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\begin{document}

\title{Molecular characterization of marine and coastal fishes of Bangladesh
through DNA barcodes}



\author[1]{Md. Sagir Ahmed}%
\author[2]{Sujan  Datta}%
\author[2]{Tonmoy   Saha }%
\author[3]{Zarif  Hossain}%
\affil[1]{University of Dhaka}%
\affil[2]{Jagannath University}%
\affil[3]{University of Dhaka Faculty of Earth and Environmental Sciences}%


\vspace{-1em}



  
  \date{May 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Abstract: This study attempted to molecular characterization of marine
and coastal fishes of Bangladesh based on mitochondrial cytochrome c
oxidase subunit I (COI) gene as a marker. A total of 376 mitochondrial
COI barcode sequences were obtained from 185 species belonging to 146
genera, 74 families, 20 orders and two classes 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
significantly lower compared to \%AT (p < \selectlanguage{english}0.005). Also, a significantly
lowered \%GC content was observed in second and third codon position
compared to the first codon position in all the samples
(1st\textgreater{}2nd\textgreater{}3rd, p-value < \selectlanguage{english}0.005). In
Elasmobranchii (Sharks and rays) the average Kimura two parameter (K2P)
distances within species, genera, families and orders were 1.2\%,
6.07\%, 11.08\% and 14.68\%, respectively and for Actinopterygii, the
average K2P distances within species, genera, families and orders were
0.40\%, 6.36\%, 14.10\% and 24.07\%, respectively. The mean interspecies
distance was 16-fold higher than the mean intraspecies distance. The K2P
neighbor-joining (NJ) trees based on the sequences generally clustered
species in accordance with their taxonomic position. A total of 21
species were newly recorded in Bangladesh. High efficiency and fidelity
in species identification and discrimination were demonstrated in the
present study by DNA barcoding, and we concluded that COI sequencing can
be used as an authentic identification marker for Bangladesh marine fish
species. Key words: COI, Barcoding, Elasmobranchii, Actinopterygii,
Genetic diversity, Phylogeny%
\end{abstract}%



\sloppy


\textbf{ABSTRACT}

This study attempted to molecular characterization of marine and coastal
fishes of Bangladesh based on mitochondrial cytochrome c oxidase subunit
I (COI) gene as a marker. A total of 376 mitochondrial COI barcode
sequences were obtained from 185 species belonging to 146 genera, 74
families, 20 orders and two classes 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 significantly lower
compared to \%AT (\emph{p} < \selectlanguage{english}0.005). Also, a significantly lowered \%GC
content was observed in second and third codon position compared to the
first codon position in all the samples
(1\textsuperscript{st}\textgreater{}2\textsuperscript{nd}\textgreater{}3\textsuperscript{rd},\emph{p-}
value < \selectlanguage{english}0.005). In Elasmobranchii (Sharks and rays\textbf{)}the average
Kimura two parameter (K2P) distances within species, genera, families
and orders were 1.2\%, 6.07\%, 11.08\% and 14.68\%, respectively and for
Actinopterygii, the average K2P distances within species, genera,
families and orders were 0.40\%, 6.36\%, 14.10\% and 24.07\%,
respectively. The mean interspecies distance was 16-fold higher than the
mean intraspecies distance. The K2P neighbor-joining (NJ) trees based on
the sequences generally clustered species in accordance with their
taxonomic position. A total of 21 species were newly recorded in
Bangladesh. High efficiency and fidelity in species identification and
discrimination were demonstrated in the present study by DNA barcoding,
and we concluded that COI sequencing can be used as an authentic
identification marker for Bangladesh marine fish species.

Key words: COI, Barcoding, Elasmobranchii, Actinopterygii, Genetic
diversity, Phylogeny

\textbf{INTRODUCTION}

Bangladesh has vast coastal and marine resources along its south edge as
the Bay of Bengal is situated in the south of Bangladesh. There is a
total of 166,000 square km water areas including the Exclusive Economic
Zone (EEZ) which is larger than the country's total land area of 147,500
square km. The country is rich not only in terms of its vast water areas
but also in terms of biological diversity. The marine fisheries sector
plays an important role in the economy of Bangladesh in terms of
nutrition, income, employment and foreign exchange earnings (DoF, 2018).
Fish provides about 60\% of animal protein in the daily dietary
requirement of 160 million people of the country. Marine fisheries alone
contributes 654,687 metric tons which is 15.31\% of the country's total
fish production (DoF, 2018).

