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The complete mitochondrial genome of Calappa bilineata: The first representative from the family Calappidae and its phylogenetic position within Brachyura
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Genomics journal homepage: www.elsevier.com/locate/ygeno
Original Article
The complete mitochondrial genome of Calappa bilineata: The first representative from the family Calappidae and its phylogenetic position within Brachyura ⁎
Xinting Lua,b, Li Gonga,b,c, ,1, Ying Zhanga,b, Jian Chenb, Liqin Liua,b, Lihua Jianga,b, ⁎ Zhenming Lüa,b, Bingjian Liua,b, Guixiang Tongd, Xinxian Weid, ,1 a
National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, 316022 Zhoushan, China Marine Science and Technology College, Zhejiang Ocean University, 316022 Zhoushan, China Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, China d Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Academy of Fishery Sciences, Nanning, Guangxi 530021, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Box crab Mitogenome Calappoidea Phylogenetic analysis
In this study, we determined the complete mitogenome sequence of Calappa bilineata, which is the first mitogenome of Calappidae up to now. The total length is 15,606 bp and includes 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and one control region. The genome composition is highly A + T biased (68.7%), and exhibits a negative AT-skew (−0.010) and GC-skew (−0.267). As with other invertebrate mitogenomes, the PCGs start with the standard ATN and stop with the standard TAN codons or incomplete T. Phylogenetic analysis showed that C. bilineata was most closely related to Matuta planipes (Matutidae), and these two species formed a sister clade, constituting a Calappoidea group and forming a sister clade with part of Eriphioidea. The existence of the polyphyletic families raised doubts over the traditional classification system. These results will help to better understand the features of the C. bilineata mitogenome and lay foundation for further evolutionary relationships within Brachyura.
1. Introduction
suggesting close relationships. Recent studies, however, have found a number of inconsistent features within the family, indicating controversial phylogenetic relationships [10–13]. Calappidae, the box crabs, appeared relatively early in the evolution of crustaceans and played an important ecological role in marine ecosystem. Due to the high morphological similarity and extreme ecological diversity, classification and phylogenetic relationships within this family are complicated and controversial [10,14,15]. To date, more than 90 Brachyura mitogenomes have been sequenced (https://www.ncbi.nlm.nih.gov). However, no complete mitogenome from Calappidae has ever been reported. Most studies of this family focused on the classification based on morphological features [14,16]. Although there are few researches on molecular level, most of them were based on partial mitochondrial and nuclear ribosomal RNA gene sequences (e.g. 28S rDNA) [15,17]. Therefore, in the present study, we determined the first complete mitogenome sequence of Calappa bilineata, representative from the family Calappidae. Additionally,
Metazoan mitochondrial genome (mitogenome) is usually 14–20 kb in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (12S and 16S) and an AT-rich region (also called control region, CR) [1]. Mitochondrial DNA is featured by maternal inheritance, simple structure, conserved organization, small genome size, and high mutation rate [2,3]. With the rapid development of sequencing technologies, complete mitogenome sequence is becoming increasingly common used in population genetics, comparative and evolutionary genomics, and reconstruction of phylogenetic relationships [4–6]. Brachyura, the true crab, is the largest clade Decapod Crustacea, with over 7250 described species in 98 families inhabiting marine, freshwater, and terrestrial habitats, most of which are economically important [7–9]. Brachyuran taxonomists have traditionally placed the various genera together, either as a family or as a super-family,
⁎
Corresponding authors. E-mail addresses: [email protected], [email protected] (L. Gong), [email protected] (X. Wei). 1 These authors contributed equally to this paper. https://doi.org/10.1016/j.ygeno.2020.02.003 Received 3 December 2019; Received in revised form 8 January 2020; Accepted 7 February 2020 0888-7543/ © 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Xinting Lu, et al., Genomics, https://doi.org/10.1016/j.ygeno.2020.02.003
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Table 1 (continued)
Table 1 List of 84 Brachyura and two outgroup used in this paper. Species
Family
Length (bp)
Accession no.
Scylla olivacea Scylla serrata Scylla tranquebarica Charybdis feriata Charybdis natator Charybdis bimaculata Thalamita sima Thalamita crenata Portunus sanguinolentus Portunus pelagicus Callinectes sapidus Monomia gladiator Ovalipes punctatus Chaceon sp. Myomenippe fornasinii Pseudocarcinus gigas Calappa bilineata Matuta planipes Atergatis integerrimus Atergatis floridus Etisus anaglyptus Leptodius sanguineus Epixanthus frontalis Pilumnus vespertilio Echinoecus nipponicus Gandalfus puia Gandalfus yunohana Austinograea rodriguezensis Austinograea alayseae Segonzacia mesatlantica Majo squinado Maja crispata Damithrax spinosissimus Sinopotamon xiushuiense Longpotamon yangtsekiense Longpotamon kenliense Sinopotamon yaanense Geothelphusa dehaani Huananpotamon lichuanense Somanniathelphusa boyangensis Uca arcuata Uca capricornis Uca polita Uca borealis Uca inversa Uca lactea Ocypode cordimanus Hemigrapsus sanguineus Hemigrapsus penicillatus Eriocheir sinensis Eriocheir hepuensis Eriocheir japonica Neoeriocheir leptognathus Varuna yui Helice latimera Helice tientsinensis Helicana wuana Pseudohelice subquadrata Metaplax longipes Macrophtalmus japonicus Mictyris longicarpus Parasesarma tripectinis Parasesarma affine Nanosesarma minutum Clistocoeloma sinense Metopaulias depressus Sesarma neglectum Chiromantes dehaani Sesarmops sinensis Chiromantes haematocheir Gecarcoidea natalis Cardisoma carnifex Iloplax deschampsi
Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Polybiidae Geryonidae Menippidae Eriphiidae Calappidae Matutidae Xanthidae Xanthidae Xanthidae Xanthidae Oziidae Pilumnidae Pilumnidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Majidae Majidae Mithracidae Potamidae Potamidae Potamidae Potamidae Potamidae Potamidae Parathelphusidae
15,723 15,775 15,833 15,660 15,664 15,714 15,831 15,787 16,024 16,157 16,263 15,878 16,084 16,126 15,658 15,515 15,606 15,760 15,924 16,180 16,435 15,480 15,993 16,222 16,173 15,548 15,567 15,611 15,620 15,521 16,598 16,592 15,817 18,480 17,885 18,499 17,126 18,197 15,380 17,032
NC_012569 NC_012565 NC_012567 NC_024632 MF285241 MG489891 MG840650 MH425338 NC_028225 KR153996 AY363392 MG770549 MH802052 KU507298 LK391943 NC_006891 MN562587 MG756601 NC_037172 NC_037201 MG751773 NC_029726 MF457404 MF457402 MG574831 NC_027414 NC_013713 JQ035658 JQ035660 NC_035300 NC_035425 NC_035424 NC_025518 NC_029226 NC_036946 MK584299 NC_036947 NC_007379 NC_031406 NC_032044
Ocypodidae Ocypodidae Ocypodidae Ocypodidae Ocypodidae Ocypodidae Ocypodidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Macrophthalmidae Mictyridae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Gecarcinidae Gecarcinidae Dotillidae
15,955 15,629 15,672 15,662 15,677 15,659 15,604 16,275 16,486 16,354 16,335 16,352 16,143 15,915 16,246 16,212 16,359 16,898 16,424 16,170 15,548 15,612 15,638 15,637 15,706 15,765 15,920 15,917 15,905 15,899 15,545 15,597 15,460
KX911977 NC_039107 NC_039106 MH796170 NC_039111 KY865330 NC_029725 NC_035307 MG751772 NC_006992 FJ455506 NC_011597 MH593561 NC_037155 NC_033865 NC_030197 NC_034995 MH718959 MF198248 NC_030048 NC_025325 NC_030046 MH310444 NC_040977 NC_033866 NC_030535 NC_031851 MH593563 NC_030196 NC_042142 NC_039811 NC_039105 NC_020040
Species
Family
Length (bp)
Accession no.
