DNA Barcodes Reveal Hidden Identity of Three Endangered Pakistani Catfish

Deep in the river systems of Punjab, Pakistan, three catfish species are slipping toward extinction while remaining surprisingly difficult to tell apart. Now, a team of researchers has combined old-fashioned morphology with cutting-edge DNA barcoding to confirm what conservationists feared and needed to know: these are three genetically distinct species, each requiring urgent protection. The findings, published in Acta Ichthyologica et Piscatoria, offer a powerful template for identifying endangered freshwater fish worldwide.

The study focused on Bagarius bagarius, locally known as Mujahid; Clupisoma garua, called Garua Bachcha; and Eutropiichthys vacha, known as Challi. All three are economically significant food fish prized across Pakistan, India, Bangladesh, Nepal, and beyond. Yet all three are threatened. Bagarius bagarius is designated critically endangered in Pakistan by the IUCN, while the other two face mounting pressure from habitat degradation, pollution, and intensified fishing. Accurate identification has long been a stumbling block for their management, because morphological traits can vary with environment and overlap between species.

Between 2022 and 2024, the researchers collected 72 specimens—24 of each species—from the Chenab, Indus, and Jhelum rivers, working with local fishermen at sites including Head Muhammad Wala, Chashma Barrage, and Rasul Barrage. The Indus, Pakistan’s longest watercourse and the world’s twelfth longest, flows from the Tibetan plateau to the Arabian Sea. The Jhelum stretches 774 kilometers from Kashmir, while the Chenab runs roughly 960 kilometers through the Himalayas and Punjab. These rivers form the backbone of Pakistan’s freshwater fisheries, which support food security and rural livelihoods nationwide.

Samples were first immobilized with clove oil and transported to the Molecular Research Laboratory at Government College University Faisalabad. There, the team measured eight morphometric features—total length, fork length, standard length, snout length, body depth, head length, head depth, and eye diameter—using vernier calipers. Five meristic traits, including dorsal, anal, caudal, pectoral, and pelvic fin ray counts, were recorded. To remove the confounding effect of body size, all linear measurements were converted to size-independent ratios by dividing by standard length.

Principal component analysis revealed striking morphological separation among the three species. PC1 accounted for 81.9% of variance, while PC2 explained 18.9%. Bagarius bagarius stood apart due to larger size traits and head dimensions, reflecting its unique benthic profile. Clupisoma garua showed distinctive caudal and pectoral fin ray counts, while Eutropiichthys vacha was unique in fork length, head length, and anal fin rays. Cluster analysis placed Bagarius bagarius at a morphological distance of approximately 55 from the other two, while Clupisoma garua and Eutropiichthys vacha clustered closely at roughly 22—suggesting a closer evolutionary relationship between the latter pair.

For genetic analysis, DNA was extracted from ethanol-preserved tissue using the QIAamp DNA Mini Kit. The mitochondrial cytochrome c oxidase subunit I gene—the standard animal DNA barcode—was amplified using universal primers Fish F1 and R1. Polymerase chain reaction conditions included an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation, annealing, and extension. Products were purified and sequenced unidirectionally using Sanger sequencing, with 18 sequences successfully deposited in GenBank and BOLD databases.

The COI sequences ranged from 584 to 665 base pairs, with a mean consensus length of 650 base pairs. No deletions, insertions, or stop codons were detected, confirming amplification of functional mitochondrial DNA rather than nuclear pseudogenes. Nucleotide composition showed the expected AT bias, with mean AT content at 55.3% and GC at 44.7%. BLAST annotations matched morphological identifications at 99.02% to 100% similarity, validating the team’s visual assessments with molecular precision.

Genetic divergence told a compelling story. Mean interspecific K2P distance was a substantial 0.112, while intraspecific variation was minimal at just 0.001 to 0.002—a clear barcode gap separating species. Haplotype analysis revealed 7 haplotypes with haplotype diversity of 0.84 and nucleotide diversity of 0.099. Tajima’s D test returned a significant positive value of 2.176, indicating strong selective pressure without recent population expansion. Fu and Li’s D* and F* statistics were also statistically significant, supporting selective neutrality.

Maximum Likelihood phylogenetic trees, constructed in MEGA 11 under the K2P model with Gamma distribution and 1000 bootstrap replicates, confirmed three distinct genetic groups. All Bagarius bagarius sequences formed a strongly supported clade at 99% bootstrap support, positioned as sister to its congener Bagarius yarrelli at 94%. Clupisoma garua clustered with full support at 100%, sister to Clupisoma naziri at 91%. Eutropiichthys vacha formed an exclusive clade at 98%, sister to Eutropiichthys murius at 90%. Labeo rohita served as a distant outgroup, rooting the entire tree.

The integrated approach proved decisive. Morphology alone could separate the species, but the molecular data provided quantitative confirmation—especially crucial for Clupisoma garua and Eutropiichthys vacha, whose morphological similarity makes field identification challenging. The study practically rules out misclassification or cryptic species within the sampled populations. Beyond taxonomy, the baseline data on population structure and genetic variation offer essential foundations for conservation planning, fisheries management, and stock assessment across Punjab’s rivers. The framework described here offers a replicable model for evaluating freshwater genetic diversity worldwide, where fish mislabeling, fillet fraud, and habitat destruction threaten aquatic biodiversity on a global scale.

Subject of Research: Genetics

Article Title: Phylogenetics of three endangered freshwater catfish species ( Actinopterygii , Siluriformes ) inferred from COI gene barcoding and morpho-meristic traits

Article References: Imran, M., & Jabeen, F. (2026). Phylogenetics of three endangered freshwater catfish species (Actinopterygii, Siluriformes) inferred from COI gene barcoding and morpho-meristic traits. Acta Ichthyologica et Piscatoria, 56, 101-110. https://doi.org/10.3897/aiep.56.157418

Image Credits: AI Generated

DOI: 10.3897/aiep.56.157418

Keywords: DNA barcoding, catfish, conservation genetics, phylogenetics, Pakistan, freshwater fish, COI gene, three, endangered, freshwater, species, Actinopterygii