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. 2017 Nov 10;7:15299. doi: 10.1038/s41598-017-15702-0

Sequencing and characterization of the complete mitochondrial genome of Japanese Swellshark (Cephalloscyllium umbratile)

Ke-Cheng Zhu 1,2,3, Yin-Yin Liang 1, Na Wu 1, Hua-Yang Guo 1,2, Nan Zhang 1,2, Shi-Gui Jiang 1,2,4, Dian-Chang Zhang 1,2,3,✉
PMCID: PMC5681689  PMID: 29127415

Abstract

To further comprehend the genome features of Cephalloscyllium umbratile (Carcharhiniformes), an endangered species, the complete mitochondrial DNA (mtDNA) was firstly sequenced and annotated. The full-length mtDNA of C. umbratile was 16,697 bp and contained ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs), 23 transfer RNA (tRNA) genes, and a major non-coding control region. Each PCG was initiated by an authoritative ATN codon, except for COX1 initiated by a GTG codon. Seven of 13 PCGs had a typical TAA termination codon, while others terminated with a single T or TA. Moreover, the relative synonymous codon usage of the 13 PCGs was consistent with that of other published Carcharhiniformes. All tRNA genes had typical clover-leaf secondary structures, except for tRNA-Ser (GCT), which lacked the dihydrouridine ‘DHU’ arm. Furthermore, the analysis of the average Ka/Ks in the 13 PCGs of three Carcharhiniformes species indicated a strong purifying selection within this group. In addition, phylogenetic analysis revealed that C. umbratile was closely related to Glyphis glyphis and Glyphis garricki. Our data supply a useful resource for further studies on genetic diversity and population structure of C. umbratile.

Introduction

Cephalloscyllium umbratile (Cephaloscyllium, Scyliorhinidae, Chondrichthyes), belonging the Carcharhiniformes order, is one of the most important aquarium and reef fish, and mainly distribute in the coastwise of China, Vietnam and Japan. Due to small amount, it is regarded as endangered species, and absorbed in red list of International Union for Conservation of Nature (IUCN)1. Since the information about C. umbratile has been generally scarce, with the development of offshore fishery, increasing research interest has been developed in conservation as well as in scientific and economic topics regarding reef fish2,3.

In Chondrichthyes, the typical complete mitochondrial DNA (mtDNA) was circular and approximately 17 kb in length with correspondingly conserved gene content which encoded 37 genes, including 22 transfer RNA (tRNA), 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA), a major non-coding control region (D-loop region), and an A + T-rich region4,5. Furthermore, genomic information is considered to be reliable for the efficient implementation strategies to study evolutionary relationships, phylogeography and phylogeny6,7. Due to its conserved gene content, maternal inheritance, a small genome size, relatively fast evolutionary rate, high copy number and lack of intermolecular genetic recombination8–10, mtDNA has been broadly adopted in species identification11,12, genome evolution13–16 and nonsynonymous (Ka) and synonymous (Ks) substitutions of many species17–23.

Moreover, Carcharhiniformes include about 49 genera and over 200 species, and many of them are important economic categories. Nevertheless, several evidences gathered with genome synteny analysis have revealed a number of shared unique mitochondrial gene features in Chondrichthyes, towards a better understanding of the functions and evolution of Chondrichthyes24–27. So far, there was still a notably lack of mtDNA information in Carcharhiniformes. In order to provide a theoretical foundation for the conservation strategy of C. umbratile within Scyliorhinidae and new sight for further studies of phylogenetically-informative sequence data, in the current study the complete mtDNA of C. umbratile was sequenced, assembled and annotated, and compared with other members of Carcharhiniformes.

Results and Discussion

Genome size and organization

About 1.5 G raw data is generated with reads length 125 bp. Sequencing coverage and depth (X) of mtDNA data is 100% and approximately 394.23, respectively. Reads number is 52,660 and total bases (bp) is 6,582,500. The mtDNA of C. umbratile was a closed-circular DNA molecule of 16,697 bp in length (GenBank: KX354996; Fig. 1, Table 1), which was comparable to other Carcharhiniformes mtDNA ranging from 16,697 bp in Scyliorhinus canicula 25 to 16,719 bp in Carcharhinus acronotus 28. Nucleotide BLAST (blastn) of the whole C. umbratile mtDNA against other Carcharhiniformes revealed sequence identities with closely related species of 88% (S. canicula), 84% (Proscyllium habereri), and 84% (Pseudotriakis microdon) and with distantly related species of 82% (Scoliodon laticaudus), 82% (Hemigaleus microstoma), 82% (Hemipristis elongata) (Supplementary Table 1). The mtDNA of C. umbratile contained 2 rRNA genes, 13 PCGs, 22 tRNA genes and D-loop region. The arrangement of the genes was identical to that of other Scyliorhinidae mtDNA (Table 1)29,30. Among these genes, 29 genes (12 PCGs, 2 rRNA genes and 15 tRNA genes) are located on the heavy strand (H-strand) and the others (1 PCGs and 8 tRNA genes) are located on the light strand (L-strand) (Table 1). These obvious features have also been reported in other Carcharhiniformes species31,32 and could be regarded as effective markers for authentication at genus and species level.

