Cryptic biodiversity of freshwater fish species in Bangladesh

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 1 / 18 OPEN ACCESS Citation: Hasan M, Kambayashi C, Anik ZH, Islam MS (2025) Cryptic biodiversity of freshwater fish species in Bangladesh. PLoS One 20(4): e0318982. https://doi.org/10.1371/ journal.pone.0318982 Editor: Amaal Gh. Yasser, Griffith University, AUSTRALIA Received: August 31, 2024 Accepted: January 26, 2025 Published: April 28, 2025 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal. pone.0318982 Copyright: © 2025 Hasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data availability statement: All DNA sequences files are available from the GenBank RESEARCH ARTICLE Cryptic biodiversity of freshwater fish species in Bangladesh Mahmudul Hasan 1,2*, Chiaki Kambayashi 3, Zahid Hasan Anik2, Md. Saiful Islam 1 1 Department of Fisheries, Jamalpur Science and Technology University, Jamalpur, Bangladesh, 2 Evolution and Diversity Research Laboratory, Jamalpur Science and Technology University, Jamalpur, Bangladesh, 3 Faculty of Science, Niigata University, Niigata, Japan * mhasan@jstu.ac.bd Abstract

Unrecognized cryptic species impede conservation planning and biodiversity assess- ments. DNA barcoding has tremendously expanded the number of novel and cryptic species in biological science. Despite few sporadic studies, the exact number of freshwa- ter species found in Bangladesh is not known. To assess this biodiversity, we sequenced the COI gene of 124 freshwater specimens, which were gathered from various localities around Bangladesh. Seven cryptic species hidden among the currently studied specimens were identified based on the findings of phylogenetic and species delimitation analyses. The preliminary assessment also encompassed a restricted morphological examination of these cryptic taxa. The appearance of cryptic species, some of them possibly endemic, has been hypothesized. This raises concerns regarding the true diversity and evolutionary history of freshwater species in Bangladesh, which are significantly underrepresented in the current systematic frameworks that do not account for DNA data. Our current study provides baseline data that might aid local ichthyologists in their quest to identify addi- tional new species by combining several variables (morphology and ecology). Further research is warranted to protect the priceless freshwater species in Bangladesh. Introduction Bangladesh is situated in the oriental region of the Indo-Himalayan and Indo-Chinese sub-regions [1]. This country is home to the Ganges-Brahmaputra river delta, which is the largest delta in the world. Approximately 230 rivers crisscross through the lands of Bangla-

desh. Among these rivers, 57 are transboundary rivers (54 rivers are shared with India and three are shared with Myanmar) [2,3]. The downstream region consisting of many different rivers, canals, lakes with both flowing as well as static water (beels), and harbors are home to several freshwater fish. Approximately 265 species of freshwater fish are found in this lowland nation [4]. Freshwater fish are found in Bangladesh’s biggest river systems, the Ganges, Brah- maputra, and Meghna, which are collectively equal to 4,339,694 hectares of inland water [5]. Approximately 30.2% of all fish species found worldwide are freshwater fish [6] and these are very valuable both nutritionally and economically [7]. The fishing industry is unquestionably important for Bangladesh’s economy, culture, and nutrition [8,9].

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.

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 3 / 18 PLOS One Cryptic biodiversity of freshwater fish Table 1. Specimens used and identified COI haplotypes found in this study. Voucher Number Scientific Name Family Local Name Location Latitude and Longitude Sample Collection Date Accession number MHBSFMSTU Fish 1 Cabdio morar Cyprinadae Pieli Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823243 MHBSFMSTU Fish 2 Tenualosa ilisha Clupeidae Ilish Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823244 MHBSFMSTU Fish 3 Ailia sp. Ailiidae Bash patari/ Kajuli Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823245 MHBSFMSTU Fish 5 Pisodonophisboro Ophichthidae Bamush/ Kharu Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823246 MHBSFMSTU Fish 6 Awaous sp. Gobiidae Bochabele Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823247 MHBSFMSTU Fish 7 Mastacembelusar- matus Mastacembelidae ShalBaim Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823248 MHBSFMSTU Fish 8 Botiadario Cobitidae Boumach Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823249 MHBSFMSTU Fish 9 Channaorientalis Channidae Raga Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823250 MHBSFMSTU Fish 10 Aplocheiluspanchax Aplocheilidae Kanpona DangarBeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823251 MHBSFMSTU Fish 11 Trichogaster lalius Osphronemidae Khoilsha DangarBeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823252

MHBSFMSTU Fish 12 Trichogaster lalius Osphronemidae Borokhoilsha DangarBeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823253 MHBSFMSTU Fish 13 Trichogaster chuna Osphronemidae Lalkholisha DangarBeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823254 MHBSFMSTU Fish 14 Colisachuna Osphronemidae ChunaKholisha DangarBeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823255 MHBSFMSTU Fish 15 Trichogaster chuna Osphronemidae Lal Kholisa DangarBeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823256 MHBSFMSTU Fish 16 Macrognathusaral Mastacembelidae Tara Baim Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823257 MHBSFMSTU Fish 17 Lepidocephal- ichthyssp. Cobitidae Morichapuiya/ Gutum Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823258 MHBSFMSTU Fish 18 Ompokpabda Siluridae Pabda Gaglajur bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 25-08-2018 LC823259 MHBSFMSTU Fish 19 Mystus cavasius Bagridae Gulsha Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823260 MHBSFMSTU Fish 20 Mystus tengara Bagridae Tengra Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823261 MHBSFMSTU Fish 22 Mystus carcio Bagridae Bujuri Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823262 MHBSFMSTU Fish 23 Channamarulius Channidae Gojar Ghaglajur, Netrokona 24°52’36.2"N 91°31’38.4"E 15-12-2018 LC823263 MHBSFMSTU Fish 25 Nandus nandus Nandidae Bheda Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823264

