DNA BARCODING AND GENETIC DIVERSITY OF THE CLUPEIDAE FISH FAMILY FROM THE FISH LANDING FACILITIES ACROSS THE COASTAL AREAS OF PAKISTAN

INTRODUCTION

Pakistan has a 1046 km long coastline in the northern Arabian Sea, which is shared between Sindh and Balochistan provinces (Laghari, 2018). It is a fascinating coastal ecosystem with a large number of commercially important species, Such as Clupeidae family members, sardines and shads (Naveed et al., 2019). The family Clupeidae is one of the most economically and ecologically important groups of pelagic fishes worldwide due to its abundance, extensive distribution, and central role in marine food webs (Abbas and Khan, 2023; Rahimi, 2013). These are small to medium-sized fishes (2-75 cm) that occasionally swim in large schools and are considered to be a paramount part of the aquatic food webs as they feed on plankton. Among the Clupeidae fish family, Anodontostoma chacunda, Escualosa thoracata, Hilsa kelee, Nematalosa nasus, Nematalosa arabica, Sardinella gibbosa, Sardinella longiceps, and Tenualosa toli are abundant in the coastal area of Pakistan (Naveed et al., 2019).

Modern molecular techniques have significantly advanced the field of taxonomy, species identification, and genetic diversity assessment. Specifically, DNA barcoding is a powerful tool for molecular-based identification, solving taxonomic ambiguities, understanding haplotype distribution and basic genetic distance differences (Hebert et al., 2003; Khan et al., 2024; Raza et al., 2025; Sial et al., 2024). DNA barcoding provides a rapid, accurate, and cheap strategy for species identification by comparing sequences against known reference sequence libraries (Hebert et al., 2003). Besides this, genetic diversity and other studies with the help of COI data can be considered to get critical information related to the haplotypes, genetic distances, and phylogenetic relationships, which are paramount in terms of biodiversity conservation and adaptation to changing environments (Rahman et al., 2025; Raza et al., 2025).

Although the Clupeidae fish family is an important economic and ecological group for the coastal waters of Pakistan, the molecular research related to this group, especially from the Pakistani area, is very limited. The recent studies were based on morphological data, which can lead to misidentification, especially between cryptic species like H. kelee and T. toli, leading to uncertainty in species-level identification and management decisions (Naveed et al., 2019). DNA barcoding has been considered a robust and fast method for detecting cryptic diversity and distinguishing closely related species within regional ichthyofaunas; however, its application to marine clupeids in Pakistan remains limited (Amir et al., 2022; Karim et al., 2024). Studies from neighbouring regions highlight the urgent need for molecular characterization of clupeids (Mazumder and Alam, 2009; Sarker et al., 2021). Significantly, T. toli has been identified as a genetically distinct species in Southeast Asian waters (Ahmad Tarmizi, 2018); however, no comparable studies have been conducted in the Pakistan region. In the regional markets of Bangladesh, DNA barcoding has revealed cases of mislabeling, highlighting the need for accurate identification tools for vulnerable stocks like Tenualosa spp. (Afrin et al., 2024). Overall, the above scenario points to the lack of fine-scale molecular studies in Pakistan and the urgent implications of DNA barcoding studies for the Clupeidae fish family in addressing the successful identification and management of these fish.

Keeping in mind the critical gaps, the present study aimed to address these gaps by providing a detailed assessment of the Clupeidae family from the coastal areas of Pakistan using DNA barcoding. The main objectives are to identify the available clupeids at the molecular level and to evaluate their genetic distances, phylogenetic relationships, haplotype networks of threatened species, and barcode gap across sampled specimens from the coastal area of Pakistan. The present research will contribute to the global fish barcode reference with novel genetic data from an understudied area of Pakistan.