Figure 1.

Figure 1

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Map of the Cephalloscyllium umbratile mitochondrial genome. The genes outside the circle are transcribed clockwise, while the genes inside are transcribed counterclockwise. Gene blocks are filled with different colors as the cutline shows. The inner ring shadow indicates the GC content of the genome.

Table 1.

Sequence characteristics of Cephalloscyllium umbratile mitochondrial genome.

Locus name One Letter code From to Size Strand Nr.of Aminao Acids Anti-Coden Inferred Initiation Coden Inferred Termination Coden GC_Percent Intergenic nucleotides*
tRNA-Phe F 1 69 69 H GAA 37.68% 0
12S-rRNA 70 1023 954 H 42.98% 0
tRNA-Val V 1024 1095 72 H TAC 40.28% 0
16S-rRNA 1096 2764 1669 H 36.01% 0
tRNA-Leu L 2765 2839 75 H TAA 44.00% 0
ND1 2840 3814 975 H 324 ATG TAA 38.97% 3
tRNA-Ile I 3818 3886 69 H GAT 39.13% 1
tRNA-Gln Q 3888 3959 72 L TTG 29.17% 0
tRNA-Met M 3960 4029 70 H CAT 40.00% 0
ND2 4030 5075 1046 H 348 ATG TA 37.86% 0
tRNA-Trp W 5076 5144 69 H TCA 33.33% 1
tRNA-Ala A 5146 5214 69 L TGC 31.88% 0
tRNA-Asn N 5215 5287 73 L GTT 34.25% 36
tRNA-Cys C 5324 5389 66 L GCA 51.52% 1
tRNA-Tyr Y 5391 5460 70 L GTA 47.14% 1
COXI 5462 7015 1554 H 517 GTG TAA 38.61% 0
tRNA-Ser S 7016 7086 71 L TGA 45.07% 3
tRNA-Asp D 7090 7159 70 H GTC 32.86% 7
COXII 7167 7857 691 H 230 ATG T 38.35% 0
tRNA-Lys K 7858 7932 75 H TTT 44.00% 1
ATP8 7934 8101 168 H 55 ATG TAA 30.95% −22
ATP6 8080 8774 695 H 231 ATG TA 37.55% 0
COXIII 8775 9560 786 H 261 ATG TAA 42.88% 2
tRNA-Gly G 9563 9632 70 H TCC 27.14% 0
ND3 9633 9981 349 H 116 ATG T 40.97% 0
tRNA-Arg R 9982 10051 70 H TCG 32.86% 0
ND4L 10052 10348 297 H 98 ATG TAA 38.72% −7
ND4 10342 11722 1381 H 460 ATG T 37.73% 0
tRNA-His H 11723 11791 69 H GTG 18.84% 0
tRNA-Ser S 11792 11858 67 H GCT 37.31% 0
tRNA-Leu L 11859 11930 72 H TAG 48.61% 0
ND5 11931 13760 1830 H 609 ATG TAA 35.85% −4
ND6 13757 14278 522 L 173 ATG TAA 36.97% 0
tRNA-Glu E 14279 14348 70 L TTC 32.86% 2
Cytb 14351 15495 1145 H 381 ATG TA 39.91% 0
tRNA-Thr T 15496 15567 72 H TGT 51.39% 2
tRNA-Pro P 15570 15638 69 L TGG 49.28% 0
D-loop 15639 16697 1059 H 31.35% 0
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+ and − correspond to the H and L strands, respectively.

The nucleotide composition of the mtDNA is biased toward A + T nucleotides (52.9%), which made up of 61.8%, 61.4%, 61.5% and 68.7% in the PCGs, tRNA, rRNA and D-loop region, respectively (Table 2). However, the A + T nucleotide composition in C. umbratile was the lowest among Carcharhiniformes. The positive AT skew (0.025) observed here with the presence of more As than Ts, was similar to that only in Sphyrna tiburo (0.031), nevertheless, mtDNA in majority of Carcharhiniformes showed negative AT skew (Table 2). The GC skew ranged from −0.324 in S. tiburo to 0.040 in C. macloti (Table 2). The C. umbratile mtDNA was negative (−0.245), indicating the presence of more Cs than Gs.