MHBSFMSTU Fish 26 Channapunctata Channidae Taki Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823265 MHBSFMSTU Fish 27 Mystus cavasius Bagridae Kabashi Tengra Mawaghat Munshigonj 23°27’59.3"N 90°17’22.1"E 25-08-2018 LC823266 MHBSFMSTU Fish 28 Bagariusbagarius Sisoridae Bagha air Kongso, Netrokona 24°52’50.2"N 90°44’03.2"E 25-08-2018 LC823267 MHBSFMSTU Fish 30 Sperata aorella Bagridae Air Kongso, Netrokona 24°52’50.2"N 90°44’03.2"E 25-08-2018 LC823268 (Continued)

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 4 / 18 PLOS One Cryptic biodiversity of freshwater fish Voucher Number Scientific Name Family Local Name Location Latitude and Longitude Sample Collection Date Accession number MHBSFMSTU Fish 31 Trichopsisvittata Osphronemidae PokaKholisha MawaghatMunshigonj 23°27’59.3"N 90°17’22.1"E 25-08-2018 LC823269 MHBSFMSTU Fish 32 Parambassislala Ambassidae Lalchanda Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823270 MHBSFMSTU Fish 34 Xenentodoncancila Belonidae Kakila Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823271 MHBSFMSTU Fish 36 Macrognathus pancalus Mastacembelidae Chikra/ Gujibaim Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823272 MHBSFMSTU Fish 37 Badis badis Badidae Napit koi Jaliarhaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823273 MHBSFMSTU Fish 38 Chacachaca Chacidae Bengachaca Gaglajur Bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 25-08-2018 LC823274 MHBSFMSTU Fish 39 Notopterusnotopterus Notopteridae Foli Balikhola, Kishorganj 24°13’27.8"N 91°06’32.2"E 25-08-2018 LC823275 MHBSFMSTU Fish 40 Labeorohita Cyprinidae Rui Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 17-04-2019 LC823276 MHBSFMSTU Fish 41 Labeocalbasu Cyprinidae Kali baush Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 17-04-2019 LC823277 MHBSFMSTU Fish 46 Esomusdanrica Cyprinidae Darkina Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 25-08-2018 LC823278 MHBSFMSTU Fish 47 Setipinnaphasa Engraulidae Chibuk chela/ Phasha

Guthail, Jamalpur 25°05’13.3"N 89°42’49.0"E 25-08-2018 LC823279 MHBSFMSTU Fish 48 Catlacatla Cyprinidae Katol Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 17-04-2019 LC823280 MHBSFMSTU Fish 49 Cirrhinusmrigala Cyprinidae Mrigal Sanondobari, Jamalpur 25°22’51.3"N 89°44’40.8"E 17-04-2019 LC823281 MHBSFMSTU Fish 50 Daniorerio Cyprinidae Zebra anju/ Zebra Kongsho, Sherpur 24°59’52.2"N 90°00’29.6"E 17-04-2019 LC823282 MHBSFMSTU Fish 51 Coricasoborna Clupeidae Subornakaski Kongso, Sherpur 24°59’52.2"N 90°00’29.6"E 15-03-2019 LC823283 MHBSFMSTU Fish 52 Salmostoma phulo Cyprinidae Narikeli Chela/ Katari Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 02-02-2019 LC823284 MHBSFMSTU Fish 53 Hyporhamphus limbatus Hemiramphidae Ekthota Kendua, Netrokona 24°39’49.9"N 90°50’28.7"E 15-03-2019 LC823285 MHBSFMSTU Fish 54 Channastriata Channidae Shol Kendua, Netrokona 24°39’49.9"N 90°50’28.7"E 02-02-2019 LC823286 MHBSFMSTU Fish 55 Pseudeutropiusathe- rinoides Schilbeidae Batai Kendua, Netrokona 24°39’49.9"N 90°50’28.7"E 22-04-2019 LC823287 MHBSFMSTU Fish 56 Ophichthys sp. Synbranchidae kuchia Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 02-02-2019 LC823288 MHBSFMSTU Fish 57 Wallagoattu Siluridae Boal Kongso, Netrokona 24°52’50.2"N 90°44’03.2"E 25-08-2018 LC823289 MHBSFMSTU Fish 59 Heteropneustesfossilis Heteropneustidae Shing Dangarbeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 02-02-2019 LC823290 MHBSFMSTU Fish 61 Puntius sophore Cyprinidae Jatpunti/ Bhadiputi JaliarHaor, Netrokona 24°41’31.3"N 90°51’46.5"E 25-08-2018 LC823291

MHBSFMSTU Fish 63 Pethia conchonius Cyprinidae Tit punti Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 17-04-2019 LC823292 MHBSFMSTU Fish 64 Chandanama Ambassidae Chanda (lomba) Balikhula, Kishorgonj 24°13’27.8"N 91°06’32.2"E 17-04-2019 LC823293 Table 1. (Continued) (Continued)

