3.3.5 Order-Beloniformes
Fifteen samples were sequenced belonging to four families, six genera and seven species. The overall mean nucleotide base frequencies observed for these sequences were—T: 32.40%, C: 25.90%, A: 24.90% and G: 16.80%. The AT content (57.30%) was higher than the GC content (42.70%). The GC contents at the first, second and third codon positions were 55.10%, 42.60% and 30.60% respectively. The K2P distances of the COI sequence within species, genus and family were 0.24, 4.77, and 9.67 respectively.
DISCUSSION
DNA barcoding has been adopted as a global bio-scanner to provide an efficient molecular technique for species-specific identification using the partial sequence of the mitochondrial COI gene. It is evident from the decade long studies (Ward et al., 2005; Hubert et al., 2008; Zhang et al., 2011; Lakra et al., 2011; Chang et al., 2017; Thu et al., 2019) that the DNA barcoding can discriminate the marine fish species from the different geographic regions, including Australia, Canada, China, India, Taiwan and Vietnam. Here, we have profiled the barcode of marine fishes collected from the coast of Bangladesh and also have demonstrated the promise of barcoding to identify these, exploiting the partial sequence of mitochondrial COI genes. Barcodes were generated for 185 species of Elashmobranchii and Actinopterygii from Bangladesh belonging to 146 genera and 74 families and 20 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, the 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.
Within the Elasmobranchii, a total of 12 rays and nine sharks species including two new records (Chiloscyllium burmensis andChiloscyllium hasseltii ) were confirmed through barcoding. The overall AT and GC content was 58.60% and 41.40%, respectively. But the mean GC content of the 11 barcoded ray species was higher than the 8 shark species (43.67% versus 38.59%). This was largely due to the GC variation in the 3rd codon position (33.59% versus 20.38%).
In this study, the COI barcode sequences for 164 teleost fish species were successfully amplified (Table 1, Fig. 2). The base composition analysis of the COI sequences revealed AT content (53.50%) to be higher than GC content (46.50%), similar to the pattern 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 54.62%, 43.66% and 38.04%, respectively. At the first codon position, the usage of G (20.00%) was the lowest, and the usages of the other bases were 28.60%, 27.90% and 27.00% for C, A and T, respectively. At the second codon position, the content of T (32.00%) was highest, and the contents of the other bases were—C: 29.50%, A: 20.20% and G: 18.30. At the third codon position, the base usage was—T: 30.00%, C: 26.40%, A: 23.50% and G:16.50% (Fig. 4). There was a significantly higher overall GC content in the 164 species of bony fish compared to the 21 species of sharks and rays (46.50% versus 41.40% with a p -value ˂0.005). This difference was attributable to the GC content at the 2nd (47.80% versus 43.60%) and, especially, the 3rd codon base (42.80% versus 27.70%). The pattern of %GC content at different codons for all these fishes was invariably 1st>2nd>3rd(p -value < 0.005) and for Pleuronectiforms 1st>2nd>3rd(p -value < 0.05, n=6).
Kimura 2-parameter distance values of 6.36 ± 0.008%, 14.10 ± 0.01% and 24.07 ± 0.02% were obtained for within genus, within family and within order respectively (Table 3, Fig 5). Consistent with previously published fish barcoding data, pairwise genetic distance values were increasing at higher taxonomic levels. This increase in the genetic distance through the higher taxonomic levels supports the significant change in genetic divergence at the species boundaries (Hubert et al., 2008; Lakra et al., 2011).
In this study, the average within species K2P distance was 0.40%, compared with 6.36% for within genera. The mean interspecific distance was found to be 16-fold higher than the mean intraspecific distance. More than 13.9-fold difference was observed in the marine fishes commonly encountered in the Canadian Atlantic (Steinke et al., 2009), Indian (Lakra et al., 2011) and Australian marine fishes (Ward et al., 2005). This result 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 0.40± 0.002%. Mean intraspecific genetic distance was calculated as <1% in previous studies; Hubert et al. (2008) found 0.30% (0–7.42%) for 194 fish species from Canadian ichthyofauna; Ward et al. (2005) 0.39% (0–14.08%) for 207 marine fish species from Australia; Thu et al. (2019) 0.34% for 458 ray finned species in Vietnam and Bingpeng et al. (2018) found 0.21% for 85 genera in Taiwan strait (Table 4).
Phylogenetic relationship of barcoded species of Elasmobranchii and Actinopterygii were shown in separate NJ tree (Fig. 1 & 2). 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.
Our study suggests that DNA barcoding has been successful in identifying and discriminating the vast majority of marine ichthyofauna. The DNA barcoding method has been proven to be an effective tool for species identification, particularly with specimens that are damaged, incomplete, or consisting of several morphologically distinct stages (Pečnikar & Buzan, 2014; Bingpeng et al., 2018). Nevertheless, DNA barcoding also has its limitations. In some cases, related species may present identical sequences making DNA barcodes useless for species discrimination. Therefore, DNA barcoding can serve as a complementary tool for species identification, though it cannot replace the traditional morpho-taxonomy. Through this study, a reliable DNA barcode reference library for the marine fish in the Bay of Bengal, Bangladesh has been established, which could be used to assign fish species by screening sequences against it in the future. We hope this would appreciably contribute to achieving better monitoring, conservation, and management of fisheries in this over exploited region.