Research Article |
Corresponding author: Wei-Kuang Wang ( wkwang@fcu.edu.tw ) Academic editor: Maria Elina Bichuette
© 2020 I-Chen Wang, Hung-Du Lin, Chih-Ming Liang, Chi-Chun Huang, Rong-Da Wang, Jin-Quan Yang, Wei-Kuang 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:
Wang I-C, Lin H-D, Liang C-M, Huang C-C, Wang R-D, Yang J-Q, Wang W-K (2020) Complete mitochondrial genome of the freshwater fish Onychostoma lepturum (Teleostei, Cyprinidae): genome characterization and phylogenetic analysis. ZooKeys 1005: 57-72. https://doi.org/10.3897/zookeys.1005.57592
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The cyprinid genus Onychostoma Günther, 1896 consists of 24 valid species distributed in Southeast Asia, including Taiwan, Hainan, mainland China and the Indochina region. In the present study, we determined the complete mitochondrial genome of O. lepturum, which is 16,598 bp in length, containing 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a typical control region (D-loop). To verify the molecular phylogeny of the subfamily Acrossocheilinae, we provide new insights to better understand the taxonomic status of Acrossocheilus, Onychostoma and Folifer brevifilis. The phylogenetic trees presented three major clades based on the 13 protein-coding genes from 28 Acrossocheilinae species. Clades I and II represent the Onychostoma and Acrossocheilus groups, respectively. Species of Acrossocheilus, Onychostoma and F. brevifilis are included in Clade III, which is considered as an ancestral group. This work provides genomic variation information and improves our understanding of the Acrossocheilinae mitogenome, which will be most valuable in providing new insights for phylogenetic analysis and population genetics research.
Cyprinid, mitogenome, Onychostoma lepturum, phylogeny, population genetics, Southeast Asia
The cyprinid genus Onychostoma Günther, 1896 consists of 24 valid species distributed in Southeast Asia, including Taiwan, Hainan, mainland China and the Indochina region (
The Cyprinidae family has the most species of any freshwater fish family. The family encompasses 11 subfamilies, with the genus Onychostoma belonging to the Acrossocheilinae subfamily (
Folifer brevifilis is closely related to O. simum (Sauvage & Dabry de Thiersant, 1874) based on the mitochondrial and nuclear markers (
Recent population analyses suggested that the nucleotide diversity of cyprinids on Hainan Island was lower than that of cyprinids in mainland China (
Previous studies suggest an inclusive phylogenetic clade including species from Acrossocheilus, Onychostoma, and Folifer brevifilis based on molecular markers (
The sample of Onychostoma lepturum was caught from the Lingshui River in Baoting County of Hainan in China (18°42'07"N, 109°40'44"E). Samples were collected from the field sites with seines, fatally anesthetized with MS-222 (Sigma, St. Louis, MO) and fixed and stored in 100% ethanol. All specimens are lodged in the laboratory of Jin-Quan Yang, Shanghai Ocean University, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources. All animal experiments were carried out in accordance with the guidelines and with approval of the Animal Research and Ethics Committee of Shanghai Ocean University (permissions, SHOU-DW-2018-021). Total genomic DNA was extracted from muscle tissue using the Genomic DNA Purification Kit (Gentra Systems, Valencia, CA) in the laboratory.
The complete mitogenome of O. lepturum was obtained from high-throughput sequencing of whole-genome DNA with a HiSeqX Ten platform (Illumina, San Diego, CA) with a paired-end 150 bp approach. Next-generation sequencing (NGS) was used to perform low-coverage whole-genome sequencing to obtain the complete mitogenome according to a previous protocol (
Phylogenetic analyses using a total of 11 mitogenomes of Onychostoma species, 16 mitogenomes of Acrossocheilus species and one mitogenome of F. brevifilis were performed based on Neighbor-joining (NJ), Maximum-likelihood (ML), and Bayesian (BI) methods, with Spinibarbus hollandi as the outgroup. Twenty-nine mitogenomes were downloaded from NCBI and were aligned using MEGA X (alignment with CLUSTALW) with default settings (
In the present study, the complete mitochondrial genome sequence of O. lepturum derived by NGS was found to be 16,598 bp and was deposited in GenBank (accession MT258556). The mitogenome contained 37 typical mitochondrial genes with 13 typical vertebrate protein-coding genes, 2 ribosomal RNA (rRNA) genes, 22 tRNAs, and a control region (D-Loop) (Fig.
