Research Article |
Corresponding author: Ying Wang ( xinyuanwangying@163.com ) Academic editor: Maria Elina Bichuette
© 2024 Min Zhou, Cheng Wang, Ziyue Xu, Zhicun Peng, Yang He, Ying Wang.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Zhou M, Wang C, Xu Z, Peng Z, He Y, Wang Y (2024) Complete mitochondrial genome of Lepidocephalichthys berdmorei and its phylogenetic status within the family Cobitidae (Cypriniformes). ZooKeys 1221: 51-69. https://doi.org/10.3897/zookeys.1221.129136
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In this study, the complete mitochondrial genome of Lepidocephalichthys berdmorei was first determined by the primer walking sequence method. The complete mitochondrial genome was 16,574 bp in length, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (D-loop). The gene arrangement pattern was identical to that of other teleosts. The overall base composition was 29.9% A, 28.5% T, 25.5% C, and 16.1% G, with an A+T bias of 58.4%. Furthermore, phylogenetic analyses were conducted based on 13 PCGs from the mitochondrial genomes of 18 cobitid species using with three different methods (Neighbor-joining, Maximum likelihood, and Bayesian inference). All methods consistently showed that the four species of the genus Lepidocephalichthys form a monophyletic group. This study would provide effective molecular information for the Lepidocephalichthys species as well as novel genetic marker for the study of species identification.
Gene arrangement pattern, Lepidocephalichthys berdmorei, mitochondrial genome, phylogenetic analysis
Lepidocephalichthys berdmorei (
The mitochondrial genome (mtDNA) is a circular double-stranded molecule consisting of 13 PCGs, 22 tRNAs, two rRNAs, and a control region (D-loop) (
In this study, the complete mitochondrial genome of L. berdmorei was sequenced for the first time. The variation in tRNA length, position, and size of the control region, and the codon usage bias were analyzed. Subsequently, the 13 PCGs were concatenated and utilized, with those of other cobitids, to confirm the phylogenetic position of L. berdmorei. Therefore, these findings will provide valuable information and contribute to future species comparison and evolutionary research.
An adult individual of L. berdmorei was obtained in 2020 from the Mengla town, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China (21°57'70"N, 101°60'54"E) (Suppl. material
Eight pairs of primers (Suppl. material
Sequences were assembled manually by the Seqman program using DNAstar v. 7.1 software (
The base composition and relative synonymous codon usage (RSCU) of the mitogenome were calculated and produced using PhyloSuite v. 1.2.3 (
To verify the phylogenetic position of L. berdmorei, 17 mitogenome sequences from GenBank were retrieved (Suppl. material
A NJ phylogenetic tree was constructed using MEGA 7 (
Mitogenome, mitochondrial genome; mtDNA, mitochondrial DNA; PCGs, protein-coding genes; tRNA, transfer RNA; rRNA, ribosomal RNA; atp6 and atp8, ATPase 6 and ATPase 8; cox1–3, cytochrome oxydasec subunits I–III; cytb, cytochrome b; LA-PCR, long and accurate polymerase chain reaction; nd1–6, NADH dehydrogenase subunits 1–6; nd4l, NADH dehydrogenase subunits 4L; A+T, A+T rich region; RSCU, relative synonymous codon usage; trnA, tRNAAla; trnC, tRNACys; trnD, tRNAAsp; trnE, tRNAGlu; trnF, tRNAPhe; rrnS, 12S rRNA; rrnL, 16S rRNA; trnG, tRNAGly; trnH, tRNAHis; trnI, tRNAIle; trnK, tRNALys; trnL1, tRNALeu(TAA); trnL2, tRNALeu(TAG); trnM, tRNAMet; trnN, tRNAAsn; trnP, tRNAPro; trnQ, tRNAGln; trnR, tRNAArg; trnS1, tRNASer(TGA); trnS2, tRNASer(GCT); trnT, tRNAThr; trnV, tRNAVal; trnW, tRNATrp; trnY, tRNATyr; DHU, Dihydrouracil; NJ, Neighbor-joining; ML, Maximum likelihood; BI, Bayesian inference.
