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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

The Complete Mitochondrial Genome of Schistura fasciolata: Phylogenetic and Evolutionary Implications within Tribe Nemacheilini

C
Chao-Yang Luo1
M
Mei-Zhong Li2
Y
Yekang Sun1
Y
Yuan Mu1,3,*
https://orcid.org/0009-0008-6515-3100
W
Wen-Xian Hu4,*
1Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali, Yunnan 671003, China.
2Baoshan Aquaculture Workstation, Yunnan Baoshan 678000, China.
3Collaborative Innovation Center for Biodiversity and Conservation in the Three Parallel Rivers Region of China, Dali, Yunnan 671006, China.
4Kunming University of Science and Technology, Kunming 650500, Yunnan, China.

ABSTRACT

Background: The mitochondrial genome is widely used to understand the phylogeny and evolution of fish. This study aims to deepen the understanding of the mitochondrial genome of Schistura fasciolata and the evolutionary implications within Nemacheilini.

Methods: This study utilized high-throughput sequencing technology and bioinformatics analysis to investigate the mitochondrial genome of the Schistura fasciolata. The aim was to reveal its structural or compositional characteristics, phylogenetic location and evolutionary rate.

Result: We conducted a comprehensive sequence analysis of the complete mitochondrial genome of S. fasciolata, in which it contains 16,570 base pairs (bp) including 37 genes, i.e., 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs). The base composition analysis indicated an A+T content of 56.51% and a G+C content of 43.49%. Phylogenetic analysis based on the 13 PCGs revealed extensive polyphyly within Nemacheilini. Furthermore, accelerated evolution in ND4, ND5 and CYTB genes were observed in Homatula and Troglonectes, suggesting an adaptation to specific environmental conditions. This study provides critical insights into the phylogeny and adaptive evolution within Nemacheilini.

INTRODUCTION

Nemacheilidae, an ecologically significant and taxonomically complex family of freshwater fish, mainly distributed across mainland Asia and its adjacent islands, Europe and northeast Africa (Kottelat, 2012; Basudha et al., 2025). The species in this lineage live in various habitats, including swamps, rapids, rivers, streams, caves and lakes (Kottelat, 2012; Eschmeyer, 2020). The family consists of nearly 760 species in 47 genera (Prokofiev, 2010; Zhang and Zhao, 2016; YoGurtCuoGlu et al., 2020).
       
Prokofiev performed a detailed comparative morphological analysis (Prokofiev, 2010), which divided Nemacheilidae into five tribes: Vaillantellini, Lefuini, Yunnanilini, Triplophysini and Nemacheilini (Zhang and Zhao, 2016; Lan et al., 2013; Zhu and Zhu, 2014; Sgouros et al., 2019; Du et al., 2021; Min et al., 2023). However, the challenges of valid species identification exist due to morphological and genetic diversity, especially within the tribe Nemacheilini (Sgouros et al., 2019; Chen et al., 2019). Besides, the distribution also has led to taxonomic issues, especially in Homatula, Nemacheilus and Schistura (Sgouros et al., 2019; Chen et al., 2019; Liu et al., 2012). Thus, the species identification, taxonomy, phylogenetic relationship has been always controversial within tribe Nemacheilini. Moreover, the extensive polyphyly has been observed in some genera, such as Homatula, Nemacheilus and Schistura, which further complicates the taxonomy (Liu et al., 2012; Min et al., 2012). Many studies focus on the complex phylogenetic relationships usually based on some mitochondrial gene fragments and/or morphological traits (Sember et al., 2015; Sgouros, 2016). However, the confusion has been always discussed due to incomplete sample coverage, inadequate analysis methods and lack of data, especially the short molecular fragments.
               
Mitochondrial DNA (mtDNA), known for its simple structure, maternal inheritance, rapid evolution and short coalescence time, is crucial for species identification and phylogenetic analysis in fish (Satoh et al., 2016), which helps assess genetic diversity, phylogeny and evolution (Xiao and Zhang, 2000; Yue et al., 2006; Zhu and Yue, 2008; Vani et al., 2022). In this study, we obtained the complete mitochondrial genome of S. fasciolata and then constructed the phylogenetic tree of tribe Nemacheilini based on 13 PCGs, meanwhile, the evolutionary rate of each genus in Nemacheilini was evaluated. This study aims to: (1) analysis the mitogenomic structure of S. fasciolata and (2) explore its phylogeny and adaptation within this lineage.

MATERIALS AND METHODS

Sample collection and genomic DNA extraction
 
Adult specimens of S. fasciolata were collected from Jianchuan, Yunnan, China (26.25788065°N, 99.82247990°E) in May 25, 2023. Identification of adult specimens was based on morphological characteristics based on FishBase (https://fishbase.de/search.php). The samples were stored in 100% ethanol and then were sent to Shanghai Personal bio technology Co. for genomic DNA extraction and sequencing. The Whole Genome Shotgun (WGS) strategy was used to construct a library of different insertions with a length of 400 bp. These libraries were Paired-end reads (PE, 150 bp in length) sequenced using Next-Generation Sequencing (NGS), based on the Illumina NovaSeq sequencing platform. Subsequently, the raw sequencing data was filtered to generate high-quality sequences.
 