Locus | Position | Codon | ||||||
---|---|---|---|---|---|---|---|---|
start | stop | size(bp) | start | stop | anti-codon | intergenic nucleotide* | strand+ | |
tRNAPhe | 1 | 69 | 69 | GAA | 0 | H | ||
12s rRNA | 70 | 1030 | 961 | 0 | H | |||
tRNAVal | 1031 | 1102 | 72 | TAC | 0 | H | ||
16s rRNA | 1103 | 2783 | 1681 | 0 | H | |||
tRNALeu | 2784 | 2859 | 76 | TAA | 0 | H | ||
ND1 | 2860 | 3834 | 975 | ATG | TAA | 0 | H | |
tRNAlle | 3840 | 3911 | 72 | GAT | 4 | H | ||
tRNAGln | 3910 | 3980 | 71 | TTG | -2 | L | ||
tRNAMet | 3983 | 4051 | 69 | CAT | 2 | H | ||
ND2 | 4052 | 5096 | 1045 | ATG | T– | 0 | H | |
tRNATrp | 5097 | 5168 | 72 | TCA | 0 | H | ||
tRNAAla | 5171 | 5239 | 69 | TGC | 2 | L | ||
tRNAAsn | 5241 | 5313 | 73 | GTT | 1 | L | ||
tRNACys | 5349 | 5415 | 67 | GCA | 35 | L | ||
tRNATyr | 5415 | 5486 | 72 | GTA | -1 | L | ||
COI | 5488 | 7038 | 1551 | GTG | TAA | 1 | H | |
tRNASer | 7039 | 7109 | 71 | TGA | 0 | L | ||
tRNAAsp | 7113 | 7184 | 72 | GTC | 3 | H | ||
COII | 7197 | 7887 | 691 | ATG | T– | 12 | H | |
tRNALys | 7888 | 7963 | 76 | TTT | 0 | H | ||
ATP8 | 7965 | 8129 | 165 | ATG | TAG | 1 | H | |
ATP6 | 8123 | 8805 | 683 | GTG | TA- | -7 | H | |
COIII | 8806 | 9590 | 785 | ATG | TAA | 0 | H | |
tRNAGly | 9591 | 9662 | 72 | TCC | 0 | H | ||
ND3 | 9663 | 10011 | 349 | ATG | T– | 0 | H | |
tRNAArg | 10012 | 10081 | 70 | TCG | 0 | H | ||
ND4L | 10082 | 10378 | 297 | ATG | TAA | 0 | H | |
ND4 | 10372 | 11752 | 1381 | ATG | T– | -7 | H | |
tRNAHis | 11753 | 11821 | 69 | GTG | 0 | H | ||
tRNASer | 11822 | 11890 | 69 | GCT | 0 | H | ||
tRNALeu | 11892 | 11964 | 73 | TAG | 1 | H | ||
ND5 | 11965 | 13788 | 1824 | ATG | TAA | 0 | H | |
ND6 | 13785 | 14306 | 522 | ATG | TAG | -4 | L | |
tRNAGlu | 14307 | 14375 | 69 | TTC | 0 | L | ||
Cytb | 14381 | 15521 | 1141 | ATG | T– | 5 | H | |
tRNAThr | 15522 | 15594 | 73 | TGT | 0 | H | ||
tRNAPro | 15594 | 15663 | 70 | TGG | -1 | L | ||
D-loop | 15664 | 16598 | 935 | 0 | H |
We assessed the amino acid (AA) codon usage by calculating the relative synonymous codon usage (RSCU) values in 13 PCGs, which are shown in Fig.
Comparison of codon usage in mitochondrial genomes of Onychostoma lepturum a Relative synonymous codon usage (RSCU) in the Onychostoma lepturum mitogenome. Codon families are provided on the X-axis, and the RSCU values, on the Y-axis b Codon distribution in the Onychostoma lepturum mitogenome. CDspT, codons per thousand codons. Codon families are provided on the X-axis.
Comparative analysis of nucleotide base composition showed that the composition of O. lepturum is identical to that of the other Onychostoma fishes, and most of the genes within these Onychostoma species maintain a consistent position and direction (Table
Nucleotide composition of the Onychostoma lepturum mitochondrial genome.
