Research Article |
Corresponding author: Yu Wang ( yuwang@zjnu.edu.cn ) Academic editor: Robert Jadin
© 2022 Shuangshuang Shan, Yu Wang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Shan S, Wang Y (2022) Complete mitochondrial genomes of Boiga kraepelini and Hebius craspedogaster (Reptilia, Squamata, Colubridae) and their phylogenetic implications. ZooKeys 1124: 191-206. https://doi.org/10.3897/zookeys.1124.87861
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The complete sequence of the mitochondrial genome is a powerful tool for studying phylogenetic relationships and molecular evolution in various species. In this work, the mitogenomes of Boiga kraepelini and Hebius craspedogaster were sequenced and characterized for the first time. The lengths of the B. kraepelini and H. craspedogaster mitogenomes were 17,124 bp and 17,120 bp, respectively, and both included 13 protein-coding genes, 22 tRNAs, two rRNAs and two control regions. The arrangements of these mitochondrial genes were the same in B. kraepelini and H. craspedogaster. In addition, both genome compositions showed A+T bias (59.03%, 60.93%) and had positive AT skews (0.179, 0.117) and negative GC skews (-0.397, -0.348). The phylogenetic results illustrated a close relationship between B. kraepelini and the genus Lycodon. Moreover, H. craspedogaster was clustered with other Hebius snakes and closely related to other Natricinae species. These results will provide references for further research on the phylogeny of Colubridae.
Colubrinae, mitogenomes, Natricinae, phylogenetic analysis, protein-coding genes
Colubridae is a family with high species diversity in the suborder Serpentes, which is distributed on almost all continents (
The mitochondrial genomes of snakes are circular molecules that contain 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and one or two duplicate control regions. Due to the advantages of small size, matrilineal inheritance, relatively stable genetic structure, easy amplification and high evolutionary rate, partial or full sequences of the mitogenome have been extensively used in molecular evolution, comparative and evolutionary genomics, phylogenetics and population genetics research in various animal species (
Specimens of B. kraepelini and H. craspedogaster were collected from Jinhua, China (29°12'N, 119°37'E). Total genomic DNA (gDNA) was extracted from tail muscle using a Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, China) according to the manufacturer’s instructions.
Conventional polymerase chain reaction (PCR) assays were conducted to amplify the complete mitogenomes of B. kraepelini and H. craspedogaster. The specific primers were designed based on the known nucleotide sequences (Suppl. material
The obtained sequences were identified using the Basic Local Alignment Search Tool (BLAST) from NCBI and were assembled using SeqMan software (DNAStar Inc., USA). The complete mitochondrial sequences were annotated by the MITOS web server (http://mitos.bioinf.uni-leipzig.de/index.py) (
To understand the phylogenetic positions of B. kraepelini and H. craspedogaster, the complete mitochondrial sequences of 13 PCGs in 38 previously available species of Colubridae and two outgroups (Naja atra and Hypsiscopus plumbea) were obtained from GenBank (Table
Mitochondrial genome sequences with GenBank accession numbers used in this study.
Family | Species | Accession No. |
---|---|---|
Colubridae | Boiga kraepelini | This study |
Elaphe anomala | KP900218 | |
Elaphe bimaculata | KM065513 | |
Elaphe dione | MH460961 | |
Elaphe carinata | KU180459 | |
Elaphe davidi | KM401547 | |
Elaphe poryphyracea | GQ181130 | |
Elaphe quadrivirgata | AB738958 | |
Elaphe quatuorlineata | MK334307 | |
Elaphe sauromates | MK070315 | |
Elaphe schrenckii | KP888955 | |
Elaphe taeniurus | KC990021 | |
Euprepiophis perlacea | KF750656 | |
Gonyosoma frenatum | MW413812 | |
Lycodon flavozonatus | KR911720 | |
Lycodon rufozonatum | KF148622 | |
Lycodon ruhstrati | MK867843 | |
Lycodon semicarinatus | AB008539 | |
Oligodon chinensis | MK347418 | |
Oocatochus rufodorsatus | KC990020 | |
Orientocoluber spinalis | MT304473 | |
Pantherophis slowinskii | DQ523162 | |
Pituophis catenifer sayi | KU833245 | |
Ptyas dhumnades | KF148621 | |
Ptyas major | KF148620 | |
Ptyas mucosa | KT982276 | |
Thermophis baileyi | MF326642 | |
Thermophis shangrila | MF066951 | |
Thermophis zhaoermii | GQ166168 | |
Hebius craspedogaster | This study | |
Hebius optatum | MN427890 | |
Hebius vibakari ruthveni | KP684155 | |
Nerodia sipedon | JF964960 | |
Opisthotropis guangxiensis | MT571495 | |
Opisthotropis latouchii | MK570292 | |
Pseudagkistrodon rudis | MW327508 | |
Rhabdophis tigrinus | KU641019 | |
Pseudoxenodon stejnegeri | MW018358 | |
Sibynophis chinensis | KF360246 | |
Sibynophis collaris | JN211315 | |
Elapidae | Naja atra | EU913475 |
Homalopsidae | Hypsiscopus plumbea | DQ343650 |
The complete mitogenomes of B. kraepelini and H. craspedogaster (GenBank accession numbers: MW699848 and MW699847, respectively) were closed double stranded DNA molecules 17,124 bp and 17,120 bp in length, respectively (Fig.
