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MOLECULAR DIVERSITY OF FRESHWATER FISHES OF BANGLADESH ASSESSED BY DNA BARCODING

This study represents the comprehensive molecular identification of freshwater fishes of Bangladesh based on a fragment of the cytochrome c oxidase subunit I (COI) gene in the mitochondrial genome. A total of 315 mitochondrial COI barcode sequences were obtained from 153 species,114 genera, 49 families and 16 orders of fishes. The mean length of the sequences was 652 base pairs. For all the samples, %G was significantly lower compared to the other nucleotides and %GC was lower compared to %AT (p-value ˂ 0.05). Also, a significantly lower %GC content was observed in second and third codon position compared to the first one in all the samples (1st>2nd>3rd, p-value˂ 0.05). The average K2P distances within species, genera, families and orders were 0.38%, 7.02%, 12.75% and 18.68%, respectively. The mean interspecific distance was 18-fold higher than the mean intraspecific distance. The K2P neighbor-joining (NJ) trees based on the sequences generally clustered species according to their taxonomic position. A total of 12 species were newly recorded in Bangladesh. High efficiency in species identification were demonstrated in the present study by DNA barcoding, and concluded that COI sequencing can be used as an authentic identification marker for freshwater fish species.


INTRODUCTION
Bangladesh is a deltaic country that emerged on the confluence of the three mighty river systems, Padma-Ganges, Jamuna-Brahmaputra and the Surma-Meghna. This unique geophysical condition constitutes about 46,99,387 ha of diverse inland water areas comprises more than 250 native freshwater fish species (IUCN 2015). Inland fisheries play an important role in the economy of the country in terms of nutrition, income, employment and foreign exchange earnings. More than 11 percent of the total population is engaged in this sector in full time and part time basis for their livelihoods. And fish itself contributes about 60% of animal protein in the daily dietary requirement of 160 million population of the country (DoF 2018). Inland capture fishery production remarkably declined in the past few decades. Currently, inland capture fishery contributes only 29% of the country's total fish production which was 63% in *Author for correspondence: ˂sagir@du.ac.bd˃, 2 Department of Zoology, Jagannath University, Dhaka1100, Bangladesh ©2020 Zoological Society of Bangladesh DOI: https://doi.org/10.3329/bjz.v48i1.47872 1983-84 (DoF 2018. This dramatic change in production was due to the continuous habitat destruction, unregulated fishing and aquatic pollution from industrial, domestic and agricultural sources. That is also leading to at least 64 species of freshwater fishes at different categories of threatened with extinction (IUCN 2015).
Nonetheless, description and information of freshwater fishes of Bangladesh are scattered throughout a wide range of publications. The first complete description of freshwater fishes of Bangladesh was compiled by Rahman (1989) which corroborated 260 species including migratory and estuarine species. Siddiqui et al. (2007) described 251 species which is in fact reproduce the previous works.
Published books on diversity of Bangladeshi fishes (Bhuyian 1964, Rahman 2005, Shafi and Quddus 1982, IUCN 2000, IUCN 2015) evident that the information is inconsistent and taxonomy used is not updated.
The accurate identification of fish species is a pivotal component to protect the extant ichthyofaunal biodiversity and to perform regular assessments of local fish faunas for conservation planning. As an alternative to the traditional species identification based on morphological characters, Hebert et al. (2003) has been suggested partial cytochrome c oxidase subunit I (COI) sequences (DNA barcodes) for standardized and routine species identification. The DNA-based barcoding method has been proven to be a valuable molecular tool for species identification and it is accessible to non-specialists (Hebert et al. 2003, Fre´zal and Leblois 2008, Leray et al. 2015. This barcoding technique has been successfully identified ichthyofauna in many geographic regions, such as Australia (Ward et al. 2005), Canada (Hubert et al. 2008), India (Lakra et al. 2015), China (Zhang 2011, Wang et al. 2018, Japan (Zhang andHanner 2011), Portugal (Costa et al. 2012), Germany (Knebelsberger et al. 2015), Taiwan (Bingpeng et al. 2018, Chang et al. 2017 and Vietnam (Thu et al. 2019), thus enriched the barcode reference library. New specimens and products can confidently be identified by comparing their DNA barcode sequences against this barcode reference library. Although barcodes for almost two-third (19,000) of all described fish species are already available (BOLD 2020), the permanent addition of new barcode data is essential to increase the taxonomic resolution.