Length (bp) | T% | C% | A% | G% | A+T% | AT-skew% | GC-skew% | |
---|---|---|---|---|---|---|---|---|
Genome | 16598 | 24 | 28.6 | 31.3 | 16.2 | 55.3 | 0.132 | 0.277 |
PCGs | 11409 | 25.7 | 29.4 | 29 | 15.8 | 54.7 | 0.06 | -0.3 |
1st codon position | 3807 | 20.3 | 27 | 26.8 | 25.8 | 47.1 | 0.138 | -0.023 |
2nd codon position | 3802 | 40.1 | 27.7 | 18.6 | 13.6 | 58.7 | -0.366 | -0.341 |
3rd codon position | 3800 | 16.8 | 33.6 | 41.7 | 8 | 58.5 | 0.423 | -0.615 |
rRNA | 2642 | 19.1 | 25.8 | 34.5 | 20.6 | 53.6 | 0.287 | -0.112 |
tRNA | 1566 | 26.6 | 21.5 | 28.2 | 23.7 | 54.8 | 0.029 | 0.049 |
D-loop | 935 | 32.5 | 20.3 | 34 | 13.2 | 66.5 | 0.022 | -0.212 |
The 22 tRNA genes in the O. lepturum mitogenome are interspersed between rRNA and protein-coding genes, with sizes ranging from 67 to 76 bp; tRNACys was the shortest (67 bp), while tRNALeu and tRNALys were the longest (76 bp). The arrangement of 8 L-strand encoded and 14 H-strand-encoded tRNA genes is similar to the distributions observed in other Onychostoma species. Two rRNA genes were identified on the L-strand in O. lepturum, which is similar to the other Onychostoma species, with a total length of 2642 bp. The 16S rRNA is located between tRNAVal and tRNALeu, with a length of 1681 bp, whereas the 12S rRNA is located between tRNAPhe and tRNAVal, with a length of 961 bp. Regarding the two rRNA genes, the GC-skew is slightly negative (–0.112), but the AT-skew is strongly positive (0.287). The total A+T content of the rRNA genes (53.1%) is lower than those of the total tRNA genes (55.0%) and the total PCG genes (55.6%).
To further investigate the phylogenetic position of O. lepturum within the genera Acrossocheilus and Onychostoma, the concatenated set of nucleotide sequences of available whole mitochondrial genomes from 10 Onychostoma species, 16 Acrossocheilus species and F. brevifilis were derived for phylogenetic reconstruction, along with S. hollandi as an outgroup. The ML, NJ and BI analyses showed the same topology, representing the three main lineages (Clades I, II and III). The phylogenetic tree revealed that Clade I is the Onychostoma group, which can be separated into two subclades (subclades I-A and I-B) with strong support (Fig.
The total length of the Onychostoma mitogenomes ranged between 16,590 (O. rarum) and 16,601 bp (O. simum and O. gerlachi), while that of the O. lepturum is a typical closed circular DNA molecule with a length of 16,598 bp (Fig.
Species | ND1 | ND2 | COI | COII | ATP8 | ATP6 | COIII | ND3 | ND4L | ND4 | ND5 | ND6 | Cytb | GenBank |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
O. lepturum | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | GTG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | MT258556 |
O. macrolepis | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAA | ATG/T– | KF999680.1 |
O. lini | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAA | ATG/T– | JQ343982.1 |
O. barbatum | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAA | ATG/T– | JX646870.1 |
O. fangi | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAA | ATG/T– | AP011193.1 |
O. barbatulum | ATG/TAG | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | KC896762.1 |
O. alticorpus | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | GTG/TAA | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | KC791686.1 |
O. rarum | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | GTG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAA | ATG/T– | KF626377.1 |
O. simum | ATG/TAG | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | GTG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | KF021233.1 |
O. gerlachi | ATG/TAG | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | GTG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | KP244449.1 |
O. meridionale | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | GTG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | AP011351.1 |
O. brevibarba | ATG/TAA | ATG/T– | GTG/TAA | ATG/T– | ATG/TAG | ATG/TA- | ATG/TA- | ATG/T– | ATG/TAA | ATG/T– | ATG/TAA | ATG/TAG | ATG/T– | MG523272.1 |
Comparison was made between the phylogenetic trees constructed by
Recent studies have revealed similar scenarios in the genetic patterns of Garra orientalis (
This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 108-2221-E-035-057 and MOST 109-2221-E-035-034). There are no conflicts of interest in this study.
Table S1
Data type: DNA
Explanation note: Composition and skewness in the genus Onychostoma mitogenomes.