DNA barcoding and phylogenetics of Wallago attu using mitochondrial COI gene from the River Indus

1 Introduction

In Pakistan, the number of freshwater fish species is not more than 193 (Abro et al., 2020). In River Indus Pakistan, more than 180 freshwater fish species have been recorded (Sheikh et al., 2017). In its tributaries, a high range of diverse, commercially important and representative fish fauna has been found annually. Many scientists have huge data on the diversity and distribution pattern of fish but they still, need a lot of attention (Hussain et al., 2016). Pakistan has varied water resources comprising dams, rivers, streams, and canals. The River Indus system is the largest river system in Pakistan. Near Kailas Mount, River Indus originates in the Tibet Gangdise Range. The Chenab, Sutlej, Jhelum, and Ravi also drain into River Indus in the Punjab plains (Mirza and Mirza, 2014). It had a diverse variety of fish species. Almost 43 native fish species are economically and commercially important (Sherzada et al., 2020). Among all these species, Wallago attu is important economic fish belonging to the family Siluridae under the order Siluriforms. Malee is a common name for W. attu with Vulnerable conservation status (Ng et al., 2019). W. attu is widely distributed in Asian countries, including Pakistan, Nepal, India, Bangladesh, Indonesia, Vietnam, Sri Lanka, and Afghanistan (Siraj et al., 2016). It is a catfish with a fast growth rate and a good food fish with high nutritional value (Gupta, 2015). It has high protein content. Due to its importance and environmental changes, it is near to being threatened (Hussain et al., 2016). According to researchers, there is a need to improve conservation and its protection.

The study of the morphology of fishes plays a vital role in the betterment of conservation, evolution, ecology, and environmental variations (Ozcan and Altun, 2015). Morphological relationships play an important role between fish stock identification and species population and distribution. But it is difficult to identify this species exactly based on morphology due to the lack of some morphological features and also taxonomic expertise. DNA barcoding can offer a precise and rapid method of identification of Wallago attu (Bhattacharjee et al., 2012).

DNA barcoding is the most commonly used genetic technique for species identification and has also been used for species discovery in different organism groups (Ude et al., 2020; Kamran et al., 2020; Tsoupas et al., 2022). DNA barcoding method in which a small mitochondrial gene fragment was used for species identification was developed by Hebert and his colleagues at the University of Guelph in Canada (Rahman et al., 2019). In this method, small fragments of mitochondrial gene almost 655 bp of COI gene fragment from the 5′ were used as a standard marker for the identification of eukaryotic species including vertebrates and invertebrates (Keskİn and Atar, 2013). Due to the advancement in computational techniques, DNA sequencing becomes the major source to understand genetics and evolutionary relationships (Hajibabaei et al., 2007). DNA barcode system is applicable for all fish species identification and is a simple, reliable, accurate, and cheap method for the identification of species (Lakra et al., 2011; Becker et al., 2011). A good barcode has less intraspecific distance than interspecific (Hajibabaei et al., 2007). DNA barcoding can accurately differentiate between the species which have highly similar morphological features. This method can identify species at any developmental stage. Cryptic species can also be identified using this technology (Bingpeng et al., 2018).

For the discrimination between closely resembled species across dissimilar animal phyla mitochondrial Cytochrome c oxidase subunit I is best for molecular analysis of marine and freshwater fishes (Hebert et al., 2003). For differentiation of a vast variety of animal species presently, there is no such great data on W. attu ecology, biology, and molecular analysis in the area of the River Indus, Punjab, Pakistan. Keeping in view the above facts, the study was planned to assess morphology, meristic characteristics, and DNA barcoding of W. attu from the River Indus. The study will be a great addition to the research and conservation of W. attu in the River Indus, Punjab, Pakistan.

2 Materials and methods

Ethic statement:

All procedures regarding the sampling were according to the protocols of the ethical review committee of Government College University Faisalabad, Pakistan in line with the international standards on animal experimentation.

2.2

2.2 Morphological analysis

Identification of all morphometric characters was done by using a special key of the fishes of Punjab, Pakistan (Mirza and Sharif, 1996). All morphometric measurements of each sample were done with the help of scales, magnifying glass, and vernier calipers were done at room temperature (Fig. 1). After the identification based on morphology samples were further processed for DNA barcoding.

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Fig. 1

Wallago attu.

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3 Statistical and sequence analysis

Morphological parameters were analyzed using Minitab 17 software by one-way ANOVA and t-test. The sequencing data were converted into FASTA format. BLASTS of the COI sequences were done at the NCBI to determine the best match homology. Evolutionary analysis of the aligned sequences was conducted in the software MEGA 11. The phylogenetic tree was rooted using Ompok bimaculatus as an out group. The history of evolution was directed using the Maximum Likelihood method (Nei and Kumar, 2000). Evolutionary divergence was calculated using the Kimura 2-parameter distance model (Kimura, 1980). Sequenced data was used to generate unique QR code by using an online QR code generator for precise detection of this specie.