Dotilla wichmanni Pachygrapsus crassipes Pachygrapsus marmoratus Grapsus tenuicrustatus Metopograpsus quadridentatus Metopograpsus frontalis Homologenus malayensis Moloha majora Dynomene pilumnoides Umalia orientalis Lyreidus brevifrons Pagurus japonicus Pagurus nigrofascia
Dotillidae Grapsidae Grapsidae Grapsidae Grapsidae Grapsidae Homolidae Homolidae Dynomenidae Raninidae Raninidae Paguridae Paguridae
15,600 15,662 15,406 15,858 15,520 15,587 15,793 15,903 16,475 15,446 16,112 16,401 15,392
NC_038180 NC_021754 MF457403 NC_029724 MH310445 MH028874 NC_026080 NC_029361 KT182070 NC_026688 NC_026721 LC222532 LC222531
the phylogenetic relationship of Brachyuran (Crustacea: Decapoda) was reconstructed based on 13 PCGs for the first time, which involved the most brachyuran crabs so far. These results will help to better understand the features of the C. bilineata mitogenome and lay foundation for further evolutionary relationships within Brachyura. 2. Materials and methods 2.1. Specimen collection, DNA extraction and primer design An individual specimen of C. bilineata was collected from Danzhou, Hainan Province, China (19°43′3.90”N, 109°13′0.14″E). A portion of the epaxial musculature was excised and the samples were immediately preserved in 95% ethanol. Total genomic DNA was extracted using the SQ Tissue DNA Kit (OMEGA) following the manufacturer's protocol and stored at −20 °C until needed for PCR amplification. A set of universal primers for invertebrata mitochondrial genomes [18–20] and 12 specific primer pairs were used to amplify the complete mitogenome (Table S1). 2.2. PCR amplification and sequencing The polymerase chain reaction (PCR) was carried out in a 25 ul reaction volume containing 2.0 mM MgCl2, 0.4 mM of each dNTP, 0.5 uM of each primer, 1.0 U of Taq polymerase (Takara, China), 2.5 ul of 10 × Taq buffer, and approximately 50 ng of DNA template. PCR cycling conditions included an initial denaturation at 95 °C for 3 min, 35 cycles at 95 °C for 30 s, an annealing temperature at 48–52 °C for 50 s, elongation at 68 °C for 1–4 min, and a final extension at 68 °C for 10 min. Fragments generated from PCR amplification were sequenced by primer walking directly or if necessary, the purified PCR products were inserted into the pMD19-T vector (TaKaRa) then transformed in E. coli competent cells and sequenced. The sequences were determined using an ABI genetic analyzer (Applied Biosystems, China). 2.3. Sequence assembly, annotation and analysis Mitochondrial fragments were assembled to create the complete mitochondrial genomes using CodonCode Aligner 5.1.5 (CodonCode Corporation, Dedham, MA). The complete mitogenome was annotated using the software of Sequin (version 15.10, http://www.ncbi.nlm.nih. gov/Sequin). The boundaries of rRNA genes were performed using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov). Transfer RNA genes and their potential cloverleaf structures were identified using tRNAscan-SE 1.21 [21], with cut-off value set to 1 when necessary. The gene map of the C. bilineata mitogenome was generated using CGView [22].The base composition and the relative synonymous codon usage (RSCU) was obtained using MEGA 7.0 [23]. Strand asymmetry was calculated using the formulae: AT-skew = (A − T) / (A + T); GC-skew = (G − C) / (G + C) [24]. 2
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Fig. 1. Gene map of the C. bilineata mitogenome.
using Modeltest 3.7 [30] from 56 models for ML analyses and MrModeltest 2.2 [31] from 24 models for BI analyses both based on the Akaike information criterion (AIC). Bootstrap analyses (1000 replicates) were performed to evaluate relative levels of support for the ML analyses [32,33]. Bayesian phylogenetic analyses were performed using “Lset” and “Prset”, and the program was allowed to converge on the best estimates of the model parameters. Other parameter settings were as follows: Each Markov chain was initiated from a random tree and run for 2,000,000 generations, sampling trees every 100 generations (20,000 total trees sampled) to assure independence of the samples. Four chains, three heated (temperature = 0.5) and one cold, were simultaneously run using Metropolis-coupled Markov chain Monte
2.4. Phylogenetic analysis Eighty-three complete mitogenome sequences were downloaded from Genbank database (https://www.ncbi.nlm.nih.gov/genbank) to reconstruct the phylogenetic relationships among Brachyura, adding two Anomura species to serve as the outgroup (Table 1). The 13 PCGs were concatenated for phylogenetic analysis. Sequences were aligned using Clustal X 2.0 [25] with the default parameters and manually checked with BioEdit [26]. Ambiguous sequences were eliminated using Gblock [27]. The dataset was used for maximum likelihood (ML) analyses implemented in PhyML [28] and Bayesian inference (BI) in MrBayes 3.2.6 [29]. The best-fit evolutionary models were determined 3
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Table 2 Features of the mitochondrial genome of C. bilineata. Gene
COI Leu COII Lys Asp ATP8 ATP6 COIII Gly ND3 Ala Arg Asn Ser Glu His Phe ND5 ND4 ND4L Thr Pro ND6 Cyt b Ser ND1 Leu 16S Val 12S CR Ile Gln Met ND2 Trp Cys Tyr
Position
Length(bp)
From
To
1 1535 1618 2303 2373 2438 2593 3264 4059 4121 4479 4546 4607 4675 4741 4829 4894 4955 6709 8037 8350 8415 8484 8990 10,125 10,214 11,173 11,240 12,566 12,637 13,458 14,167 14,250 14,322 14,390 15,399 15,477 15,543
1539 1598 2302 2372 2437 2596 3264 4055 4120 4474 4542 4606 4674 4740 4806 4893 4957 6689 8043 8339 8414 8481 8990 10,126 10,191 11,149 11,239 12,551 12,636 13,457 14,166 14,234 14,320 14,389 15,400 15,466 15,541 15,606
Amino acid
1539 64 685 70 65 159 672 792 62 354 64 61 68 66 66 65 64 1735 1335 303 65 67 507 1137 67 936 67 1312 71 821 709 68 71 68 1011 68 65 64
Codon
512
Anticodon
Star
Stop
ATG
TAA TAA
228
ATG
T TTT GTC
52 223 263
ATG ATA ATG
TAG TAA TAG
117
ATT
TAA
TCC TGC TCG GTT TCT TTC GTG GAA 578 444 100
ATG ATG ATG
T TAG TAA TGT TGG
168 378
ATC ATG
TAA TAA
311
ATA
TAG
TGA TAG TAC
GAT TTG CAT 336
ATG
TAG GCA GTA
Intergenic region
Strand
−5 19 0 0 0 −4 −1 3 0 4 3 0 0 0 22 0 −3 19 −7 10 0 2 −1 −2 22 23 0 14 0 0 0 15 1 0 −2 10 1 −1
H H H H H H H H H H H H H H H L L L L L H L H H H L L L L L H H L H H H L L
Table 3 Composition and skewness of C. bilineata mitogenome.
Mitogenome PCGs COI COII ATP8 ATP6 COIII ND3 ND5 ND4 ND4L ND6 CYTB ND1 ND2 tRNAs rRNAs CR
A%
T%
G%
C%
A + T%
AT-skew
GC-skew
Length(bp)
34.0 27.8 28.0 29.9 33.3 29.0 27.4 24.3 29.2 27.5 26.1 25.4 28.1 25.3 27.1 36.3 37.0 41.7
34.7 39.2 35.3 35.0 40.9 38.8 35.0 42.7 40.2 40.4 43.2 44.0 38.1 44.6 39.9 36.5 35.0 34.4
11.5 16.2 16.4 13.6 7.5 11.5 16.0 12.4 20.7 21.9 22.4 7.5 14.1 20.3 9.3 15.4 17.7 9.4
19.8 16.8 20.3 21.5 18.2 20.7 21.6 20.6 9.8 10.2 8.3 23.1 19.8 9.8 23.7 11.7 10.3 14.4
68.7 67.0 63.3 65.0 74.2 67.9 62.4 66.9 69.5 67.9 69.3 69.4 66.1 69.9 67.0 72.9 72.0 76.2
−0.010 −0.172 −0.115 −0.079 −0.102 −0.145 −0.121 −0.274 −0.159 −0.191 −0.248 −0.267 −0.152 −0.275 −0.191 −0.003 0.027 0.096
−0.267 −0.019 −0.108 −0.225 −0.415 −0.287 −0.148 −0.248 0.358 0.364 0.462 −0.510 −0.169 0.348 −0.437 0.134 0.263 −0.207
15,606 11,165 1539 685 159 672 792 354 1735 1335 303 507 1137 936 1011 1456 2133 709
3. Results and discussion
Carlo (MCMCMC) to enhance the mixing capabilities of the Markov chains. To guarantee the stationarity had been reached, the average standard deviation of split frequencies was set below 0.01.
3.1. Mitogenome organization The complete 15,606 bp closed-circular mitogenome of C. bilineata has been deposited in GenBank under accession number MN562587.
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Fig. 2. Amino acid composition (A) and Relative synonymous codon usage (B) in C. bilineata mitogenome.
in ND6 and Cyt b) (Table 2), which is commonly identified in other crabs [18,36,37].
The gene structure of C. bilineata is the same as most other published decapods, including 13 PCGs, two rRNAs, 22 tRNAs, as well as a putative CR (Fig. 1, Table 2) [34,35]. The majority of 37 genes are encoded by the heavy (H-) strand, except four PCGs (ND5, ND4, ND4L and ND1), eight tRNAs (tRNA-His, Phe, Pro, Leu, Val, Gln, Cys and Tyr) and two rRNAs which are encoded by the light (L-) strand (Table 2). The nucleotide composition of the complete mitogenome is as follows: 34.0% A, 34.7% T, 11.5% G, and 19.8% C, respectively, with a high AT bias (68.7%). Both AT-skew and GC-skew of the mitogenome are negative, −0.010 and–0.267, respectively (Table 3), indicating Ts and Cs are more abundant than As and Gs. Generally, the mitogenome is quite compact, whereas a total of 168 base pairs in 15 intergenic spacers are found in the C. bilineata mitogenome, ranging from 1 to 23 bp in length. Simultaneously, nine overlapping sites (totally 26 bp) are observed, including the four typical overlaps between protein-coding genes (4 bp in ATP8 and ATP6, 1 bp in ATP6 and COIII, 7 bp in ND4L and ND4, 1 bp
3.2. PCGs and codon usage The mitogenome of C. bilineata contains 13 PCGs in the typical order found in Brachyuran species. The total PCGs are 11,165 bp in size, consisting of seven NADH dehydrogenases (ND1-ND6 and ND4L), three cytochrome c oxidases (COI-COIII), two ATPases (ATP6 and ATP8) and one cytochrome b (Cyt b). As with other invertebrate mitogenomes [38–40], the mitogenome of C. bilineata starts with the standard ATN codons: most of them start with ATG (COI, COII, ATP8, COIII, ND5, ND4, ND4L, Cyt b, and ND2), ATP6 and ND1 with ATA, ND3 with ATT, ND6 with ATC. The majority of the PCGs terminate with TAA or TAG; however, two other PCGs (COII and ND5) stop with an incomplete stop codon T (Table 2). Such immature stop codon is commonly found in 5
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Fig. 3. Phylogenetic tree of Brachyuran species inferred from the 13 PCGs based on Bayesian inference (BI) and maximum likelihood (ML) analysis. Node marked with solid circle indicates 100% supporting value and 100 maximum likelihood bootstrap value. The number after the species name is the GenBank accession number. 6
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animal mitochondrial genes [41,42]. The most frequently used amino acids are Leu (15.18%), Ser (10.11%), Phe (8.95%) and Ile (8.17%). In comparison, the least common amino acids are Cys (0.97%), Arg (1.56%), Gln (1.83%) and Asp (1.97%) (Fig. 2A). The RSCU (relative synonymous codon usage) value for C. bilineata for the third positions was shown in Fig. 2B. The usage of both two- and four-fold degenerate codons is biased toward the use of codons abundant in A or T, which is consistent with other crabs [36,43,44]. The AT content of the 13 PCGs is 67.0%. The AT-skew and GC-skew are −0.172 and − 0.019, respectively (Table 3).