Table 2.

Nucleotide composition of the mitochondrial genome in different Carcharhiniformes mtDNA.

Species Size (bp) A% T% G% C% A + T % AT skewness GC skewness
Whole mitogenome
C.umbratile 16896 27.08 25.78 17.81 29.34 52.86 0.025 −0.245
S. canicula 16697 30.80 31.20 14.12 23.87 62.00 −0.006 −0.257
S. tiburo 16723 31.26 29.38 13.24 25.94 60.64 0.031 −0.324
P. habereri 16708 30.88 31.19 14.18 23.75 62.07 −0.005 −0.252
C. acronotus 16719 31.48 30.22 13.18 25.20 61.65 −0.311 0.017
C.amblyrhynchoides 16705 31.40 30.34 13.15 25.03 61.79 −0.313 0.020
C. amboinensis 16704 31.57 30.42 13.06 24.95 62.00 −0.313 0.019
C. brevipinna 16706 31.35 30.13 13.24 25.28 61.47 −0.313 0.020
C. leucas 16704 31.47 31.10 13.11 24.32 62.57 −0.300 0.006
C.longimanus 16706 31.49 30.01 13.12 25.38 61.50 −0.318 0.024
C.macloti 16701 31.61 29.19 13.02 26.18 60.80 −0.336 0.040
C.melanopterus 16706 31.28 30.06 13.32 25.33 61.35 −0.311 0.020
C. plumbeus 16706 31.25 29.89 13.32 25.54 61.14 −0.314 0.022
C. sorrah 16707 31.45 29.60 13.17 25.77 61.05 −0.323 0.030
L.tephrodes 16705 31.43 29.77 13.02 25.70 61.25 −0.328 0.027
L.macrorhinus 16702 31.71 29.36 13.14 25.80 61.06 −0.325 0.039
P. microdon 16700 31.30 32.32 13.63 22.75 63.62 −0.251 −0.016
T. obesus 16700 31.38 29.65 13.19 25.78 61.03 −0.323 0.028
Protein-coding genes
C.umbratile 11440 28.73 33.02 13.74 24.51 61.75 −0.282 −0.069
S. canicula 11430 28.71 33.15 13.85 24.30 61.85 −0.274 −0.072
S. tiburo 11430 28.85 31.09 13.06 26.99 59.95 −0.348 −0.037
P. habereri 11430 28.83 33.25 13.74 24.18 62.08 −0.275 −0.071
C. acronotus 11429 29.44 31.95 12.58 26.02 61.40 −0.348 −0.041
C.amblyrhynchoides 11430 29.45 32.30 12.59 25.66 61.75 −0.342 −0.046
C. amboinensis 11430 29.58 32.32 12.49 25.61 61.90 −0.344 −0.044
C. brevipinna 11430 29.36 31.92 12.65 26.06 61.29 −0.346 −0.042
C. leucas 11430 29.43 33.08 12.55 24.94 62.51 −0.331 −0.058
C.longimanus 11430 29.55 31.85 12.53 26.07 61.40 −0.351 −0.038
C.macloti 11430 29.42 30.83 12.62 27.13 60.25 −0.365 −0.023
C.melanopterus 11430 29.32 31.96 12.77 25.93 61.29 −0.340 −0.043
C. plumbeus 11430 29.22 31.72 12.81 26.25 60.94 −0.344 −0.041
C. sorrah 11430 29.34 31.41 12.74 26.52 60.74 −0.351 −0.034
L.tephrodes 11247 29.23 31.52 9.29 26.69 62.80 −0.484 −0.038
L.macrorhinus 11430 29.51 30.84 12.69 26.96 60.35 −0.360 −0.022
P. microdon 11496 29.51 34.71 13.21 22.57 64.21 −0.262 −0.081
T. obesus 11430 29.22 31.35 12.78 26.65 60.57 −0.352 −0.035
tRNA
C.umbratile 1538 32.51 28.87 17.43 21.20 61.38 −0.098 0.059
S. canicula 1551 31.53 30.82 20.12 17.54 62.35 0.068 0.011
S. tiburo 1551 32.62 27.98 17.21 22.18 60.61 −0.126 0.077
P. habereri 1553 30.71 29.75 21.31 18.22 60.46 0.078 0.016
C. acronotus 1552 30.86 29.70 21.20 30.86 60.57 0.075 0.019
C.amblyrhynchoides 1551 32.62 27.92 17.28 32.62 60.54 −0.124 0.078
C. amboinensis 1548 32.62 27.78 17.31 32.62 60.40 −0.126 0.080
C. brevipinna 1550 30.77 29.55 21.35 30.77 60.32 0.076 0.020
C. leucas 1552 0.069 32.73 28.48 17.14 61.21 −0.116 0.069
