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. 2025 Sep 13;10(10):937–941. doi: 10.1080/23802359.2025.2559714

Complete mitochondrial genome of schistura yingjiangensis (Zhu 1982) (cypriniformes: nemacheilidae): insights into its features and phylogenetic relationships

Chunxiang Yang a,b, Xiangxing Lu a,b, Guangming Wu c, Chunpeng Li b,d, Jianyu Song a,b,✉, Junjie Wu a,b,✉
PMCID: PMC12434845  PMID: 40959690

Abstract

This study sequenced and analyzed the complete mitochondrial genome (16,571 bp) of Schistura yingjiangensis, a species endemic to Yunnan, China. The genome contains 13 protein-coding genes, 22 tRNAs, and 2 rRNAs. Phylogenetic analysis revealed a close relationship between S. yingjiangensis, S. polytaenia, and S. longa. This work helps clarify the evolution of the large, paraphyletic genus Schistura and contributes to understanding the evolutionary dynamics of Cypriniformes.

Keywords: Schistura yingjiangensis, mitochondrial genome, phylogenetics, evolutionary biology

Introduction

Mitochondrial DNA (mtDNA) has been widely used in phylogenetic studies due to its maternal inheritance, high mutation rate, and lack of recombination (Alvarenga et al. 2024). The complete mitochondrial genome provides a comprehensive resource for understanding the evolutionary history and genetic diversity of species (Boore 1999). Schistura yingjiangensis (Zhu 1982) is an small endemic freshwater loach distributed in the Mengdian River and Daying River of China, classified to the Cypriniformes, family Nemacheilidae (Zhu 1982; Chen 2013). A decline in the taxonomic diversity of fish assemblages has been observed in the Daying River. In response, the Binlangjiang Aquatic Germplasm Resource Protection Area was established in 2008 to specifically protect fish species in the Daying River, including S. yingjiangensis (Cao 2000; Yang et al. 2016). Although the mitochondrial genomes of other Schistura have been analyzed in previous studies (Siva et al. 2018), ; Sharma et al. 2020; Peng et al. 2024; the mitochondrial genome of S. yingjiangensis has not been previously characterized. Here, we present the complete mitochondrial genome of S. yingjiangensis and explore its phylogenomic and evolutionary relationships within the Nemacheilidae.

Materials and methods

Sample collection and DNA extraction

The specimens of S. yingjiangensis was collected in Yingjiang River (N: 24°44′39′′, E: 98°3′1′′), Yingjiang, Yunnan, China. The status of the collected specimens was recorded, comprising 3 live individuals. The three live specimens ranged from 20.6 to 22.7 cm in total length (TL) and 17.9 to 19.5 cm in standard length (SL). Total genomic DNA was extracted from muscle tissue using TIANamp Genomic DNA Kit (TIANGEN Co., Ltd, Beijing, China) following the manual’s protocol. The quality and quantity of the DNA were assessed using agarose gel electrophoresis and spectrophotometry. Three samples were deposited in the Yunnan Institute of Fishery Sciences Research (contact person: Chunxiang Yang, miehuoqi1995@outlook.com) under voucher number YJNQ-01-03. The descriptive image of a sample was captured by Junjie Wu and Jianyu Song (corresponding authors) (Figure 1).

Figure 1.

Figure 1.

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A reference image of S. yingjiangensis used in this study was collected by Junjie Wu in the Daying River (N: 24°44′39″, E: 98°3′1″), yingjiang, Yunnan, China. The reference image was photographed by Junjie Wu and Jianyu Song.

Mitogenome sequencing and assembly

Genomic DNA was extracted from muscle tissue using the TIANamp Genomic DNA Kit (TIANGEN Co., Ltd, Beijing, China) following the manufacturer’s protocol. The abundance and quality of DNA were assessed using the NanoPhotometer® N60 spectrophotometer. The Illumina library was constructed following the manufacturer’s protocols with the TruSeq™ Nano DNA Sample Preparation Kit (Illumina, U.S.) (Meyer and Kircher 2010). Subsequently, the NovaSeq platform was employed to generate raw sequencing reads (Modi et al. 2021), with the sequencing services provided by Tsingke Co., Ltd. (Beijing, China). The raw reads were assembled using MitoZ 3.6 (Meng et al. 2019), and the mitogenome was annotated using MITOS with the default parameters (Bernt et al. 2013).