Sequence assembly, annotation and analyses
 
High quality second-generation sequencing data were reconstructed from scratch using A5-miseq v20150522 (David et al., 2014) and SPAdes v3.9.0 (Bankevich et al., 2012) to construct contig and scaffold sequences. Moreover, the sequences were extracted according to the sequencing depth of the splicing sequences and the sequences with high sequencing depth were compared with the NT library on NCBI by blastn (BLAST v2.2.31 +) and the mitochondrial sequences of each splicing result were selected. Secondly, mummer v3.1 (Delcher et al., 2003) software was used to carry out collinearity analysis of mitochondrial splicing results obtained by the above different software, so as to determine the position relationship between contigs and fill the gap between contigs. The results were corrected using pilon v1.18 (Walker et al., 2017) software to obtain the final mitochondrial sequence, the “fasta” file. Then, the whole mitochondrial sequence was submitted to NCBI (Accession No. OR797719.1). Subsequently, the complete mitochondrial genome sequence obtained by splices was uploaded to the MITOS web server (http://mitos.bioinf.uni-leipzig.de/) for functional annotation (Matthias et al., 2012). The Genetic Code selection is set to 02-vertebrate and the rest of the settings follow the default parameters set by MITOS. At the same time, in the MITOS web server, click Download each tRNA, structure download svg format file, you can view each predicted tRNA secondary structure.
       
In addition, PhyloSuite v1.2.3 software was used to compute the mitochondrial whole genome, the protein-coding genes and the base composition of the rRNA genes, respectively (Zhang et al., 2020; Xiang et al., 2023). The strand asymmetry was calculated by using the formulas:
 
The whole mitochondrial genome was mapped using CGView visualization software (Paul and David, 2005).
 
Phylogenetic analysis
 
To investigate the position of S. fasciolata within Nemacheilini and its phylogenetic relationships, we retrieved mitochondrial genome sequences of 12 Nemacheilini genera and used Triplophysa bombifrons and T. labiata as outgroups from NCBI. We utilized the sequences of 13 protein-coding genes (PCGs) from 58 species to construct the phylogenetic tree. Initially, sequences were processed using MEGA 7 software (Kumar et al., 2018). Following alignment of PCG sequences with the MAFFT algorithm (Katoh et al., 2019), we conducted Bayesian inference (BI) analyses using PhyloSuite v1.2.3 (Zhang et al., 2020). Four independent runs were performed, each for 20 million generations, sampling the phylogenetic trees every 1,000th generation, which resulted in 20,000 trees. The first 25% of the trees were discarded as burn-ins. BI Posterior Probability (PP) ≥0.8 was deemed significant. Convergence of the BI analyses was assessed by the average standard deviation of split frequencies <0.01 and Tracer v1.5 (Rambaut et al., 2018) was used to investigate the convergence of the BI analyses, ensuring that the effective sample size (ESS) for all parameters was >200. We also reconstructed phylogenetic relationships using Maximum Likelihood (ML) methods in PhyloSuite v1.2.3. ML phylogenetic trees were generated in IQ-TREE (Nguyen et al., 2015) using the ultrafast bootstrap (BS) algorithm with 1000 replications. ML bootstrap values (BS) ≥75 were deemed significant, indicating that higher values correlate with increased topology reliability (Wang et al., 2023). All analyses were performed in PhyloSuite v1.2.3 (Zhang et al., 2020). Finally, the phylogenetic trees were visualized and refined using FigTree v1.4.4 software (http://tree.bio.ed.ac.uk/software/Figtree/). This phylogenetic tree was used in next-step selective analysis.
 
Selection analyses
 
We used the CodeML program in PAML v4.9 (Yang, 2007) to assess the selection pressure on 13 PCGs sequences of mitochondria from 58 species and calculated the evolution rate (namely, the ratio of nonsynonymous substitution and synonymous substitution, dN/dS) of each branch by using the free-ratio model. In order to evaluate whether there were significant differences in the evolution rate of 13 PCGs of each main genus in Nemacheilini, the dN/dS values of 56 extant species were extracted. In order to evaluate the discrepancy accurately, the data of dN or dS = 0 and dN/dS ≥1 were not considered and then Kruskal-Wallis H test was implemented in SPSS to test the difference (IBM Corp N, 2017).

RESULTS AND DISCUSSION

Mitogenome organisation and nucleotide composition
 
The mitochondrial genome of S. fasciolata, a double-stranded, circular molecule measuring 16,570 bp, aligns with the size range typical for the genus Schistura (from S. incerta: 16,561 bp to S. geisleri: 16,819 bp). This mitogenome consists of a canonical set of 13 PCGs, two rRNAs (12S rRNA and 16S rRNA), 22 tRNAs and a control region known as the D-loop. Positioned between the tRNA-Pro and tRNA-Phe genes, the D-loop region serves as a crucial regulatory element for initiating replication and transcription. This organization is typical of metazoan mitochondrial genomes (Wolstenholme, 1992; Watanabe et al., 2014). Notably, the mitochondrial genome analysis reveals the presence of 23 intergenic spacers of variable lengths, including a significant -10 base pair overlap between the ATP8 and ATP6 genes, indicative of direct genetic contiguity (Watanabe et al., 2014; Ballard and Whitlock, 2004). Analysis of the S. fasciolata mitogenome nucleotide composition shows 30.14% adenine (A), 16.69% guanine (G), 26.37% thymine (T) and 26.80% cytosine (C). The A + T content (56.51%) predominates over the G + C content (43.49%), highlighting the genome’s nucleotide bias and providing insights into its evolutionary adaptations and constraints.
       