C.longimanus 1553 0.077 32.39 27.75 17.51 60.14 −0.121 0.077
C.macloti 1542 0.074 32.49 28.02 17.32 60.51 −0.123 0.074
C.melanopterus 1551 0.076 32.43 27.85 17.54 60.28 −0.117 0.076
C. plumbeus 1551 0.071 32.17 27.92 17.73 60.09 −0.111 0.071
C. sorrah 1552 0.003 27.90 27.71 17.53 58.23 −0.121 0.003
L.tephrodes 1551 0.080 32.75 27.92 17.21 60.67 −0.125 0.080
L.macrorhinus 1552 31.25 30.15 20.75 17.85 61.4 0.075 0.018
P. microdon 1551 31.85 28.76 17.73 21.66 60.61 −0.100 0.051
T. obesus 1552 32.73 27.90 17.27 22.10 60.63 −0.123 0.080
rRNA
C.umbratile 2623 34.77 26.69 17.69 20.85 61.46 −0.082 0.132
S. canicula 2630 34.26 26.50 18.02 21.22 60.76 −0.081 0.128
S. tiburo 2623 35.46 26.12 17.35 21.08 61.57 −0.097 0.152
P. habereri 2619 35.01 26.42 17.83 20.73 61.44 −0.075 0.140
C. acronotus 2629 35.34 26.21 17.15 21.30 61.54 −0.108 0.148
C.amblyrhynchoides 2624 35.21 25.88 17.34 21.57 61.09 −0.109 0.153
C. amboinensis 2627 35.40 26.19 17.17 21.24 61.59 −0.106 0.150
C. brevipinna 2626 35.15 25.89 17.40 21.55 61.04 −0.107 0.152
C. leucas 2624 35.18 26.68 17.38 20.77 61.85 −0.089 0.137
C.longimanus 2625 35.20 25.71 17.33 21.75 60.91 −0.113 0.156
C.macloti 2622 35.28 25.36 17.28 22.08 60.64 −0.122 0.164
C.melanopterus 2626 35.03 25.55 17.48 21.93 60.59 −0.113 0.157
C. plumbeus 2629 35.26 25.45 17.27 22.02 60.71 −0.121 0.162
C. sorrah 2627 35.25 25.58 17.24 21.93 60.83 −0.120 0.159
L.tephrodes 2624 35.37 25.69 17.15 21.72 61.10 −0.118 0.159
L.macrorhinus 2625 35.73 26.10 17.10 21.07 61.83 −0.104 0.156
P. microdon 2624 35.02 26.64 17.72 20.62 61.66 −0.076 0.136
T. obesus 2622 35.51 25.55 17.09 21.85 61.06 −0.122 0.163
Control region
C.umbratile 1059 34.09 34.56 12.94 18.41 68.65 −0.175 −0.007
S. canicula 1051 33.21 33.59 13.23 19.89 66.86 −0.201 −0.006
S. tiburo 1087 31.83 32.84 12.60 21.07 65.76 −0.251 −0.016
P. habereri 1067 32.61 33.55 13.96 19.87 66.17 −0.175 −0.014
C. acronotus 1076 31.69 35.13 13.57 19.61 66.82 −0.182 −0.051
C.amblyrhynchoides 1067 31.40 35.05 13.59 19.96 66.45 −0.190 −0.055
C. amboinensis 1067 31.68 35.43 13.40 19.49 67.10 −0.185 −0.056
C. brevipinna 1068 31.74 35.11 13.67 19.48 66.85 −0.175 −0.050
C. leucas 1066 32.27 35.08 13.32 19.32 67.35 −0.184 −0.042
C.longimanus 1066 31.24 35.27 13.51 19.98 66.51 −0.193 −0.061
C.macloti 1066 33.40 34.80 12.38 19.42 68.20 −0.221 −0.021
C.melanopterus 1067 31.58 34.58 13.40 20.43 66.17 −0.208 −0.045
C. plumbeus 1063 31.14 35.47 13.55 19.85 66.60 −0.189 −0.065
C. sorrah 1066 31.99 34.80 13.23 19.98 66.79 −0.203 −0.042
L.tephrodes 1069 32.18 34.89 13.38 19.27 67.26 −0.181 −0.040
L.macrorhinus 1063 32.64 34.24 13.26 19.85 66.89 −0.199 −0.024
P. microdon 1058 33.74 34.03 11.81 20.42 67.77 −0.267 −0.004
T. obesus 1064 31.48 35.53 13.72 19.27 67.01 −0.168 −0.060
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Note: The A + T biases of whole mitogenome, protein-coding genes, tRNA, rRNA and control regions were calculated by AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G + C), respectively.

