The Beki River (known as the Kurissu River in Bhutan) is an important right-bank tributary of the Brahmaputra River, originating from the Kula Kangri glacier and draining an area of approximately 19,354.35 km² before joining the Brahmaputra at 26.48^o N and 91.02^o E (Mazumdar et al., 2023). The river system forms part of the Indo-Burma biodiversity hotspot, globally recognized for its exceptional biological richness and high levels of endemism (Myers et al., 2000). The Beki River supports diverse aquatic fauna, particularly ichthyofaunal assemblages comprising several economically and ecologically important freshwater fishes. Among these, the ray- finned catfishes belonging to the family Bagridae (order Siluriformes) constitute a significant component of inland fish diversity in Northeast India (Chakravorty et al., 2016). Members of Bagridae are widely distributed throughout Asian and African freshwater ecosystems which  represent a significant component of inland fish diversity in Northeast India. Siluriformes, comprising more than 30 families, represent one of the most evolutionarily diverse groups of freshwater teleosts, while ray-finned fishes (Actinopterygii) constitute the largest and most species-rich group of vertebrates (Nelson et al., 2016).

Accurate species identification is critical for fisheries management and conservation, yet morphology-based taxonomy relying on morphometric and meristic traits (Rosso et al., 2012) is often unreliable due to phenotypic plasticity, cryptic diversity and ontogenetic variation (Chen et al., 2015). With limited taxonomic expertise, molecular tools provide standardized and reproducible alternatives (Steinke et al., 2009; de Carvalho et al., 2011). In this context, biotechnological approaches, particularly those based on mitochondrial DNA (mtDNA) sequences, carrying the hereditary information have emerged as reliable methods for species-level identification, assessment of population structure and for elucidating evolutionary and taxonomic relationships among diverse taxa (Kaur et al., 2021; Naseer et al., 2018; Ujjania et al., 2026). Among these approaches, DNA barcoding, particularly using the mitochondrial cytochrome c oxidase subunit I (COI) gene, has emerged as a robust approach for species identification and phylogenetic inference due to its conserved function in oxidative phosphorylation and high interspecific variability (Hebert et al., 2003a, 2003b; Hebert et al., 2004; Hajibabaei et al., 2007; Ward et al., 2009; Robideau et al., 2011).

Beyond taxonomy, COI-derived protein sequences provide structural and functional insights through computational modelling when experimental structures are unavailable. Protein three-dimensional (3D) structure is crucial in evolutionary biology because protein function depends on both amino acid sequence and spatial folding. Although protein sequences may diverge during evolution, structural folds often remain conserved due to functional constraints. This structural conservation provides important phylogenetic signals for detecting deep evolutionary relationships beyond sequence similarity alone. In fish phylogenetics and DNA barcoding, mitochondrial protein-coding genes such as COI, Cyt b and ATPase are widely used because their encoded proteins are functionally essential and evolutionarily informative. Comparative analyses of fish mitochondrial genomes further show that structural variation in mitochondrial proteins and genome organization contributes significantly to understanding fish evolution and phylogenetic diversification. Satoh et al., 2016 reported that comparative analyses of fish mitochondrial genomes revealed important structural aspects of the mt genome and the encoded genes, highlighting their evolutionary significance.

Similarly, Miya and Nishida, (2015) emphasized that fish mitogenomics has substantially advanced molecular phylogenetics and evolution of fishes through analysis of mitochondrial gene structure and organization. Structural biology studies on fish proteins also show that evolutionary adaptation can be traced through changes in protein folding patterns. For example, research on ancestral fish galectins demonstrated that protein-fold evolution occurred in specific evolutionary lineages, linking structural divergence with adaptive evolution in fish immune proteins (Konno et al., 2011). Furthermore, modern evolutionary structural biology recognizes that protein structures evolve more slowly than sequences, making them especially valuable for resolving distant phylogenetic relationships and identifying functional conservation across taxa (Illergård et al.,  2009).

The present study aims to integrate molecular and bioinformatics approaches to confirm species identity, to assess phylogenetic relationships and genetic divergence and to evaluate evolutionary relatedness among four Bagridae catfishes viz. Mystus tengara, Mystus vittatus, Mystus cavasius and Rita rita from the Beki River, Assam, using partial COI sequences, while also predicting the 3D structure of cytochrome c oxidase I to examine its functional conservation and structural stability, thereby supporting its significance in evolutionary studies and future conservation-oriented research.