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 5 / 18 PLOS One Cryptic biodiversity of freshwater fish Voucher Number Scientific Name Family Local Name Location Latitude and Longitude Sample Collection Date Accession number MHBSFMSTU Fish 65 Parambassis ranga Ambassidae Chanda (Gol)/ Phopa Kendua, Netrokona 24°39’49.9"N 90°50’28.7"E 17-04-2019 LC823294 MHBSFMSTU Fish 66 Macrognathus pancalus Mastacembelidae Guchibaim Mawaghat, Munshigonj 23°27’59.3"N 90°17’22.1"E 17-04-2019 LC823295 MHBSFMSTU Fish 67 Amblypharyngodon mola Cyprinidae Fakasemola JaliarHaor, Netrokona 24°41’31.3"N 90°51’46.5"E 17-04-2019 LC823296 MHBSFMSTU Fish 69 Channagachua Channidae Cheng Dangarbeel, Jamalpur 24°55’06.5"N 89°51’03.6"E 17-04-2019 LC823297 MHBSFMSTU Fish 70 Salmostoma bacalia Cyprinidae Ful chela Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 17-04-2019 LC823298 MHBSFMSTU Fish 71 Eutropiichthysvacha Ailiidae Bacha Ghaglajur, Netrokona 24°52’36.2"N 91°31’38.4"E 25-08-2018 LC823299 MHBSFMSTU Fish 72 Gudusia chapra Clupeidae GoniChapila Sariakandi, Bogra 24°55’08.4"N 89°38’26.0"E 01-07-2018 LC823300 MHBSFMSTU Fish 73 Clupisomagarua Ailiidae Ghaura Mawaghat, Munshigonj 23°27’59.3"N 90°17’22.1"E 17-04-2019 LC823301 MHBSFMSTU Fish 74 Rita rita Bagridae Rita Balikhola, Kishorgonj 24°13’27.8"N 91°06’32.2"E 01-07-2018 LC823302 MHBSFMSTU Fish 75 Hemibagrusmenoda Bagridae Hugli/ Arwari/ Ghagla Ghaglajur bazaar, Netrokona 24°52’36.2"N 91°31’38.4"E 01-07-2018 LC823303

MHBSFMSTU Fish 77 Leiodon cutcutia Tetraodontidae Patipotka Bramaputra river, Jamalpur 24°55’37.0"N 89°57’42.6"E 10-11-2018 LC823304 MHBSFMSTU Fish 78 Devariodevario Cyprinidae Hingra kata punti/ Debari/ Chapchela Ghaglajur Bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 02-02-2019 LC823305 MHBSFMSTU Fish 79 Osteobramacotio Cyprinidae Dhela Gaglajur Bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 02-02-2019 LC823306 MHBSFMSTU Fish 80 Mylopharyngodon piceus Cyprinidae Mohashol Sokal Bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 25-08-2019 LC823307 MHBSFMSTU Fish 81 Labeo gonius Cyprinidae Gonia Sokal Bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 25-08-2019 LC823308 MHBSFMSTU Fish 82 Labeo gonius Cyprinidae Tatkini/ Bhagna/ Raik/ Lacho/bata Balikhula, Kishorgonj 24°13’27.8"N 91°06’32.2"E 25-08-2019 LC823309 MHBSFMSTU Fish 83 Erethistes sp. Sisoridae Kutakanti Ghaglajur Bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 25-08-2019 LC823310 MHBSFMSTU Fish 84 Gagatayoussoufi Sisoridae Gum/ Gagata Ghaglajur, Netrokona 24°52’36.2"N 91°31’38.4"E 25-08-2019 LC823311 MHBSFMSTU Fish 85 Pethia gelius Cyprinidae Gilipunti/ puti Ghaglajur Bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 07-01-2020 LC823312 MHBSFMSTU Fish 86 Rhinomugil corsula Mugilidae Folla, khalla, halla, khorsula Balikhula, Kishorgonj 24°13’27.8"N 91°06’32.2"E 01-01-2020 LC823313 MHBSFMSTU Fish 87 Chandramara chandramara Bagridae Jolbujuri Ghaglajur Bazar, Netrokona 24°52’36.2"N 91°31’38.4"E 07-01-2020 LC823314 MHBSFMSTU Fish 90 Silonia silondia Ailiidae Shilong Kawniar char, Dewangonj 25°23’59.3"N

89°47’49.0"E 15-01-2020 LC823315 MHBSFMSTU Fish 91 Glyptothorax telchitta Sisoridae Lalkutakanti Kawniar char, Dewangonj 25°23’59.3"N 89°47’49.0"E 15-01-2020 LC823316 MHBSFMSTU Fish 92 Gagatacenia Sisoridae Kutakanti, Cenia, Jan- gla, Kaowa, Gang tengra Sanondabar, Jamalpur 25°22’51.3"N 89°44’40.8"E 25-01-2020 LC823317 MHBSFMSTU Fish 93 Gogangra laevis Sisoridae Gang Tengra Dewangonj bazar, Jamalpur 25°10’01.7"N 89°45’57.5"E 15-01-2020 LC823318 Table 1. (Continued) (Continued)