Phylogenetic analysis

The phylogenetic relationship of S. yingjiangensis was inferred by using the 13 protein-coding gene (PCGs) sequences from 63 related species. Multiple sequence alignment was performed using MAFFT with default parameters (Katoh et al. 2019), and phylogenetic trees were constructed using the maximum likelihood (ML) methods as implemented in IQ-TREE with the an effective stochastic algorithm and 5000 bootstrap duplications (Nguyen et al. 2015; Kalyaanamoorthy et al. 2017).

Results

Mitogenome organization and composition

The complete mitochondrial genome of S. yingjiangensis is 16571 bp in length with a depth of 678.9x (Supplementary Figure 1) and exhibits the typical circular structure found in vertebrates. It contains 13 PCGs, 22 tRNA genes, 2 rRNA genes, and a control region (D-loop). The overall base composition is 30.82%A, 24.92%T, 16.51%G, 27.75%C, with an AT skew. Among the 13 protein-coding genes (PCGs), 12 initiated with the start codon ATG, while COI is transcribed with GTG. Six PCGs (ND1, ND4L, ND5, ND6, COI, and ATP8) had complete stop codons (TAA). Incomplete stop codons were identified in ND2, ND3, ND4, ATP6, COII, COIII, and CYTB. Additionally, 12 PCGs (excluding ND6) and 14 tRNA genes were encoded on the heavy strand (Figure 2). The gene order and orientation are consistent with other fish species in the genus Schistura.

Figure 2.

Figure 2.

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The complete mitochondrial genome map of S. yingjiangensis (GenBank: PP114298) is presented. Encoded genes and RNAs are depicted in different colors. The light and heavy strands are shown on the inner and outer sides of the circle, respectively.

Phylogenetic analysis

The maximum likelihood (ML) tree revealed that all members of the genus Schistura formed a monophyletic group, such as S. balteata, S. geisleri, S. pridii, S. jarutanini, S. longa, S. polytaenia, S. reticulofasciata, S. scaturigina, S. sikmaiensis, and S. notostigma, were clustered within the Schistura clade, with strong bootstrap support. Phylogenetic analysis indicated that S. yingjiangensis is closely associated with the Schistura clade that includes S. polytaenia and S. longa (Figure 3). These findings offer novel perspectives on the evolutionary relationships within the Nemacheilidae family.

Figure 3.

Figure 3.

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Phylogenetic tree of S. yingjiangensis and other 62 species based on the complete mitogenomes from NCBI. Misgurnus anguillicaudatus and misgurnus bipartitus were treated as outgroups. The bootstrap values were marked near the nodes. Accession numbers for each species were listed following the name of the species. The following sequences were used: B.barbatula (NC 027192, (Murienne et al. 2016)), B. nuda (NC 022858, (Zhao et al. 2015)), B. toni (NC 008670, (Saitoh et al. 2006)), T. alticeps (KT213585, (Wang et al. 2016)), T. chondrostoma (KT213589, (Wang et al. 2016)), T. stoliczkai (NC 017890, (Li et al. 2013)), T. bombifrons (OP499856), T. tenuis (NC 030511), T. dorsalis (NC 029423, (Lei et al. 2016)), T. labiata (NC 073586), T. strauchii (NC 026714, (Kanu et al. 2016)), T. scleroptera (KT213588, (Wang et al. 2016)), T. nujiangensa (KT213598, (Wang et al. 2016)), T. stenura (NC 032692), T. stewarti (NC 030506), T. grahami (PP114297), T. cuneicephala (NC 037924), T. robusta (NC 025632, (Yan et al. 2016)), T. pseudostenura (KT213601, (Wang et al. 2016)), T. nanpanjiangensis (NC 072346), T. nasobarbatula (NC 058005), T. rosa (NC 019587, (Wang et al. 2012)), H. berezowskii (NC 040302), H. potanini (NC 025321, (Que et al. 2016)), H. laxiclathra (NC 046753), H. vatiegata (NC 020095), H. wenshanensis (NC 063102), H. pycnolepis (NC 056344), H. wuliangensis (PP175348), L. costata (NC 029385), L. nikkonis (NC 027662, (Miya et al. 2015)), L.echigonia (NC 004696, (Saitoh et al. 2003)), O.guananensis (NC 073134, (Luo et al. 2023)), O. luochengensis (NC 073128, (Luo et al. 2023)), O. platycephalus (NC 031579), O. polystigmmus (ON116517, (Luo et al. 2023)), T. barbatus (NC 073130, (Luo et al. 2023)), T. donglanensis (NC 073132, (Luo et al. 2023)), T. translucens (NC 073136, (Luo et al. 2023)), T. macrolepis (NC 073129, (Luo et al. 2023)), T. microphthalmus (NC 073127, (Luo et al. 2023)), T. furcocaudalis (NC 073138, (Luo et al. 2023)), T. elongatus (NC 073131, (Luo et al. 2023)), T. jiarongensis (NC 073145, (Luo et al. 2023)), T. longibarbatus (NC 058004), T. lihuensis (NC 077595), T. shuilongensis (NC 073144, (Luo et al. 2023)), S. balteata (NC 008679, (Saitoh et al. 2006)), S.geisleri (NC 029437), S. pridii (NC 031638), S. jarutanini (NC 031584), S. longa (KT213583, (Wang et al. 2016)), S. polytarnia (NC 084194), S. reticulofasciata (KY379150), S. scaturigina (NC 031378), S. sikmaiensis (NC 034746 (Sharma et al. 2020)), S. notostigma (NC 031585), M. anguillicaudatus (NC 011209 (He et al. 2008)), M. mizolepis (NC 038151 (Lee 2016)).