Study area and sample collection
 
The study was conducted in the Beki River (26^o20' 00" N, 90^o56' 00" E), Assam, India. A total of 20 adult specimens belonging to the family Bagridae were collected from Narayunguri Ghat (26^o39' 56" N, 90^o59' 48" E) in the Baksa district using cast nets, gill nets and traps with the assistance of local fishermen (Fig 1). The collected specimens comprised five individuals each of Mystus tengara (Hamilton-Buchanan, 1822), Mystus vittatus (Bloch, 1797), Mystus cavasius (Hamilton-Buchanan, 1822) and Rita rita (Hamilton, 1822). Species identification was performed following the taxonomic keys and diagnostic characters described by Talwar and Jhingran (1991) and Jayaram (1999). For molecular analyses, one healthy and morphologically well-characterized representative specimen from each species was selected. Fresh tissue samples (20-50 mg) were aseptically excised from the selected specimens and preserved in 95% molecular-grade ethanol at -20^oC until further use. The preserved tissues were subsequently utilized for high-fidelity PCR amplification and bidirectional DNA sequencing.


 
DNA extraction, PCR amplification and sequencing
 
Genomic DNA was extracted from all the specimens using the CTAB method with the Macherey-Nagel Nucleospin kit (Ref. No. 740952.250) (Macherey-Nagel, 2019). The COI gene was amplified with primers COI-Fish-F1 (5' -TCAACCAACCACAAAGACATTGGGAC-3') and COI-Fish-R1 (5' -TAGACTTCTGGGTGGCCAAAGAATCA-3') (Ward et al., 2005) in a VeritiPro™ thermal cycler. PCR reactions (12.5 µL) contained DNA (2.5 µL), Premix Ex Taq™ (6.25 µL), primers (0.25 µL each) and nuclease-free water (9.75 µL) (Takara Bio Inc., 2018). Cycling conditions: 95^oC for 30 s; 45 cycles of 95^oC for 45 s, 60^oC for 45 s, 72^oC for 1 min; final extension at 72^oC for 7 min. Amplicons (~600-800 bp) were verified on 2% agarose gel (0.5X TBE buffer, Labsafe dye) with gScale DNA ladder, visualized under UV and documented using BIO-RAD GelDoc-XR (Fig 2). Double stranded PCR products were purified with ExoSAP-IT™ (Thermo Fisher Scientific, 2017) and sequenced using ABI BigDye® Terminator v3.1 kit (Applied Biosystems, 2016). Sequencing was performed on an ABI 3500 Genetic Analyzer (Applied Biosystems, 2015) and FASTA sequences were generated with SeqA 8. Species confirmation was done via BLAST (NCBI) based on query cover (%) against GenBank references.


 
Sequence analysis and phylogenetics
 
Physicochemical properties and multiple sequence alignments of COI sequences were analyzed using CLC Genomics Workbench v25.0.3 (QIAGEN, 2023). Pairwise alignment scores were calculated with ClustalW (Thompson et al., 1994). Phylogenetic trees were constructed using the Maximum Likelihood (ML) method (Tamura and Nei, 1993) with 1000 bootstraps in MEGA v12 (Kumar et al., 2024). Pairwise genetic distances were estimated using the Kimura 2-parameter (K2P) model (Kimura, 1980).
 
Protein homology modelling and validation
 
Homology-based three-dimensional (3D) structures of COI proteins were generated using SWISS-MODEL (Waterhouse et al., 2018) and structural quality was validated by PROCHECK using Ramachandran plot assessment (Laskowski et al., 1993).
 
Structural analysis
 
Solvent-accessible surface area (SASA) and active site predictions were performed using Discovery Studio Visualizer v25.1.0.24284 (BIOVIA, Dassault Systèmes, 2023). Potential cavities and binding pockets were identified with DoGSiteScorer (Volkamer et al., 2012) and amino acid composition of the largest predicted pocket was analyzed.

Species identification and COI sequence properties
 
Morphological and molecular analyses confirmed four Bagridae catfishes in the Beki River- Mystus tengara (Hamilton- Buchanan, 1822), Mystus vittatus (Bloch, 1797), Mystus cavasius (Hamilton-Buchanan, 1822) and Rita rita (Hamilton, 1822). COI nucleotide sequence lengths were 685 bp in M. tengara, 671 bp in M. vittatus, 668 bp in M. cavasius and 667 bp in R. rita, with molecular weights 423.271 kDa, 414.618 kDa, 412.748 kDa and 412.124 kDa for M. tengara, M. vittatus, M. cavasius and R. rita, respectively. Base composition analysis revealed highest AT content in R. rita (57.12%) and highest GC content in M. tengara (46.71%) (Table 1). Melting temperature (Tm) increased with salt concentration, indicating ionic stabilization of the DNA duplex (Table 2).




