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Genetic Characterization of Freshwater Fishes in Bangladesh Using DNA Barcodes

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Genetic Characterization of Freshwater Fishes in Bangladesh Using DNA Barcodes Genetic characterization of freshwater fishes in Bangladesh using DNA barcodes 1 2 2 1 Md. Mizanur Rahman ,Sven O. Kullander , Michael Norén and Abdur Rob Mollah ID 718 1Department of Zoology, University of Dhaka, Bangladesh. 2Department of Zoology, Swedish Museum of Natural History Stockholm, Sweden. IBOL 2017 Abstract The project focuses on genetic characterization of Bangladesh’s freshwater fish fauna in the form of a DNA barcode library composed of standardized well identified mitochondrial cytochrome c oxidase subunit I (COI) sequences and taxonomic revision. Development of a DNA based reference database is in progress. To date, >175 species of freshwater fishes was identified through obtained barcode sequences (COI) sequences in combination with classical taxonomic validation. Two new species, namely Danio annulosus (3.4% p- distance from the most similar species) and Garra mini (12 % p-distance from closely related taxa) were described and a good number of species are yet to be described as new species. A rapid expansion of several alien species (e.g. Trichopsis vittata, Pterygoplichthys disjunctivus) was also been detected. The barcode sequences from the present study along with traditional taxonomy have also confirmed the existence of many misidentifications in current literature. Background Study area: Covered different regions across country considering Bangladesh is a biogeographically important area in the heart of the diverse habitat including lowland and upland freshwater water hyper-diverse Indo-Burman region of South Asia but has one of the ecosystems of Bangladesh (shown on map). most taxonomically unresolved freshwater fish fauna in the world. Specimens collected water bodies: Rivers, streams, creeks, Although substantial progress has been made in documenting fish canals, pond and ditches species diversity Bangladesh based on morphological studies, the Collecting methods: Beach seine nets and local fishing gear and diversity of fish species has not been fully explored especially in the crafts, Fishermen catch and Fish market upland streams and creeks. This project aims for genetic characterization of Bangladesh’s freshwater through DNA barcodes composed of standardized well identified mitochondrial COI sequences and for taxonomic revision combining morphological and molecular data. We also aim to establish a stable nomenclature with Bangladeshi barcode voucher specimens through taxonomic revision. Morphological Identification Fish Specimens Storage: NRM, Swedish Museum of Natural History, Stockholm; DU, Zoology Department, University of Dhaka, Dhaka. Molecular Identification Measurements : Taken with digital callipers to a precision of 0.1 mm DNA Extraction: Thermo Scientific™ KingFisher™ fully automated following Fang (1997) and Kullander (2015). liquid-handling instrument, Counts: Fin-ray and vertebral counts were taken from X-radiographs Primer: The COI fragment was amplified using the fish barcoding made with a Philips MG-105 low voltage X-ray unit and Kodak EDR2 primers Fish-F1 and Fish-R1 (Ward et al. 2005). plates. Amplification: Performed with the puReTaq Ready-To-Go PCR kit Identification : Confirmed by existing taxonomic keys and relevant PCR cycling: 94°C 4min; 35 * (94°C 30s; 52°C 30s; 72°C 30s); Statistics were calculated using SYSTAT v. 13 (Systat Software, 2009) 72°C 8min). Sanger Sequencing: Sequencing of both strands of all fragments was Results carried out by Macrogen Europe (Holland). Currently, we have 504 completed Sequence Analysis: Proofread and assembly done using the software COI barcodes of Bangladeshi Geneious R8 (Kearse et al., 2012) and BLASTed against GenBank freshwater fishes, representing and BOLD nucleotide database. Sequences were aligned with 175 named species belong to 47 the MUSCLE (Edgar, 2004) and edges were manually trimmed families (Table 1) plus a minimum to around 650 basepairs. Phylogenetic analysis was performed of 5 currently undescribed using the software MrBayes v3.3 (Huelsenbeck & Ronquist, species. 