Genetic characterization of freshwater fishes in Bangladesh using DNA barcodes

Md. Mizanur Rahman1,Sven O. Kullander2, Michael Norén2 and Abdur Rob Mollah1

1Department of Zoology, University of Dhaka, Bangladesh.2Department of Zoology, Swedish Museum of Natural History Stockholm, Sweden.

ID 718IBOL 2017

AbstractThe project focuses on genetic characterization of Bangladesh’s freshwater fish fauna in the form of a DNA barcode library composedof standardized well identified mitochondrial cytochrome c oxidase subunit I (COI) sequences and taxonomic revision. Development ofa DNA based reference database is in progress. To date, >175 species of freshwater fishes was identified through obtained barcodesequences (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 numberof species are yet to be described as new species. A rapid expansion of several alien species (e.g. Trichopsis vittata, Pterygoplichthysdisjunctivus) was also been detected. The barcode sequences from the present study along with traditional taxonomy have alsoconfirmed the existence of many misidentifications in current literature.

Bangladesh is a biogeographically important area in the heart of the hyper-diverse Indo-Burman region of South Asia but has one of the most taxonomically unresolved freshwater fish fauna in the world. Although substantial progress has been made in documenting fish species diversity Bangladesh based on morphological studies, the diversity of fish species has not been fully explored especially in the 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.

SignificanceThis is the first comprehensive attempt to develop a DNA based reference library for freshwater fishes of Bangladesh that provides a several new species, new records, and high taxonomic resolution of existing taxa improving on previous taxonomic identifications. This study also underscores the scope of further investigation into surveillance of fish species composition and invasive alien species using environmental DNA.

Morphological IdentificationFish Specimens Storage: NRM, Swedish Museum of Natural History, Stockholm; DU, Zoology Department, University of Dhaka, Dhaka. Measurements : Taken with digital callipers to a precision of 0.1 mm following Fang (1997) and Kullander (2015). Counts: Fin-ray and vertebral counts were taken from X-radiographs made with a Philips MG-105 low voltage X-ray unit and Kodak EDR2 plates.Identification : Confirmed by existing taxonomic keys and relevant Statistics were calculated using SYSTAT v. 13 (Systat Software, 2009)

Background

Results

Study area: Covered different regions across country considering

diverse habitat including lowland and upland freshwater water ecosystems of Bangladesh (shown on map).

Specimens collected water bodies: Rivers, streams, creeks,

canals, pond and ditches

Collecting methods: Beach seine nets and local fishing gear and

crafts, Fishermen catch and Fish market

Table: Barcoded freshwater fishes of Bangladesh

Currently, we have 504 completed COI barcodes of Bangladeshi freshwater fishes, representing 175 named species belong to 47 families (Table 1) plus a minimum of 5 currently undescribed species. New Species Described:Danio annulosus: Diagnosed by much shorter pectoral and pelvic fins, and a humeral spot that is slightly wider than deep instead of round or deeper than wide. The mitochondria (COI) sequence separates it from the most similar species, D. catenatus by a p-distance of 3.4%. and phylogenetic tree (Photo B).

(Kullander et al., 2015)Garra mini Diagnosed by the numerous small predorsal scales and the presence of acontrasted dark stripealong the middle

of the side, and also by the DNA barcode sequence ((12 % p-distance from closely related taxa) (Photo A)(Rahman et al., 2016)

Molecular IdentificationDNA Extraction: Thermo Scientific™ KingFisher™ fully automated

liquid-handling instrument,Primer: The COI fragment was amplified using the fish barcoding

primers Fish-F1 and Fish-R1 (Ward et al. 2005).Amplification: Performed with the puReTaq Ready-To-Go PCR kit

PCR cycling: 94°C 4min; 35 * (94°C 30s; 52°C 30s; 72°C 30s); 72°C 8min).

Sanger Sequencing: Sequencing of both strands of all fragments was carried out by Macrogen Europe (Holland).

Sequence Analysis: Proofread and assembly done using the software Geneious R8 (Kearse et al., 2012) and BLASTed against GenBank and BOLD nucleotide database. Sequences were aligned with the MUSCLE (Edgar, 2004) and edges were manually trimmed to around 650 basepairs. Phylogenetic analysis was performed using the software MrBayes v3.3 (Huelsenbeck & Ronquist,

2001; Ronquist et al., 2014).

Family Name No. of

Species

No. of

Barcodes

Family Name No. of

Species

No. of

Barcodes

Adrianichthyidae 02 06 Latidae 01 04

Ambassidae 02 08 Loricariidae 01 05

Amblycipitidae 01 06 Mastacembelidae 03 20

Anabantidae 02 06 Megalopidae 01 05

Anguillidae 01 04 Mugilidae 02 08

Aplocheilidae 01 06 Nandidae 01 04

Badidae 02 10 Nemacheilidae 03 06

Bagridae 08 20 Notopteridae 02 09

Balitoridae 05 16 Olyridae 02 07

Belonidae 01 04 Ophichthidae 01 03

Chacidae 01 03 Osphronemidae 04 07

Channidae 04 15 Pangasiidae 02 07

Cichlidae 01 03 Poeciliidae 01 04

Clariidae 02 05 Polynemidae 01 07

Clupeidae 05 16 Psilorhynchidae 04 21

Cobitidae 09 30 Schilbeidae 04 15

Cynoglossidae 01 03 Siluridae 02 08

Cyprinidae 65 180 Sisoridae 05 20

Eleotrididae 02 06 Soleidae 01 03

Engraulididae 01 04 Syngnathidae 01 04

Erethistidae 02 07 Teraponidae 01 03

Gobiidae 10 30 Tetraodontidae 01 03

Hemiramphidae 01 07 Zenarchopteridae 01 05

Horabagridae 01 07

F ig. Bayesian majority-rule tree from analysis of mitochondrial COI DNA data

Distribution of Trichopsis vittata and Pseudosphromenus cupanusTrichopsis vittata: Spread of this Croaking Gourami has been confirmed in Myanmar and Bangladesh. (Noren et al., 2017)Pseudosphromenus cupanus: This fish has also been clarified that this species never existed in Bangladesh. (Kullander et al., 2015)

ReferencesRahman, M.M., Mollah, A. R., Norén, M.and Kullander, S. O. 2016. Garra mini, a new small species of rheophilic cyprinid fish(Teleostei: Cyprinidae) from southeastern hilly areas of Bangladesh. Ichthyol. Explor. Freshwaters, Vol. 27, No. 2, pp. 173-181.Kullander S. O., Rahman M. M., Norén M. & Mollah, A R. 2015. Why is Pseudosphromenus cupanus(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. 3994 (1): 053–068.Norén M, Kullander S.O, Rahman M.M, Mollah, A.R 2017).First records of Croaking Gourami, Trichopsis vittata (Cuvier, 1831) (Teleostei: Osphronemidae), from Myanmar and Bangladesh. Check List 13 (4): 81–85.

Acknowledgements

Swedish Research Council (Vetenskapsrådet)