 
Sequence editing, submission and alignment
 
The raw nucleotide sequences obtained from all specimens were manually verified and edited using the software BioEdit (Hall, 1999). The finalized sequences were then deposited in NCBI (http://www.ncbi.nlm.nih.gov) and accession numbers were assigned for each species (Table 1). Multiple sequence alignment using CLUSTAL W, revealed conserved regions across all taxa, particularly in central sequence portions and variable regions with substitutions, insertions and deletions (Fig 3). R. rita exhibited the greatest divergence, with unique 52  insertions and 32 deletions. Pairwise similarity scores were highest between M. tengara and M. vittatus (88), moderate for M. cavasius (79-80)  and lowest for R. rita (74-79). The cumulative alignment score was 23,063, which indicate better overall alignment quality and similarity among the sequences.


 
Genetic distance and phylogenetic analysis
 
To resolve the evolutionary relationships among the studied bagrid catfishes, a Maximum Likelihood (ML) phylogenetic tree was constructed using MEGA version 12 (Fig 4). The resulting topology showed that Mystus vittatus and Mystus tengara formed a closely related sister clade with strong bootstrap support (92%), indicating high sequence similarity and a recent common ancestry. This M. vittatus-M. tengara clade further clustered with Mystus cavasius, forming a robust monophyletic group supported by a bootstrap value of 93%, thereby reinforcing the evolutionary relatedness within the genus Mystus. In contrast, Rita rita diverged earlier and occupied a basal position relative to the Mystus cluster, indicating substantial evolutionary divergence. The comparatively longer branch length (0.13) observed for R. rita further reflects its greater genetic differentiation from the remaining taxa, whereas the shorter branch lengths within the Mystus cluster indicate lower sequence divergence among those species. Overall, the ML phylogeny provided a well-resolved and robust framework for understanding the evolutionary relationships of the studied members of the family Bagridae.



Complementing the phylogenetic analysis, the Kimura 2-parameter (K2P) genetic distance analysis revealed distinct interspecific variation among all four taxa (Table 3). Pairwise genetic divergence values ranged from 0.109 to 0.228. The lowest genetic distance was observed between M. vittatus and M. tengara (0.109), confirming their close genetic affinity. In contrast, the highest genetic divergence was recorded between R. rita and M. vittatus (0.228), indicating considerable evolutionary separation between the two genera. Intermediate divergence values were observed between R. rita and the other Mystus species, namely M. cavasius (0.200) and M. tengara (0.201). Within the genus Mystus, the genetic distance between M. cavasius and M. tengara was 0.153, whereas M. vittatus and M. cavasius exhibited a divergence value of 0.191. Collectively, the K2P distance estimates clearly delineate the genetic boundaries among the studied species and strongly support their taxonomic distinctiveness and evolutionary divergence.


 
COI protein physicochemical properties
 
The COI protein sequences of the four studied species showed slight variation in length: Mystus tengara (228 amino acids), Mystus vittatus (223 amino acids) and both Mystus cavasius and Rita rita (222 amino acids each). The molecular weight of the COI protein was 24.4 kDa in M. tengara (pI 5.29; aliphatic index 120.702), 23.857 kDa in M. vittatus (pI 5.91; aliphatic index 120.359), 23.602 kDa in M. cavasius (pI 5.29; aliphatic index 124.414) and 23.632 kDa in Rita rita (pI 5.56; aliphatic index 120). Physicochemical properties of the selected bagrid catfish COI protein sequences were analysed using CLC Genomic Workbench v25.0.3 (Table 4).


 
Model prediction and validation
 
The three-dimensional structures of the COI proteins from four selected Bagridae catfish species were predicted using the SWISS-MODEL server, employing the template 8ijn.1.A (Bovine heart cytochrome c oxidase in the nitric oxide-bound fully reduced state at 100 K) (Fig 5). The predicted models exhibited high sequence identity/coverage values of 95.18% for Mystus tengara, 95.95% for Mystus cavasius, 94.14% for Mystus vittatus  and 95.48%  for Rita rita.