included only one representative, which might produce unstable phylogenetic relationships. For example, our phylogenetic relationship of most families were accordance with previous results except the interrelationships among Polybiidae, Geryonidae, Menippidae, Eriphiidae, Matutidae and Oziidae, which included only one representative [5,46,51]. Therefore, larger number of brachyuran species is required to be explored and more complete mitogenomes are needed to be sequenced to better resolve the phylogeny of Brachyuran.
3.3. Transfer RNAs, ribosomal RNAs, and CR
In this study, we determined and described the complete mitogenome of C. bilineata, which is the first mitogenome of the family Calappidae (Decapoda: Brachyuran: Calappoidea). The 15,606 bp mitogenome contains 37 genes and one AT-rich region, as is typical of metazoan mitogenome. The genome composition is highly A + T biased (68.7%), and exhibits a negative AT-skew (−0.010) and GCskew (−0.267). As with other invertebrate mitogenomes, the PCGs start with the standard ATN and stop with the standard TAN codons or incomplete T. Phylogenetic analysis showed that C. bilineata had the most closely relationship with Matuta planipes (Matutidae), supporting a Calappoidea group. The existence of the polyphyletic families raised doubts over the traditional classification system. More brachyuran mitogenomes are needed to be sequenced to better resolve the phylogeny of Brachyuran. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ygeno.2020.02.003.
4. Conclusion
A total of 22 tRNAs were identified in the mitogenome of C. bilineata. Fourteen of them are encoded by the H-strand and the remaining are encoded by the L-strand (Fig. 1, Table 2). All of them possess the typical cloverleaf structure except for tRNA-Ser (TCT), which lacks the entire dihydrouridine arm (Fig. S1). The abnormal structure of the Ser is commonly found in both vertebrate and invertebrate mitogenomes [36,41,45]. The tRNAs range from 61 to 71 bp and the total length is 1456 bp and an obvious AT bias (72.9%) is presented. The AT-skew and GC-skew are −0.003 and 0.134, respectively, showing a slight bias toward the use of Ts and an obvious bias toward Cs (Table 3). The 12S and 16S rRNA genes are 821 and 1312 bp, respectively, which are typically separated by tRNA-Val (Fig. 1, Table 2). The AT content of the rRNAs is 72.0%. Both the AT-skew (0.027) and GC-skew (0.263) are positive, suggesting clearly that more As and Gs than Ts and Cs (Table 3). As most typical mitogenomes of other crabs, CR is also located between 12S rRNA and IQM gene cluster (tRNA-Ile, Gln, Met) [18,19,38]. The 709 bp CR is obviously AT biased (76.2%). The ATskew and GC-skew are 0.096 and − 0.207, respectively (Table 3), indicating an obvious bias toward the use of As and Cs.
Acknowledgements This work was supported by the Basic Scientific Research Operating Expenses of Zhejiang Provincial Universities (2019 J00022) and Guangxi innovation-driven development special fund project (AA17204081-4).
3.4. Phylogenetic analysis
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To further investigate the phylogenetic position of C. bilineata within Brachyuran, two phylogenetic trees (ML tree and BI tree) were constructed including 84 species of Brachyuran belonging to 26 families and two Anomura species (Pagurus japonicas and P. nigrofascia) as outgroup. The results showed that both trees were largely congruent in the topological structure; therefore, only one topology (BI) with both support values was displayed, including the bootstrap values for the ML tree and the posterior probability for Bayesian analysis (Fig. 3). It is obvious that C. bilineata was most closely related to Matuta planipes (Matutidae), and these two species formed a sister clade with high support value (Fig. 3), constituting a Calappoidea group and forming a sister clade with part of Eriphioidea. Of the 26 families included in this phylogeny, each family in the tree formed a monophyletic clade. However, in the super-family level, the current phylogenetic analysis of Brachyuran recovered three polyphyletic clades: Eriphioidea, Ocypodoidea and Grapsoidea, which was in concordance with previous studies [5,46,47]. It showed that Eriphioidea was divided into two clades ((Menippidae + Eriphiidae) + Oziidae); Ocypodoidea was divided into three clades (Ocypodidae + ((Macrophthalmidae + Mictyridae) + Dotillidae)); Grapsoidea was divided into three clades (Varunidae + ((Sesarmidae + Gecarcinidae) + Grapsidae)). Early Brachyuran taxonomists placed numerous divergent genera together based on the complicated morphological characteristics, either as the same family or super-family, implying close relationships. Recent studies, however, have presented inconsistent viewpoints on the traditional classification system [48–50]. For example, some sesarmid crabs are originally considered as members of Sesarminae, Grapsidae, while recent researches have classified them into Sesarmidae, Grapsoidea [48,50]. Our phylogenetic trees showed the phylogeny of most brachyuran crabs published so far. Nevertheless, almost half of the families (12/26)
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8
Contents lists available at ScienceDirect
Genomics journal homepage: www.elsevier.com/locate/ygeno
Original Article
The complete mitochondrial genome of Calappa bilineata: The first representative from the family Calappidae and its phylogenetic position within Brachyura ⁎
Xinting Lua,b, Li Gonga,b,c, ,1, Ying Zhanga,b, Jian Chenb, Liqin Liua,b, Lihua Jianga,b, ⁎ Zhenming Lüa,b, Bingjian Liua,b, Guixiang Tongd, Xinxian Weid, ,1 a
National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, 316022 Zhoushan, China Marine Science and Technology College, Zhejiang Ocean University, 316022 Zhoushan, China Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, China d Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Academy of Fishery Sciences, Nanning, Guangxi 530021, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Box crab Mitogenome Calappoidea Phylogenetic analysis
In this study, we determined the complete mitogenome sequence of Calappa bilineata, which is the first mitogenome of Calappidae up to now. The total length is 15,606 bp and includes 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and one control region. The genome composition is highly A + T biased (68.7%), and exhibits a negative AT-skew (−0.010) and GC-skew (−0.267). As with other invertebrate mitogenomes, the PCGs start with the standard ATN and stop with the standard TAN codons or incomplete T. Phylogenetic analysis showed that C. bilineata was most closely related to Matuta planipes (Matutidae), and these two species formed a sister clade, constituting a Calappoidea group and forming a sister clade with part of Eriphioidea. The existence of the polyphyletic families raised doubts over the traditional classification system. These results will help to better understand the features of the C. bilineata mitogenome and lay foundation for further evolutionary relationships within Brachyura.
1. Introduction
suggesting close relationships. Recent studies, however, have found a number of inconsistent features within the family, indicating controversial phylogenetic relationships [10–13]. Calappidae, the box crabs, appeared relatively early in the evolution of crustaceans and played an important ecological role in marine ecosystem. Due to the high morphological similarity and extreme ecological diversity, classification and phylogenetic relationships within this family are complicated and controversial [10,14,15]. To date, more than 90 Brachyura mitogenomes have been sequenced (https://www.ncbi.nlm.nih.gov). However, no complete mitogenome from Calappidae has ever been reported. Most studies of this family focused on the classification based on morphological features [14,16]. Although there are few researches on molecular level, most of them were based on partial mitochondrial and nuclear ribosomal RNA gene sequences (e.g. 28S rDNA) [15,17]. Therefore, in the present study, we determined the first complete mitogenome sequence of Calappa bilineata, representative from the family Calappidae. Additionally,
Metazoan mitochondrial genome (mitogenome) is usually 14–20 kb in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (12S and 16S) and an AT-rich region (also called control region, CR) [1]. Mitochondrial DNA is featured by maternal inheritance, simple structure, conserved organization, small genome size, and high mutation rate [2,3]. With the rapid development of sequencing technologies, complete mitogenome sequence is becoming increasingly common used in population genetics, comparative and evolutionary genomics, and reconstruction of phylogenetic relationships [4–6]. Brachyura, the true crab, is the largest clade Decapod Crustacea, with over 7250 described species in 98 families inhabiting marine, freshwater, and terrestrial habitats, most of which are economically important [7–9]. Brachyuran taxonomists have traditionally placed the various genera together, either as a family or as a super-family,
⁎
Corresponding authors. E-mail addresses: [email protected], [email protected] (L. Gong), [email protected] (X. Wei). 1 These authors contributed equally to this paper. https://doi.org/10.1016/j.ygeno.2020.02.003 Received 3 December 2019; Received in revised form 8 January 2020; Accepted 7 February 2020 0888-7543/ © 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Xinting Lu, et al., Genomics, https://doi.org/10.1016/j.ygeno.2020.02.003
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Table 1 (continued)
Table 1 List of 84 Brachyura and two outgroup used in this paper. Species
Family
Length (bp)
Accession no.
Scylla olivacea Scylla serrata Scylla tranquebarica Charybdis feriata Charybdis natator Charybdis bimaculata Thalamita sima Thalamita crenata Portunus sanguinolentus Portunus pelagicus Callinectes sapidus Monomia gladiator Ovalipes punctatus Chaceon sp. Myomenippe fornasinii Pseudocarcinus gigas Calappa bilineata Matuta planipes Atergatis integerrimus Atergatis floridus Etisus anaglyptus Leptodius sanguineus Epixanthus frontalis Pilumnus vespertilio Echinoecus nipponicus Gandalfus puia Gandalfus yunohana Austinograea rodriguezensis Austinograea alayseae Segonzacia mesatlantica Majo squinado Maja crispata Damithrax spinosissimus Sinopotamon xiushuiense Longpotamon yangtsekiense Longpotamon kenliense Sinopotamon yaanense Geothelphusa dehaani Huananpotamon lichuanense Somanniathelphusa boyangensis Uca arcuata Uca capricornis Uca polita Uca borealis Uca inversa Uca lactea Ocypode cordimanus Hemigrapsus sanguineus Hemigrapsus penicillatus Eriocheir sinensis Eriocheir hepuensis Eriocheir japonica Neoeriocheir leptognathus Varuna yui Helice latimera Helice tientsinensis Helicana wuana Pseudohelice subquadrata Metaplax longipes Macrophtalmus japonicus Mictyris longicarpus Parasesarma tripectinis Parasesarma affine Nanosesarma minutum Clistocoeloma sinense Metopaulias depressus Sesarma neglectum Chiromantes dehaani Sesarmops sinensis Chiromantes haematocheir Gecarcoidea natalis Cardisoma carnifex Iloplax deschampsi
Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Portunidae Polybiidae Geryonidae Menippidae Eriphiidae Calappidae Matutidae Xanthidae Xanthidae Xanthidae Xanthidae Oziidae Pilumnidae Pilumnidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Bythograeidae Majidae Majidae Mithracidae Potamidae Potamidae Potamidae Potamidae Potamidae Potamidae Parathelphusidae
15,723 15,775 15,833 15,660 15,664 15,714 15,831 15,787 16,024 16,157 16,263 15,878 16,084 16,126 15,658 15,515 15,606 15,760 15,924 16,180 16,435 15,480 15,993 16,222 16,173 15,548 15,567 15,611 15,620 15,521 16,598 16,592 15,817 18,480 17,885 18,499 17,126 18,197 15,380 17,032
NC_012569 NC_012565 NC_012567 NC_024632 MF285241 MG489891 MG840650 MH425338 NC_028225 KR153996 AY363392 MG770549 MH802052 KU507298 LK391943 NC_006891 MN562587 MG756601 NC_037172 NC_037201 MG751773 NC_029726 MF457404 MF457402 MG574831 NC_027414 NC_013713 JQ035658 JQ035660 NC_035300 NC_035425 NC_035424 NC_025518 NC_029226 NC_036946 MK584299 NC_036947 NC_007379 NC_031406 NC_032044
Ocypodidae Ocypodidae Ocypodidae Ocypodidae Ocypodidae Ocypodidae Ocypodidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Varunidae Macrophthalmidae Mictyridae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Sesarmidae Gecarcinidae Gecarcinidae Dotillidae
15,955 15,629 15,672 15,662 15,677 15,659 15,604 16,275 16,486 16,354 16,335 16,352 16,143 15,915 16,246 16,212 16,359 16,898 16,424 16,170 15,548 15,612 15,638 15,637 15,706 15,765 15,920 15,917 15,905 15,899 15,545 15,597 15,460
KX911977 NC_039107 NC_039106 MH796170 NC_039111 KY865330 NC_029725 NC_035307 MG751772 NC_006992 FJ455506 NC_011597 MH593561 NC_037155 NC_033865 NC_030197 NC_034995 MH718959 MF198248 NC_030048 NC_025325 NC_030046 MH310444 NC_040977 NC_033866 NC_030535 NC_031851 MH593563 NC_030196 NC_042142 NC_039811 NC_039105 NC_020040
Species
Family
Length (bp)
Accession no.
