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 À80 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 nextgeneration 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 þ 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 Lstrand, 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). 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 þ GþI) 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.

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; writingoriginal draft, XJ Chen, L Song; writingreview 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.