Discussion

In this study, The mitogenome of S. yingjiangensis exhibits high conservation in gene order, base composition, and AT bias (55.74% AT content), consistent with the characteristics of most freshwater fish mitogenomes (Wang et al. 2016; Siva et al. 2018;). Notably, the COI gene in S. yingjiangensis starts with GTG, while other PCGs use ATG as the initiation codon, a feature shared with other Schistura species such as S. fasciolata, S. sikmaiensis and S. reticulofasciata (Siva et al. 2018; Sharma et al. 2020; Peng et al. 2024; Additionally, the incomplete stop codons ‘T’ in ND2, ND3, ND4, ATP6, COII, COIII, and CYTB genes are likely completed to TAA through post-transcriptional polyadenylation, a common phenomenon in fish mitogenomes (Broughton et al. 2001).

Phylogenetic analysis reveals that the evolutionary relationship of S. yingjiangensis is close to S. polytaenia and S. longa, which corroborates the results of previous study (Peng et al. 2024). These findings contribute vital genetic data crucial for elucidating the evolution and taxonomy within the genus Schistura.(Wang et al. 2016)

Conclusion

This study presents the first complete mitogenome of S. yingjiangensis, enriching the Schistura genetic database and providing valuable insights into the species’ phylogenetic relationships, evolutionary biology, and conservation, thereby contributing to the broader understanding of fish mitochondrial genome evolution and laying a foundation for future research.

Supplementary Material

Supplemental Material
TMDN_A_2559714_SM2245.tif (18.3MB, tif)

Acknowledgments

The authors would like to thank Tsingke Biotechnology Co., Ltd. (Beijing, China) for providing excellent services and support in the generation of the genome skimming data.

Funding Statement

This work was supported by the first systematic survey of aquaculture germplasm resources in Yunnan Province and Selection of sex-specific molecular markers using chromosomal characteristics in the Parachromis managuensis and new variety breeding (202401AT070091).

Ethical approval

Animals were live and well cared for before and after the experiment. All experimental procedures involving animals were approved (animal protocol number: 202302) by the Ethics Committee and Animal welfare of Yunnan Institute of Fishery Sciences Research. The sample in this study did not involve protected and endangered animals. Obtaining the sample was legal and the process of this research was in line with the Chinese Association for the Laboratory Animal Sciences and the Institutional Animal Care and Use Committee (IACUC) protocols.

Author contribution statement

CRediT: Chunxiang Yang: Conceptualization, Data curation, Formal analysis, Resources, Validation, Visualization, Writing – original draft; Xiangxing Lu: Resources, Supervision; Guangming Wu: Investigation, Resources; Chunpeng Li: Formal analysis, Investigation; Jianyu Song: Investigation, Resources, Supervision, Validation; Junjie Wu: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The complete mitochondrial genome assembly data of S. yingjiangensis was available in GenBank database under the accession number PP114298, the associated BioProject, BioSample and SRA numbers are PRJNA1123854, SAMN41829188 and SRR29409023, respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
TMDN_A_2559714_SM2245.tif (18.3MB, tif)

Data Availability Statement

The complete mitochondrial genome assembly data of S. yingjiangensis was available in GenBank database under the accession number PP114298, the associated BioProject, BioSample and SRA numbers are PRJNA1123854, SAMN41829188 and SRR29409023, respectively.

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