MATERIALS AND METHODS

Sample Collection and DNA Extraction:  Samples were collected from 2019 to 2023 from the major fish landing facilities along the coastal area of Pakistan, including Karachi Fisheries Harbour, Sindh (24.848° N, 66.975° E), and Gawadar Fish Harbour, Balochistan (25.126° N, 62.322° E), located on the Arabian Sea coast. Once samples were collected, they were transported to the Centre of Excellence in Marine Biology (CEMB), University of Karachi. After washing, morphological identification was done using the FAO's field identification guide (Psomadakis et al., 2015). Overall, 30 specimens representing eight species of the Clupeidae family were collected (details are given in Table 1). Upon identification, each specimen was labelled with a specific laboratory code. Each specimen was placed in its natural pose with the common pins, and the specimens were photographed using a high-resolution Camera. Later on, the samples were placed in an ice box and transferred to the Department of Biotechnology at the University of Sargodha for molecular work. The detailed information about each specimen, including collection dates, taxonomy, etc, was noted in a standardized Excel template and was uploaded to global databases (GenBank and BOLDsystems). After thawing, the tissue samples were excised from each of the specimens using sterilized conditions and instruments, and were stored in 95% ethanol and at -20°C for subsequent molecular analysis. All the specimens were stored in 95 % ethanol and are now available at the Department of Biotechnology, University of Sargodha. Every specimen is linked to a distinct BOLD Systems identifier and a voucher-linked reference, allowing access to the metadata, images, and available sequence data of the specimen. To make them traceable, the identifiers are provided in Table 1. All procedures involving animal handling were approved by the Institutional Bioethics Committee, University of Karachi (Approval No: IBC-KU-284/2023). DNA was extracted using the GENEkit extraction protocol (Catalogue No. K0721).

Table 1: Metadata for each specimen

PCR Amplification, Purification, and Sequencing: The PCR reaction was carried out using the genomic DNA in 25 µL reaction volumes with Universal fish COI primers FishF1 and FishR1 (Karim et al., 2016), which were used for PCR amplification. FishF1: 5ʹ-TCA ACC AAC CAC AAA GAC ATT GGC AC-3ʹ; FishR1: 5ʹ-TAG ACT TCT GGG TGG CCA AAG AAT CA-3ʹ. The 25µL reaction for one sample had 15.75µL of PCR water, 2µL of MgCl2, 2.5µL of dNTPs, 2.5µL of Taq buffer, 0.25µL of F1, 0.25µL of R1, 0.25µL of Taq polymerase and 1.5µL of DNA sample.  An initial denaturation at 94°C for 10 minutes, followed by the final denaturation at 94°C for 30 seconds, was set for the denaturation step. The annealing temperature was set at 55°C for 45 seconds, and the extensions were done at 72°C for 1 minute, followed by the final extension at 72°C for 10 minutes. All PCR reactions included negative controls to detect possible contamination. The amplified PCR products were visualized on a 1.5% agarose gel. The specific band of 650bp was cut from the gel, and the gel extraction was done using the Thermo Scientific GeneJET Gel Extraction Kit (Catalogue No. K0691). The purified product was sent for Sanger sequencing at the Canadian Centre for DNA Barcoding (CCDB). Quality of the sequences obtained was assessed through visual examination of chromatograms on Geneious Prime 2021.1 (Biomatters, Auckland, New Zealand) to see clear and unambiguous resolution of peaks. Poor-quality areas and unclear bases were removed before downstream analyses. All quality sequences were then uploaded to the BOLD Systems and NCBI GenBank databases.

Barcode Gap and Genetic Distance Analysis: The obtained sequences were trimmed and assembled using the Geneious Prime 2021.1 (Biomatters, Auckland, New Zealand) software. A total of 30 sequences representing eight species were considered for analysis (Table 1). The processed sequences were aligned using MEGA11 software. The resulting alignment sequence was used to calculate the pairwise genetic distances among Clupeidae species using the Kimura 2-parameter (K2P) model. The Barcode gap analysis was done via BOLDsystems analytical tools, utilizing both intra- and interspecific K2P distances.

(https://www.boldsystems.org/index.php/Login/page?destination=MAS_Management_UserConsole)

Haplotype Network Analysis: A haplotype network analysis was performed for T. toli using experimental and publicly available COI sequences retrieved from NCBI GenBank using the search term: “Tenualosa toli AND COI”. Missing data, ambiguous nucleotides and short reads were filtered out. All the sequences were aligned with MEGA11; moreover, the alignment was verified and trimmed to a fixed length of 560bp for the comparison. The final curated dataset was then used to construct the haplotype network. The detailed information is given in Supplementary Table 1. Upon sequence alignment and trimming, DNAsp v6 (Librado and Rozas, 2009) was used for the haplotype calling. The obtained haplotype data were then converted to NEXUS format for visualization using PopART v1.7. The TCS network method (Leigh and Bryant, 2015) was employed to infer haplotype connectivity and potential ancestral types. Accession numbers and metadata of all sequences are provided in Supplementary Table 1.