4 Results

The species were first identified by shape, size, fin ray count, color, and other morphometric and meristic characteristics (Table 1). All these characters showed significant differences (p < 0.005) between and within groups. Pectoral fins of fish belonging to the family Siluridae, genus wallago were considered for DNA barcode creation. Amplification of the mitochondrial COI gene of 650 bp was done by using Fish F1 and Fish R1 primers.

There were 605 of 675 conserved sites, 61 of 675 variable sites, 33 of 675 parismony informative sites, and 28 of 675 singleton variable sites found in the barcodes. Transversional substitutions (sv = 71.36) were found to be more common than transitional substitutions (si = 28.65), with R ratio of 0.40 for the dataset. BLAST analysis revealed that all the sequences of COI gene of W. attu specimens have a maximum identity (100%) with the respective sequence in the mitochondrial region in the GenBank database (Table 2). After editing the consensus length of all barcode sequences was 655 bp and no deletions, insertions, or stop codons were observed in any sequence. All analyzed sequences were more than 600 bp which confirmed that NUMT (Nuclear DNA sequence originated from mDNA sequences and are less than 600 bp in length) were not sequenced in the present study.

A Phylogenetic tree was constructed by using the maximum likelihood (ML) method based on the K2P method (Fig. 2). The analysis involved 21 nucleotide sequences of experimental species. To compare with experimental specie some reference sequences from NCBI with accession numbers from the BLAST search were downloaded to minimize error for identification. After this, experimental and reference sequences were arranged in FASTA format. Then, all sequences were uploaded to MEGA 11 software for advance alignment, construction and analysis, of the phylogenetic tree. The analysis concerned 39 consensus sequences. Positions of codon were included 1st + 2nd + 3rd + Noncoding. Gaps and missing data from all sequences were eliminated. After elimination of all gaps total 572 positions were remained in the final sheet. By using MEGA 11 software all evolutionary analyses were accomplished. The evolutionary tree between specie exposed that the majority of the specie clustered simultaneously indicating less divergence between them. Similarly, these species showed less pairwise genetic distance (Table 3).

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Fig. 2

Maximum likelihood tree representing genetic relationship among Wallago attu.

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The Percentage base composition of all 4 bases in COI sequences of all W. attu from seven sites was calculated (Table 4). The overall mean nucleotide base frequencies observed for these sequences were T (28.6%), C (27.9%), A (25.1%), and G (18.3%). The overall AT content (53.7%) was higher than GC content (46.2%).

This is the first study of this specie from the selected sites to generate QR codes for accurate identification of W. attu. This specie could be identified with their unique QR code (Fig. 3).

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Fig. 3

QR codes generated using Fasta Sequences of Wallago attu.

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5 Discussion

It is very important to identify fish biodiversity accurately before the utilization of fish in any field. The standard and traditional approach for fish identification is based on morphological characters, but when there is phenotypic plasticity this approach is not reliable. To understand the speciation and phylogenetic relationship of fishes in the past, the only tools were meristic and morphometric. By the investigation of a small fragment of the whole genome, the discrimination of biodiversity is allowed by the micro genomic identification system. Mitochondrial DNA of animals is the best technology for the identification of species analysis than nuclear DNA due to the lack of haploid and introns approach of inheritance. (Cytochrome c oxidase subunit I) COI gene is used as a standard marker for the bio-identification of species (Hebert et al., 2003).

In the current study, thirty-five (35) individuals of Wallago attu (catfish: Siluridae family) were collected from River Indus Punjab, Pakistan. 21 out of 35 samples were processed for DNA barcoding. These samples were initially identified based on morphological characters and to resolve the taxonomic problems; a molecular study has also been done considering the effectiveness of DNA barcoding for the identification of W. attu. Therefore, this study shows so as to the results obtained from the morphometric and molecular analysis is similar.

W. attu is rare and ranked near threatened (Khan et al., 2008) in this area due to which a limited number of samples were taken for this study. Identification based on morphology is a basic method to know about demographic characteristics, growth, and systematic variation of fish. Morphometric analysis performs a vital role in the estimation of the relationship between different body parts. The Mean and standard deviation of all the samples showed that all relationships of TL with HL, SL, SnL, FL, ED, PrDL, and PoDL are extremely significant (p < 0.005), showed that all (length-length relationships) LLRs are considerably interrelated (Tables 1, 2). These extremely significant relationships in the current study for length-length relationships are in universal harmony with other studies described on Tor potitura (Naeem et al., 2011b), Oreochromis mossambicus (Naeem et al., 2011a).

The morphological parameters demonstrated a comparative constructive increase with an increase in the length of fish. (Ujjania et al., 2012) too revealed that with an enhancement in fish length morphometric parameters were also recorded with positive growth. Meristic counts were almost invariable with different body lengths in all the collected samples of fish, so it concluded that the meristic counts were independent of body length (Ishtiaq and Naeem, 2016).

6 Conclusions

In the current study, the identification of catfish Wallago attu was done based on morphology which was further confirmed through DNA barcoding. Morphological identification has many limitations due to which DNA barcoding has enough variability to differentiate among species. This research will be helpful for future researchers and this data has a great addition to River Indus biodiversity and conservation.