The length of the complete mitochondrial genome of L. berdmorei is 16,574 bp (GenBank accession number: OP651767). The complete mitochondrial genome of L. berdmorei shares high similarity in gene arrangement, base composition, and codon usage pattern with those of other teleosts, indicating that the mitochondrial genome is highly conserved in evolution (
Organization of the mitochondrial genome of Lepidocephalichthys berdmorei.
Locus | Position | Size (bp) | Intergenic nucleotidesa | Codon | Anti-codon | Strandb | ||
---|---|---|---|---|---|---|---|---|
From | To | Start | Stop | |||||
tRNAPhe (S) | 1 | 69 | 69 | 0 | – | – | GAA | H |
12S rRNA | 70 | 1019 | 950 | 0 | – | – | – | H |
tRNAVal (V) | 1020 | 1091 | 72 | 0 | – | – | TAC | H |
16S rRNA | 1092 | 2767 | 1676 | 0 | – | – | – | H |
tRNALeu ( TAA )(L1) | 2768 | 2842 | 75 | 1 | – | – | TAA | H |
nd1 | 2844 | 3818 | 975 | 5 | ATG | TAA | – | H |
tRNAAIle (I) | 3824 | 3895 | 72 | -2 | – | – | GAT | H |
tRNAGln (Q) | 3894 | 3964 | 71 | 1 | – | – | TTG | L |
tRNAMet (M) | 3966 | 4034 | 69 | 0 | – | – | CAT | H |
nd2 | 4035 | 5081 | 1047 | -2 | ATG | TAG | – | H |
tRNATrp (W) | 5080 | 5148 | 69 | 2 | – | – | TCA | H |
tRNAAla (A) | 5151 | 5219 | 69 | 1 | – | – | TGC | L |
tRNAAsn (N) | 5221 | 5293 | 73 | 30 | – | – | GTT | L |
tRNACys (C) | 5324 | 5390 | 67 | 0 | – | – | GCA | L |
tRNATyr (Y) | 5391 | 5459 | 69 | 1 | – | – | GTA | L |
cox1 | 5461 | 7011 | 1551 | 2 | GTG | TAA | – | H |
tRNASer ( TGA )(S1) | 7014 | 7084 | 71 | 1 | – | – | TGA | L |
tRNAAsp (D) | 7086 | 7158 | 73 | 13 | – | – | GTC | H |
cox2 | 7172 | 7862 | 691 | 0 | ATG | T | – | H |
tRNALys (K) | 7863 | 7938 | 76 | 1 | – | – | TTT | H |
atp8 | 7940 | 8107 | 168 | -10 | ATG | TAA | – | H |
atp6 | 8098 | 8781 | 684 | -1 | ATG | TAA | – | H |
cox3 | 8781 | 9566 | 786 | -1 | ATG | TAA | – | H |
tRNAGly (G) | 9566 | 9638 | 73 | 0 | – | – | TCC | H |
nd3 | 9639 | 9989 | 351 | -2 | ATG | TAG | – | H |
tRNAArg (R) | 9988 | 10056 | 69 | 0 | – | – | TCG | H |
nd4l | 10057 | 10353 | 297 | -7 | ATG | TAA | – | H |
nd4 | 10347 | 11729 | 1383 | -1 | ATG | TAG | – | H |
tRNAHis (H) | 11729 | 11797 | 69 | 0 | – | – | GTG | H |
tRNASer ( GCT )(S2) | 11798 | 11866 | 69 | 1 | – | – | GCT | H |
tRNALeu ( TAG )(L2) | 11868 | 11940 | 73 | 0 | – | – | TAG | H |
nd5 | 11941 | 13779 | 1839 | -4 | ATG | TAA | – | H |
nd6 | 13776 | 14297 | 522 | 0 | ATG | TAA | – | L |
tRNAGlu (E) | 14298 | 14366 | 69 | 5 | – | – | TTC | L |
cytb | 14372 | 15512 | 1141 | 0 | ATG | T | – | H |
tRNAThr (T) | 15513 | 15584 | 72 | -2 | – | – | TGT | H |
tRNAPro (P) | 15583 | 15652 | 70 | 0 | – | – | TGG | L |
D-loop | 15653 | 16574 | 922 | 0 | – | – | – | H |
Gene map and organization of the mitochondrial genome of Lepidocephalichthys berdmorei. Photograph of L. berdmorei from https://fishbase.se/summary/Lepidocephalichthys-berdmorei.html.