Nonetheless, description and information of marine fishes of Bangladesh
are scattered throughout a wide range of scientific publications (Shafi
\& Quddus, 1982; Rahman et al., 1995; Rahman et al., 2009; Hussain et
al., 1971). Estimates of total fish species vary from 170 (Shafi \&
Quddus 1982) to 402 (Rahman et al., 2009), 442 (IUCN, 2000) or 475
(Hussain et al., 1971) including the migratory and estuarine species. If
we compare this number with our neighboring countries, diversity
estimates for Bangladeshi marine fishes seems to be underestimated. It
is evident from the published articles, books and review papers on
Bangladeshi marine fishes (Ahmed et al., 2019a; Rahman et al., 2009;
Shafi \& Quddus 1982; Hussain et al., 1971; IUCN, 2000) that the
ichthyofaunal diversity statistics are incomplete. Moreover, no
systematic survey or in-depth taxonomic study was undertaken on marine
fish faunal diversity since 1969 (Hussain et al., 1971).

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 (Ahmed et
al., 2019a). Currently, partial cytochrome c oxidase subunit I (COI)
sequences (DNA barcodes) are applying as a standardized and routine
species identification (Hebert et al. 2003; Shehata et al., 2019; Chin
et al., 2016; Filonzi et al., 2010) instead of traditional mopho
taxonomy. The marked divergence and lack of overlap between
intraspecific and interspecific genetic distances is the primary reason
for the selection of COI as the standard barcode gene (Hebert et al.
2003). More importantly, COI evolution is sufficiently rapid to allow
the discrimination of very closely related species in most groups, as
well as taxonomically significant intraspecific variation associated
with geographic structure (Buckling et al., 2010).

DNA barcoding has been successfully identified marine ichthyofauna and
provided the wealth of DNA barcode information in many places, such as
Australia (Ward et al., 2005), Canada (Hubert et al., 2008; Steinke et
al., 2009), India (Lakra et al, 2011), China (Zhang et al., 2011; Zhang
\& Hanner, 2012; Wang et al., 2018), Portugal (Costa, et al., 2012),
Germany (Knebelsberger et al., 2014), Taiwan (Bingpeng et al., 2018;
Chang et al., 2017) and Vietnam (Thu et al., 2019).

Considering the economic importance of marine fishery and the expected
richness of the fish fauna and in the absence of an expert based
taxonomy, DNA barcoding may be an important component in biological
conservation and management of biodiversity and fishery of Bangladeshi
marine fishes. As a highly overpopulated country, anthropogenic
activities, overfishing, habitat destruction and natural disasters have
generated significant impacts on the biodiversity and structure of the
fish community in Bangladesh. Unfortunately, the marine ichthyofauna of
Bangladesh remains unexplored due to the lack of taxonomists. Hence,
adopting an authentic and quick identification method is essential to
assist fishery managers, scientists and policy makers for sustainable
management of this invaluable marine resources. This study aims to build
a DNA based barcode library of the morphologically identified marine and
coastal fish species of Bangladesh using partial COI gene sequence.

\textbf{MATERIALS AND METHODS}

\textbf{2.1 Study area and specimen collection}

Fish samples were collected from marine and coastal habitats, fish
landing centers, fish markets or from the local fishermen from July 2015
to June 2019. Most of the specimens were collected from the Cox's Bazar
and Patuakhali regions. At least three specimens were collected for each
species. In case of rare ones single specimen were analyzed. Personal
fishing was also conducted to collect some rare and non-commercial fish
species whenever necessary. Digital photographs of all the fishes were
taken immediately and taxonomic identification of specimens was done
following previous reports (Talwar and Jhingran, 1991; Rahman et al.,
2009; Siddiqui et al., 2007; Nakabo, 2002; Carpenter et al., 2002a,
2002b; Last et al., 2016). 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 Dhaka University Zoology Museum (DUZM).