Considering the economic importance of inland fishery and the expected richness of the fish fauna and in the absence of an expert-based taxonomy, the first attempt for barcoding of freshwater fishes of Bangladesh was initiated in 2014 and consequently partially published barcode data of 81 small indigenous fish species (SIS) by Ahmed et al. (2019). This paper deals with the molecular characterization of morphologically identified freshwater species of Bangladesh using partial COI gene sequence.

Study area and specimen collection:
Fish samples were collected from rivers, haor, baor, beels, floodplain, fish landing centers, fish markets or from the local fishermen during July 2014 to June 2018. Personal fishing was also conducted to collect some rare and non-commercial fish species whenever necessary. Photographs of all the fishes were taken immediately and taxonomic identification of specimens were done following previous reports (Talwar and Jhingran 1991, Rahman 2005, Siddiqui et al. 2007. 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 Professor Kazi Zaker Hussain Museum at the Department of Zoology, University of Dhaka. DNA barcoding: Genomic DNA was extracted from the muscle tissue samples by the standard Proteinase-K/Phenol-Chloroform-isoamyl alcohol method (Green andSambrook 2012, Ahmed et al. 2019). The quality and quantity of the extracted DNA was measured using Nanodrop™ spectrophotometer. Approximately 658bp was amplified from the 5′ region of the MT-COI gene using the following primers: FishF1 5′TCAACCAACCACAAAGACATTGGCAC3′ and FishR1 5′ TAGACTTCTGGGTGGCCAAAGAATCA3′ only when failed to amplified the FishF2 5′TCGACTAATCATAAAGATATCGGCAC3′ and FishR2 5′ACTTCAGGGTGACCGAAGAATCAGAA3′ were used (Ward et al. 2005). For this, 25 µl PCR reaction mixtures were prepared which included 17.25-18.75 µl of ultrapure water, 2.5 µl of 10× PCR buffer, 1.25 µl of MgCl2 (50 mM), 0.25 µl of each primer (0.01 mM), 0.125 µl of each dNTP (0.05 mM), 1 µl (0.625 U) of Taq polymerase, and 0.5-2.0 µl of DNA template. Amplifications were performed using ABI thermal cycler (Thermo Fisher Scientific). The thermal regime consists of an initial step of 2 min at 95°C followed by 35 cycles of 0.5 min at 94°C, 0.5 min at 54°C, and 1 min at 72°C, followed in turn by 10 min at 72°C and then held at 4°C. PCR products were visualized on 1% agarose gel. The PCR products were purified using PureLink™ PCR purification kit and sequenced from First BASE Laboratories, Sdn Bhd, Malaysia. All sequences were translated into amino acids to confirm the effectiveness of the sequences and to detect the presence of nuclear DNA pseudo genes, insertions, deletions, or stop codons. Sequences were checked and aligned using Sequencer v5.4.6 and were submitted to GenBank with referred accession numbers. All the data including taxonomic characteristics and GenBank accession numbers were tagged with the voucher specimens preserved at the Professor Kazi Zaker Husain Museum of Department of Zoology, University of Dhaka.

Bioinformatic and statistical analyses:
Bioinformatic analyses of the sequences were performed using CLC Workbench v7.7.1, Mega X, Clustal Omega, and T-Coffee. Base compositions were analyzed using CLC Workbench v7.7.1 and Mega X. Genetic distance and sequence divergences were calculated using the Kimura two parameter (K2P) distance model (Kimura 1980). Neighbor-joining (NJ) trees of K2P distances were created to provide a graphic representation of divergence pattern between species (Saitou and Nei 1987). Bootstrapping was performed in MEGA X (Tamura and Nei 1993) with 1000 replications (Felsenstein 1985). Necessary statistical analyses were performed in Excel 2013 and RStudio.

RESULTS
A total of 550 tissue samples were collected, during the study period among which 315 COI sequences were obtained (Table 1). Based on morphological and molecular identifications, these samples represented 153 species of 114 genera, 49 families and 16 orders (Table 1). Among the collected species, Order Cypriniformes was recorded as the most diversified fish group in terms of both number of species and individuals observed followed by Perciformes and Siluriformes (Table1). Among the 153 species, 12 fish species were newly recorded in Bangladesh. The average length of all barcode sequences was 652 bp ranging from 352 to 696 bp where 95% were longer than 600bp. No stop codon, insertion, or deletion was observed in any of the obtained sequences.  The lack of stop codons in these sequences indicates that they are functional mitochondrial COI sequences, together with the fact that each of the amplified sequence was about 658 bp in length. Hence, it suggests that Nuclear DNA Sequences Originating from Mitochondrial DNA Sequences (NUMTs) were not sequenced, as vertebrate NUMTS are typically less than 600 bp (Zhang and Hewitt 1996).