The overall base composition is 29.9% for A, 16.1% for G, 25.5% for C, and 28.5% for T, which is consistent with the lowest frequency for G among the four bases in fish mitochondrial genomes, and revealing the A+T-rich content (58.4%) (
Cobitidae mitogenomes range from 16,574 bp (L. berdmorei) to 16,646 bp (Cobitis striata (Ikeda, 1936)) in length (Suppl. material
The length of PCGs was 11,413 bp (68.86%) and it blanketed 7 NADH dehydrogenases (nd1–6 and nd4l), three cytochrome coxidases (cox1–3), two ATPases (atp6 and atp8) and one cytochrome b (cytb). The size of PCGs ranged from nd4l (297 bp) to nd5 (1839 bp). As in other vertebrates, the nd6 and eight tRNA genes (tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer, tRNAPro, and tRNAGlu) are encoded on the light strand, and the others are encoded on the heavy strand (Fig.
Further analysis revealed that among 13 PCGs, most mitochondrial genes of L. berdmorei started with codon ATG, while only the cox1 gene began with codon GTG. Unconventional start codons are a common phenomenon within the mitogenomes of fish (
Codon usage in the mitochondrial genome of Lepidocephalichthys berdmorei.
Codon | Count | RSCU | Codon | Count | RSCU | Codon | Count | RSCU | Codon | Count | RSCU |
---|---|---|---|---|---|---|---|---|---|---|---|
UUU(F) | 201 | 1.28 | UCU(S) | 103 | 1.37 | UAU(Y) | 118 | 1.19 | UGU(C) | 40 | 0.95 |
UUC(F) | 113 | 0.72 | UCC(S) | 110 | 1.46 | UAC(Y) | 80 | 0.81 | UGC(C) | 44 | 1.05 |
UUA(L) | 188 | 1.68 | UCA(S) | 68 | 0.9 | UAA(*) | 156 | 1.66 | UGA(W) | 75 | 1.18 |
UUG(L) | 71 | 0.64 | UCG(S) | 40 | 0.53 | UAG(*) | 99 | 1.06 | UGG(W) | 52 | 0.82 |
CUU(L) | 164 | 1.47 | CCU(P) | 110 | 1.01 | CAU(H) | 95 | 0.95 | CGU(R) | 21 | 0.65 |
CUC(L) | 73 | 0.65 | CCC(P) | 134 | 1.23 | CAC(H) | 104 | 1.05 | CGC(R) | 37 | 1.15 |
CUA(L) | 111 | 0.99 | CCA(P) | 137 | 1.26 | CAA(Q) | 156 | 1.34 | CGA(R) | 49 | 1.52 |
CUG(L) | 63 | 0.56 | CCG(P) | 55 | 0.5 | CAG(Q) | 76 | 0.66 | CGG(R) | 22 | 0.68 |
AUU(I) | 170 | 1.27 | ACU(T) | 102 | 1.06 | AAU(N) | 134 | 1.06 | AGU(S) | 49 | 0.65 |
AUC(I) | 98 | 0.73 | ACC(T) | 120 | 1.25 | AAC(N) | 118 | 0.94 | AGC(S) | 82 | 1.09 |
AUA(M) | 98 | 1.18 | ACA(T) | 128 | 1.33 | AAA(K) | 172 | 1.36 | AGA(*) | 75 | 0.8 |
AUG(M) | 68 | 0.82 | ACG(T) | 35 | 0.36 | AAG(K) | 81 | 0.64 | AGG(*) | 45 | 0.48 |
GUU(V) | 73 | 1.6 | GCU(A) | 82 | 1.13 | GAU(D) | 63 | 0.92 | GGU(G) | 44 | 0.81 |
GUC(V) | 26 | 0.57 | GCC(A) | 114 | 1.57 | GAC(D) | 74 | 1.08 | GGC(G) | 53 | 0.98 |
GUA(V) | 56 | 1.22 | GCA(A) | 75 | 1.03 | GAA(E) | 83 | 1.06 | GGA(G) | 70 | 1.3 |
GUG(V) | 28 | 0.61 | GCG(A) | 20 | 0.27 | GAG(E) | 74 | 0.94 | GGG(G) | 49 | 0.91 |
The complete mitogenome of L. berdmorei contains 22 tRNA genes with a size of 1,559 bp, 14 of which are located on the H-strand while the others are on the L-strand (Table
The most prevalent non-Watson-Crick base pairs in the secondary structure of tRNAs are A-C (e.g., trnI, trnH, trnM, trnV, trnS1, trnT, trnW, and trnF), followed by T-T (trnQ and trnN), which are mostly located in the DHU, anticodon stems and acceptor (Fig.