Additionally, the mitogenome comprises two ribosomal RNAs, 12S rRNA (952 bp) and 16S rRNA (1,657 bp) (Table 1). Positioned between tRNA-Phe and tRNA-Leu and separated by tRNA-Val, these rRNAs have high A/T contents of 50.32% and 55.94%, respectively. Their positioning and nucleotide composition, indicated by a positive AT-skew and a negative GC-skew, are essential for the structural and functional integrity of ribosomal RNA, crucial for protein synthesis (Xiao and Zhang, 2000).

Table 1: Analysis of mitochondrial genome characteristics of S. fasciolata.


 
Transfer RNAs, ribosomal RNAs
 
The S. fasciolata mitochondrial genome contains 22 tRNA genes, ranging in size from 66 bp (tRNA-Cys) to 76 bp (tRNA-Lys), each displaying the classic cloverleaf secondary structure (Table 1; Fig 1). This set highlights the structural integrity and functional importance of mitochondrial tRNAs, particularly the trnA gene’s retention of its dihydrouridine (DHU) arm, demonstrating these molecules conservation across mitochondrial genomes. Despite some tRNAs lacking the D-arm, notably in trnS for AGY/N codons, this trait is widespread, signifying evolutionary adaptations for mitochondrial efficiency (Garey and Wolstenholme, 1989). Further analysis of tRNA base-pairing shows mostly classical Watson-Crick A-U and G-C matches, with deviations like unmatched pairs U-U, U-C, A-A and C-C in various tRNAs, indicating specific evolutionary adaptations. Additionally, a rare C-A pairing in trnF, trnH, trnM and trnR was noted (Fig 1). These observations highlight the nuanced balance between conserving structural features essential for function and introducing variations that may confer advantages in the mitochondrial context.

Fig 1: The predicted secondary structure of 22 tRNAs in the mitochondrial genome of S. fasciolata.


 
Protein-coding genes and codon usage
 
The S. fasciolata mitochondrial genome contains 13 PCGs for mitochondrial function: seven NADH dehydrogenases (ND1-6, ND4L), three cytochrome c oxidases (COI, COII, COIII), two ATP synthase subunits (ATP6, ATP8) and one cytochrome b (CYTB), totaling 16,570 bp. All genes are similar in length and identical in gene arrangement to those in other Schistura species. This arrangement, with 12 PCGs on the major strand and ND6 on the minor strand, exemplifies the coding efficiency and conservation of the mitochondrial genome. The PCGs employ standard start codons, primarily ATN and TTG and their stop codons exhibit specificity for precise gene expression: TAA for COI, ATP8, ATP6, ND1, ND4L and ND5; TAG for ND6; and a unique TA dinucleotide or a single T for the others, suggesting post-transcriptional modifications to complete the standard stop codons (Fig 2).

Fig 2: The complete mitochondrial genome circle map of S. fasciolata.


       
Analysis of Relative Synonymous Codon Usage (RSCU) values provides further insight into the mitogenome’s evolutionary dynamics, revealing codon usage biases that favor genes encoding Ala, Arg, Pro, Ser2, Thr and Leu1 over those encoding Asn, Asp and Cys (Fig 3). This codon usage bias reflects adaptive evolutionary strategies aimed at enhancing translational efficiency and accuracy (Du et al., 2008), underscoring the nuanced interplay between genetic code specificity and mitochondrial function optimization in S. fasciolata.

Fig 3: Relative synonymous codon usage (RSCU) in the mitochondrial genomes of S. fasciolata.


 
Phylogenetic relationships
 
In this study, we reconstructed phylogenetic relationships using ML methods and BI methods, yielding a phylogenetic tree with a single topology and high support rate (with Bootstrap Support (BS) values consistently above 75 and BI posterior probabilities all exceeding 0.85), significantly enhancing our understanding of their phylogenetic relationships (Fig 4).

Fig 4: Phylogenetic analysis of 58 species using 13 PCGs via ML and BI, with node numbers indicating BS and PP values.


       
Phylogenetic analyses show that Acanthocobitis, Barbatula, Nemacheilus and Homatula form a distinct monophyletic lineage, respectively (Fig 4), which was in line with previous studies (Kottelat, 2012; Min et al., 2012; Sgouros et al., 2019; Chen et al., 2019). On the other hand, our study reveals a widespread paraphyly within the tribe Nemacheilini, which is also prevalent across the entire Nemacheilidae family (Zhang and Zhao, 2016; Du et al., 2021). Specifically, Schistura, Troglonectes and Heminoemacheilus form a paraphyletic lineage. Luo et al., (2023) uncovered paraphyly between Heminoemacheilus and Paranemachilus, recommending merging them to ensure the monophyly of Paranemachilus. Conversely, our research challenges the classification of Heminoemacheilus, particularly highlighting H. zhengbaoshani placement within Troglonectes, which also questions Troglonectes previously accepted monophyly (Lan et al., 2013; Du et al., 2008).
       