Graphical maps of Boiga kraepelini and Hebius craspedogaster mitogenomes. Thirteen protein-coding genes (PCGs) and two ribosomal RNA genes (rrnS and rrnL) are shown with standard abbreviation. Twenty-two transfer RNA (tRNA) are abbreviated by a single letter. CR1 and CR2 are two putative control regions.
Summary of the mitogenomes of Boiga kraepelini and Hebius craspedogaster.
Gene | Strand | Boiga kraepelini | Hebius craspedogaster | Anti-codon | ||||
---|---|---|---|---|---|---|---|---|
Location | Size (bp) | Start / Stop codon | Location | Size (bp) | Start / Stop codon | |||
trnF | H | 1–61 | 61 | – | 1–63 | 63 | – | GAA |
rrnS | H | 62–978 | 917 | – | 64–988 | 925 | – | – |
trnV | H | 979–1042 | 64 | – | 989–1052 | 64 | – | TAC |
rrnL | H | 1043–2498 | 1456 | – | 1053–2497 | 1445 | – | – |
nad1 | H | 2515–3478 | 964 | ATA/T | 2519–3482 | 964 | ATA/T | – |
trnI | H | 3479–3544 | 66 | – | 3483–3546 | 64 | – | GAT |
CR2 | – | 3545–4556 | 1012 | – | 3547–4537 | 991 | – | – |
trnL2 | H | 4557–4629 | 73 | – | 4538–4610 | 73 | – | TAA |
trnQ | L | 4631–4701 | 71 | – | 4611–4681 | 71 | – | TTG |
trnM | H | 4703–4764 | 62 | – | 4682–4744 | 63 | – | CAT |
nad2 | H | 4765–5794 | 1030 | ATT/T | 4745–5771 | 1027 | ATG/T | – |
trnW | H | 5795–5859 | 65 | – | 5772–5838 | 67 | – | TCA |
trnA | L | 5860–5922 | 63 | – | 5841–5905 | 65 | – | TGC |
trnN | L | 5923–5994 | 72 | – | 5906–5978 | 73 | – | GTT |
OL | – | 5997–6031 | 35 | – | 5981–6015 | 35 | – | – |
trnC | L | 6030–6089 | 60 | – | 6014–6072 | 59 | – | GCA |
trnY | L | 6090–6151 | 62 | – | 6074–6135 | 62 | – | GTA |
cox1 | H | 6144–7754 | 1611 | ATA/AGG | 6128–7738 | 1611 | ATA/AGG | – |
trnS2 | L | 7745–7811 | 67 | – | 7729–7795 | 67 | – | TGA |
trnD | H | 7812–7875 | 64 | – | 7796–7860 | 65 | – | GTC |
cox2 | H | 7876–8560 | 685 | ATG/T | 7862–8546 | 685 | ATG/T | – |
trnK | H | 8561–8624 | 64 | – | 8547–8609 | 63 | – | TTT |
atp8 | H | 8626–8784 | 159 | ATG/TAA | 8610–8774 | 165 | ATG/TAA | – |
atp6 | H | 8775–9455 | 681 | ATG/TAA | 8765–9445 | 681 | ATG/TAA | – |
cox3 | H | 9455–10238 | 784 | ATG/T | 9445–10228 | 784 | ATG/T | – |
trnG | H | 10239–10299 | 61 | – | 10229–10289 | 61 | – | TCC |
nad3 | H | 10300–10642 | 343 | ATT/T | 10290–10632 | 343 | ATA/T | – |
trnR | H | 10643–10707 | 65 | – | 10633–10696 | 64 | – | TCG |
nad4L | H | 10708–10998 | 291 | ATG/TAA | 10697–10987 | 291 | ATG/TAA | – |
nad4 | H | 10998–12335 | 1338 | ATG/TAA | 10987–12321 | 1335 | ATG/TAA | – |
trnH | H | 12336–12400 | 65 | – | 12322–12386 | 65 | – | GTG |
trnS1 | H | 12402–12458 | 57 | – | 12388–12444 | 57 | – | GCT |
trnL1 | H | 12456–12526 | 71 | – | 12442–12512 | 71 | – | TAG |
nad5 | H | 12527–14290 | 1764 | ATG/TAA | 12514–14295 | 1782 | ATG/TAA | – |
nad6 | L | 14286–14798 | 513 | ATG/AGG | 14291–14809 | 519 | ATG/AGA | – |
trnE | L | 14799–14860 | 62 | – | 14810–14872 | 63 | – | TTC |
cob | H | 14861–15977 | 1117 | ATG/T | 14873–15989 | 1117 | ATG/T | – |
trnT | H | 15978–16043 | 66 | – | 15990–16053 | 64 | – | TGT |
trnP | L | 16044–16105 | 62 | – | 16054–16115 | 62 | – | TGG |
CR1 | – | 16106–17124 | 1019 | – | 16116–17120 | 1005 | – | – |
Nucleotide composition of Boiga kraepelini and Hebius craspedogaster mitogenomes; the values for B. kraepelini are shown before the slash (/) and of H. craspedogaster are listed after the slash.