\textbf{DNA barcoding}

DNA was isolated from muscle sample using the QIAGEN DNeasy Blood \&
Tissue Kit following the manufacturer's protocol, under sterile
condition. The concentration of the isolated DNA was measured in
NanoDrop spectrophotometer to evaluate its quality and quantity. A 658
bp long fragment from the 5' region of the COI gene was PCR-amplified by
the primer pair FishF2 5'TCGACTAATCATAAAGATATCGGCAC3' and FishR2
5'ACTTCAGGGTGACCGAAGAATCAGAA3'. The primer pair FishF1
5'TCAACCAACCACAAAGACATTGGCAC3' and FishR1 5'TAGACTTCTGGGTGGCCAAAGAATCA3'
were used for the amplification of COI that failed to amplify using
FishF2/FishR2. The PCR amplification of each sample was conducted in a
25 \selectlanguage{greek}µ\selectlanguage{english}l volume comprised of 23 \selectlanguage{greek}µ\selectlanguage{english}l of PCR Master Mix and 2 \selectlanguage{greek}µ\selectlanguage{english}l of template
DNA that was subsequently spun for 30 seconds for homogenization. The
components of the PCR Master Mix were as such: 12.5 \selectlanguage{greek}µ\selectlanguage{english}l Taq Polymerase,
8.5 \selectlanguage{greek}µ\selectlanguage{english}l Nano Pure water, 1 \selectlanguage{greek}µ\selectlanguage{english}l forward primer and 1 \selectlanguage{greek}µ\selectlanguage{english}l reverse primer. The
PCR amplification was carried out in the ASTEC Thermal Cycler GeneAtlas~
(Astec Co. Ltd.) where the thermocycling profile was customized as such:
an initial denaturation at 95 for 5 minutes followed by 41 cycles of
denaturation at 95 for 30 s, primer annealing at 54 for 30 seconds,
extension for 72 for 1 minute, and a final extension at 72 for 5
minutes. The PCR products were kept at room temperature for 15 minutes,
and preserved afterward at -20 until further downstream application.
The amplicons were further identified through electrophoresis in a 1\%
agarose gel. Then they were purified using PureLink PCR purification
kit. The good quality amplicons were then parceled for sequencing to the
First BASE laboratories, Malaysia where they conducted the Sanger's
dideoxy sequencing using~ABI PRISM 3730xl Genetic Analyzer~exploiting
the BigDye\selectlanguage{ngerman}® Terminator v3.1 cycle sequencing kit chemistry. The
assembled contigs were prepared by the CAP3 DNA assembly program. These
newly obtained sequences were uploaded in the BLASTn suite to check
whether they meet the threshold value of \selectlanguage{english}[?] 97\% for both the percent
identity and query coverage. The sequences of the high-fidelity
amplicons were then deposited in the GenBank with the help of the
Barcode Submission Tool with detailed source information and feature
annotation.

\textbf{2.3 Bioinformatic and statistical analyses}

DNA sequences were translated in-silico by the Translation Tool provided
in the ExPASy portal to check for any pseudogenes and avoid sequencing
errors. The composition of nucleotides was calculated on the CLC
Workbench v7.7.1 and Mega X. The alignment of the amplicons was carried
out in MUSCLE with the default specifications. Kimura two parameter
(K2P) model was employed to calculate pairwise genetic distance at the
level of species, genus and family (Kimura, 1980). Using this distance
matrix, a Neighbor-Joining (NJ) algorithm was implemented to generate
the phylogenetic tree topology in Mega X with a bootstrapping value of
1000 replications (Saitou and Nei, 1987, Felsenstein, 1985). Basic
statistical analyses were done in Excel 2013 and the hypotheses testing
(e.g.: \emph{t} -test, \emph{F} -test, etc.) was done in
RStudio-1.2.5001.

\textbf{3. RESULTS}

\textbf{3.1 General inference}

A total of 748 tissue samples were collected from Bangladesh coast,
among which 376 COI sequences were obtained (Table 1). Based on
morphological and molecular identifications, these samples represented
185 species of 146 genera, 74 families, 20 orders and two classes (Table
1). Among the 185 species, 21 fishes were new records in Bangladesh. The
length of all barcode sequences ranged from 477 to 683 bp, with an
average of 652 bp and 89\% of sequences were longer than 600 bp. 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 sequences 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 \& Hewitt 1996).

\textbf{3.2 Elasmobranchii (Sharks and rays)}

A total of 52 samples were sequenced belonging to 5 order, 8 families,
13 genera and 21 species (Table 1). Among these 21 species, two species
have been found as endangered (EN), five as vulnerable (VU) and seven as
near threatened (NT). One species was least concern (LC), two were not
evaluated (NE) and the remaining four species were data deficient (DD)
(IUCN, 2020).\emph{Chiloscylliumburmensis} and\emph{C.~hasseltii}were
reported as two new records (Ahmed et al., 2019b) in this area. The
sequence analysis indicated the average nucleotide frequencies to be- A:
25.90\%, T:32.70\%, G:16.20\% and C:25.20\%. The base composition
analysis for the COI sequence showed that the average percent T content
was the highest and the average percent G content was the lowest; the AT
content (58.60\%) was higher than the GC content (41.40\%). The GC
contents at the first, second and third codon positions for the
Elasmobranchii were 53.30\%, 43.60\% and 27.70\%, respectively. At the
first codon position, the usage of T (20.00\%) was the lowest, and the
usages of the other bases were 23.50\%, 27.40\% and 29.50\% for C, A and
G respectively. At the second codon position, the content of T (42.00\%)
was highest, and the percentage of the other bases were 29.40\%, 14.50\%
and 14.20\% for C, A and G respectively. At the third codon position,
the base usage was T: 37.00\%, C: 22.80\%, A: 35.70\% and G: 4.90\%; the
G content being the lowest, exhibited a clear pattern of anti-G bias.

The K2P genetic distances within each taxonomic level are summarized in
Table 2. The average genetic distance within species, genus, family and
order were 1.2+-0.0007\%, 6.07+-0.014\%, 11.08+-0.018\% and 14.68+-0.04\%,
respectively. The NJ tree clearly distinguished all the species and the
species belonging to 8 families were represented by eight distinct
clades (Fig. 1).

\textbf{3.3 Actinopterygii (Ray-finned fishes)}

A total of 324 sequences were generated belonging to 15 orders, 66
families, 133 genera and 164 species (Table 1). The sequence analysis
revealed average nucleotide frequencies to be A: 23.9\%, T: 29.6\%, G:
18.3\% and C: 28.2\% (Fig. 3). 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 (53.50\%) was higher
than the GC content (46.50\%). The GC contents at the first, second and
third codon positions for the fish species were 54.62\%, 43.66\% and
38.04\% respectively (Fig. 4). The pattern of \%GC content at different
codons was
1\textsuperscript{st}\textgreater{}2\textsuperscript{nd}\textgreater{}3\textsuperscript{rd}(\emph{p}
-value \textless{}0.005\textbf{)} The K2P genetic distances within each
taxonomic level are summarized in Table 3. The average genetic distance
within species, genus, family and order were 0.40 +- 0.002\%, 6.36 +-
0.008\%, 14.10 +- 0.01\% and 24.07 +- 0.02\%, respectively (Table 3).

The NJ tree of all generated sequences included 164 species is provided
in Figure 2. Most of the specimens of the same species were clustered
together, which reflected the prior taxonomic assignment based on
morphology. No taxonomic deviation was detected at the species level,
indicating that the majority of the examined species could be
authenticated by the barcode approach.

\textbf{3.3.1} \textbf{Order Clupeiformes}

This order includes many of the most important forage and food fish. A
total of 45 samples were sequenced belonging to three families, 12
genera and 19 species. Among the three families Clupeidae is the most
valuable family where a single species \emph{Tenualosa
ilisha}contributes over 12\% of total fish production of the country
(DoF, 2018). The overall mean nucleotide base frequencies observed for
these sequences were T: 28.60\%, C: 27.80\%, A: 24.20\% and G 19.40\%.
The AT content (52.80\%) was higher than the GC content (47.20\%) (Fig.
3). The GC contents at the first, second and third codon positions were
49.10\%, 48.20\% and 44.40\% respectively (Fig. 4). The K2P distances of
the COI sequence within species, genus and family were 1.81, 6.55 and
13.41, respectively (Table 3). The NJ tree clearly distinguished all the
species (Supplementary Fig.1). The species belonging to family
Clupeidae, Engraulidae and and Pristigasteridae were represented by
three distinct clades.

\textbf{3.3.2 Order Perciformes}

Perciformes is the most dominant order among the marine fishes of
Bangladesh and contributes to over 50\% of total exploited species. A
total of 195 samples were sequenced belonging to 35 families, 79 genera
and 100 species. Among the 35 families, Gobiidae was the most dominant
one followed by Carangidae, Sciaenidae and Polynemidae. The overall mean
nucleotide base frequencies observed for these sequences were- T:
29.90\%, C: 28.10\%, A: 23.80\% and G: 18.20\%. The AT content (53.70\%)
was higher than the GC content (46.30\%) (Fig. 3). The GC contents at
the first, second and third codon positions were 56.70\%, 42.70\% and
39.50\% respectively. The K2P distances of the COI sequence within
species, genus and family were 0.58, 6.26, and 15.62, 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 (Supplementary Fig. 2).

\textbf{3.3.3 Order Siluriformes}

A total of 14 samples were sequenced belonging to four families, six
genera and six species. The overall mean nucleotide base frequencies
observed for these sequences were---T: 29.30\%, C: 28.10\%, A: 24.80\%
and G: 17.70\%. The AT content (54.10\%) was higher than the GC content
(45.90\%). The GC contents at the first, second and third codon
positions were 56.70\%, 42.70\% and 38.10\% respectively. The K2P
distances of the COI sequence within species and between species were
0.14 and 26.62 respectively.

\textbf{3.3.4 Order Pleuronectiformes}

Fourteen samples were sequenced belonging to three families, five genera
and six species. The overall mean nucleotide base frequencies observed
for these sequences were---T: 30.80\%, C: 26.70\%, A: 24.10\% and G:
18.30\%. The AT content (54.90\%) was higher than the GC content
(45.10\%). The GC contents at the first, second and third codon
positions were 55.50\%, 42.10\% and 37.60\%, respectively. The K2P
distances of the COI sequences within species and family were 0.36 and
17.65 respectively.

\textbf{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.

\textbf{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 (\emph{Chiloscyllium burmensis}
and\emph{Chiloscyllium~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 3\textsuperscript{rd} 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 \emph{p} -value \selectlanguage{english}<0.005). This difference was
attributable to the GC content at the 2\textsuperscript{nd} (47.80\%
versus 43.60\%) and, especially, the 3\textsuperscript{rd} codon base
(42.80\% versus 27.70\%). The pattern of \%GC content at different
codons for all these fishes was invariably
1\textsuperscript{st}\textgreater{}2\textsuperscript{nd}\textgreater{}3\textsuperscript{rd}(\emph{p}
-value \textless{} 0.005) and for
Pleuronectiforms~1\textsuperscript{st}\textgreater{}2\textsuperscript{nd}\textgreater{}3\textsuperscript{rd}(\emph{p}
-value \textless{} 0.05, n=6).

Kimura 2-parameter distance values of 6.36 \selectlanguage{ngerman}± 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
\textless{}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
(Pe\selectlanguage{polish}č\selectlanguage{english}nikar \& 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.

\textbf{Acknowledgements}

We acknowledge the financial support from the Ministry of Education,
Government of the People's Republic of Bangladesh as a grant for
advanced research in Education (Grants No. MRS2017448). We are also
thankful to Dr. Mohammad Abdul Baki, Nusrat Jahan Sanzida, JBM Aysha
Akter, Ayesha Akhter Zhilik, Nishat Jahan Chowdhury, Sumaiya Salam,
Tamim Afrin, Noor Aida Arfin, Mysha Mahjabin, Sajid Ahmed and Afroza
Kaonine Haque for their cooperation during this research.

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\textbf{Ethics statement}

All fish species were caught in the offshore area (not national parks,
other protected areas, or private areas, etc.), so no specific
permissions were required for these locations/activities. However,
specimen collection was conducted under a permit to the University of
Dhaka, and approval of the Ethical Review Committee, Faculty of
Biological Sciences, University of Dhaka.

\textbf{Conflict of interest}

The authors declare no conflict of interest.

\textbf{Data availability}

All the voucher specimens with their respective Voucher ID are keeping
at the Museum of Department of Zoology, University of Dhaka, Bangladesh,
and has public access with permision. The DNA barcoding data can be
retrieving from the NCBI GenBank as have open public access.

\textbf{Contribution}

Ahmed MS initiated the project, acquired funding, managed project
administration, specimen curation and internal reports, performed the
taxonomic analyses, and prepared the whole manuscript. Datta SK and Saha
T performed field collection, generated and analyzed molecular
sequences, curated tissue samples, and participated in the writing of
the final manuscript. Hossain Z contributed laboratory analyses,
participated in generation of sequences and made contribution to the
manuscript. All authors reviewed the manuscript.

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