Protein-coding gene features

The PCG region formed 68.5% of the C. umbratile mitogenome, and was 11,440 bp long. Furthermore, a contrast of nucleotide composition, AT-skew, and GC-skew of Carcharhiniformes PCGs were exhibited in Table 2. A + T content of the rRNA genes was 61.75%. The AT skew value (−0.282) of the PCG region in the C. umbratile mtDNA was higher than that of several reported mtDNA, nevertheless the negative GC skew (−0.069) was similar to that observed in other fish33,34.

Each PCG was initiated by a canonical ATN codon, except for COXI, which was initiated by a GTG codon (Table 1). Similar results have been documented in other Carcharhiniformes35,36. Seven of 13 PCGs (ND1, COXI, ATP8, COXIII, ND4L, ND5, ND6) used a typical TAA termination codon, which was typical for Carcharhiniformes mtDNA35,36; whereas COXII, ND3 and ND4 terminated with a single T and ATP6, ND2 and Cytb terminated with TA (Table 1). It was akin to sequenced mtDNA of Carcharhiniformes, including Triaenodon obesus 37, Carcharhinus macloti 38, Mustelus griseus 39, S. canicula 25 and C. acronotus 28.

A total of 3,803 amino acids of PCGs are encoded in C. umbratile. In addition, the codon usage is shown in Table 3. The most frequent amino acids in the PCGs of C. umbratile were Leucine (17.3%), Isoleucine (9.02%) and Alanine (7.45%) (Table 3). Relative synonymous codon usage (RSCU) analysis of PCGs in C. umbratile revealed that the codons encoding Leu, Thr, Ala, Arg, Gln, Gly, Pro and Ser were the most frequently present, nevertheless those encoding Asn, Asp, Cys and Lys were rare (Fig. 2). In the PCGs of the eight species examined, codon distributions and amino acid content were corresponding among species (Fig. 3). It was declared that conserved amino acid sequences were present among those fish28,32,40. Moreover, codons with A or T in the third position were overused in comparison to other synonymous codons, for example, the codons for glutamine CAG and GAG were rare, while the synonymous codons CAA and GAA were prevalent (Fig. 4), which is consistent with previous observations of Carcharhiniformes36.

Table 3.

Codon usage of Cephalloscyllium umbratile mitochondrial protein-coding genes.

Amino acid Codon Number Frequency (%) RSCU Amino acid Codon Number Frequency (%) RSCU
Ala GCC 117 3.07 1.65 CAC 56 1.47 1.19
GCA 87 2.28 1.23 CAT 38 0.99 0.81
GCT 76 1.99 1.07 Ile ATT 246 6.45 1.43
GCG 4 0.11 0.06 ATC 98 2.57 0.57
Arg CGA 40 1.05 2.19 Leu TTA 229 6.01 2.08
CGT 16 0.42 0.88 CTA 153 4.02 1.39
CGC 13 0.34 0.71 CTT 145 3.81 1.32
CGG 4 0.10 0.22 CTC 90 2.36 0.82
Asn AAT 96 2.52 1.28 TTG 24 0.63 0.22
AAC 54 1.42 0.72 CTG 18 0.47 0.16
Asp GAT 44 1.15 1.31 Lys AAA 77 2.02 1.90
GAC 23 0.60 0.69 AAG 4 0.10 0.10
Cys TGT 16 0.42 1.19 Met ATA 136 3.57 1.53
TGC 11 0.29 0.81 ATG 42 1.10 0.47
Gln CAA 89 2.34 1.85 Phe TTT 146 3.83 1.24
CAG 7 0.18 0.15 TTC 89 2.34 0.76
GAA 89 2.34 1.71 Pro CCA 87 2.28 1.67
GAG 15 0.39 0.29 CCC 76 1.99 1.45
Gly GGA 88 2.31 1.53 CCT 42 1.10 0.80
GGC 57 1.50 0.99 CCG 4 0.10 0.08
GGT 51 1.34 0.89 Ser TCA 89 2.34 1.99
GGG 34 0.89 0.59 TCT 62 1.63 1.38
His CAC 56 1.47 1.19 TCC 58 1.52 1.29
Amino acid Codon Number Frequency (%) RSCU Amino acid Codon Number Frequency (%) RSCU
AGC 34 0.89 0.76 ACG 7 0.18 0.1
AGT 21 0.55 0.47 Trp TGA 107 2.81 1.78
TCG 5 0.13 0.11 TGG 13 0.34 0.22
Stp* TAA 7 0.18 4 Tyr TAT 88 2.31 1.45
AGA 0 0 0 TAC 33 0.87 0.55
AGG 0 0 0 Val GTA 80 2.10 1.76
TAG 0 0 0 GTT 52 1.36 1.14
Thr ACA 117 3.07 1.67 GTC 31 0.81 0.68
ACC 99 2.60 1.41 GTG 19 0.50 0.42
ACT 57 1.50 0.81
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Figure 2.