Phylogenetic Tree Construction: The phylogenetic tree was constructed using a total of 30 sequences (29 from the experimental species and one outgroup). Due to the small sample size (only one), S. longiceps was removed from the phylogenetic tree to improve the interpretation of results. The sequences were aligned and curated in MEGA11. Ilisha elongata (Family: Pristigasteridae; Accession Number: HM030778) was selected as the outgroup. The Best DNA/Protein Models (ML) function in MEGAX was utilized to identify the optimal model for phylogenetic reconstruction. Based on the lowest BIC, AIC, and AICc values, the Hasegawa-Kishino-Yano (HKY) model was chosen. The Maximum-Likelihood analysis was conducted using this model with 1000 bootstrap replicates to assess node support. The final tree was annotated and visualized using the iTOL (Interactive Tree of Life) online platform (Dinov et al., 2008; Tamura et al., 2021).

RESULTS

Barcode Gap: A clear gap between intra- and inter-specific genetic distances, for each of the eight Clupeidae species, was revealed, confirming the presence of a barcode gap in all cases. Maximum intra-specific distances ranged from 0% to 1.53%, with S. gibbosa exhibiting the highest divergence (1.53%) while most other species showed little to no variation.

Figure 1: Max intraspecific distance vs nearest neighbour

The distance between neighbouring species was significantly higher than intra-specific distances, ranging from 0.1476 to 0.2241, ensuring that all points fell below the diagonal in both the Max and Mean Intra-Specific vs Nearest Neighbour plots. Moreover, the Individuals per Species vs Max Intra-Specific graph showed that increasing the sampling did not significantly affect intra-specific divergence, except in the case of S. gibbosa (Figure 1). Overall, the data confirm effective species delineation using DNA barcoding within the studied group.

Table 2: Summary of barcode gap analysis for Clupeidae species

Pairwise distance analysis: The genetic distance between representative sequences of each species was calculated using the Kimura 2-Parameters (K2P) model (Table 3). The analysis revealed an evident interspecific divergence among all species of Clupeidae. The greatest genetic distance was found between T. toli and E. thoracata (25.4), and comparatively lower genetic distance was observed between H. kelee and S. gibbosa (14.6). In general, the distance matrix shows that there is a significant genetic separation between species, which is consistent with the barcode gap analysis. An entire sample-level heatmap is included in Supplementary Figure S1.

 Table 3. Pairwise interspecific genetic distances (%) based on the K2P model for eight Clupeidae species.

Haplotype Network: Considering the IUCN vulnerable status of T. toli, a haplotype network (Figure 2) was created to explore the genetic variation among the individuals from four countries (green = India, pink = Malaysia, blue = Pakistan, orange = Bangladesh). Eight haplotypes (Hap1 to Hap8) were found. Hap1 was the most frequent and central haplotype shared among individuals from India, Pakistan, and Bangladesh. Indian individuals were further distributed in Hap2 and Hap3, which were only a few mutational steps away from Hap1. The Malaysian haplotypes (Hap4-Hap7) constituted a distinct group from Hap1 and were separated from it by a few mutational steps. Likewise, Hap8 consisted of individuals from Bangladesh and exhibited relatively greater variation in the network. However, due to the limited number of samples and incomplete geographic distribution, these patterns must be taken with care and warrant further investigation with more extensive sampling and other molecular markers.

Figure 2: Haplotype network of T. toli constructed from its COI sequences from India (green), Malaysia (pink), Pakistan (blue) and Bangladesh (orange). Each circle is a haplotype (1-8) with the size of the circle reflecting the number of individuals. Lines denote mutation steps, and hash marks denote the number of mutations between haplotypes. The network shows the genetic relationships and geographical distribution of the haplotypes in the sampled populations.