The extensive paraphyly across tribe Nemacheilini, even family Nemacheilidae, might be related with the rapid evolution, extensive radiation and remarkable adaptability of species, reflected in their diversity and wide ecological niche occupation (Kottelat, 2012; Eschmeyer, 2020; Prokofiev, 2010), as well as geological event (Wang et al., 2016; Wu et al., 2020), niche competition (Fischer, 2000), hybridism (Saitoh et al., 2010) and rapid adaptive evolution in response to environmental pressures (Zhang et al., 2024). These comprehensive factors collectively contribute to the rapid evolution within the family Nemacheilidae. Additionally, the genus Schistura was clustered into two clades in this study (clade 1 and clade 2 in Fig 4), among which the S. incerta (No. MK361215.1) was nested with S. fasciolata (clade 2 in Fig 4), our results align with prior studies (Liu et al., 2012; Min et al., 2012) which imply the paraphyletic origin for genus Schistura. Of cause, it possibly resulted from the complex gene flow (Min et al., 2023) or need redefinition of species. Meanwhile, the same situation for Acanthocobitis botia in genus Acanthocobitis and Barbatula toni in Barbatu.
 
Selection analyses
 
In this study, we examined the evolutionary dynamics of 13 mitochondrial PCGs of five genera in Nemacheilini: Acanthocobitis, Barbatula, Homatula, Schistura and Troglonectes. By applying the M0 model, we found omega (ù) ratios significantly below 1, ranging from 0.00659 (COI) to 0.12080 (ATP8), across the tribe Nemacheilini. This result emphasizes the role of purifying selection in the oxidative phosphorylation (OXPHOS) pathway. Notably, the ATP8 gene showed the highest dN/dS value, indicating potential accelerated evolution, similar with prior research on species such as Glyptothorax macromaculatus (Lv et al., 2018), Orthoptera insects (Chang et al., 2020), Desis jiaxiangi (Li et al., 2021) and Lamprologus (Wang et al., 2023). In contrast, the COI gene displayed the lowest dN/dS value, suggests it may have faced much stronger evolutionary constraints, which consist with (Singh et al., 2017); Yang et al., (2018) and Li (2018). This evidence underlines the extensive use of the COI gene in phylogenetic reconstructions across species groups due to its low mutation rate. Furthermore, M1 model analysis revealed significant dN/dS ratio variability among mitochondrial PCGs, indicating a diversity of selective pressures in Nemacheilini (Table 2). This variability suggests that while some genes are highly conserved due to their critical roles in cellular metabolism and energy production, others may evolve more rapidly, possibly reflecting adaptations to different ecological niches or life-history strategies within the tribe (Zhang et al., 2025).

Table 2: The results of evolution rate of tribe Nemacheilini lineages detected by free ratio model.


       
In order to further evaluate the significance of evolutionary rates among different genus, the Kruskal-Wallis H test was implemented. The results uncovered significance of evolutionary rates for the ND4, ND5, ATP6 and CYTB genes (Table 3). Specifically, the ND4, ND5 and CYTB genes have undergone the accelerated evolution in cave-adapted genera Homatula and Troglonectes. It might be related to the environmental pressures of their cave-dwelling lifestyle, such as low temperatures and low oxygen levels, where it needs to much more accurate energy consumption (Li, 2018; Woo et al., 2013). The studies have confirmed that ND5 interacts with protein subunits, playing a crucial role in regulating respiration (Wu et al., 2016). And, the transmembrane helices in ND4, crucial for the proton transfer process in complex I, interact with at least one lipid molecule (Wu et al., 2016). Consequently, the accelerated evolution of ND4 in Homatula may significantly influence mitochondrial regulation of lipid synthesis and metabolism, potentially conferring a survival advantage in environments with irregular resources. However, the ATP6, ND4, ND5 and CYTB genes exhibit lower evolutionary rates in Acanthocobitis. In summary, our findings underscore the complexity of adaptive evolution within the tribe Nemacheilini, with various taxa exhibiting unique evolutionary trajectories in mitochondrial genes, reflecting their diverse ecological adaptations (Fig 5).

Table 3: Statistical test of evolutionary rates in different genes.



Fig 5: Boxplot of dN/dS of the13 PCGs for 5 main genera.

CONCLUSION

The mitochondrial genome of S. fasciolata was sequenced and analyzed in this study, shedding light on the mitochondrial genome of the tribe Nemacheilini. Notably, the phylogenetic analysis reveals widespread polyphyly within the tribe Nemacheilini. Furthermore, the evolutionary dynamics highlight the intricate nature of adaptive evolution within the tribe and each taxon displaying distinct evolutionary trajectories in mitochondrial genes, indicative of their unique ecological adaptations. Our results have established a foundation for further exploration of evolutionary mechanisms in Nemacheilini, even for Nemacheilidae.
 
Supplementary information
 
The supporting materials in this paper will be obtained in Figshare (https://figshare.com/) based on this DOI: 10.6084/m9.figshare.25285282; https://github.com/qwrtyrev686tfstff/Project-The-Complete-Mitochondrial-Genome-of-Schistura-fasciolata.git.

ACKNOWLEDGEMENT

The authors thanked the anonymous reviewers and the editor for their valuable comments.
 
Fundings
 
Grants supporting this research were received from the Erhai Watershed Ecological Environment Quality Testing Engineering Research Center of Yunnan Provincial Universities to WH and YM (No. DXDGCZX03), as well as the start-up projects on high-level talent introduction of Dali University to YM (No. KY1916101940).
 
Ethical approval
 
This research project was conducted in accordance with the ethical guidelines. And the animal study was reviewed and approved by Laboratory Animal Welfare Ethics Review Committee, Dali University, for research project (No. DXDGCZX03). The ethical certification was shown in “Related files”.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interests regarding the publication of this article.