Figure 2

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Comparison of codon usage within the mitochondrial genome of members of the Carcharhiniformes. Species (Sphyrna tiburo, Proscyllium habereri, Lamiopsis tephrodes, Pseudotriakis microdon, Cephalloscyllium umbratile, Carcharhinus acronotus, Triaenodon obesus, Loxodon macrorhinus) represent the superfamily to which the species belongs (Sphyrna, Proscyllium, Lamiopsis, Pseudotriakis, Cephaloscyllium, Carcharhinus, Triaenodon, Loxodon).

Figure 3.

Figure 3

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Codon distribution in members of eight superfamilies in the Carcharhiniformes. CDspT = codons per thousand codons.

Figure 4.

Figure 4

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Relative Synonymous Codon Usage (RSCU) of the mitochondrial genome of eight superfamilies in the Carcharhiniformes. Codon families are plotted on the x-axis. Codons indicated above the bar are not present in the mitogenome.

Transfer RNAs and ribosomal RNAs

The representative complement structures of 22 tRNAs were identified in the C. umbratile mtDNA, ranging from 62 bp (tRNAThr) to 76 bp (tRNALys)35,36 for 1,538 bp in total (Table 1). Of those, the highest A + T content of tRNAs was S. canicula and the lowest was C. sorrah. Fifteen tRNA genes were encoded on the H strand while the remains were located in the L strand (Table 1). The overall A+T content of tRNAs was 61.38% which was approximate to that observed in Loxodon macrorhinus (61.4%). The negative AT skew (−0.098) and positive GC skew (0.059) showed in the C. umbratile mtDNA were also analogous with several sequenced Carcharhiniformes (Table 2).

The forecasted tRNAs were shown in Fig. 5. All of the tRNAs could be folded into classic clover-leaf secondary structures in C. umbratile, except for tRNA-Ser (GCT), which lacked the dihydrouridine ‘DHU’ arm (Fig. 5). The ‘DHU’ arm of this tRNA was a large loop instead of the conserved stem-and-loop structure. Due to a representative characteristics41, it was also observed in other Chondrichthyes mtDNA, including Chiloscyllium griseum 42 T. obesus 37 and so on. Fifteen of the tRNA genes were each observed to have at least one G-T mismatches in their respective secondary structures, which forming a weak bond. Five T-T mismatches were present in the respective amino acid acceptor stems of tRNA Asp(GTC), tRNA Cys(GCA), tRNA His(GTG), tRNA Ile(GAT) and tRNAMet(CAT) (Fig. 5). Interestingly, A-G mismatch was also present in tRNA-Leu (TAA). Unmatched base pairs perceived in tRNA sequences can be amended by RNA-editing mechanisms that were well known for vertebrate mtDNA43.

Figure 5.

Figure 5

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Putative secondary structures for 22 tRNA genes in mitochondrial genome of Cephalloscyllium umbratile. Watson-Crick and GT bonds are illustrated by “−” and “+”, respectively.

The A + T content of the rRNA genes was 61.46%, indicating an A+C-rich trend as in other Scyliorhinidae fish25. AT and GC skews were negative (−0.082) and positive (0.132), respectively (Table 2). The 12S rRNA and 16S rRNA subunit gene of C. umbratile was 954 bp and 1,668 bp in length, respectively. As in other vertebrates44, both two genes are separated by the tRNA Val gene, and located between tRNA Phe and tRNA Leu(UUR) (Fig. 1, Table 1). The overall content of the rRNA was analogous to that observed for other Carcharhiniformes.