Phylogenetic Tree: Ilisha elongata (Family: Pristigasteridae) was included as an outgroup to reconstruct and re-root the COI-based phylogenetic tree to clarify evolutionary relationships of seven Clupeidae species (Figure 3). The ingroup and the outgroup species are distinctly depicted by the rooted rectangular tree. Each species has a distinctive colour-coded clade: T. toli (red), E. thoracata (green), S. gibbosa (blue), A. chacunda (yellow), H. kelee (gray), N. nasus (pink), and N. arabica (light orange). Each species showed a well-supported monophyletic cluster, and there was no overlap between individuals of different species. The tree topology showed a clear differentiation between the species.

Figure 3: Rooted phylogenetic tree of Clupeidae species based on COI sequences, labelled by GenBank accession numbers (PV426823-PV426852). The tree is rooted using I. elongata (Family: Pristigasteridae) as an outgroup. Species colour codes are as follows: T. toli (red), E. thoracata (green), S. gibbosa (blue), A. chacunda (yellow), H. kelee (gray), N. nasus (pink), and N. arabica (light orange), while the outgroup (I. elongata) is shown in cyan. Bootstrap support values are indicated at the nodes.

DISCUSSION

The present study aimed to explore genetic diversity and characterization of the Clupeidae family members from Pakistan through DNA barcoding. The results show clear differentiation between different species, including the morphologically similar species such as H. kelee and T. toli, due to the presence of a distinct barcode gap, clustering in phylogenetic analyses, and a clear difference in genetic distances. The integration of genetic distances, phylogenetics, and geographic haplotype analysis of T. toli offers a comprehensive molecular framework for understanding species diversity within the Clupeidae family. Although specimens were collected over five years from major landing sites, the sampling was opportunistic rather than fully random and resulted in uneven representation among species. Some species (e.g., S. gibbosa) were represented by multiple individuals, whereas others (e.g., S. longiceps) were represented by a single specimen. This imbalance limits the robustness of intraspecific genetic distance estimates and may influence comparisons among taxa. Therefore, interpretations for species with low sample sizes, particularly singleton records, should be treated with caution.

The barcode gap analysis showed a clear difference between intra- and interspecific genetic distance among the studied Clupeidae species, which validates the existence of a barcode gap. The level of intraspecific divergence was relatively low (ranging from 0.00% to 1.53%), compared to the level of interspecific genetic distances (14.6% to 25.4%). This pattern supports effective species-level discrimination among the studied taxa. However, it should be noted that some species, e.g. N. arabica, showed intraspecific variation of 0.00%. This can be due to low sample size or genetic variations in the sampled individuals. Correspondingly low or negligible intraspecific variation has been similarly observed in studies with limited sampling and does not always reflect the complete genetic diversity of a species. The observed interspecific divergence values are within the expected range of marine clupeid fishes. Similar studies for the clupeids from the Bay of Bengal have reported values of interspecific divergence reaching up to 20 percent with very low values of intraspecific variation (Haque, 2019). Similarly, research in the northern Arabian Sea has reported interspecific divergence of over 17%, which supports species-level resolution within clupeids (Amir et al., 2022). In general, the patterns of genetic distance identified in this study support the validity of COI-based DNA barcoding as an effective method to differentiate closely related species within the Clupeidae family. However, due to the uneven sampling of taxa, especially those that have few representatives, these results are to be regarded as tentative, and additional research with larger sample sizes is necessary to prove these trends.