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 6 / 18 PLOS One Cryptic biodiversity of freshwater fish Voucher Number Scientific Name Family Local Name Location Latitude and Longitude Sample Collection Date Accession number MHBSFMSTU Fish 96 Rasboradaniconius Cyprinidae Darkina Kendua, Netrokna 24°39’49.9"N 90°50’28.7"E 22-01-2020 LC823319 MHBSFMSTU Fish 97 Botialohachata Cobitidae Bou/Rani Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 22-01-2020 LC823320 MHBSFMSTU Fish 98 Labeo boggut Cyprinidae Vangon/ Gonari Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 22-01-2020 LC823321 MHBSFMSTU Fish 99 Paracanthocobitis abutwebi Nemacheilidae Balichata/ Puiya Sanondabari, Jamalpur 25°22’51.3"N 89°44’40.8"E 25-01-2020 LC823322 MHBSFMSTU Fish 100 Schistura sp. Nemacheilidae Balikhura/ Puiya Sanondabari, Jamalpur 25°22’51.3"N 89°44’40.8"E 25-01-2020 LC823323 MHBSFMSTU Fish 101 Anodontostomacha- cunda Clupeidae Chacunda Defla Bazar, Jamalpur 25°01’17.1"N 89°50’16.3"E 25-01-2020 LC823324 MHBSFMSTU Fish 102 Salmostoma bacalia Cyprinidae Chela Soal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 21-12-2020 LC823325 MHBSFMSTU Fish 103 Badis badis Badidae Napit koi Sokal bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 01-02-2020 LC823326 MHBSFMSTU Fish 104 Rhinomugil corsula Mugilidae Flathead mullet Sokal Bazar, Jamalpur 24°55’47.4"N 89°56’45.2"E 01-02-2020 LC823327 MHBSFMSTU Fish 105 Pethia conchonius Cyprinidae Kanchonputi/ Taka puti Guthail, Jamalpur 25°05’13.3"N 89°42’49.0"E 05-02-2020 LC823328 MHBSFMSTU Fish 106

Neoeucirrhichthys maydelli Cobitidae GualParapuiya Guthail, Jamalpur 25°05’13.3"N 89°42’49.0"E 05-02-2020 LC823329 MHBSFMSTU Fish 107 Chela laubuca Cyprinidae Chep chela Guthail, Jamalpur 25°05’13.3"N 89°42’49.0"E 05-02-2020 LC823330 MHBSFMSTU Fish 108 Tariqilabeo latius Cyprinidae Matikhora/kalabata Guthail, Jamalpur 25°05’13.3"N 89°42’49.0"E 05-02-2020 LC823331 MHBSFMSTU Fish 109 Pangio Pangia Cobitidae Kuttapuiya/Panga/ Kolipuiya Guthail, Jamalpur 25°05’13.3"N 89°42’49.0"E 05-02-2020 LC823332 MHBSFMSTU Fish 110 Odontamblyopus rubicundus Gobiidae Lalchewa Mawaghat, Munshigonj 23°27’59.3"N 90°17’22.1"E 08-01-2020 LC823333 MHBSFMSTU Fish 111 Apocryptes bato Gobiidae Chewa/ Chiring Mawaghat, Munshigonj 23°27’59.3"N 90°17’22.1"E 08-01-2020 LC823334 MHBSFMSTU Fish 112 Sperata aor Bagridae Guizza air Mawaghat, Munshigonj 23°27’59.3"N 90°17’22.1"E 08-01-2020 LC823335 MHBSFMSTU Fish 118 Ompok bimaculatus Siluridae Kala pabda Rangamati 22°58’00.8"N 92°12’05.6"E 11-01-2020 LC823336 MHBSFMSTU Fish 121 Latescalcarifer Centropomidae Deshivetki PaikariMotshoArot, Barishal 22°42’13.4"N 90°22’32.4"E 27-01-2021 LC823337 MHBSFMSTU Fish 126 Cynoglossus sp. Cynoglossidae Kukurjib Fishery Ghat, Chittagong 22°19’43.9"N 91°50’49.6"E 11-02-2021 LC823338 MHBSFMSTU Fish 130 Polynemusparadiseus Polynemidae Taposhi PaikariMotshoArot, Barishal 22°42’13.4"N 90°22’32.4"E 27-01-2021 LC823339 MHBSFMSTU Fish 134 Tariqilabeo latius Cyprinidae Kalobata Shampurnagarghat, Rajshahi 24°20’55.7"N 88°39’26.5"E 22-12-2020 LC823340 MHBSFMSTU Fish 135

Psilorhynchus sucatio Psilorhynchidae Titari CharlokkhipurShibgonj, Chapainawabgonj 24°35’56.2"N 88°05’20.1"E 21-12-2020 LC823341 MHBSFMSTU Fish 136 Glossogobius giuris Gobiidae Bele Narayanpur, Shibgonj, Chapainawabgonj 24°34’05.7"N 88°05’48.8"E 21-12-2020 LC823342 Table 1. (Continued) (Continued)

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 7 / 18 PLOS One Cryptic biodiversity of freshwater fish Table 1. (Continued) Voucher Number Scientific Name Family Local Name Location Latitude and Longitude Sample Collection Date Accession number MHBSFMSTU Fish 137 Setipinna phasa Engraulidae Pasha Charlokkhipur, Shibgonj, Chapai Nawabgonj 24°35’56.2"N 88°05’20.1"E 21-12-2020 LC823343 MHBSFMSTU Fish 138 Terapon jarbua Terapontidae Rekha Shondhya bazar, Moylaputa,Khulna 22°48’46.7"N 89°33’25.6"E 28-01-2021 LC823344 MHBSFMSTU Fish 139 Cynoglossus sp. Cynoglossidae Kukurjib RupshaGhat, Khulna 22°48’05.5"N 89°34’52.1"E 28-01-2021 LC823345 MHBSFMSTU Fish 140 Maculabatis pastinacoides Dasyatidae Shaplapata PaikarimothsoArot, Barishal 22°42’13.4"N 90°22’32.4"E 27-01-2021 LC823346 MHBSFMSTU Fish 141 Stigmatogobius sadanandio Gobiidae Nondibaila RupshaGhat, Khulna 22°48’26.0"N 89°34’49.5"E 28-01-2021 LC823347 MHBSFMSTU Fish 142 Acentrogobius viridipunctatus Gobiidae Shobujfutkibaila Sondha bazar, Rupsha, Khulna 22°48’47.1"N 89°33’25.0"E 28-01-2021 LC823348 MHBSFMSTU Fish 143 Apocryptes bato Gobiidae Chiring Sondha bazar, Rupsha, Khulna 22°48’47.1"N 89°33’25.0"E 28-01-2021 LC823349 MHBSFMSTU Fish 145 Trypauchen vagina Gobiidae Raja chewa RupshaGhat, Khulna 22°48’05.5"N 89°34’52.1"E 28-01-2021 LC823350 MHBSFMSTU Fish 146 Platycephalusindicus Platycephalidae Mur baila RupshaGhat, Khulna 22°48’05.5"N 89°34’52.1"E 28-01-2021 LC823351 MHBSFMSTU Fish 147 Eleutheronematetrad- actylum