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    Full Research Article

    The Complete Mitochondrial Genome of Schistura fasciolata: Phylogenetic and Evolutionary Implications within Tribe Nemacheilini

    C
    Chao-Yang Luo1
    M
    Mei-Zhong Li2
    Y
    Yekang Sun1
    Y
    Yuan Mu1,3,*
    https://orcid.org/0009-0008-6515-3100
    W
    Wen-Xian Hu4,*
    1Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali, Yunnan 671003, China.
    2Baoshan Aquaculture Workstation, Yunnan Baoshan 678000, China.
    3Collaborative Innovation Center for Biodiversity and Conservation in the Three Parallel Rivers Region of China, Dali, Yunnan 671006, China.
    4Kunming University of Science and Technology, Kunming 650500, Yunnan, China.

    ABSTRACT

    Background: The mitochondrial genome is widely used to understand the phylogeny and evolution of fish. This study aims to deepen the understanding of the mitochondrial genome of Schistura fasciolata and the evolutionary implications within Nemacheilini.

    Methods: This study utilized high-throughput sequencing technology and bioinformatics analysis to investigate the mitochondrial genome of the Schistura fasciolata. The aim was to reveal its structural or compositional characteristics, phylogenetic location and evolutionary rate.

    Result: We conducted a comprehensive sequence analysis of the complete mitochondrial genome of S. fasciolata, in which it contains 16,570 base pairs (bp) including 37 genes, i.e., 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs). The base composition analysis indicated an A+T content of 56.51% and a G+C content of 43.49%. Phylogenetic analysis based on the 13 PCGs revealed extensive polyphyly within Nemacheilini. Furthermore, accelerated evolution in ND4, ND5 and CYTB genes were observed in Homatula and Troglonectes, suggesting an adaptation to specific environmental conditions. This study provides critical insights into the phylogeny and adaptive evolution within Nemacheilini.

    INTRODUCTION

    Nemacheilidae, an ecologically significant and taxonomically complex family of freshwater fish, mainly distributed across mainland Asia and its adjacent islands, Europe and northeast Africa (Kottelat, 2012; Basudha et al., 2025). The species in this lineage live in various habitats, including swamps, rapids, rivers, streams, caves and lakes (Kottelat, 2012; Eschmeyer, 2020). The family consists of nearly 760 species in 47 genera (Prokofiev, 2010; Zhang and Zhao, 2016; YoGurtCuoGlu et al., 2020).
           
    Prokofiev performed a detailed comparative morphological analysis (Prokofiev, 2010), which divided Nemacheilidae into five tribes: Vaillantellini, Lefuini, Yunnanilini, Triplophysini and Nemacheilini (Zhang and Zhao, 2016; Lan et al., 2013; Zhu and Zhu, 2014; Sgouros et al., 2019; Du et al., 2021; Min et al., 2023). However, the challenges of valid species identification exist due to morphological and genetic diversity, especially within the tribe Nemacheilini (Sgouros et al., 2019; Chen et al., 2019). Besides, the distribution also has led to taxonomic issues, especially in Homatula, Nemacheilus and Schistura (Sgouros et al., 2019; Chen et al., 2019; Liu et al., 2012). Thus, the species identification, taxonomy, phylogenetic relationship has been always controversial within tribe Nemacheilini. Moreover, the extensive polyphyly has been observed in some genera, such as Homatula, Nemacheilus and Schistura, which further complicates the taxonomy (Liu et al., 2012; Min et al., 2012). Many studies focus on the complex phylogenetic relationships usually based on some mitochondrial gene fragments and/or morphological traits (Sember et al., 2015; Sgouros, 2016). However, the confusion has been always discussed due to incomplete sample coverage, inadequate analysis methods and lack of data, especially the short molecular fragments.
                   
    Mitochondrial DNA (mtDNA), known for its simple structure, maternal inheritance, rapid evolution and short coalescence time, is crucial for species identification and phylogenetic analysis in fish (Satoh et al., 2016), which helps assess genetic diversity, phylogeny and evolution (Xiao and Zhang, 2000; Yue et al., 2006; Zhu and Yue, 2008; Vani et al., 2022). In this study, we obtained the complete mitochondrial genome of S. fasciolata and then constructed the phylogenetic tree of tribe Nemacheilini based on 13 PCGs, meanwhile, the evolutionary rate of each genus in Nemacheilini was evaluated. This study aims to: (1) analysis the mitogenomic structure of S. fasciolata and (2) explore its phylogeny and adaptation within this lineage.

    MATERIALS AND METHODS

    Sample collection and genomic DNA extraction
     
    Adult specimens of S. fasciolata were collected from Jianchuan, Yunnan, China (26.25788065°N, 99.82247990°E) in May 25, 2023. Identification of adult specimens was based on morphological characteristics based on FishBase (https://fishbase.de/search.php). The samples were stored in 100% ethanol and then were sent to Shanghai Personal bio technology Co. for genomic DNA extraction and sequencing. The Whole Genome Shotgun (WGS) strategy was used to construct a library of different insertions with a length of 400 bp. These libraries were Paired-end reads (PE, 150 bp in length) sequenced using Next-Generation Sequencing (NGS), based on the Illumina NovaSeq sequencing platform. Subsequently, the raw sequencing data was filtered to generate high-quality sequences.
     