The control region

The length of D-loop region of C. umbratile was 1,059 bp, which was less long than majority of Carcharhiniformes. The A + T content was 68.65%, and equal with other Carcharhiniformes (Table 2), which was consistent with the findings of previous reports on other teleosts33,45,46. Moreover, both of the AT-skew and GC-skew were strongly negative (Table 2).

Overlapping and intergenic spacer regions

There were three gene boundaries where bases overlapped between adjacent genes, ranging from 4–22 bp in size. The longest overlapping region was 22 bp between ATP8 and ATP6 (Table 1) which has been documented in several other Chondrichthyes mtDNA4,25,32. Moreover, intergenic spacers of C. umbratile were spread over 12 locations and ranged from 1–36 bp, making up 60 bp in total, and the longest intergenic spacer region (36 bp) was between tRNA Asn and tRNA Cys (Table 1).

Synonymous and nonsynonymous substitutions

The ratio of Ka/Ks is generally regarded as a pointer of selective pressure and evolutionary relations at the molecular level among homogenous or heterogeneous species47,48. It is reported that Ka/Ks > 1, Ka/Ks = 1, and Ka/Ks < 1 popularly declared positive selection, neutral mutation and negative selection, respectively49. To investigate the evolutionary rate differences in three Carcharhiniformes mtDNA (C. umbratile, S. canicula and P. habereri), sequence divergences by counting Ka and Ks substitution rates were next calculated. The Ka/Ks values of 13 PCGs varied from 0.0198 (COXI) to 0.5322 (ATP8) and were less than 0.6 (Ka was lower than Ks) for all other genes which indicated a strong purifying and negative selection in those fishes (Fig. 6). Our result of the Ka/Ks ratio illustrated that the multitudinous genes evolved under strong negative selection which meant natural selection against profitless mutations with negative selective coefficients50. The percentages of variable sites of SC/PH were the highest in COXIII and ND1 among the groups, while the percentages was the least in COXI gene, which indicated that COXIII and ND1 were under the least selective pressure, and COXI was under the most selective pressure among all mitochondrial proteins. In C. umbratile and S. canicula, the ratio of Ka/Ks was the least in all 13 protein-coding genes compared to P. habereri, implying that these two Scyliorhinidae fish had the closer phylogenetic relationship than P. habereri, which was consistent with their rozmieszczenie naturalne and ecological habit25.

Figure 6.

Figure 6

Open in a new tab

Ka/Ks ratios for the 13 mitochondrial protein-coding genes among the reference Cephalloscyllium umbratile (CU), Scyliorhinus canicula (SC), Proscyllium habereri (PH).

Phylogeny

To understand the phylogenetic relationships among Carcharhiniformes, base on Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) methods, a dataset of 25 species containing the concatenated nucleic acid and amino acid sequences of 13 PCGs was used to generate phylogenetic relationships (Fig. 7). The topologies of the 6 phylogenetic trees were analogical in our study. The results implied that strong statistics supported for the following relationship among the 5 Superfamily (Scyliorhinidae, Carcharhinidae, Hemigaleidae, Proscylliidae, Pseudotriakidae) (Fig. 7A,B). This clustered pattern of 5 Superfamily was broadly consistent with previous studies32,42,51–53. Furthermore, based on all of ML, NJ and BI methods, 5 superfamily divided into 13 closely genera, and C. umbratile (Cephaloscyllium) was most closely related to S. canicula (Scyliorhinus) in Scyliorhinidae, which was accord with the tendency of nucleotide sequence identity and a recent study51,54–57. Scyliorhinidae was most closely related to Proscylliidae. Additionally, further taxon sampling within Scyliorhinidae and related superfamilies is required to resolve the location of Scyliorhinidae in Carcharhiniformes.

Figure 7.

Figure 7

Open in a new tab

Phylogenetic trees of Cephalloscyllium umbratile relationships from the nucleotide (A) and amino acid datasets (B). Sequences alignment of mtDNA were analyzed using the MEGA 6.0 and Phylobayes 3.3 f software with Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian inference (BI) method, respectively. The accession numbers of the sequences used in the phylogenetic analysis are listed in Supplementary Table 1.

Materials and Methods

Sample collection and mitochondrial DNA extraction

C. umbratile juveniles were collected from South China Sea (Longitude 5°20.267′ N and latitude 109°48.435′ E) in September 2014 and directly frozen. Muscle tissues were used for DNA extraction according to the Genomic DNA Extraction Kit’s instructions (TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0, Japan). The quantity (concentration) of isolated total DNA was determined by NANODROP 2000 spectrophotometer (Thermo Scientific, USA). Furthermore, quality of extracted DNA was assessed by electrophoresis on a 1% agarose gel stained with Gel Red™ (Biotium).