The lengths of 12S rRNA and 16S rRNA genes were 950 bp and 1,676 bp,which are located on the H strand (Table
The only large control region of L. berdmorei mitogenome is the D-loop, located between the tRNAPro and tRNAPhe, with a length of 922 bp (Fig.
In addition to gene duplication and insertion/deletion events, the main cause of mitochondrial genome size variation is differences in control region length (
Cobitidae belongs to Osteichthyes, Cypriniformes, and has three subfamilies: Nemacheilinae, Botiinae and Cobitinae (
Phylogenetic tree of Cobitidae and two outgroups based on the NJ, ML and BI analysis of 13 concatenated protein-coding genes. Tree topologies produced by NJ, ML methods, and BI analysis were equivalent. The numbers at the nodes represent bootstrap support values for NJ and ML analyses and Bayesian posterior probability, sequentially, and the red branch represents the specie in this study.
Firstly, the phylogenetic tree revealed that L. guntea, L. hasselti, and L. berdmorei clustered as a monophyletic clade, followed by a clade with L. micropogon with high bootstrap support. Secondly, the genus Lepidocephalichthys and Pangio which formed a sister branch with high bootstrap support and posterior probability values, which was consistent with the previous study (
In conclusion, the complete mitochondrial DNA sequence of L. berdmorei is determined for the first time by the primer walking sequence method. The mitogenome is 16,574 bp in length, and encodes all of the 37 genes that are typical for Cobitidae fish. We compared mtDNA from L. berdmorei with that of other teleost and analyzed mitogenome composition, PCGs, and codon usage, transfer and ribosomal RNA genes, and noncoding regions (control region, intergenic spacers). The generated phylogenetic trees yielded convincing evidence that the genus Lepidocephalichthys formed a monophyletic group. These findings will provide new insights into better understanding the phylogenetic status of this intriguing and ecologically important group.
This work was supported by grants from the National Natural Science Foundation of China (31702016), and Jianghan University Research Project Funding Plan (2023KJZX42). We greatly appreciate the valuable suggestions of two anonymous referees.
The authors have declared that no competing interests exist.
All animal protocols have been reviewed and approved by the experimental animal welfare and ethics review committee of Jianghan University, Qinghai Normal University, and Chinese Academy of Sciences.
This work was supported by the National Natural Science Foundation of China (Grant number 31702016) and Jianghan University Research Project Funding Plan (Grant number 2023KJZX42).
Ying Wang contributed to the concept and design of the study. Sample collection and preparation of materials were done by Cheng Wang and Zhicun Peng. Ziyue Xu, Yang He and Min Zhou performed the data analysis and interpretation, and Min Zhou wrote the first draft of the manuscript. Ying Wang revised this manuscript. All authors read and revised the manuscript and approved the final version. All authors agree to be accountable for all aspects of the manuscript.
Ying Wang https://orcid.org/0000-0002-8222-7510
Genome sequence data that support the findings of this study are openly available from the GenBank at https://www.ncbi.nlm.nih.gov/, under accession No. OP651767.
Supplementary files
Data type: docx
Explanation note: figure S1. Images of biological sample for this study. table S1. Primers used for amplification of the mitochondrial genome of Lepidocephalichthys berdmorei. table S2. Species and GenBank accession numbers of mitogenomes used in this study. table S3. Nucleotide contents of genes and the mitochondrial genome skew of Lepidocephalichthys berdmorei.