The sequence analysis indicated that the average nucleotide frequencies to be A: 25.20%, T:29.80%, G:17.70% and C: 27.30% (Fig. 1).The base composition analysis for the COI sequence showed that the average T content was the highest and the average G content was the lowest; the AT content (55.0%) was higher than   the GC content (45.0%). The GC contents at the first, second and third codon positions for all fish were 56.09%, 42.78% and 36.02% respectively (Fig. 2). At the first codon position, the usage of T (19.10%) was the lowest, and the usages of the other bases were C: 25.40%, A: 24.80% and G: 30.60%. At the second codon position, the content of T (41.80%) was highest, and the contents of the other bases were C: 28.10%, A: 15.50% and G: 14.70%. At the third codon position, the base usage was T (28.40%), C (28.20%), A (35.60%) and G (7.80%); the G content was the lowest, exhibiting a clear pattern of anti-G bias. The average genetic distance within species, genus, family and order were 0.38±0.01%, 7.02±0.02%, 12.75±0.03% and 18.68±0.04%, respectively ( Table 2). The NJ tree of 315 generated sequences including 153 species is presented in Figure 4.
Order Cypriniformes: This order includes many of the most important forage and food fish. A total of 112 samples were sequenced belonging to five families, 37 genera and 54 species. The overall mean nucleotide base frequencies observed for these sequences were-T: 29.70%, C: 27.0%, A: 25.80% and G: 17.50%. The AT content (55.50%) was higher than the GC content (44.50%). The GC contents at the first, second and third codon positions were 49.10%, 48.20% and 44.40% respectively. The NJ tree clearly distinguished all the species (Fig. 4).
Order Perciformes: A total of 72 samples were sequenced belonging to 12 families, 27 genera and 33 species. The overall mean nucleotide base frequencies observed for these sequences were T: 29.90%, C: 28.20%, A: 23.80% and G: 18.10%. The AT content (53.70%) was higher than the GC content (46.30%). The GC contents at the first, second and third codon positions were 56.70%, 42.70% Frequency (%) K2P Distance and 39.50%, respectively. In the NJ tree, most of the specimens belonging to the same species were clustered together bolstering the prior taxonomic assignment based on morphology (Fig. 4).
Order Siluriformes: A total of 76 samples were sequenced belonging to 14 families, 25 genera and 36 species. The overall mean nucleotide base frequencies observed for these sequences were-T: 29.70%, C: 26.90%, A:25.60% and G:17.70%. The AT content (55.30%) was higher than the GC content (44.70%). The GC contents at the first, second and third codon positions were 56.70%, 42.70% and 38.10% respectively.

DISCUSSION
DNA barcoding could be considered as a global bio-scanner for rapid and authentic identification of organisms using the partial sequence of mitochondrial COI gene. Barcoding has clearly discriminated freshwater fish species from around the globe including Australia, Canada, India, Thailand, Germany (Ward et al. 2005, Hubert et al. 2008, Lakra et al. 2015, Panprommin et al. 2019, Knebelsberger et al. 2014. Here, we have complied the COI sequence profile of freshwater fishes collected from the different inland waters of Bangladesh and confirmed the efficacy of barcoding to identify these species. Barcodes were generated for153species of belonging to 114 genera and 49 families and 16 orders (Table 1). We observed no insertions/ deletions or codon stops after translating the nucleotide sequences, supporting the view that all of the amplified sequences denote functional mitochondrial COI sequences. Moreover, average length of the amplified sequences was larger than 650bp, the limit typically observed for nuclear DNA sequences originating from mtDNA (NUMTs) (Gunbin et al. 2017). All of these species were differentiable based on the individual COI barcodes. Hence, this study has strongly validated the efficiency of COI barcodes for identifying fish species.
The base composition analysis of the COI sequences revealed that AT content (55.0%) to be higher than GC content (45.0%), similar to the patterns were observed in Australian (Ward et al. 2005), Canadian (Steinke et al. 2009) and Cuban fish species (Lara et al. 2010). The GC contents in the first, second and third codon positions were 56.09%, 42.78% and 36.02%, respectively (Fig. 2). At the first codon position, the usage of G (19.00%) was the lowest, and the usages of the other bases were 23.9%, 32.6% and 24.00% for C, A and T, respectively. At the second codon position, the content of T (31.00%) was highest, and the contents of the other bases were 25.7%, 23.1% and 20.0 for C, A and G, respectively. At the third codon position, the base usage was-T: 33.00%, C:32.0%, A:20.3% and G:14.3%. There was a significantly higher overall GC content in the 153 species and this difference was attributable to the GC content at the 2 nd and the 3 rd codon base. The pattern of %GC content at different codons for all the fishes was invariably 1 st >2 nd >3 rd (p<0.005) (Fig. 2).