Genome sequencing

According to NEBNext DNA sample libraries kit (NEB, New England)‘s instructions, the normalized DNA (4 μg) was used to structure the paired-end library. Size and quantification estimation of the library were implemented by a Bioanalyzer 2100 High Sensitivity DNA chip (Agilent, USA). Illumina HiSeq. 2500 (2 × 101 bp paired-end reads) (Illumina, USA) was used to sequence the normalized library (2 nM).

Genome assembly and annotation

A de novo assembly of the paired-end HiSeq reads was performed using SeqMan NGen (http://www.dnastar.com/t-tutorials-seqman-ngen.aspx) (DNASTAR Inc., Madison, WI, USA)58. Assembly parameters minimum match percentage, match spacing, match size, gap penalty, mismatch penalty, maximum gap length and expected genome length were set to 93, 10, 50, 30, 20, 6% and 16,000, respectively. Accordance sequence was exported and ends were manually edited to remove duplicated nucleotides. Subsequently, the assembled sequences were aligned to NCBI nt database with blastn method (https://blast.ncbi.nlm.nih.gov/). Sequences that mapping to Carcharhiniformes mtDNA were considered as C. umbratile mtDNA. To verify the accuracy of the assembled mtDNA sequence, the primers (Supplementary Table 2) were used to amplify the genome sequence. The procedure of PCR amplification was referred from Sun et al.59. To determine whether this method was accurate, the sequence segments of same genomic region obtained from Sanger sequencing and shotgun assembly were compared. If they were identical, that meaning this method was precise. Moreover, the PCGs, rRNA genes, tRNA genes and D-loop region of mtDNA were annotated by MitoAnnotator (http://mitofish.aori.u-tokyo.ac.jp/annotation/input.html)60 with parameters of complete circular genome. The mtDNA sequence of C. umbratile has been deposited in the GenBank database under accession numbers KX354996.

Genome sequence analysis

tRNAscan-SE Search Server 1.21 program was used to primordially determine Transfer RNAs61,62. The gene map of C. umbratile mtDNA was built by OGDRAW1.2 and embellished manually63. The strand skew values were reckoned in terms of the formulae by Perna and Kocher (1995)64. The mode of “models- > Compute Codon Usage Bias” was chose to obtain RSCU in MEGA 6.065. To determine the evolutionary branching of the Carcharhiniformes lineage, codon usage in the 13 PCGs and the rates of Ka/Ks substitutions in the mtDNA of Carcharhiniformes were calculated by DnaSP 5.10.0166. To describe base composition, we analyzed skew as described as below: AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G+C)67.

Phylogenetic analysis

To discuss the phylogenetic position of Carcharhiniformes, a total of 25 species of 13 PCG sequences were used to perform phylogenetic analysis, including those of C. umbratile. Alignments of the 13 concatenated PCGs nucleotide and amino acid sequences were conducted using ClustalX version 2.0 with default parameters68. Phylogenetic analyses for each concatenated dataset was performed using ML, MP and BI methods with MEGA 6.0 and Phylobayes 3.3 f, respectively65,69. The methods of ML and MP analysis were performed with GTR+I+G model and Subtree-Purning-Regrafting (SPR) model using MEGA 6.0, respectively. The evaluation of node accuracy was done by using 1,000 bootstrap replicates in MEGA 6.0 with default parameters. Furthermore, BI analysis was selecting the CAT-GTR model, two independent Markov chain Monte Carlo (MCMC) chains were run for 10,000 cycles. The phylogenetic tree was embellished using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

Electronic supplementary material

Supplementary Information (211.4KB, pdf)

Acknowledgements

This work was supported by the Special Scientific Research Funds for Central Non-profit Institute, Chinese Academy of Fishery Sciences (2016HY-JC0304), the Science and Technology Infrastructure Construction Project of Guangdong Province (2014A030305005, 2015A030303008), National Infrastructure of Fishery Germplasm Resource Project (2017DKA30470).

Author Contributions

K.C.Z. and D.C.Z. designed the research and wrote the paper. N.W. and H.Y.G. performed the research. Y.Y.L. and N.Z. analyzed the data, S.G.J. contributed reagents/materials/analysis tools.

Competing Interests

The authors declare that they have no competing interests.

Footnotes

Electronic supplementary material

Supplementary information accompanies this paper at 10.1038/s41598-017-15702-0.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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