Considering the IUCN vulnerable status of Tenualosa toli, the haplotype diversity was determined from the experimental sequences and all available sequences on NCBI (Supplementary Table 1). The haplotype diversity can play a paramount role in revealing the marked geographic and genetic differentiation among species populations (Du et al., 2009; Zhao et al., 2021). Hap1 was the most prevalent and centrally positioned haplotype in the network and was observed in individuals from Bangladesh, India and Pakistan, indicating possible genetic similarity among these populations. Indian individuals were subdivided into Hap2 and Hap3 close to Hap1, suggesting low genetic divergence. On the other hand, the Malaysian haplotypes (Hap4-Hap7) were grouped in a distinct cluster in the network and were not shared by Hap1 and Hap8, which comprised solely of Bangladeshi individuals, exhibited relatively high internal network divergence. These patterns might be a result of structuring due to geographical variations or low population connectivity. Partial barriers to gene flow may exist due to geographic barriers like the Bay of Bengal and the Andaman Sea, possibly reducing gene flow between populations. Another factor contributing to the observed genetic differentiation may be life history traits of T. toli such as spawning behaviour, habitat preference, and restricted long-distance dispersal. However, the interpretations presented here should be viewed as tentative because they may not be representative at the geographic level or have small sample sizes from the countries involved. Several regions of Southeast Asia, such as Myanmar, Thailand and Indonesia, did not have sequences available to be included in the present analysis. Thus, more comprehensive geographic sampling and the use of more molecular markers will be needed to fully understand regional genetic structure and population connectivity in T. toli. However, the observed patterns of haplotype are generally similar to previous studies that found high haplotype diversity and population differentiation at T. toli in geographically isolated regions. (Habib et al., 2022; Sultana et al., 2022). 

The phylogenetic tree depicted non-overlapping and monophyletic clades for each Clupeidae species. The close relations of N. nasus and N. arabica are supported by COI-based molecular evidence, as they are from the same genus and occupy the same ecological niche (Lavoué et al., 2007; Wang et al., 2022). Additionally, species like T. toli and H. kelee, believed to diverge early in the tree of life, show anadromous life histories and possess distinct reproductive behaviours. Morphologically, these species are similar and often lead to misidentification in the field. The present study provides the first COI-based identification from the coastal area of Pakistan. Nevertheless, numerous other similar studies have been conducted from other parts of the world (Afrin et al., 2024; Sarker et al., 2021).

Overall, DNA barcoding, COI-based genetic distance metrics, haplotype diversity, and COI-based phylogenetic reconstruction provide strong justification for the taxonomic resolution and evolutionary distinctiveness of Clupeidae species in the northern Arabian Sea. However, DNA barcoding also has some limitations, particularly when using DNA barcoding to identify species at the species level within the Clupeidae family. In some lineages, mitochondrial markers may have a reduced resolution because of relatively slow rates of evolution, which can impair their capacity to differentiate newly diverged species. Also, other aspects like the lack of lineage sorting and possible introgression can further blur genetic boundaries. Hence, whereas COI will be useful as a first-level measurement of species identity, future investigations involving nuclear markers or genomic techniques will be advisable in order to obtain a more resolute picture of evolutionary connections and population framework in clupeids.

Conclusion: Overall, these findings provide a paramount COI-based background knowledge on clupeid diversity, which can be used to facilitate better biodiversity monitoring, fisheries management, and conservation measures in the information-limited Pakistani coastal regions where the species continue to be essential in food security and livelihood. The one mitochondrial COI gene was actually useful to delimit species boundaries and expose haplotype diversity, but with a single-marker approach, there are certain limitations. Future investigations with multi-locus datasets or genomic data will be a crucial study to be performed to gain specific information about evolutionary relationships, demographic history and adaptive variability of Clupeidae, especially from the Pakistani regions.

Conflict of Interest: The Authors declare that there is no conflict of interest

Author Contributions Statement:  MR (Performed research, wrote the manuscript, analyzed data), AMK (Designed research, contributed reagents, analyzed data), AAK (Designed and performed research), SA (Analyzed data, reviewed manuscript), AM (Performed research), MI (Analyzed data, reviewed manuscript) and MA (Analyzed sequence data).

Data Availability Statement: The COI barcode sequences generated during this study are publicly available in the NCBI GenBank repository under accession numbers PV426823-PV426852. Voucher-associated specimen information and metadata are available through the BOLD Systems database. Publicly retrieved sequences used for haplotype network analysis, along with their metadata and sequence lengths, are provided in Supplementary Table 1. Sequence alignments and additional analytical data are available from the corresponding author upon reasonable request.

Acknowledgment: We are delighted to accomplish the current research with the help of the Higher Education Commission of Pakistan (NRPU-10403). Sequencing work was performed at the Canadian Centre for DNA Barcoding (CCDB), University of Guelph, Canada.