Dotilla wichmanni Pachygrapsus crassipes Pachygrapsus marmoratus Grapsus tenuicrustatus Metopograpsus quadridentatus Metopograpsus frontalis Homologenus malayensis Moloha majora Dynomene pilumnoides Umalia orientalis Lyreidus brevifrons Pagurus japonicus Pagurus nigrofascia
Dotillidae Grapsidae Grapsidae Grapsidae Grapsidae Grapsidae Homolidae Homolidae Dynomenidae Raninidae Raninidae Paguridae Paguridae
15,600 15,662 15,406 15,858 15,520 15,587 15,793 15,903 16,475 15,446 16,112 16,401 15,392
NC_038180 NC_021754 MF457403 NC_029724 MH310445 MH028874 NC_026080 NC_029361 KT182070 NC_026688 NC_026721 LC222532 LC222531
the phylogenetic relationship of Brachyuran (Crustacea: Decapoda) was reconstructed based on 13 PCGs for the first time, which involved the most brachyuran crabs so far. These results will help to better understand the features of the C. bilineata mitogenome and lay foundation for further evolutionary relationships within Brachyura. 2. Materials and methods 2.1. Specimen collection, DNA extraction and primer design An individual specimen of C. bilineata was collected from Danzhou, Hainan Province, China (19°43′3.90”N, 109°13′0.14″E). A portion of the epaxial musculature was excised and the samples were immediately preserved in 95% ethanol. Total genomic DNA was extracted using the SQ Tissue DNA Kit (OMEGA) following the manufacturer's protocol and stored at −20 °C until needed for PCR amplification. A set of universal primers for invertebrata mitochondrial genomes [18–20] and 12 specific primer pairs were used to amplify the complete mitogenome (Table S1). 2.2. PCR amplification and sequencing The polymerase chain reaction (PCR) was carried out in a 25 ul reaction volume containing 2.0 mM MgCl2, 0.4 mM of each dNTP, 0.5 uM of each primer, 1.0 U of Taq polymerase (Takara, China), 2.5 ul of 10 × Taq buffer, and approximately 50 ng of DNA template. PCR cycling conditions included an initial denaturation at 95 °C for 3 min, 35 cycles at 95 °C for 30 s, an annealing temperature at 48–52 °C for 50 s, elongation at 68 °C for 1–4 min, and a final extension at 68 °C for 10 min. Fragments generated from PCR amplification were sequenced by primer walking directly or if necessary, the purified PCR products were inserted into the pMD19-T vector (TaKaRa) then transformed in E. coli competent cells and sequenced. The sequences were determined using an ABI genetic analyzer (Applied Biosystems, China). 2.3. Sequence assembly, annotation and analysis Mitochondrial fragments were assembled to create the complete mitochondrial genomes using CodonCode Aligner 5.1.5 (CodonCode Corporation, Dedham, MA). The complete mitogenome was annotated using the software of Sequin (version 15.10, http://www.ncbi.nlm.nih. gov/Sequin). The boundaries of rRNA genes were performed using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov). Transfer RNA genes and their potential cloverleaf structures were identified using tRNAscan-SE 1.21 [21], with cut-off value set to 1 when necessary. The gene map of the C. bilineata mitogenome was generated using CGView [22].The base composition and the relative synonymous codon usage (RSCU) was obtained using MEGA 7.0 [23]. Strand asymmetry was calculated using the formulae: AT-skew = (A − T) / (A + T); GC-skew = (G − C) / (G + C) [24]. 2
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Fig. 1. Gene map of the C. bilineata mitogenome.