Nandus nandus Nandidae Meni Ilisharot, Rangamati 22°38’54.8"N 92°11’05.0"E 30-01-2021 LC823364 MHBSFMSTU Fish 301 Amblypharyngodon mola Cyprinidae Lalmola Mawaghat, Munshigonj 23°27’59.3"N 90°17’22.1"E 08-01-2020 LC823365 MHBSFMSTU Fish 302 Awaous sp. Gobiidae Bele Mach Tepakhula Bazar, Faidpur 23°36’48.0"N 89°51’22.8"E 26-01-2021 LC823366 https://doi.org/10.1371/journal.pone.0318982.t001

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 8 / 18 PLOS One Cryptic biodiversity of freshwater fish Phylogenetical analysis The resulting COI gene nucleotide sequences were matched with additional COI sequences of Bangladeshi fish from INSD [14] and the MAFFT (Multiple Alignment using Fast Fourier Transform) v. 7.427 with the L-INS-I option [21]. Phylogenetic trees were inferred using maximum likelihood (ML) and Bayesian inference (BI) techniques. IQ-TREE v. 1.6.12 was used to estimate nucleotide substitution models for ML and BI analyses using AICc [22]. The IQ-TREE was used to determine the ML phylogeny, and 1000 pseudo replicates were used for an ultra-fast bootstrapping (BS). MrBayes v. 3.2.6 was used to obtain the Bayesian poste- rior probabilities and BI tree [23]. A tree sample was obtained after every 1000 generations, throughout two separate runs of four Markov chains totaling 100 million generations. Tracer v. 1.7.1 [24] was used to examine the parameter estimates and convergence, and the first 10% trees were eliminated in light of the findings. MEGA11’spairwise-deletion option was used for computing pairwise comparisons of the uncorrected p-distance [25] (S1 Table). Species delimitation analyses and morphology measurement Species delimitation investigations were conducted using Assemble Species by Automatic Par- titioning (ASAP) [26] based on 712 COI sequences used in phylogenetic analysis (S2 Table). The ASAP analysis was conducted using the online ASAP version (https://bioinfo.mnhn.

fr/abi/public/asap/asapweb.html) using the uncorrected pairwise distances with the default parameters. We looked at morphological parameters under both a microscope and a magnify- ing glass. We only took morphometric measurements of the chosen cryptic fish using a digital vernier caliper (Mitutoyo, accuracy ±0.02 mm). Ethics statement The specimens were obtained from either from markets or fishermen, or they were collected from the wild using a beach seine or hand net. The fish were then euthanized by immersion in buffered tricaine-methanesulphonate (MS 222) for thirty minutes after the animals stopped moving, in accordance with the guidelines outlined in the permits issued by Jamalpur Science and Technology University’s Ethics Committee (Letter no. 37.01.0044.064.05.001.24.553). Results Fish specimens were gathered nationwide from more than 28 locations (Table 1), and ana- lyzed the mitochondrial COI sequences of 124 individuals representing 121 species, 94 genera, 39 families, and 19 orders. Sequences with ≥1 nucleotide change were considered distinct haplotypes. Our COI alignment matrix consisted of 655 bp representing 119 haplotypes. Table 1 provides a summary of all haplotypes. Based on COI sequences, nearly identical topologies were observed in the ML (ln L = −59788.54) and BI (ln L = −60808.92) trees (S1 Fig). Uncorrected p-distance and species delimitation analysis demonstrated 243 groups with the best score. Most of the OTUs (operational taxonomy units) sequenced in this study were

assigned to groups with known species; however, some specimens formed their own unique groups. In particular, seven OTUs had to be assigned to different groups, despite the presence of the same species identified morphologically, indicating that these specimens were cryptic species (Table 2). Specifically, the specimen of Ailia sp. (Ailiidae) had been morphologically identified as Ailia coila, but genetically it was found to belong to a different species (Ailia sp.) compared to its nearest congener (COI divergence 8.2%) (Fig 1). This specimen was collected from the area adjacent to the Jamuna River (Sariakandi, Bogura). Our examined specimens of genus Awaous

