2.1 Study area and specimen collection
Fish samples were collected from marine and coastal habitats, fish landing centers, fish markets or from the local fishermen from July 2015 to June 2019. Most of the specimens were collected from the Cox’s Bazar and Patuakhali regions. At least three specimens were collected for each species. In case of rare ones single specimen were analyzed. Personal fishing was also conducted to collect some rare and non-commercial fish species whenever necessary. Digital photographs of all the fishes were taken immediately and taxonomic identification of specimens was done following previous reports (Talwar and Jhingran, 1991; Rahman et al., 2009; Siddiqui et al., 2007; Nakabo, 2002; Carpenter et al., 2002a, 2002b; Last et al., 2016). Immediately after collecting the specimens, tissue samples were excised and stored in 90% ethanol. Voucher specimens were fixed with 10% formalin and then transferred to 70% ethanol solution for preservation. Voucher specimens were transported to Dhaka and deposited in the Dhaka University Zoology Museum (DUZM).
DNA barcoding
DNA was isolated from muscle sample using the QIAGEN DNeasy Blood & Tissue Kit following the manufacturer’s protocol, under sterile condition. The concentration of the isolated DNA was measured in NanoDrop™ spectrophotometer to evaluate its quality and quantity. A 658 bp long fragment from the 5′ region of the COI gene was PCR-amplified by the primer pair FishF2 5′TCGACTAATCATAAAGATATCGGCAC3′ and FishR2 5′ACTTCAGGGTGACCGAAGAATCAGAA3′. The primer pair FishF1 5′TCAACCAACCACAAAGACATTGGCAC3′ and FishR1 5′TAGACTTCTGGGTGGCCAAAGAATCA3′ were used for the amplification of COI that failed to amplify using FishF2/FishR2. The PCR amplification of each sample was conducted in a 25 µl volume comprised of 23 µl of PCR Master Mix and 2 µl of template DNA that was subsequently spun for 30 seconds for homogenization. The components of the PCR Master Mix were as such: 12.5 µl Taq Polymerase, 8.5 µl Nano Pure water, 1 µl forward primer and 1 µl reverse primer. The PCR amplification was carried out in the ASTEC Thermal Cycler GeneAtlas  (Astec Co. Ltd.) where the thermocycling profile was customized as such: an initial denaturation at 95℃ for 5 minutes followed by 41 cycles of denaturation at 95℃ for 30 s, primer annealing at 54℃ for 30 seconds, extension for 72℃ for 1 minute, and a final extension at 72℃ for 5 minutes. The PCR products were kept at room temperature for 15 minutes, and preserved afterward at -20℃ until further downstream application. The amplicons were further identified through electrophoresis in a 1% agarose gel. Then they were purified using PureLink™ PCR purification kit. The good quality amplicons were then parceled for sequencing to the First BASE laboratories, Malaysia where they conducted the Sanger’s dideoxy sequencing using ABI PRISM 3730xl Genetic Analyzer exploiting the BigDye® Terminator v3.1 cycle sequencing kit chemistry. The assembled contigs were prepared by the CAP3 DNA assembly program. These newly obtained sequences were uploaded in the BLASTn suite to check whether they meet the threshold value of ≥ 97% for both the percent identity and query coverage. The sequences of the high-fidelity amplicons were then deposited in the GenBank with the help of the Barcode Submission Tool with detailed source information and feature annotation.