using Modeltest 3.7 [30] from 56 models for ML analyses and MrModeltest 2.2 [31] from 24 models for BI analyses both based on the Akaike information criterion (AIC). Bootstrap analyses (1000 replicates) were performed to evaluate relative levels of support for the ML analyses [32,33]. Bayesian phylogenetic analyses were performed using “Lset” and “Prset”, and the program was allowed to converge on the best estimates of the model parameters. Other parameter settings were as follows: Each Markov chain was initiated from a random tree and run for 2,000,000 generations, sampling trees every 100 generations (20,000 total trees sampled) to assure independence of the samples. Four chains, three heated (temperature = 0.5) and one cold, were simultaneously run using Metropolis-coupled Markov chain Monte
2.4. Phylogenetic analysis Eighty-three complete mitogenome sequences were downloaded from Genbank database (https://www.ncbi.nlm.nih.gov/genbank) to reconstruct the phylogenetic relationships among Brachyura, adding two Anomura species to serve as the outgroup (Table 1). The 13 PCGs were concatenated for phylogenetic analysis. Sequences were aligned using Clustal X 2.0 [25] with the default parameters and manually checked with BioEdit [26]. Ambiguous sequences were eliminated using Gblock [27]. The dataset was used for maximum likelihood (ML) analyses implemented in PhyML [28] and Bayesian inference (BI) in MrBayes 3.2.6 [29]. The best-fit evolutionary models were determined 3
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Table 2 Features of the mitochondrial genome of C. bilineata. Gene
COI Leu COII Lys Asp ATP8 ATP6 COIII Gly ND3 Ala Arg Asn Ser Glu His Phe ND5 ND4 ND4L Thr Pro ND6 Cyt b Ser ND1 Leu 16S Val 12S CR Ile Gln Met ND2 Trp Cys Tyr
Position
Length(bp)
From
To
1 1535 1618 2303 2373 2438 2593 3264 4059 4121 4479 4546 4607 4675 4741 4829 4894 4955 6709 8037 8350 8415 8484 8990 10,125 10,214 11,173 11,240 12,566 12,637 13,458 14,167 14,250 14,322 14,390 15,399 15,477 15,543
1539 1598 2302 2372 2437 2596 3264 4055 4120 4474 4542 4606 4674 4740 4806 4893 4957 6689 8043 8339 8414 8481 8990 10,126 10,191 11,149 11,239 12,551 12,636 13,457 14,166 14,234 14,320 14,389 15,400 15,466 15,541 15,606
Amino acid
1539 64 685 70 65 159 672 792 62 354 64 61 68 66 66 65 64 1735 1335 303 65 67 507 1137 67 936 67 1312 71 821 709 68 71 68 1011 68 65 64
Codon
512
Anticodon
Star
Stop
ATG
TAA TAA
228
ATG
T TTT GTC
52 223 263
ATG ATA ATG
TAG TAA TAG
117
ATT
TAA
TCC TGC TCG GTT TCT TTC GTG GAA 578 444 100
ATG ATG ATG
T TAG TAA TGT TGG
168 378
ATC ATG
TAA TAA
311
ATA
TAG
TGA TAG TAC
GAT TTG CAT 336
ATG
TAG GCA GTA
Intergenic region
Strand
−5 19 0 0 0 −4 −1 3 0 4 3 0 0 0 22 0 −3 19 −7 10 0 2 −1 −2 22 23 0 14 0 0 0 15 1 0 −2 10 1 −1
H H H H H H H H H H H H H H H L L L L L H L H H H L L L L L H H L H H H L L
Table 3 Composition and skewness of C. bilineata mitogenome.
Mitogenome PCGs COI COII ATP8 ATP6 COIII ND3 ND5 ND4 ND4L ND6 CYTB ND1 ND2 tRNAs rRNAs CR
A%
T%
G%
C%
A + T%
AT-skew
GC-skew
Length(bp)
34.0 27.8 28.0 29.9 33.3 29.0 27.4 24.3 29.2 27.5 26.1 25.4 28.1 25.3 27.1 36.3 37.0 41.7
34.7 39.2 35.3 35.0 40.9 38.8 35.0 42.7 40.2 40.4 43.2 44.0 38.1 44.6 39.9 36.5 35.0 34.4
11.5 16.2 16.4 13.6 7.5 11.5 16.0 12.4 20.7 21.9 22.4 7.5 14.1 20.3 9.3 15.4 17.7 9.4
19.8 16.8 20.3 21.5 18.2 20.7 21.6 20.6 9.8 10.2 8.3 23.1 19.8 9.8 23.7 11.7 10.3 14.4
68.7 67.0 63.3 65.0 74.2 67.9 62.4 66.9 69.5 67.9 69.3 69.4 66.1 69.9 67.0 72.9 72.0 76.2
−0.010 −0.172 −0.115 −0.079 −0.102 −0.145 −0.121 −0.274 −0.159 −0.191 −0.248 −0.267 −0.152 −0.275 −0.191 −0.003 0.027 0.096
−0.267 −0.019 −0.108 −0.225 −0.415 −0.287 −0.148 −0.248 0.358 0.364 0.462 −0.510 −0.169 0.348 −0.437 0.134 0.263 −0.207
15,606 11,165 1539 685 159 672 792 354 1735 1335 303 507 1137 936 1011 1456 2133 709
3. Results and discussion
Carlo (MCMCMC) to enhance the mixing capabilities of the Markov chains. To guarantee the stationarity had been reached, the average standard deviation of split frequencies was set below 0.01.
3.1. Mitogenome organization The complete 15,606 bp closed-circular mitogenome of C. bilineata has been deposited in GenBank under accession number MN562587.
4
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Fig. 2. Amino acid composition (A) and Relative synonymous codon usage (B) in C. bilineata mitogenome.
in ND6 and Cyt b) (Table 2), which is commonly identified in other crabs [18,36,37].
The gene structure of C. bilineata is the same as most other published decapods, including 13 PCGs, two rRNAs, 22 tRNAs, as well as a putative CR (Fig. 1, Table 2) [34,35]. The majority of 37 genes are encoded by the heavy (H-) strand, except four PCGs (ND5, ND4, ND4L and ND1), eight tRNAs (tRNA-His, Phe, Pro, Leu, Val, Gln, Cys and Tyr) and two rRNAs which are encoded by the light (L-) strand (Table 2). The nucleotide composition of the complete mitogenome is as follows: 34.0% A, 34.7% T, 11.5% G, and 19.8% C, respectively, with a high AT bias (68.7%). Both AT-skew and GC-skew of the mitogenome are negative, −0.010 and–0.267, respectively (Table 3), indicating Ts and Cs are more abundant than As and Gs. Generally, the mitogenome is quite compact, whereas a total of 168 base pairs in 15 intergenic spacers are found in the C. bilineata mitogenome, ranging from 1 to 23 bp in length. Simultaneously, nine overlapping sites (totally 26 bp) are observed, including the four typical overlaps between protein-coding genes (4 bp in ATP8 and ATP6, 1 bp in ATP6 and COIII, 7 bp in ND4L and ND4, 1 bp
3.2. PCGs and codon usage The mitogenome of C. bilineata contains 13 PCGs in the typical order found in Brachyuran species. The total PCGs are 11,165 bp in size, consisting of seven NADH dehydrogenases (ND1-ND6 and ND4L), three cytochrome c oxidases (COI-COIII), two ATPases (ATP6 and ATP8) and one cytochrome b (Cyt b). As with other invertebrate mitogenomes [38–40], the mitogenome of C. bilineata starts with the standard ATN codons: most of them start with ATG (COI, COII, ATP8, COIII, ND5, ND4, ND4L, Cyt b, and ND2), ATP6 and ND1 with ATA, ND3 with ATT, ND6 with ATC. The majority of the PCGs terminate with TAA or TAG; however, two other PCGs (COII and ND5) stop with an incomplete stop codon T (Table 2). Such immature stop codon is commonly found in 5
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Fig. 3. Phylogenetic tree of Brachyuran species inferred from the 13 PCGs based on Bayesian inference (BI) and maximum likelihood (ML) analysis. Node marked with solid circle indicates 100% supporting value and 100 maximum likelihood bootstrap value. The number after the species name is the GenBank accession number. 6
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animal mitochondrial genes [41,42]. The most frequently used amino acids are Leu (15.18%), Ser (10.11%), Phe (8.95%) and Ile (8.17%). In comparison, the least common amino acids are Cys (0.97%), Arg (1.56%), Gln (1.83%) and Asp (1.97%) (Fig. 2A). The RSCU (relative synonymous codon usage) value for C. bilineata for the third positions was shown in Fig. 2B. The usage of both two- and four-fold degenerate codons is biased toward the use of codons abundant in A or T, which is consistent with other crabs [36,43,44]. The AT content of the 13 PCGs is 67.0%. The AT-skew and GC-skew are −0.172 and − 0.019, respectively (Table 3).