A % | T % | G % | C % | A+T % | AT-skew | GC-skew | |
---|---|---|---|---|---|---|---|
Mitogenome | 34.81 / 34.04 | 24.22 / 26.89 | 12.36 / 12.73 | 28.61 / 26.34 | 59.03 / 60.93 | 0.18 / 0.12 | -0.40 / -0.35 |
PCGs | 35.48 / 34.72 | 23.53 / 27.19 | 11.00 / 11.15 | 29.99 / 26.94 | 59.01 / 61.92 | 0.20 / 0.12 | -0.46 / -0.42 |
tRNAs | 33.38 / 32.82 | 24.39 / 25.32 | 16.80 / 17.60 | 25.44 / 24.26 | 57.77 / 58.13 | 0.16 / 0.13 | -0.21 / -0.16 |
rrnS | 36.75 / 36.97 | 18.54 / 20.11 | 17.78 / 17.84 | 26.94 / 25.08 | 55.29 / 57.08 | 0.33 / 0.30 | -0.21 / -0.17 |
rrnL | 40.80 / 39.65 | 20.33 / 21.25 | 15.38 / 16.40 | 23.49 / 22.70 | 61.13 / 60.90 | 0.34 / 0.30 | -0.21 / -0.16 |
rRNAs | 39.23 / 38.61 | 19.64 / 20.80 | 16.31 / 16.96 | 24.82 / 23.63 | 58.87 / 59.41 | 0.33 / 0.30 | -0.21 / -0.16 |
CR1 | 27.67 / 26.37 | 33.17 / 33.43 | 11.68 / 12.64 | 27.48 / 27.56 | 60.84 / 59.80 | -0.09 / -0.12 | -0.40 / -0.37 |
CR2 | 27.17 / 26.24 | 33.20 / 33.00 | 11.86 / 12.92 | 27.77 / 27.85 | 60.38 / 59.23 | -0.10 / -0.11 | -0.40 / -0.37 |
CRs | 27.42 / 26.30 | 33.19 / 33.22 | 11.77 / 12.78 | 27.62 / 27.71 | 60.61 / 59.52 | -0.10 / -0.12 | -0.40 / -0.37 |
The lengths of 13 PCGs of B. kraepelini and H. craspedogaster varied from 159 bp (atp8) to 1764 bp (nad5) and from 165 bp (atp8) to 1782 bp (nad5), respectively (Table
Amino acid composition and relative synonymous codon usage (RSCU) in the mitogenome of Boiga kraepelini and Hebius craspedogaster; RSCU values of B. kraepelini are shown before the slash (/) and of H. craspedogaster are listed after the slash.