    Sequence assembly, annotation and analyses
     
    High quality second-generation sequencing data were reconstructed from scratch using A5-miseq v20150522 (David et al., 2014) and SPAdes v3.9.0 (Bankevich et al., 2012) to construct contig and scaffold sequences. Moreover, the sequences were extracted according to the sequencing depth of the splicing sequences and the sequences with high sequencing depth were compared with the NT library on NCBI by blastn (BLAST v2.2.31 +) and the mitochondrial sequences of each splicing result were selected. Secondly, mummer v3.1 (Delcher et al., 2003) software was used to carry out collinearity analysis of mitochondrial splicing results obtained by the above different software, so as to determine the position relationship between contigs and fill the gap between contigs. The results were corrected using pilon v1.18 (Walker et al., 2017) software to obtain the final mitochondrial sequence, the “fasta” file. Then, the whole mitochondrial sequence was submitted to NCBI (Accession No. OR797719.1). Subsequently, the complete mitochondrial genome sequence obtained by splices was uploaded to the MITOS web server (http://mitos.bioinf.uni-leipzig.de/) for functional annotation (Matthias et al., 2012). The Genetic Code selection is set to 02-vertebrate and the rest of the settings follow the default parameters set by MITOS. At the same time, in the MITOS web server, click Download each tRNA, structure download svg format file, you can view each predicted tRNA secondary structure.
           
    In addition, PhyloSuite v1.2.3 software was used to compute the mitochondrial whole genome, the protein-coding genes and the base composition of the rRNA genes, respectively (Zhang et al., 2020; Xiang et al., 2023). The strand asymmetry was calculated by using the formulas:
     
    The whole mitochondrial genome was mapped using CGView visualization software (Paul and David, 2005).
     
    Phylogenetic analysis
     
    To investigate the position of S. fasciolata within Nemacheilini and its phylogenetic relationships, we retrieved mitochondrial genome sequences of 12 Nemacheilini genera and used Triplophysa bombifrons and T. labiata as outgroups from NCBI. We utilized the sequences of 13 protein-coding genes (PCGs) from 58 species to construct the phylogenetic tree. Initially, sequences were processed using MEGA 7 software (Kumar et al., 2018). Following alignment of PCG sequences with the MAFFT algorithm (Katoh et al., 2019), we conducted Bayesian inference (BI) analyses using PhyloSuite v1.2.3 (Zhang et al., 2020). Four independent runs were performed, each for 20 million generations, sampling the phylogenetic trees every 1,000th generation, which resulted in 20,000 trees. The first 25% of the trees were discarded as burn-ins. BI Posterior Probability (PP) ≥0.8 was deemed significant. Convergence of the BI analyses was assessed by the average standard deviation of split frequencies <0.01 and Tracer v1.5 (Rambaut et al., 2018) was used to investigate the convergence of the BI analyses, ensuring that the effective sample size (ESS) for all parameters was >200. We also reconstructed phylogenetic relationships using Maximum Likelihood (ML) methods in PhyloSuite v1.2.3. ML phylogenetic trees were generated in IQ-TREE (Nguyen et al., 2015) using the ultrafast bootstrap (BS) algorithm with 1000 replications. ML bootstrap values (BS) ≥75 were deemed significant, indicating that higher values correlate with increased topology reliability (Wang et al., 2023). All analyses were performed in PhyloSuite v1.2.3 (Zhang et al., 2020). Finally, the phylogenetic trees were visualized and refined using FigTree v1.4.4 software (http://tree.bio.ed.ac.uk/software/Figtree/). This phylogenetic tree was used in next-step selective analysis.
     
    Selection analyses
     
    We used the CodeML program in PAML v4.9 (Yang, 2007) to assess the selection pressure on 13 PCGs sequences of mitochondria from 58 species and calculated the evolution rate (namely, the ratio of nonsynonymous substitution and synonymous substitution, dN/dS) of each branch by using the free-ratio model. In order to evaluate whether there were significant differences in the evolution rate of 13 PCGs of each main genus in Nemacheilini, the dN/dS values of 56 extant species were extracted. In order to evaluate the discrepancy accurately, the data of dN or dS = 0 and dN/dS ≥1 were not considered and then Kruskal-Wallis H test was implemented in SPSS to test the difference (IBM Corp N, 2017).

    RESULTS AND DISCUSSION

    Mitogenome organisation and nucleotide composition
     
    The mitochondrial genome of S. fasciolata, a double-stranded, circular molecule measuring 16,570 bp, aligns with the size range typical for the genus Schistura (from S. incerta: 16,561 bp to S. geisleri: 16,819 bp). This mitogenome consists of a canonical set of 13 PCGs, two rRNAs (12S rRNA and 16S rRNA), 22 tRNAs and a control region known as the D-loop. Positioned between the tRNA-Pro and tRNA-Phe genes, the D-loop region serves as a crucial regulatory element for initiating replication and transcription. This organization is typical of metazoan mitochondrial genomes (Wolstenholme, 1992; Watanabe et al., 2014). Notably, the mitochondrial genome analysis reveals the presence of 23 intergenic spacers of variable lengths, including a significant -10 base pair overlap between the ATP8 and ATP6 genes, indicative of direct genetic contiguity (Watanabe et al., 2014; Ballard and Whitlock, 2004). Analysis of the S. fasciolata mitogenome nucleotide composition shows 30.14% adenine (A), 16.69% guanine (G), 26.37% thymine (T) and 26.80% cytosine (C). The A + T content (56.51%) predominates over the G + C content (43.49%), highlighting the genome’s nucleotide bias and providing insights into its evolutionary adaptations and constraints.
           