REFERENCES

Abbas, G. and M.W. Khan (2023). Studies on the Use of Aquatic Food in Pakistan. J. Zool. Syst. 1: 40-57. https://doi.org/10.56946/jzs.v1i2.246

Afrin, S., M.A. Baki, M. Chowdhury, N. Sultana, S. Saha, A. Sarker and M. Begum (2024). DNA barcoding of mislabeled juvenile Tenualosa spp. as Gudusia chapra in the fish markets of Bangladesh. Bangladesh J. Sci. Ind. Res. 59(2): 105-114. https://doi.org/10.3329/bjsir.v59i2.71655

Ahmad Tarmizi, N.N. (2018). Genetic structure of longtail shad Tenualosa macrura (Bleeker, 1852) populations in Sarawak and phylogenetic relationships among clupeids. M.Sc. thesis (unpublished). Universiti Putra Malaysia, Serdang (Malaysia).

Amir, S.A., B. Zhang, R. Masroor, Y. Li, D.-X. Xue, S. Rashid, N. Ahmad, S. Mushtaq, J.-D. Durand and J. Liu (2022). Deeper in the blues: DNA barcoding of fishes from Pakistani coast of the Arabian Sea reveals overlooked genetic diversity. Mar. Biodivers. 52: 37. https://doi.org/10.1007/s12526-022-01272-6

Dinov, I.D., D. Rubin, W. Lorensen, J. Dugan, J. Ma, S. Murphy, B. Kirschner, W. Bug, M. Sherman, A. Floratos, D. Kennedy, H.V. Jagadish, J. Schmidt, B. Athey, A. Califano, M. Musen, R. Altman, R. Kikinis, I. Kohane, S. Delp, D.S. Parker and A.W. Toga (2008). iTools: A Framework for Classification, Categorization and Integration of Computational Biology Resources. PLoS ONE 3(5): e2265. https://doi.org/10.1371/journal.pone.0002265

Du, X., Z. Chen, Y. Deng and Q. Wang (2009). Comparative Analysis of Genetic Diversity and Population Structure of Sipunculus nudus as Revealed by Mitochondrial COI Sequences. Biochem. Genet. 47(11): 884-891. https://doi.org/10.1007/s10528-009-9291-x

Habib, K.A., K. Nam, Y. Xiao, J. Sathi, M.N. Islam, S.K. Panhwar and A.H.M.S. Habib (2022). Population structure, phylogeography and demographic history of Tenualosa ilisha populations in the Indian Ocean region inferred from mitochondrial DNA sequence variation. Reg. Stud. Mar. Sci. 54: 102478. https://doi.org/10.1016/j.rsma.2022.102478

Haque, A.K. (2019). DNA barcoding of marine clupeid fishes (Order-Clupeiformes) of Bangladesh. PhD thesis (unpublished). Brac University, Dhaka (Bangladesh).

Hebert, P.D.N., A. Cywinska, S.L. Ball and J.R. deWaard (2003). Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B 270: 313-321. https://doi.org/10.1098/rspb.2002.2218

Karim, A., R. Saif, A. Ali, B. Nadeem, H.A. Ilyas and W. Sajjad (2024). DNA Barcoding Application in Study of Icthyo-Biodiversity in Rivers of Pakistan. Int. J. Multidiscip. Res. 6(2). https://doi.org/10.36948/ijfmr.2024.v06i02.15854

Khan, A.M., B. Sial, A.A. Khan, M.K. Hanif, M. Raza, S.K. Panhwar and M. Ashfaq (2024). Genetic Diversity and DNA Barcoding of Cyprinidae Habiting Lotic and Lentic Ecosystem of Pakistan. Pak. J. Agri. Sci. 61(2): 463-474. https://doi.org/10.21162/PAKJAS/24.263

Laghari, M.Y. (2018). Aquaculture in Pakistan: Challenges and opportunities. Int. J. Fish. Aquat. 6(2): 56-59.