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 9 / 18 PLOS One Cryptic biodiversity of freshwater fish formed clades with other Awaous sp. (Gobiidae) and demonstrated poor degrees of diver- gence (COI divergence 0.3%) (Fig 2). These specimens were collected from the Bogura and Faridpur districts which are linked with the rivers Jamuna and Padma. Lepidocephalichthys sp. (Cobitidae) collected from Netrokona (24°41’31.3“N 90°51’46.5”E), formed clades with other known sequences (MK572291, MK572294) and demonstrated15.50% COI divergence from its nearest congeners (Fig 1). Ophichthys sp.(Synbranchidae) demonstrated 7.7% genetic divergence from its near congeners (Fig 1). Erethistes sp. (Sisoridae), belonging to the genus Table 2. Possible cryptic species found in this study. Voucher Number Scientific Name (Family) Morphological Characteristics Total length (mm) Ratios Accession Number MHBSFMSTU Fish 3 Ailia sp. (Ailiidae) 1. Body elongate, deeply compressed and silvery in color 2. Four pairs of barbells present. 3. Adipose fin present. 4. Lateral line extends up to tip of the caudal fin. 78 TL:SL=7.8:7.2 TL:HB=7.8:1.3 TL:LBD=7.8:0.4 TL:HL=7.8:1.1 TL:ED=7.8:0.3 TL:PROL=7.8:0.2 TL:PTOL=7.8:0.6 LC823245 MHBSFMSTU Fish 6 Awaous sp. (Gobiidae) 1. Elongate, sub-cylindrical anteriorly, compressed posteriorly 2. Head obtusely convex and mouth little oblique 3. Spiny dorsal fin. 4. Opercule scaled. 99 TL:SL=9.9:8.2 TL:HB=9.9:1.9 TL:LBD=9.9:0.8 TL:HL=9.9:2.1 TL:ED=9.9:0.4 TL:PROL=9.9:0.7 TL:PTOL=9.9:1.0 LC823247 MHBSFMSTU Fish 17 Lepidocephal- ichthyssp.

(Cobitidae) 1. Caudal fin rounded; a light band extends from snout to caudal 2. Deep black spots present pectoral region and these spots become irregular at the base of caudal peduncle. 3. Small pelvic fin present. 69 TL:HB=6.9:1.2 TL:LBD=6.9:0.8 TL:HL=6.9:1.1 TL:ED=6.9:0.2 TL:PROL=6.9:0.4 TL:PTOL=6.9:1.2 LC823258 MHBSFMSTU Fish 56 Ophichthys sp. (Synbranchidae) 1. Body cylindrical, elongated with rounded abdomen. 2. Small eye and head not conspicuous. 3. Tail tapering and compressed. 4. Upper jaw longer, fleshy lips. 256 TL:LBD=25.5:1.3 TL:HL=25.5:0.5 TL:ED=25.5:0.1 TL:PROL=25.:0.3 LC823288 MHBSFMSTU Fish 83 Erethistes sp. (Sisoridae) 1. Head depressed and flattened ventrally. 2. Dorsal profile arched and ventral profile nearly horizontal. 3. Mouth small inferior and upper jaw little longer. 4. Four pairs of barbells and pectoral girdle present 57 TL:SL=5.7:4.7 TL:HB=5.7:1.1 TL:LBD=5.7:0.3 TL:HL=5.7:1.5 TL:ED=5.7:0.1 TL:PROL=5.7:0.8 TL:PTOL=5.7:0.6 LC823309 MHBSFMSTU Fish 100 Schisturasp. (Nemacheilidae) 1. Snout conical and abdomen rounded. 2. Eyes dorso-lateral. 3. Nostril in front of eye separated by valve. 4. Caudal fin weakly forked. 38 TL:SL=3.8:3.3 TL:HB=3.8:0.5 TL:LBD=3.8:0.1 TL:HL=3.8:0.6 TL:ED3.8:0.1 TL:PROL=3.8:0.2 TL:PTOL=3.8:0.3 LC823323 MHBSFMSTU Fish 126 Cynoglossus sp. (Cynoglossidae) 1. Body elongated, flat and tapering posterioly. 2. Eyes close together, on the left side of the body, upper one is advanced compared to lower one 3. Snout obtusely pointed. 4. Dorsal and anal fins confluent with caudal. 291 TL:SL=29.1:27.2 TL:HB=29.1:6.1

TL:LBD=29.1:0.2 TL:HL=3.8:4.8 TL:ED=29.1:0.5 TL:PROL=29.1:0.9 TL:PTOL=29.1:3.4 LC823337 https://doi.org/10.1371/journal.pone.0318982.t002

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 10 / 18 PLOS One Cryptic biodiversity of freshwater fish Fig 1. Maximum Likelihood (ML) tree of freshwater species of Bangladesh based on nucleotide sequences of COI gene (partial portion of S1 Fig). https://doi.org/10.1371/journal.pone.0318982.g001

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 11 / 18 PLOS One Cryptic biodiversity of freshwater fish Fig 2. Maximum Likelihood (ML) tree of freshwater species of Bangladesh based on nucleotide sequences of COI gene (partial portion of S1 Fig). https://doi.org/10.1371/journal.pone.0318982.g002

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 12 / 18 PLOS One Cryptic biodiversity of freshwater fish Erethistes, exhibited a genetic variation of more than 5.9% from the most closely related E. pusilus (Fig 2). This specimen was collected from the Netrokona region. Similarly, Schistura sp. under the family Nemacheilidae (collected from Sakal Bazar, Jamalpur),belonging to the genus Schistura demonstrated a genetic variation of more than 9.2% from its closest related species Schistura corica (Fig 3). Two specimens of genus Cynoglossus, collected from Khulna and Chittagong close to the coastal belt in Bangladesh, identified as Cynoglossus sp. (Cyno- glossidae) were found to be distantly related (COI gene divergence 17.52%) with their conge- ners Cynoglossus (MK572284) (Fig 4).The graphic quality of seven cryptic species is subpar, yet we have included images here for enhanced clarification and comprehension (see Fig 5). Discussion Bangladesh is home to 265 [4] or 293 freshwater species, including coastal fishes, since no physiological alterations transpire during their journey [27]. Rahman et al. [14] collected 694 specimens nationwide, representing 243 species-level Operational Barcode Units (OBUs), although our sampled specimens are fewer than their findings. The absence of anticipated spe- cies in our aquatic environments was one of the problems we faced throughout the sampling procedure. The current sample size is extensive and includes the majority of available species; nonetheless, it is likely incomplete.