Amino acid | Codon | RSCU | Codon | RSCU | Codon | RSCU | Codon | RSCU |
---|---|---|---|---|---|---|---|---|
Ala (A) | GCC | 1.79 / 1.68 | GCA | 1.59 / 1.56 | GCU | 0.55 / 0.63 | GCG | 0.07 / 0.13 |
Arg (R) | CGA | 2.69 / 2.26 | CGC | 0.63 / 0.52 | CGU | 0.44 / 0.84 | CGG | 0.25 / 0.39 |
Asn (N) | AAC | 1.68 / 1.15 | AAU | 0.32 / 0.85 | ||||
Asp (D) | GAC | 1.72 / 0.97 | GAU | 0.28 / 1.03 | ||||
Cys (C) | UGC | 1.33 / 0.96 | UGU | 0.67 / 1.04 | ||||
Glu (E) | GAA | 1.74 / 1.74 | GAG | 0.26 / 0.26 | ||||
Gln (Q) | CAA | 1.83 / 1.86 | CAG | 0.17 / 0.14 | ||||
Gly (G) | GGA | 1.96 / 1.60 | GGC | 0.80 / 0.88 | GGG | 0.73 / 0.70 | GGU | 0.51 / 0.82 |
His (H) | CAC | 1.66 / 1.41 | CAU | 0.34 / 0.59 | ||||
Ile (I) | AUC | 1.22 / 0.90 | AUU | 0.78 / 1.10 | ||||
Leu1 (L1) | CUA | 3.16 / 2.25 | CUC | 0.65 / 0.61 | CUU | 0.55 / 0.96 | CUG | 0.41 / 0.23 |
Leu2 (L2) | UUA | 1.09 / 1.68 | UUG | 0.15 / 0.26 | ||||
Lys (K) | AAA | 1.89 / 1.79 | AAG | 0.11 / 0.21 | ||||
Met (M) | AUA | 1.80 / 1.76 | AUG | 0.20 / 0.24 | ||||
Phe (F) | UUC | 1.28 / 1.02 | UUU | 0.72 / 0.98 | ||||
Pro (P) | CCA | 2.54 / 2.65 | CCC | 1.02 / 0.75 | CCU | 0.28 / 0.44 | CCG | 0.16 / 0.16 |
Ser1 (S1) | AGC | 0.88 / 0.66 | AGU | 0.27 / 0.33 | ||||
Ser2 (S2) | UCA | 2.47 / 2.55 | UCC | 1.58 / 1.31 | UCU | 0.63 / 1.00 | UCG | 0.16 / 0.15 |
Thr (T) | ACA | 1.96 / 1.94 | ACC | 1.56 / 1.29 | ACU | 0.41 / 0.65 | ACG | 0.08 / 0.11 |
Trp (W) | UGA | 1.68 / 1.76 | UGG | 0.32 / 0.24 | ||||
Tyr (Y) | UAC | 1.34 / 0.96 | UAU | 0.66 / 1.04 | ||||
Val (V) | GUA | 1.70 / 1.71 | GUU | 0.96 / 1.14 | GUC | 0.78 / 0.62 | GUG | 0.56 / 0.52 |
Similar to other snakes, 22 tRNA genes were recovered from the mitogenomes of B. kraepelini and H. craspedogaster. The tRNA lengths of these two species ranged from 57 bp (trnS1) to 73 bp (trnL2) (Table
As shown in Table
Additionally, similar to some snakes, there were two control regions in both species mitogenomes, in which CR1 was located between trnP and trnF, and CR2 was located between trnI and trnL (UUR). The nucleotide composition and length of the two control regions in the same species were almost identical. The AT skews and GC skews of the two CRs in B. kraepelini and H. craspedogaster were negative, indicating that T and C were more numerous than A and G (Table
Phylogenetic trees were constructed based on nucleotide sequences of 13 PCGs in 38 Colubridae species and two outgroups from the families Elapidae and Homalopsidae (Fig.
Both Boiga and Hebius are species-rich genera in the family Colubridae, with more than 30 species each (
In this study, we sequenced and characterized the complete mitochondrial genomes of B. kraepelini and H. craspedogaster for the first time. The mitogenomes of B. kraepelini and H. craspedogaster were 17,124 bp and 17,120 bp in size, respectively, including 13 PCGs, 22 tRNAs, two rRNAs and two control regions. Both (B. kraepelini and H. craspedogaster) genome compositions were A+T biased (59.03% and 60.93%, respectively) and showed positive AT skews (0.179 and 0.117, respectively) and negative GC skews (-0.397 and -0.348, respectively). All of the tRNA genes could be folded into typical cloverleaf secondary structures, with the exception of trnS1, which lacks the D arm, and trnC, which lacks the T Ψ C loop. Phylogenetic analyses were performed with 38 other species from the family Colubridae and two outgroup species. Five clades that represent five subfamilies, Colubrinae, Natricinae, Sibynophiinae, Dipsadinae and Pseudoxenodontinae, were identified. The genus Boiga was closely related to the genus Lycodon, and both genera belong to the subfamily Colubrinae. Hebius craspedogaster was clustered with the other two Hebius species and closely related to other Natricinae species. This work will be helpful for understanding the evolutionary relationships within the family Colubridae and will provide basic data for the molecular identification of these two species.
This research was supported by the National Natural Science Foundation of China under Grant No. 31400472 and Zhejiang Provincial Natural Science Foundation of China under Grant No. L19C030005.
Table S1, S2
Data type: docx file
Explanation note: Table S1. Primers used for mitogenome amplification of Boiga kraepelini and Hebius craspedogaster. Table S2. Nucleotide composition of each tRNA of Boiga kraepelini and Hebius craspedogaster.