included only one representative, which might produce unstable phylogenetic relationships. For example, our phylogenetic relationship of most families were accordance with previous results except the interrelationships among Polybiidae, Geryonidae, Menippidae, Eriphiidae, Matutidae and Oziidae, which included only one representative [5,46,51]. Therefore, larger number of brachyuran species is required to be explored and more complete mitogenomes are needed to be sequenced to better resolve the phylogeny of Brachyuran.
3.3. Transfer RNAs, ribosomal RNAs, and CR
In this study, we determined and described the complete mitogenome of C. bilineata, which is the first mitogenome of the family Calappidae (Decapoda: Brachyuran: Calappoidea). The 15,606 bp mitogenome contains 37 genes and one AT-rich region, as is typical of metazoan mitogenome. The genome composition is highly A + T biased (68.7%), and exhibits a negative AT-skew (−0.010) and GCskew (−0.267). As with other invertebrate mitogenomes, the PCGs start with the standard ATN and stop with the standard TAN codons or incomplete T. Phylogenetic analysis showed that C. bilineata had the most closely relationship with Matuta planipes (Matutidae), supporting a Calappoidea group. The existence of the polyphyletic families raised doubts over the traditional classification system. More brachyuran mitogenomes are needed to be sequenced to better resolve the phylogeny of Brachyuran. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ygeno.2020.02.003.
4. Conclusion
A total of 22 tRNAs were identified in the mitogenome of C. bilineata. Fourteen of them are encoded by the H-strand and the remaining are encoded by the L-strand (Fig. 1, Table 2). All of them possess the typical cloverleaf structure except for tRNA-Ser (TCT), which lacks the entire dihydrouridine arm (Fig. S1). The abnormal structure of the Ser is commonly found in both vertebrate and invertebrate mitogenomes [36,41,45]. The tRNAs range from 61 to 71 bp and the total length is 1456 bp and an obvious AT bias (72.9%) is presented. The AT-skew and GC-skew are −0.003 and 0.134, respectively, showing a slight bias toward the use of Ts and an obvious bias toward Cs (Table 3). The 12S and 16S rRNA genes are 821 and 1312 bp, respectively, which are typically separated by tRNA-Val (Fig. 1, Table 2). The AT content of the rRNAs is 72.0%. Both the AT-skew (0.027) and GC-skew (0.263) are positive, suggesting clearly that more As and Gs than Ts and Cs (Table 3). As most typical mitogenomes of other crabs, CR is also located between 12S rRNA and IQM gene cluster (tRNA-Ile, Gln, Met) [18,19,38]. The 709 bp CR is obviously AT biased (76.2%). The ATskew and GC-skew are 0.096 and − 0.207, respectively (Table 3), indicating an obvious bias toward the use of As and Cs.
Acknowledgements This work was supported by the Basic Scientific Research Operating Expenses of Zhejiang Provincial Universities (2019 J00022) and Guangxi innovation-driven development special fund project (AA17204081-4).
3.4. Phylogenetic analysis
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To further investigate the phylogenetic position of C. bilineata within Brachyuran, two phylogenetic trees (ML tree and BI tree) were constructed including 84 species of Brachyuran belonging to 26 families and two Anomura species (Pagurus japonicas and P. nigrofascia) as outgroup. The results showed that both trees were largely congruent in the topological structure; therefore, only one topology (BI) with both support values was displayed, including the bootstrap values for the ML tree and the posterior probability for Bayesian analysis (Fig. 3). It is obvious that C. bilineata was most closely related to Matuta planipes (Matutidae), and these two species formed a sister clade with high support value (Fig. 3), constituting a Calappoidea group and forming a sister clade with part of Eriphioidea. Of the 26 families included in this phylogeny, each family in the tree formed a monophyletic clade. However, in the super-family level, the current phylogenetic analysis of Brachyuran recovered three polyphyletic clades: Eriphioidea, Ocypodoidea and Grapsoidea, which was in concordance with previous studies [5,46,47]. It showed that Eriphioidea was divided into two clades ((Menippidae + Eriphiidae) + Oziidae); Ocypodoidea was divided into three clades (Ocypodidae + ((Macrophthalmidae + Mictyridae) + Dotillidae)); Grapsoidea was divided into three clades (Varunidae + ((Sesarmidae + Gecarcinidae) + Grapsidae)). Early Brachyuran taxonomists placed numerous divergent genera together based on the complicated morphological characteristics, either as the same family or super-family, implying close relationships. Recent studies, however, have presented inconsistent viewpoints on the traditional classification system [48–50]. For example, some sesarmid crabs are originally considered as members of Sesarminae, Grapsidae, while recent researches have classified them into Sesarmidae, Grapsoidea [48,50]. Our phylogenetic trees showed the phylogeny of most brachyuran crabs published so far. Nevertheless, almost half of the families (12/26)
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