Our research identified seven cryptic fish species in the northeastern areas of Bangladesh and has contributed to a more thorough method of clarifying species delimitation based on COI gene. Although Rahman et al. [14] developed the DNA barcoding system, our work largely differs from their work with few overlapping aspects due to the study’s inadequate cov- erage of Bangladesh’s western and southwestern regions, primarily the Sundarbans and a fig of river Ganga and its tributaries. We attempted to cover the Sherpur region and some selected areas of the Mymensingh region as well as the exclusive region of the Old Brahmaputra River, which is included in the Ganga tributaries. Although specimens from the Khulna (Sundarban) area are limited, we have included samples from this location (see Table 1). Despite the several investigation based on molecular data [14,28,29], the presence of overlooked species of fresh- water fishes in this study indicates that the hidden diversity among well-known and widely distributed fish species in Bangladesh might be more prevalent than previously believed. Despite being a low-lying country, Bangladesh’s robust river systems impede gene flow and affect the population structure of Hoplobatarchus tigerinus frogs [30]. Further, both intrinsic species characteristics, such as dispersal capability and habitat specialization, and extrinsic environmental factors, such as physical obstructions and geographic separation, often deter- mine the level of population connectivity and gene flow in the freshwater fishes [31].

The occurrence of cryptic, spatially dictated variety in fish species in Bangladesh, as indicated in this study, is not a recent revelation. Hasan et al. [2] examined the significant cryptic anuran biodiversity in Bangladesh utilizing 16S mitochondrial DNA (mtDNA); however, Raman et al. [14] contended the taxonomic ambiguity and advocated for the use of DNA barcoding (COI sequence) for the taxonomic revision of Bangladeshi freshwater fish species. We employed the COI gene in conjunction with restricted morphological data to identify the cryptic species. Identifying species merely based on physical features may prove challenging, especially when their phenotypes exhibit considerable variation [32]. Moreover, precise taxonomic identification is sometimes unattainable due to the reliance on species identification keys, which are often effective only at specific life stages. Researchers have used DNA as an alternative diagnostic tool for species, regardless of the integrated taxonomic approach [14,33]. Generally closely related vertebrate species consistently demonstrate over

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 13 / 18 PLOS One Cryptic biodiversity of freshwater fish Fig 3. Maximum Likelihood (ML) tree of freshwater species of Bangladesh based on nucleotide sequences of COI gene (partial portion of S1 Fig). https://doi.org/10.1371/journal.pone.0318982.g003

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 14 / 18 PLOS One Cryptic biodiversity of freshwater fish 2% divergence in the mitochondrial cytochrome b gene [34] and COX1 gene for conspecific genetic divergence in fish [35]. Fouquet et al. [36] demonstrated that a 3% cutoff value for 16S mtDNA yielded a greater number of potential neotropical frog species. In this work, intra- specific genetic divergence exceeds the minimum intraspecific distance of 3% (the COI gene), which is higher than the threshold value of 2% for fish species identification via DNA barcod- ing [32,37]. Based on the results of this biodiversity study, it is tempting to make conjectures about the extent of unexplored freshwater fish diversity that still exists in Bangladesh. There might still be several undiscovered cryptic species in Bangladesh. Our investigation involving the analysis of the COI gene has revealed the presence of seven likely cryptic species. These species are genetically distinct from their closely related counterparts. Ailia sp. is often wrongly identified as its closely related species, Ailia coila. Our preliminary morphological (Table 2) and genetic findings indicate that Ailia sp. is cryptic and necessitates further investigation, utilizing multiple datasets, to establish its classification as a new species. Other genera, such as Awaous, Lepidocephalichthys, Ophichthys, Erethistes, Schistura, and Cynoglossus, contain cryptic species

Fig 4. Maximum Likelihood (ML) tree of freshwater species of Bangladesh based on nucleotide sequences of COI gene (partial portion of S1 Fig). https://doi.org/10.1371/journal.pone.0318982.g004

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 15 / 18 PLOS One Cryptic biodiversity of freshwater fish as well. Furthermore, the specimens we studied are poorly preserved, with some having lost their scales and/or fins. The key morphological features of each proposed cryptic species need further check and investigation with the type locality specimen whether our speculated cryptic species have any diagnosable character or not. Because, in this study we just focused cryptic species based on molecular data along with limited morphological feature which does not give any guarantee that they are each a new species. Additionally, digital photos must be captured to accurately verify the identity. In this study, we identified several COI sequences and connected them to previously identified species, but they have recommended that the most significant aspect of the DNA barcoding is that the end users can access it and can alter taxonomies to help with identifica- tion, nomenclature, species delimitation, introgression, and multiple mitochondrial lineages [14]. However, it appears that this was only the beginning of the barcoding effort. As a result, this study is an extra effort to produce more DNA barcode data and facilitate specimen identification. A major portion of Bangladesh’s small indigenous species is a primary source of protein. Together with the recent developed deep learning-based techniques [38] and molecular-morpholgy based species/genus identification [39] our DNA barcode library can

assist the accurate species identification and contribute to their proper management for con- servation. To prevent a potential wave of extinctions, we must quickly curtail anthropological activities that are detrimental to freshwater fish populations [40]. Immediate habitat resto- ration and species-specific conservation efforts are essential to save our valuable fish species and fisheries biodiversity in Bangladesh. Conclusion Annotated species lists are essential for creating appropriate conservation policies. Our findings of seven cryptic species (Table 2, Fig 5) provide additional information regarding the freshwater species check list in Bangladesh. To figure out the taxonomic status of the Fig 5. Seven possible cryptic species found in this study. (A) Ailia sp. (Ailiidae), (B) Awaous sp. (Gobiidae), (C) Lepidocephalichthys sp. (Cobitidae), (D) Ophich- thys sp.(Synbranchidae), (E) Erethistes sp. (Sisoridae), (F) Schistura sp.(Nemacheilidae) and (G) Cynoglossus sp. (Cynoglossidae). Not in scale. https://doi.org/10.1371/journal.pone.0318982.g005

