02 −0.04 −0.02 −0.02 −0.01 −0.01 −0.02 −0.02 −0.02 −0.02 −0.02 −0.01 −0.01 −0.09 −0.04 −0.03 −0.03 −0.03 −0.03 0.04 0.04 0.04
−0.47 −0.50 −0.47 −0.46 −0.46 −0.45 −0.47 −0.48 −0.49 −0.48 −0.46 −0.45 −0.46 −0.46 −0.45 −0.54 −0.49 −0.50 −0.51 −0.48 −0.53 −0.53 −0.51 −0.44 −0.48 −0.48 −0.48 −0.49 −0.46 −0.48 −0.48 −0.49 −0.49 −0.48 −0.48 −0.48 −0.48 −0.48 −0.49 −0.49 −0.43 −0.46 −0.47 −0.47 −0.47 −0.47 −0.54 −0.54 −0.54
Potamidae, Bythograeidae, and Portunidae was also identified and relationship on these families is also consistent with previous researches [38–40].
and one outgroup. The results of the BI and ML analyses generated consistent tree topologies (Fig. 5). In this study, both the BI and ML analyses showed that each superfamily in the tree formed a monophyletic clade. It is obvious that P. pictum and P. tripectinis clustered in one branch in the phylogenetic tree with high nodal support values (BI posterior probabilities [PP] = 1; ML bootstrap [BP] = 100) (Fig. 5), indicating that P. pictum and P. tripectinis have a sister group relationship. Additionally, the results of the phylogenetic analyses revealed Metopaulias depressus and Clistocoeloma sinense were grouped into one clade with high nodal support value. These two pairs of Sesarmide apparently have a stable sister relationship which is consistent with previous study [38]. This result supported that P. pictum belongs to Grapsoidea, Sesarmidae. Further, Xenograpsus testudinatus, which was originally placed in Varunidae, has been transferred to its own family (Xenograpsidae) [39,40]. From the phylogenetic tree, we also found that X. testudinatus and Sesarmidae species formed a group and showed close relationships. Additionally, the relationship on Sesarmidae, Xenograpsidae, Grapsidae, Varunidae,
5. Conclusion In this study, we firstly sequenced the complete 15,613 bp mitogenome of P. pictum, in which 37 genes (13 PCGs, 22 tRNA genes and 2 rRNA genes) and one control region are located as a typical of Brachyura mitogenome. With all PCGs are initiated by ATN codon, the cox1, cox2 and cob genes have incomplete stop codons consisting of just a T, and the other 10 PCGs stop with the canonical TAA or TAG. The AT-skew and the GC-skew are both negative in the mitogenomes of P. pictum which are consistent with most sequenced brachyuran crabs. The gene order of P. pictum is the same as P. tripectinis which is consistent of Sesarmidae, indicating the relationship between P. pictum and P. tripectinis. Futhermore, the phylogenetic analyses support that P. pictum belongs to Grapsoidea, Sesarmidae.
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Fig. 3. Secondary structures of the 22 transfer RNA genes of P. pictum. tRNAs are labeled with the abbreviations of their corresponding amino acids. Dashes (−) indicate Watson-Crick pairing.
Fig. 4. Linear representation of gene rearrangements of Ancestor of Brachyura, Sesarmidae, Parasesarma tripectinis and Parasesarma pictum. All genes are transcribed from left to right. tRNA genes are represented by the corresponding single-letter amino acid code. S1 (AGN), S2 (UCN), L1 (CUN), L2 (UUR), CR (control region). rrnL and rrnS are the large and small ribosomal RNA subunits.
Fig. 5. Phylogeny of P.pictum based on nucleotide sequences. The phylogenetic tree was inferred from the nucleotide sequences of 13 mitogenome PCGs using BI and 8 ML methods. Numbers on branches indicate posterior probability (BI) and bootstrap (ML).
