Complete mitochondrial genome and phylogenetic analysis of Mystacoleucus lepturus (Teleostei, Cypriniformes, Cyprinidae)

Abstract

In this study, we aimed to sequence and annotate the complete mtDNA genome sequence of Mystacoleucus lepturus, which were collected from Luosuojiang River in Menglun area, Yunnan Province, China. The mitochondrial genome was 16,592 bp in length, comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and two non-coding regions (origin of L-strand replication and control region). The whole genome contained C (26.5%), A (32.5%), T (25.3%), and G (15.7%), with an obvious A + T bias (57.8%). Based on the concatenated amino acids sequences of 13 PCGs of M. lepturus and other 22 fishes, a phylogenetic tree was reconstructed using the maximum-likelihood method. The result of phylogenetic analysis supported a close relationship between M. lepturus and M. marginatus. The fundamental genetic data of M. lepturus would be useful for conservation and phylogeny.

Mystacoleucus lepturus (Huang 1979) belongs to the subfamily Poropuntiinae (Cypriniformes, Cyprinidae). It is widely distributed in Yunnan Province of China; Lao People's Democratic Republic and Thailand. M. lepturus has most recently been assessed for The IUCN Red List of Threatened Species in 2007, and is listed as vulnerable under criteria A2ce (Jenkins et al. 2009). The typical features that distinguish M. lepturus from morphology perspective are dorsal fin rays iv-8; anal fin rays iii-8-9; pectoral fins i-12-15; ventral fin rays ii-8; predorsal scales 8–10; pericaudal handle scale 14; half-moon shaped black brown spots at the base of redorsal scales; black dorsal fin thorn and outer edge of dorsal fin; black narrow edge for inner edge of caudal fin (Wu et al. 1977; Le 2000). Kong et al. (2003) analyzed the Cytb and HVSI sequences of M. lepturus to determine its taxonomic status, and the conclusion was consistent with Huang (1979). This project first determined the complete mitochondrial genome sequence of M. lepturus, and a phylogenetic analysis was accomplished with the available mitogenomes sequences among subfamily Poropuntiinae.

Studies involving laboratory animals followed the ARRIVE guidelines (https://arriveguidelines.org/). The specimens of M. lepturus (voucher number: ASTIH-21B1108D17) were obtained from Luosuojiang River in Menglun area, Yunnan Province, China (21.660199 N, 101.343714 E) on 15 October 2021. The specimen was preserved in 95% ethanol and stored at Aquatic Science and Technology Institution Herbarium (https://www.jsahvc.edu.cn/, XJ Chen, email: 2007020030@jsahvc.edu.cn). Total genomic DNA was extracted from muscle tissues using Tguide Cell/tissue genomic DNA Extraction Kit (OSR-M401) (Tiangen, Beijing, China) and stored in a deep freezer at −80 °C. The extracted DNA was subjected to sample quality control, DNA library was subsequently constructed and amplified by PCR, followed by size selection and library quality check, finally library pooling and sequencing were carried out on Illumina Hiseq platform 2500 (Genesky Biotechnologies Inc., Shanghai, China). The next-generation sequencing raw data were assembled using MetaSPAdes 3.13.0 (Nurk et al. 2017), and the assembled mitogenome sequences were annotated using the MitoMaker 1.14 (Bernt et al. 2013).

The complete mitogenome of M. lepturus was 16,592 bp in length, containing 13 protein-coding genes (PCGs, 3795 bp), 22 transfer RNA genes (tRNAs, 1565), two ribosomal RNA genes (rRNAs, 2,605 bp), and two non-coding regions (an origin of L-strand replication, 32 bp, and a control region, 817 bp). The overall base composition of the M. lepturus was C (26.5%), A (32.5%), T (25.3%), and G (15.7%), with an obvious A + T bias (57.8%). The origin of L-strand replication located between tRNA^Asn and tRNA^Cys, and the control region located between tRNA^Phe and tRNA^Pro. Among all 37 genes, nine genes (tRNA^Gln, tRNA^Ala, tRNA^Asn, tRNA^Cys, tRNA^Tyr, tRNA^Ser(UCN), tRNA^Glu, tRNA^Pro, and ND6) were encoded on L-strand, the other 28 genes were encoded on the H-strand. The length of 13 PCGs ranged from 165 bp (ATP8) to 1824 bp (ND5). Most PCGs initiated with ATG except that COI gene initiated with GTG. As for stop codons, seven genes (ND1, COI, ATP6, COIII, ND4L, ND5, and ND6) used TAA, three genes (ND2, ATP8, and ND3) used TAG, and three genes (CO II, ND4, and Cyt b) contained an incomplete termination codon (T). The length of tRNA ranged from 67 bp (tRNA^Cys) to 77 bp (tRNA^Lys), while the rRNAs were 952 bp (12S rRNA) and 1653 bp (16S rRNA) (Table 1).

Table 1.

Relevant features of Mystacoleucus lepturus mitochondrial genome.