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 16 / 18 PLOS One Cryptic biodiversity of freshwater fish seven species that were found in this study but haven’t been named yet, more comprehensive sampling that combines morphological and DNA data would need to be done. Researchers and policymakers can utilize the results of our study, which reveals that Bangladesh is home to many cryptic species, as baseline data. Supporting information S1 Fig. Maximum Likelihood (ML) tree of freshwater species of Bangladesh based on nucleotide sequences of COI gene. (TRE) S1 Table. Uncorrected P distance among the analyzed taxa. (CSV) S2 Table. Result of species delimitation analyses. (TXT) Acknowledgments We are grateful to Associate Professor Dr. A. Kurabayashi, Nagahama Institute of Bio-Science and Technology for some technical support to accomplish the molecular work. Author contributions Conceptualization: Mahmudul Hasan. Data curation: Chiaki Kambayashi. Formal analysis: Chiaki Kambayashi. Funding acquisition: Mahmudul Hasan, Md. Saiful Islam. Investigation: Mahmudul Hasan. Methodology: Mahmudul Hasan, Zahid Hasan Anik. Supervision: Mahmudul Hasan. Writing – original draft: Mahmudul Hasan, Chiaki Kambayashi. Writing – review & editing: Mahmudul Hasan, Chiaki Kambayashi. References 1. Nishat A, Huq SMI, Barua SP, Reza AHMA, Khan ASM. Bio-Ecological Zones of Bangladesh. IUCN, The World Conservation Union, Bangladesh Country Office; 2002. 2. Hasan M, Islam MM, Khan MR, Alam MS, Kurabayashi A, Igawa T, et al. Cryptic anuran biodiversity

in Bangladesh revealed by mitochondrial 16S rRNA gene sequences. Zoolog Sci. 2012;29(3):162–72. https://doi.org/10.2108/zsj.29.162 PMID: 22379983 3. Afroz R, Rahman M. Transboundary river water for Ganges and Teesta Rivers in Bangladesh: an assessment. Global Sci Technol J. 2013;1:100–11. 4. Rahman AKA. Freshwater Fishes of Bangladesh. 2nd Edition. Zoological Society of Bangladesh, Department of Zoology, University of Dhaka; 2005. 5. Hussain MG, Mazid MA. Genetic improvement and conservation of carp species in Bangladesh. Ban- gladesh Fisheries Research Institute and International Center for Living Aquatic Resources Manage- ment. 2001;74. 6. Fricke R, Eschmeyer WN, Laan R. 2024. Eschmeyer’s catalog of fishes: Genera, species, references. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp 7. Ghorbani R, Abbasi F, Molaei M, Naeimi A. Identification and distribution of fish fauna in Kaboodval stream (Golestan Province, Iran). World J Fish Marine Sci. 2013;5(5):467–73. https://doi.org/10.5829/ idosi.wjfms.2013.05.05.73142

PLOS One | https://doi.org/10.1371/journal.pone.0318982 April 28, 2025 17 / 18 PLOS One Cryptic biodiversity of freshwater fish 8. Islam S, Nabi T, Laboni M, Ahammed F. Fish production of Bangladesh, it’s pattern & impact on GDP. J Survey Fisheries Sci. 2023;10(3):368–73. https://doi.org/10.53555/sfs.v10i3.1604 9. Hasan M, Islam MdS. Collection, preservation, and identification of freshwater fish species in middle to north-east Bangladesh with special notes on phenotypic plasticity of few species. World J Biol Biotechnol. 2022;7(2):7. https://doi.org/10.33865/wjb.007.02.0508 10. Shelley JJ, Swearer SE, Adams M, Dempster T, Le Feuvre MC, Hammer MP, et al. Cryptic biodiversity in the freshwater fishes of the Kimberley endemism hotspot, northwestern Australia. Mol Phylogenet Evol. 2018;127:843–58. https://doi.org/10.1016/j.ympev.2018.06.032 PMID: 29953937 11. Pramanik M, Hasan M, Bisshas S, Hossain A, Biswas T. Fish biodiversity and their present conserva- tion status in the Meghna River of Bangladesh. Int J Fisheries Aquatic Stud. 2017;5:446–55. 12. Akter T, Hossain M, Begum R, Barman P, Debnath P. Diversity of fish species in South-Eastern coast of Bangladesh. Bangladesh J Agric Res. 2017:267–79. 13. Parvez I, Alam MA, Hassan MM, Ara Y, Hoshan I, Kibria ASM. A checklist of fish species from three rivers in northwestern Bangladesh based on a seven-year survey. J Threat Taxa. 2019;11(6):13786– 94. https://doi.org/10.11609/jott.4303.11.6.13786-13794 14. Rahman MM, Norén M, Mollah AR, Kullander SO. Building a DNA barcode library for the freshwater