2001; Ronquist et al., 2014). New Species Described: Danio annulosus: Diagnosed by Table: Barcoded freshwater fishes of Bangladesh much shorter pectoral and pelvic fins, and a humeral spot that is Family Name No. of No. of Family Name No. of No. of Species Barcodes Species Barcodes slightly wider than deep instead Adrianichthyidae 02 06 Latidae 01 04 of round or deeper than wide. Ambassidae 02 08 Loricariidae 01 05 The mitochondria (COI) sequence Amblycipitidae 01 06 Mastacembelidae 03 20 separates it from the most similar Anabantidae 02 06 Megalopidae 01 05 species, D. catenatus by a p- Anguillidae 01 04 Mugilidae 02 08 Aplocheilidae 01 06 Nandidae 01 04 distance of 3.4%. and Badidae 02 10 Nemacheilidae 03 06 phylogenetic tree (Photo B). Bagridae 08 20 Notopteridae 02 09 (Kullander et al., 2015) Balitoridae 05 16 Olyridae 02 07 Garra mini Diagnosed by the Belonidae 01 04 Ophichthidae 01 03 Chacidae 01 03 Osphronemidae 04 07 numerous small predorsal scales Channidae 04 15 Pangasiidae 02 07 and the presence of a Cichlidae 01 03 Poeciliidae 01 04 contrasted dark stripe Clariidae 02 05 Polynemidae 01 07 along the middle Clupeidae 05 16 Psilorhynchidae 04 21 of the side, and also by Cobitidae 09 30 Schilbeidae 04 15 Cynoglossidae 01 03 Siluridae 02 08 the DNA barcode sequence ((12 % Cyprinidae 65 180 Sisoridae 05 20 p-distance from closely related Eleotrididae 02 06 Soleidae 01 03 taxa) (Photo A) Engraulididae 01 04 Syngnathidae 01 04 (Rahman et al., 2016) F ig. Bayesian majority-rule tree from analysis of Erethistidae 02 07 Teraponidae 01 03 mitochondrial COI DNA data Gobiidae 10 30 Tetraodontidae 01 03 Hemiramphidae 01 07 Zenarchopteridae 01 05 Distribution of Trichopsis vittata and Pseudosphromenus cupanus Horabagridae 01 07 Trichopsis vittata: Spread of this Croaking Gourami has been confirmed in Myanmar and Bangladesh. (Noren et al., 2017) Significance Pseudosphromenus cupanus: This fish has also been clarified that This is the first comprehensive attempt to develop a DNA based this species never existed in Bangladesh. (Kullander et al., 2015) reference library for freshwater fishes of Bangladesh that provides a several new species, new records, and high taxonomic resolution of References existing taxa improving on previous taxonomic identifications. This Rahman, M.M., Mollah, A. R., Norén, M.and Kullander, S. O. 2016. Garra mini, a new small species of study also underscores the scope of further investigation into rheophilic cyprinid fish(Teleostei: Cyprinidae) from southeastern hilly areas of Bangladesh. Ichthyol. Explor. Freshwaters, Vol. 27, No. 2, pp. 173-181. surveillance of fish species composition and invasive alien species Kullander S. O., Rahman M. M., Norén M. & Mollah, A R. 2015. Why is Pseudosphromenus cupanus using environmental DNA. (Teleostei: Osphronemidae) reported from Bangladesh, Indonesia, Malaysia, Myanmar, and Pakistan?Zootaxa 3990 (4): 575–583. Kullander S. O., Rahman M. M., Norén M. & Mollah, A R. 2015. Danio annulosus, a new species of chain Danio from the Shuvolong Falls in Bangladesh (Teleostei: Cyprinidae: Danioninae).Zootaxa. Acknowledgements 3994 (1): 053–068. Norén M, Kullander S.O, Rahman M.M, Mollah, A.R 2017).First records of Croaking Gourami, Swedish Research Council (Vetenskapsrådet) Trichopsis vittata (Cuvier, 1831) (Teleostei: Osphronemidae), from Myanmar and Bangladesh. Check List 13 (4): 81–85..
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