Kimura 2-parameter distance values of 7.02 ±0.02%, 12.75 ± 0.03% and 18.68 ± 0.04% were obtained for within genus, within family and within order respectively (Table 2, Fig. 3). The pairwise genetic distance values were increasing at higher taxonomic levels as expected and consistent with the previous studies that supports the significant change in genetic divergence at the species boundaries (Hubert et al. 2008, Lakra et al. 2015, Ahmed et al. 2019. In this study, the average K2P distance within species was 0.38%, compared with 7.02% for within genera. The mean interspecific distance was found to be 18-fold higher than the mean intraspecific distance. More than 20-fold difference was observed in the freshwater fishes commonly encountered in the Australian, Canadian and Indian freshwater fishes (Ward et al. 2005, Hubert et al. 2008, Lakra et al. 2015. This finding corresponds to the DNA barcoding principle that interspecific divergence sufficiently outscores intraspecific divergence. The accuracy of species identification through DNA barcoding mostly depends on both interspecific and intraspecific divergence. In our study, the average genetic distance within species was found 0.38±0.01%. Mean intraspecific genetic distance was calculated as <1% in previous studies; Hubert et al. (2008) found 0.30% Lakra et al. (2015) 0.25 % (0-0.82%) for the freshwater fishes. Phylogenetic relationships of barcoded species were shown in NJ tree (Fig. 4). Each species was associated with a specific DNA barcode cluster and the relationship among these species was clearly revealed. Closer species in terms of genetic divergence, were clustered at the same nodes and the distance between the terminal branches of the NJ tree widened as they got more distinct.
In some cases, deviations were observed. For example, out of five sequences of Labeo bata two form different cluster, one (MH087029.1) with Cirrhinus reba and another (MN083093.1) showed more close relationship with Labeo gonius (Fig. 4). We know that L. bata is widely used as an aquaculture species and its induced breeding performed almost all hatcheries of the country. Not only this, its fry and fingerlings are released in the natural habitat. So, it not unlikely to be its hybrid population in natural habitats. Moreover, among the three sequences of Anabas testudineus, one sequence deviated from the rest with 83% bootstrap value and we suspect this an exotic variety (Thai koi) which is now available everywhere in the country. Channa orientalis was documented in Bangladesh (Siddiqui et al. 2007, IUCN 2015 which is an endemic species of Sri Lanka, and it often misidentified as C. gachua (Tanomtong et al. 2014). The major morphological difference between the two species is that C. gachua has ventral fins and C. orientalis lack of ventral fin. We could not find any specimen of C. orientalis, and this led us to believe that C. orientalis not existing in Bangladesh and all the older reports are pertaining to C. gachua (Ahmed et al. 2018, Conte-Grand et al. 2017. Macrognathus of Bangladesh having been referred by virtually all authors to M. aculeatus (Rahman 2005, Siddiqui et al. 2007. M. aculeatus body usually with 14-17 oblique dark bars whereas, M. aral body typically with two or more broad pale longitudinal bands of varying width extending its entire length, never with oblique bars (Roberts 1980). Our five generated sequences of collected species showed high similarity with GB pre-existing sequences and we thus confirm the presence of M. aral instead of M. aculeatus. Loaches are another diverse group of fishes with very confusing characteristics. At least three new records (Garra nasuta, Schistura fasciolata, Botia rostrata) have been confirmed in this study and we presume that some new species/records are still to be explored under the genus Lepidocephalichthys, Botia, or Garra. A taxonomic revision on taxa of this group is urgently needed for their biodiversity conservation.
The present study revealed that DNA barcoding has been successful in identifying the freshwater fishes of Bangladesh. We have barcoded 153 species of freshwater fishes and these barcode data confirms the 12 new records from Bangladesh. When traditional morpho taxonomy does not work, this molecular tool is effective for species identification, particularly with specimens that are damaged, incomplete, or morphologically distinct stages. Nevertheless, DNA barcoding also has its limitations too. Therefore, DNA barcoding can serve as a complementary tool for species identification, but it cannot replace the traditional morpho-taxonomy. Through this study, a reliable DNA barcode reference library for Bangladeshi freshwater fish was established, which could be used to assign fish species by screening sequences against it in the future. This could enhance to achieve better monitoring, conservation, and management of fisheries in this overexploited country.