	Position					Codon
Gene	From	To	Nucleotide size (bp)	Amino acid	Space (+) overlap (–)	Initial	Terminal	Strand
tRNAPhe	1	69	69		0			H
12SrRNA	70	1021	952		0			H
tRNAVal	1024	1095	72		2			H
16SrRNA	1118	2770	1653		22			H
tRNALeu(UUR)	2788	2863	76		17			H
ND1	2864	3838	975	325	0	ATG	TAA	H
tRNAIle	3843	3914	72		4			H
tRNAGln	3913	3983	71		–2			L
tRNAMet	3985	4053	69		1			H
ND2	4054	5100	1047	348	0	ATG	TAG	H
tRNATrp	5099	5169	71		–2			H
tRNAAla	5172	5240	69		2			L
tRNAAsn	5242	5314	73		1			L
OL	5317	5348	32		2			–
tRNACys	5348	5414	67		–1			L
tRNATyr	5414	5484	71		–1			L
CO I	5486	7036	1551	516	1	GTG	TAA	H
tRNASer(UCN)	7037	7107	71		0			L
tRNAAsp	7111	7182	72		3			H
CO II	7195	7885	691	230	12	ATG	T	H
tRNALys	7886	7962	77		0			H
ATPase8	7964	8128	165	54	1	ATG	TAG	H
ATPase6	8122	8805	684	227	–7	ATG	TAA	H
CO III	8805	9590	786	261	–1	ATG	TAA	H
tRNAGly	9590	9661	72		–1			H
ND3	9662	10012	351	116	0	ATG	TAG	H
tRNAArg	10011	10080	70		–2			H
ND4L	10081	10377	297	98	0	ATG	TAA	H
ND4	10371	11751	1381	460	–7	ATG	T	H
tRNAHis	11752	11820	69		0			H
tRNASer(AGY)	11821	11889	69		0			H
tRNALeu(CUN)	11891	11963	73		1			H
ND5	11967	13790	1824	607	3	ATG	TAA	H
ND6	13787	14308	522	173	–4	ATG	TAA	L
tRNAGlu	14309	14377	69		0			L
Cyt b	14383	15523	1141	380	5	ATG	T	H
tRNAThr	15524	15596	73		0			H
tRNAPro	15596	15665	70		–1			L
Control region	15681	16497	817		0			–

In order to verify the evolutionary relationship, the whole mitochondrial genomes of 18 fish species from nine genera (Mystacoleucus, Sikukia, Poropuntius, Discherodontus, Cyclocheilichthys, Cosmochilus, Puntioplites, Puntius, and Sinocyclocheilus) were selected. Based on the connected amino acids of 13 PCGs, the phylogenetic tree was conducted using the maximum-likelihood method and by MEGA X software (Kumar et al. 2018), and the model (mtREV + G+I) with the lowest Bayesian information criterion (BIC) scores was considered to describe the substitution pattern the best (Jones et al. 1992; Adachi and Hasegawa 1996), with a bootstrap of 1000 replicates. The result of phylogenetic analysis confirmed that M. lepturus clustered with M. marginatus, and they formed a sister-group with the genus Sikukia (S. gudgeri), then the above three species formed a sister-group with the genus Poropuntius (P. bantamensis and P. normani). M. lepturus was closer to M. marginatus (Figure 1). Presently, the studies on M. lepturus were limited, and we believed that the fundamental genetic data in this study would be beneficial for further studies on population genetics and evolution of the subfamily Poropuntiinae as well as resource conservation.

Figure 1.

A phylogenetic tree was reconstructed for nine genera (Mystacoleucus, Sikukia, Poropuntius, Discherodontus, Cyclocheilichthys, Cosmochilus, Puntioplites, Puntius, and Sinocyclocheilus), Sinocyclocheilus (S. altishoulderus and S. graham) was used as outgroup, using the maximum-likelihood (ML) method based on the connected aminoacids of 13 PCGs. The tree topology was evaluated by 1000 bootstrap replicates. The species names were followed by their GenBank accession numbers, and the numbers at the nodes represented bootstrap values.

Ethical approval

Experiments were approved by the Ethical Committee for Animal Experiments of Jiangsu Agri-animal Husbandry Vocational College and conducted following the Chinese Association for the Laboratory Animal Sciences and the Institutional Animal Care and Use Committee (IACUC) protocols.

Author contributions

Conception and design, XJ Chen and L Song; data curation, XJ Chen; analysis and interpretation of the data, L Song and XJ Chen; funding acquisition, XJ Chen; writing – original draft, XJ Chen, L Song; writing – review and editing, XJ Chen, L Song. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding Statement

This work was supported by the 311 Talents Project in Taizhou City [2017III-804]; the Research Project of Jiangsu Agri-animal Husbandry Vocational College [NSF2021ZR14] and [NSFHP202102]; the Excellent Teaching Team of Jiangsu University Blue Project [2021-11].

Disclosure statement

The authors declare no potential conflict of interest.

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession no. OM617721.1. The associated ‘BioProject’, ‘Bio-Sample’, and ‘SRA’ numbers are PRJNA805112, SAMN25829074, and SRR17970748, respectively.