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Acknowledgements
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This study was funded by the National Natural Science Foundation of China (grant number 31702014), and Doctoral Scientific Research Foundation of Yancheng Teachers University to ZFW, and Open Foundation of Jiangsu Key Laboratory for Bioresources of Saline Soils (grant number JKLBS2016007), and Yancheng Teachers University Undergraduate Training Programs for Innovation and Entrepreneurship to YTT. Conflict of interest The authors declare there are no competing interests. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Authors' contributions HYG, XJS, QW, ZFW and YTT designed and conceived the experiment. YTT, ZFW, YZB and DT performed the data analysis and draft the manuscript. All authors read and approved the final manuscript. References [1] J. Krieger, A. Sombke, F. Seefluth, et al., Comparative brain architecture of the European shore crab Carcinus maenas (Brachyura) and the common hermit crab Pagurus bernhardus (Anomura) with notes on other marine hermit crabs, Cell Tissue Res. 348 (1) (2012) 47–69. [2] H. Shen, A. Braband, G. Scholtz, Mitogenomic analysis of decapod crustacean phylogeny corroborates traditional views on their relationships, Mol. Phylogenet. Evol. 66 (3) (2013) 776–789. [3] S. Cannicci, C.D. Schubart, G. Innocenti, et al., A new species of the genus Parasesarma De Man 1895 from East African mangroves and evidence for mitochondrial introgression in sesarmid crabs, Zool. Anz. 269 (2017) 89–99. [4] A.A. Flores, J. Saraiv, J. Paula, Sexual, maturity, reproductive cycles, and juvenile recruitment of Perisesarma guttatum (brachyura, sesarmidae) at ponta rasa mangrove swamp, inhaca island, mozambique, J. Crustac. Biol. 22 (1) (2002) 143–156. [5] S.A. Khan, S.M. Raffi, P.S. Lyla, Brachyuran crab diversity in natural (Pichavaram) and artificially developed mangroves (Vellar estuary), Curr. Sci. 88 (2005) 1316–1324. [6] D.L. Rahayu, P.K.L. Ng, Revision of the Parasesarma plicatum (Latreille, 1803) species-group (Crustacea: Decapoda: Brachyura: Sesarmidae), Zootaxa 2327 (2010) 1–22. [7] A. Shahdadi, P.J.F. Davie, C.D. Schubart, Perisesarma tuerkayi, a new species of mangrove crab from Vietnam (Decapoda;Brachyura;Sesarmidae),with an assessment of its phylogenetic relationships, Crustaceana 90 (7–10) (2017) 1155–1175. [8] T. Naruse, P.K.L. Ng, A new species of Chiromantes s. str. (Decapoda: Brachyura: Sesarmidae) from the Ryukyu Islands, Japan, with a note on the identity of Holometopus serenei Soh, 1978, Crustacean Res. 37 (2008) 1–13. [9] G. Guerao, K. Anger, U.W.E. Nettelmann, et al., Complete larval and early juvenile development of the mangrove crab Perisesarma fasciatum (Crustacea: Brachyura: Sesarmidae) from Singapore, with a larval comparison of Parasesarma and Perisesarma, J. Plankton Res. 26 (12) (2004) 1389–1408. [10] L.M. Surhone, M.T. Tennoe, S.F. Henssonow, Parasesarma Pictum, Betascript Publishing, 2010. [11] J.L. Boore, Animal mitochondrial genomes, Nucleic Acids Res. 27 (8) (1999) 1767–1780. [12] Q.N. Liu, B.J. Zhu, L.S. Dai, et al., The complete mitochondrial genome of the wild silkworm moth, Actias selene, Gene 505 (2) (2012) 291–299. [13] L.S. Dai, B.J. Zhu, Q.N. Liu, et al., Characterization of the complete mitochondrial genome of Bombyx mori strain H9 (Lepidoptera: Bombycidae), Gene 519 (2) (2013) 326–334.
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