Lavoué, S., M. Miya, K. Saitoh, N.B. Ishiguro and M. Nishida (2007). Phylogenetic relationships among anchovies, sardines, herrings and their relatives (Clupeiformes), inferred from whole mitogenome sequences. Mol. Phylogenet. Evol. 43(3): 1096-1105. https://doi.org/10.1016/j.ympev.2006.09.018

Leigh, J.W. and D. Bryant (2015). POPART: full‑feature software for haplotype network construction. Methods Ecol. Evol. 6(9): 1110-1116. https://doi.org/10.1111/2041-210X.12410

Librado, P. and J. Rozas (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25(11): 1451-1452. https://doi.org/10.1093/bioinformatics/btp187

Mazumder, S.K. and M.S. Alam (2009). High levels of genetic variability and differentiation in hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes) populations revealed by PCR-RFLP analysis of the mitochondrial DNA D-loop region. Genet. Mol. Biol. 32(1): 190-196. https://doi.org/10.1590/S1415-47572009005000023

Naveed, A., W. Baradi, K. Punhal, I. Malik and A. P (2019). Phenotypic Characteristics of the Clupeid Fish Tenualosa ilisha (Family: Clupeidae) collected from Manjhand Vicinity, Pakistan. Sindh Univ. Res. J. (Sci. Ser.) 51. https://doi.org/10.26692/SURJ/2019.09.611

Psomadakis, P.N., H.B. Osmany and M. Moazzam (2015). Field identification guide to the living marine resources of Pakistan. FAO, Rome (Italy).

Rahimi, P. (2013). Population genetics of three species of Clupeidae family (Sardinella sindensis, Sardinella albella, Dussumieria acuta) in the Persian Gulf and Oman Sea. M.Sc. thesis (unpublished). Islamic Azad University of Zanjan, Zanjan (Iran).

Rahman, M.M., K.A. Ahmed, M.G. Rabbane and M.S. Alam (2025). Genetic Diversity Resonates With Conservation Strategies: A Case Study of LABEO ROHITA Population. Ecol. Evol. 15: e71480. https://doi.org/10.1002/ece3.71480

Raza, M., A.M. Khan, A.A. Khan, S.A. Raza Bukhari, S.K. Panhwar and M. Ashfaq (2025). Genetic diversity of nearly threatened and vulnerable species of Serranidae fish family from the coastal area of Pakistan. J. Asia-Pac. Biodivers. 18(4): 798-806. https://doi.org/10.1016/j.japb.2025.01.011

Sarker, A., J. Jiang, H. Naher, J. Huang, K.K. Sarker, G. Yin, M.A. Baki and C. Li (2021). Cross-species gene enrichment revealed a single population of Hilsa shad (Tenualosa ilisha) with low genetic variation in Bangladesh waters. Sci. Rep. 11: 11560. https://doi.org/10.1038/s41598-021-90864-6

Sial, B., A.M. Khan, M. Raza, A.A. Khan, S.A.R. Bukhari, M.K. Hanif, S.K. Panhwar and M. Ashfaq (2024). Northern Arabian Sea: Rare Fish Diversity and Biogeographic Affinities. Punjab Univ. J. Zool. 39(2): 191-212. https://doi.org/10.17582/journal.pujz/2024/39.2.191.212

Sultana, S., M.M. Hasan, M.S. Hossain, M.A. Alim, K.C. Das, M. Moniruzzaman, M.H. Rahman, M. Salimullah and J. Alam (2022). Assessment of genetic diversity and population structure of Tenualosa ilisha in Bangladesh based on partial sequence of mitochondrial DNA cytochrome b gene. Ecol. Genet. Genomics 25: 100139. https://doi.org/10.1016/j.egg.2022.100139

Tamura, K., G. Stecher and S. Kumar (2021). MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 38: 3022-3027. https://doi.org/10.1093/molbev/msab120

Wang, Q., L. Purrafee Dizaj, J. Huang, K. Kumar Sarker, C. Kevrekidis, B. Reichenbacher, H. Reza Esmaeili, N. Straube, T. Moritz and C. Li (2022). Molecular phylogenetics of the Clupeiformes based on exon-capture data and a new classification of the order. Mol. Phylogenet. Evol. 175: 107590. https://doi.org/10.1016/j.ympev.2022.107590

Zhao, Y., X. Zhu, Y. Jiang, Z. Li, X. Li, W. Xu, H. Wei, Y. Li and X. Li (2021). Genetic diversity and variation of seven Chinese grass shrimp (Palaemonetes sinensis) populations based on the mitochondrial COI gene. BMC Ecol. Evol. 21: 167. https://doi.org/10.1186/s12862-021-01893-8