Cryptic biodiversity of freshwater fish species in Bangladesh

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 2 / 18 PLOS One Cryptic biodiversity of freshwater fish According to Shelley et al. [10], cryptic fish species present one of the main obstacles to the proper management, conservation, and planning of fish biodiversity. Cryptic species, of which are genetic variants, however, physically identical are a common occurrence and they are con- cealed as nominal species. Although morphology-based investigations have been conducted in Bangladesh [11–13], accurate identification of a species macroscopically is difficult for ichthyologists as well, leading to errors in lists of species included in the literature. It is evident from several publications and review papers that, Bangladesh’s taxonomy is out-of-date and not harmonized, when compared with that of the neighboring countries [14]. Hence, an integrative taxonomy method incorporating molecular and morphological data is required to overcome this obstacle and provide a seamless and widely recognized list of species inhabiting this nation. Researchers describe additional new species of fish each year, but it is not clear as to how many species are endemic to this nation. In the current study, the authors have conducted phylogenetic analyses on fish specimens available in fish markets and natural resources nationwide to examine freshwater fish bio- diversity in Bangladesh. Whenever necessary, morphological data has been used to identify cryptic or candidate species. The DNA-based barcoding method has proven to be an import-

ant molecular tool for non-specialists for species identification [15], and the mitochondrial cytochrome c subunit I (COI) sequence is a dependable barcode marker for measuring and assessing the taxonomic status of fish species [14,16–18]. This study attempts to review the biodiversity of Bangladesh’s freshwater fishes, and explore the possible presence of cryptic species using the DNA barcoding technique. Materials and methods Study area, duration and identification of sample A total of 124 fish specimens were collected from all regions of Bangladesh (see Table 1), photographed and their sex was recorded. All specimens were transferred at the “Evolution and Diversity Research Laboratory” at Jamalpur Science and Technology University, located in Jamalpur, Bangladesh. A voucher number was assigned to each specimen (please refer to Table 1). For subsequent molecular research, the species were maintained in either saturated DMSO/NaCl solution or 95% ethanol. Table 1 lists the study sites in detail. Initial identifi- cation of the collected fish species was based on current taxonomy knowledge and literature dealing with the fisheries science [4]. If necessary, the names of the species that have been accepted by the Catalog of Fishes online portal have been adhered to [6]. DNA extraction and sequencing Genomic DNA of all fish specimens was extracted and purified from conserved fin tissues using Chelex 100 [19]. Using the primers Fish-F1, Fish-F2, Fish-R1, and Fish-R2, the partial COI fragment was amplified by polymerase chain reaction (PCR) using EmeraldAmp PCR

Master Mix (Takara Bio) [20]. Cycle conditions described in a previously conducted study [14] were followed for performing PCR. The amplified PCR products were purified using polyethylene glycol precipitation. Cycle sequencing procedures were then performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Bioscience). Sequencing was per- formed using ABI 3500 automated sequencers. The newly discovered sequences were incor- porated in the International Nucleotide Sequence Databases (INSD) through the DNA Data Bank of Japan (Accession numbers LC823243–LC823366). database (accession numbers: LC823243 and LC823366). Other relevant data are within the paper and its supporting files. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist.