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.