    Additionally, the mitogenome comprises two ribosomal RNAs, 12S rRNA (952 bp) and 16S rRNA (1,657 bp) (Table 1). Positioned between tRNA-Phe and tRNA-Leu and separated by tRNA-Val, these rRNAs have high A/T contents of 50.32% and 55.94%, respectively. Their positioning and nucleotide composition, indicated by a positive AT-skew and a negative GC-skew, are essential for the structural and functional integrity of ribosomal RNA, crucial for protein synthesis (Xiao and Zhang, 2000).

    Table 1: Analysis of mitochondrial genome characteristics of S. fasciolata.


     
    Transfer RNAs, ribosomal RNAs
     
    The S. fasciolata mitochondrial genome contains 22 tRNA genes, ranging in size from 66 bp (tRNA-Cys) to 76 bp (tRNA-Lys), each displaying the classic cloverleaf secondary structure (Table 1; Fig 1). This set highlights the structural integrity and functional importance of mitochondrial tRNAs, particularly the trnA gene’s retention of its dihydrouridine (DHU) arm, demonstrating these molecules conservation across mitochondrial genomes. Despite some tRNAs lacking the D-arm, notably in trnS for AGY/N codons, this trait is widespread, signifying evolutionary adaptations for mitochondrial efficiency (Garey and Wolstenholme, 1989). Further analysis of tRNA base-pairing shows mostly classical Watson-Crick A-U and G-C matches, with deviations like unmatched pairs U-U, U-C, A-A and C-C in various tRNAs, indicating specific evolutionary adaptations. Additionally, a rare C-A pairing in trnF, trnH, trnM and trnR was noted (Fig 1). These observations highlight the nuanced balance between conserving structural features essential for function and introducing variations that may confer advantages in the mitochondrial context.

    Fig 1: The predicted secondary structure of 22 tRNAs in the mitochondrial genome of S. fasciolata.


     
    Protein-coding genes and codon usage
     
    The S. fasciolata mitochondrial genome contains 13 PCGs for mitochondrial function: seven NADH dehydrogenases (ND1-6, ND4L), three cytochrome c oxidases (COI, COII, COIII), two ATP synthase subunits (ATP6, ATP8) and one cytochrome b (CYTB), totaling 16,570 bp. All genes are similar in length and identical in gene arrangement to those in other Schistura species. This arrangement, with 12 PCGs on the major strand and ND6 on the minor strand, exemplifies the coding efficiency and conservation of the mitochondrial genome. The PCGs employ standard start codons, primarily ATN and TTG and their stop codons exhibit specificity for precise gene expression: TAA for COI, ATP8, ATP6, ND1, ND4L and ND5; TAG for ND6; and a unique TA dinucleotide or a single T for the others, suggesting post-transcriptional modifications to complete the standard stop codons (Fig 2).

    Fig 2: The complete mitochondrial genome circle map of S. fasciolata.


           
    Analysis of Relative Synonymous Codon Usage (RSCU) values provides further insight into the mitogenome’s evolutionary dynamics, revealing codon usage biases that favor genes encoding Ala, Arg, Pro, Ser2, Thr and Leu1 over those encoding Asn, Asp and Cys (Fig 3). This codon usage bias reflects adaptive evolutionary strategies aimed at enhancing translational efficiency and accuracy (Du et al., 2008), underscoring the nuanced interplay between genetic code specificity and mitochondrial function optimization in S. fasciolata.

    Fig 3: Relative synonymous codon usage (RSCU) in the mitochondrial genomes of S. fasciolata.


     
    Phylogenetic relationships
     
    In this study, we reconstructed phylogenetic relationships using ML methods and BI methods, yielding a phylogenetic tree with a single topology and high support rate (with Bootstrap Support (BS) values consistently above 75 and BI posterior probabilities all exceeding 0.85), significantly enhancing our understanding of their phylogenetic relationships (Fig 4).

    Fig 4: Phylogenetic analysis of 58 species using 13 PCGs via ML and BI, with node numbers indicating BS and PP values.


           
    Phylogenetic analyses show that Acanthocobitis, Barbatula, Nemacheilus and Homatula form a distinct monophyletic lineage, respectively (Fig 4), which was in line with previous studies (Kottelat, 2012; Min et al., 2012; Sgouros et al., 2019; Chen et al., 2019). On the other hand, our study reveals a widespread paraphyly within the tribe Nemacheilini, which is also prevalent across the entire Nemacheilidae family (Zhang and Zhao, 2016; Du et al., 2021). Specifically, Schistura, Troglonectes and Heminoemacheilus form a paraphyletic lineage. Luo et al., (2023) uncovered paraphyly between Heminoemacheilus and Paranemachilus, recommending merging them to ensure the monophyly of Paranemachilus. Conversely, our research challenges the classification of Heminoemacheilus, particularly highlighting H. zhengbaoshani placement within Troglonectes, which also questions Troglonectes previously accepted monophyly (Lan et al., 2013; Du et al., 2008).
           