The Global Model Quality Estimation (GMQE) scores were 0.90, 0.94, 0.90 and 0.93 for M. tengara, M. cavasius, M. vittatus and R. rita, respectively. Similarly, the QMEAN scores obtained for the respective species were -1.63, -1.63, -1.82 and -1.88. These values indicate that all predicted models possessed high structural reliability and acceptable stereochemical quality.

Model quality assessed through PROCHECK Ramachandran plot analysis showed residues in the most favoured regions for Mystus tengara (93.6%), Mystus vittatus (93.4%), Mystus cavasius (93.4%) and Rita rita (91.3%). Discovery Studio Visualizer v25.1.0.24284 revealed that M. tengara comprised 1723 atoms (24,395.5 kDa; net charge -5; C1151H1760N267O294S11), M. vittatus 1679 atoms (23,783.9 kDa; -3; C1120H1723N264O284S11), M. cavasius 1664 atoms (23,597.6 kDa; “5; C1106H1713N260O287S11) and R. rita 1656 atoms (23,481.4 kDa; “4; C1100H1699N259O286S11).
 
SASA and active site prediction
 
Analysis using Discovery Studio Visualizer v25.1.0.24284 showed that the total solvent-accessible surface area (SASA) was 11,271.2 Ų in Mystus tengara, 11,431.2 Ų in Mystus vittatus, 11,237.2 Ų in Mystus cavasius and 11,266.5 Ų in Rita rita. Active site volumes calculated using DoGSiteScorer were highest in M. vittatus (1485.12 ų), followed by M. tengara (1311.68 ų), M. cavasius (1062.40 ų) and R. rita (531.33 ų), with hydrophobicity ratios of 0.49, 0.49, 0.47 and 0.44, respectively (Table 5).



Combined morphological and molecular analyses confirmed four Bagridae catfishes in the Beki River, demonstrating the reliability of COI-based DNA barcoding for species-level identification. Minor variations in COI sequence length and molecular weight reflect interspecific differences while overall conservation underscores functional stability.

The recovered mitochondrial COI gene fragments ranged from 667 bp (R. rita) to 685 bp (M. tengara), yielding molecular weights between 412.124 kDa and 423.271 kDa. These lengths match the standard structural parameters (typically ~650 bp) established for the core teleost barcoding region (Ahmed et al., 2020; Suryawanshi et al., 2024).

Analysis of the nucleotide base profiles revealed a conspicuous bias toward Adenine and Thymine, with AT content ranging from 53.28% in M. tengara to 57.12% in R. rita. Consequently, Guanine and Cytosine (GC content) varied from 42.87% (R. rita) to 46.71% (M. tengara). This preference for AT over GC is a hallmark of the teleost mitochondrial genome, which is continuously influenced by strand-specific replication biases and asymmetrical mutational pressures during transcription (Ahmed et al., 2020). The noticeable divergence in GC percentages between the closely related Mystus species and Rita rita reflects distinct evolutionary trajectories and genetic drift within the family Bagridae, acting as excellent indicators for species-level discrimination and phylogenetic separation (Barathkumar and Thangaraj, 2020). A novel biophysical dimension explored in this study is the melting temperature (Tm) of the amplified COI sequences under varying salt concentrations (0.1 M to 0.5 M). The empirical data demonstrated a direct, proportional relationship across all four species: as ionic strength increased, the Tm rose significantly. For instance, in M. tengara, Tm escalated from 83.32^oC at  0.1 M to 94.93^oC at 0.5 M.

Multiple sequence alignment identified conserved regions under strong evolutionary constraint and variable regions reflecting divergence. Rita rita exhibited the greatest variability, consistent with its distinct phylogenetic placement outside Mystus. The K2P analysis revealed clear genetic divergence among the studied catfish species. The lowest genetic distance was observed between Mystus vittatus and Mystus tengara (0.109), indicating closer evolutionary relatedness, whereas the highest divergence occurred between Rita rita and Mystus vittatus (0.228), suggesting greater evolutionary separation. Similar patterns of interspecific genetic divergence have been reported in previous DNA barcoding studies of freshwater fishes, where K2P distances effectively distinguished closely related taxa and supported species-level differentiation (Ward et al., 2005; Hubert et al., 2008).

COI protein analyses revealed minor differences in length, molecular weight and isoelectric points, yet strong conservation overall, consistent with the essential role of cytochrome c oxidase  (COI) in mitochondrial respiration. The anionic nature of the proteins suggests enhanced interactions with positively charged cofactors and ions in electron transport. Higher negative charges in Mystus tengara and Mystus cavasius may reflect species-specific biochemical adaptations enhancing solubility, ligand binding  and protein-protein interactions.

The predicted COI protein models of the studied Bagridae catfishes exhibited high sequence identity and favorable structural quality parameters, indicating reliable homology-based model construction. High GMQE and acceptable QMEAN scores, together with favorable Ramachandran statistics (>91% residues in most favored regions), confirmed the structural stability of the models. The highly conserved COI structure observed among Mystus tengara, Mystus cavasius, Mystus vittatus and Rita rita suggests preservation of the functional regions required for efficient electron transfer from cytochrome c to molecular oxygen during mitochondrial respiration and ATP synthesis (Capaldi, 1990; Wikström et al.,  2018). However, subtle interspecific structural variations may influence electron transport efficiency and reflect species-specific metabolic adaptations. Similar observations regarding structural reliability have been reported in previous protein modeling studies using SWISS-MODEL and PROCHECK validation approaches (Waterhouse et al., 2018; Laskowski et al., 1993).

Further structural analysis through molecular surface mapping and topological cavity assessment revealed that, despite evolutionary nucleotide divergence, the overall tertiary architecture of the Cytochrome c Oxidase Subunit I (COI) protein remained highly conserved among the four studied bagrid catfishes. The total solvent-accessible surface area (SASA) values were narrowly distributed, ranging from 11,237.2 Ų to 11,431.2 Ų, indicating substantial structural conservation across species.

Among the studied taxa, Mystus vittatus exhibited the largest primary catalytic pocket volume (1485.12 ų), whereas Rita rita displayed a markedly reduced primary pocket volume (531.33 ų), representing less than half of that observed in Mystus vittatus. This structural variation may reflect species-specific ecological and metabolic adaptations. Species of the genus Mystus are generally active pelagic to mid-water column foragers that require sustained swimming activity and comparatively higher metabolic output. In this context, the expanded catalytic pocket volume and relatively higher pocket hydrophobicity ratio (0.49) observed in Mystus species may facilitate efficient electron transport and oxidative metabolic activity within the COI complex.

In contrast, Rita rita is a predominantly benthic and relatively sedentary species with lower locomotory activity and specialized feeding habits, including molluscivory. Its comparatively reduced primary catalytic pocket may therefore correspond to lower energetic demands. Interestingly, Rita rita appears to compensate structurally for this reduced primary cavity through the presence of relatively enlarged secondary sub-pockets (P2 = 309.44 ų, P3 = 265.73 ų and P4 = 194.88 ų), together with a lower hydrophobicity ratio (0.44). This distinct cavity organization may reflect localized functional variation associated with its phylogenetic divergence from the Mystus lineage.

Overall, these findings demonstrate that, despite pronounced genetic divergence, the core structural framework of the COI protein remains highly conserved among the studied Bagridae species from the Beki River. The observed differences in cavity topology and physicochemical properties may provide important insights into species-specific metabolic adaptations and establish a valuable baseline for future comparative studies in structural biology, molecular evolution and ecotoxicology within the family Bagridae.

This study integrated molecular, morphological and structural analyses to characterize four Bagridae catfishes viz. Mystus tengara, Mystus vittatus, Mystus cavasius and Rita rita from the Beki River, Assam, India. COI-based DNA barcoding confirmed species identity and genetic differentiation, with Rita rita showing the highest divergence and placement outside Mystus. Base composition and genetic distance analyses indicated close evolutionary affinity between Mystus tengara and Mystus vittatus, while Mystus cavasius showed an intermediate position. COI protein sequences were structurally conserved, with minor variations in active site volume and hydrophobicity suggesting species-specific adaptations. These findings validate COI as a reliable marker for species identification and phylogenetic inference in Bagridae and provide a baseline for future research on evolutionary relationships, functional genomics and conservation of freshwater catfishes.

The authors are grateful to the Vice-Chancellor of Bhattadev University, Bajali, for providing the necessary facilities to conduct this research. The authors also acknowledge the Head of the Department and the laboratory staff of the Department of Zoology for their technical support and thank the local fishermen for their cooperation and valuable traditional knowledge that facilitated specimen collection.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
This study did not involve live animal experimentation. The research was conducted exclusively on edible fish obtained from local fishermen and no handling or experimental procedures were performed on live animals. Therefore, approval from an animal ethics committee was not required.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.