    The extensive paraphyly across tribe Nemacheilini, even family Nemacheilidae, might be related with the rapid evolution, extensive radiation and remarkable adaptability of species, reflected in their diversity and wide ecological niche occupation (Kottelat, 2012; Eschmeyer, 2020; Prokofiev, 2010), as well as geological event (Wang et al., 2016; Wu et al., 2020), niche competition (Fischer, 2000), hybridism (Saitoh et al., 2010) and rapid adaptive evolution in response to environmental pressures (Zhang et al., 2024). These comprehensive factors collectively contribute to the rapid evolution within the family Nemacheilidae. Additionally, the genus Schistura was clustered into two clades in this study (clade 1 and clade 2 in Fig 4), among which the S. incerta (No. MK361215.1) was nested with S. fasciolata (clade 2 in Fig 4), our results align with prior studies (Liu et al., 2012; Min et al., 2012) which imply the paraphyletic origin for genus Schistura. Of cause, it possibly resulted from the complex gene flow (Min et al., 2023) or need redefinition of species. Meanwhile, the same situation for Acanthocobitis botia in genus Acanthocobitis and Barbatula toni in Barbatu.
     
    Selection analyses
     
    In this study, we examined the evolutionary dynamics of 13 mitochondrial PCGs of five genera in Nemacheilini: Acanthocobitis, Barbatula, Homatula, Schistura and Troglonectes. By applying the M0 model, we found omega (ù) ratios significantly below 1, ranging from 0.00659 (COI) to 0.12080 (ATP8), across the tribe Nemacheilini. This result emphasizes the role of purifying selection in the oxidative phosphorylation (OXPHOS) pathway. Notably, the ATP8 gene showed the highest dN/dS value, indicating potential accelerated evolution, similar with prior research on species such as Glyptothorax macromaculatus (Lv et al., 2018), Orthoptera insects (Chang et al., 2020), Desis jiaxiangi (Li et al., 2021) and Lamprologus (Wang et al., 2023). In contrast, the COI gene displayed the lowest dN/dS value, suggests it may have faced much stronger evolutionary constraints, which consist with (Singh et al., 2017); Yang et al., (2018) and Li (2018). This evidence underlines the extensive use of the COI gene in phylogenetic reconstructions across species groups due to its low mutation rate. Furthermore, M1 model analysis revealed significant dN/dS ratio variability among mitochondrial PCGs, indicating a diversity of selective pressures in Nemacheilini (Table 2). This variability suggests that while some genes are highly conserved due to their critical roles in cellular metabolism and energy production, others may evolve more rapidly, possibly reflecting adaptations to different ecological niches or life-history strategies within the tribe (Zhang et al., 2025).

    Table 2: The results of evolution rate of tribe Nemacheilini lineages detected by free ratio model.


           
    In order to further evaluate the significance of evolutionary rates among different genus, the Kruskal-Wallis H test was implemented. The results uncovered significance of evolutionary rates for the ND4, ND5, ATP6 and CYTB genes (Table 3). Specifically, the ND4, ND5 and CYTB genes have undergone the accelerated evolution in cave-adapted genera Homatula and Troglonectes. It might be related to the environmental pressures of their cave-dwelling lifestyle, such as low temperatures and low oxygen levels, where it needs to much more accurate energy consumption (Li, 2018; Woo et al., 2013). The studies have confirmed that ND5 interacts with protein subunits, playing a crucial role in regulating respiration (Wu et al., 2016). And, the transmembrane helices in ND4, crucial for the proton transfer process in complex I, interact with at least one lipid molecule (Wu et al., 2016). Consequently, the accelerated evolution of ND4 in Homatula may significantly influence mitochondrial regulation of lipid synthesis and metabolism, potentially conferring a survival advantage in environments with irregular resources. However, the ATP6, ND4, ND5 and CYTB genes exhibit lower evolutionary rates in Acanthocobitis. In summary, our findings underscore the complexity of adaptive evolution within the tribe Nemacheilini, with various taxa exhibiting unique evolutionary trajectories in mitochondrial genes, reflecting their diverse ecological adaptations (Fig 5).

    Table 3: Statistical test of evolutionary rates in different genes.



    Fig 5: Boxplot of dN/dS of the13 PCGs for 5 main genera.

    CONCLUSION

    The mitochondrial genome of S. fasciolata was sequenced and analyzed in this study, shedding light on the mitochondrial genome of the tribe Nemacheilini. Notably, the phylogenetic analysis reveals widespread polyphyly within the tribe Nemacheilini. Furthermore, the evolutionary dynamics highlight the intricate nature of adaptive evolution within the tribe and each taxon displaying distinct evolutionary trajectories in mitochondrial genes, indicative of their unique ecological adaptations. Our results have established a foundation for further exploration of evolutionary mechanisms in Nemacheilini, even for Nemacheilidae.
     
    Supplementary information
     
    The supporting materials in this paper will be obtained in Figshare (https://figshare.com/) based on this DOI: 10.6084/m9.figshare.25285282; https://github.com/qwrtyrev686tfstff/Project-The-Complete-Mitochondrial-Genome-of-Schistura-fasciolata.git.

    ACKNOWLEDGEMENT

    The authors thanked the anonymous reviewers and the editor for their valuable comments.
     
    Fundings
     
    Grants supporting this research were received from the Erhai Watershed Ecological Environment Quality Testing Engineering Research Center of Yunnan Provincial Universities to WH and YM (No. DXDGCZX03), as well as the start-up projects on high-level talent introduction of Dali University to YM (No. KY1916101940).
     
    Ethical approval
     
    This research project was conducted in accordance with the ethical guidelines. And the animal study was reviewed and approved by Laboratory Animal Welfare Ethics Review Committee, Dali University, for research project (No. DXDGCZX03). The ethical certification was shown in “Related files”.

    CONFLICT OF INTEREST

    The authors declare that there is no conflict of interests regarding the publication of this article.

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