Ecological Genetics and Genomics 17 (2020) 100067

Contents lists available at ScienceDirect

Ecological Genetics and Genomics

Genetic variation of native and introduced climbing perch Anabas

testudineus (Bloch, 1792) derived from mitochondrial DNA analyses
Imran Parvez a, *, Tanjiba Mahajebin a, Michèle L. Clarke b, Mousumi Sarker Chhanda c,
Shirin Sultana d
a Department of Fisheries Biology and Genetics, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh
b School of Geography, University of Nottingham, University Park, Nottingham, NG7 2RD, UK
c Department of Aquaculture, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh
d Fisheries Biotechnology Division, National Institute of Biotechnology, Dhaka, Bangladesh

ARTICLE INFO ABSTRACT

Keywords: The native and introduced Thai and Vietnamese Climbing perch Anabas testudineus are cultured in semi-
Genetic variation intensive and intensive systems in Bangladesh. We sampled the native and the introduced A. testudineus from
Mitochondrial DNA
Bangladesh to test genetic variations based on Cytochrome C oxidase subunit I (COI) and Cytochrome (Cytb)
Phylogeny
gene. The COI nucleotide sequences of A. testudineus of India, Thailand, and Vietnam were retrieved from
Climbing perch
Anabas testudineus
NCBI genebank and analyze. In the COI nucleotides, the higher AT content (53.6%) and lower GC content
(46.4%) with larger differences were observed (values>0). The transition/transversion bias (R) based on
Kimura 2-parameter (K2P) model was 8.44 with a higher transitional substitution (89%). The intraspecies ge-
netic distance of COI nucleotide sequences based on a K2P model ranged from 0.000 to 0.094. The lowest ge-
netic distance (0.00) was observed between the pair of reference and sequenced COI nucleotide sequences of
Thai as well as Vietnamese A. testudineus, where very little genetic distance (0.002) was found between the se-
quenced Bangladeshi and reference Indian COI nucleotide sequences. The Thai and Vietnamese A. testudineus
formed one cluster, whereas Bangladeshi and reference Indian A. testudineus formed a separate cluster in the
phylogenetic tree using maximum likelihood method and time tree extrapolated from the molecular clock hy-
pothesis. Our study provides evidence that the native and introduced A. testudineus are different lineages that
might have evolved due to ecological variations at their origin. We suggest maintaining purity of breeding and
cultured populations of the native and introduced A. testudineus for sustainable aquaculture in Bangladesh.

1. Introduction declined severely in Bangladesh [3]. To meet demand as food, initia-

Climbing perch Anabas testudineus is a native freshwater fish
species in Bangladesh which has high aquaculture potential. This
species is widely distributed in India, Myanmar, Pakistan, Sri-Lanka,
China, Thailand, Philippines, Hong Kong, and Malaysia [1]. Due to its
flavor, nutritional properties, air-breathing capacity that enables it to
keep them alive during transportation and marketing, this species is
very popular as food fish [1]. This species used to be abundant in
freshwater bodies of Bangladesh such as streams, lakes, floodplain and
beels, paddy fields, and swamps [2]. Due to anthropogenic activities
and environmental degradation, the availability of climbing perch has
tives have been taken for artificial seed production in the hatchery
[1]. However, the indigenous A. testudineus showed lower growth and
survival in cultured ponds [4]. In 2002 the Bangladesh Fisheries Re-
search Institute introduced A. testudineus from Thailand that displayed
higher growth rates. Initially, the introduced Thai climbing perch was
successful in contributing to aquaculture production, but inbreeding
has resulted in a declined growth rate [4]. In 2010, the Sharnalata
Agro-Fishery introduced Vietnamese A. testudineus that displayed
faster growth rates than native and Thai climbing perch. Although the
native and introduced climbing perch belong to the same species, they
are dissimilar in body shape, coloration, and growth rate [4]. While

Corresponding author.
*

E-mail addresses: iparvez.fbg@hstu.ac.bd, iparvez.fbg@hstu.ac.bd (I. Parvez), tanjibamahajebin@gmail.com (T. Mahajebin), indigo34@outlook.com (M.L.
Clarke), chh.sarker@hstu.ac.bd (M.S. Chhanda), shirinbau@yahoo.com (S. Sultana).

https://doi.org/10.1016/j.egg.2020.100067
Received 13 April 2020; Received in revised form 15 August 2020; Accepted 1 September 2020
Available online 6 September 2020
2405-9854/© 2020 Elsevier Inc. All rights reserved.
I. Parvez et al.
morphology continues to be used to distinguish among the climbing
perch of different origins, the mitochondrial (mt) DNA based barcod-
ing system offers higher precision in the identification of species or
lineages [5]. DNA barcoding includes the amplification and sequenc-
ing of a short mitochondrial cytochrome C oxidase subunit I (COI)
gene [6]. The use of COI sequences for the identification of fish has
been widely reported, such as Sparidae family from the Egyptian wa-
ters [7], grouper fishes [8], Cyprinidae family of India [9], Ompok
genus of Indian River [10] and Clariidae and Pangasiidae family of
the United States [6]. The use of partial sequences of the cytochrome
b (Cyt b) gene is also effective in fish species identification [11,12].
For example, cytochrome b sequences were used for catfishes in Korea
[13]; Puntius genus of Indian rivers [14]; fishes of South China Sea
[12] and Perciformes of India [15]. In this study, we aim to reveal the
genetic variations among the native and introduced A. testudineus in
Bangladesh through the analysis of mitochondrial gene cytochrome C
oxidase subunit I (COI) and cytochrome b (Cyt b).
2. Materials and methods
2.1. Sample collection and preservation
Native Bangladeshi live climbing perch were collected from paddy
fields in Dinajpur district (25.70°N, 88.68°E), exotic Thai climbing
perch were collected from the freshwater sub-station in Saidpur,
Bangladesh Fisheries Research Institute (25.47°N, 88.56°E) and the
Vietnamese climbing perch were collected from the Sharnalata Agro-
Fisheries Limited located in Fulbaria, Mymensingh (24.36°N,
90.20°E). Muscle samples were collected from the trunk region of
each fish and preserved in 95% ethanol. All specimens were tagged
accordingly and preserved in 10% buffered formalin solution.
2.2. DNA extraction
DNA was extracted from the preserved muscle tissues using the
Phenol-Chloroform-Isoamyl alcohol based on the method described by
Ref. [16]. The extracted DNA was checked through 1% agarose gel
electrophoresis with ethidium bromide (5 μg/mL) incorporated in
1 × TBE buffer. The final DNA concentration was quantified by opti-
cal density (OD) measurement using a spectrophotometer (Thermo
Scientific™ NanoDrop™ 2000: Model SIA340) at 260 nm.
2.3. Amplification of partial sequences of mitochondrial COI and Cytb
genes
The partial sequence of the COI gene targeting 648 bp, was ampli-
fied using primer pair Fish F1 (5′-TCA ACC AAC CAC AAA GAC ATT
GGC AC-3′), Fish-R1 (5′-TAG ACT TCT GGG TGG CCA AAG AAT CA-
3′) in 25 μl reactions volume containing 1 × assay buffer (100 mM
Tris, 500 mM KCL, 0.1% gelatin, at pH 9.0) with 1.5 mM MgCl2,
5 pmol of each primer, 200 μM of each dNTP, 1.5 U Taq DNA poly-
merase and 20 ng of template DNA. The thermal profile started with
95 °C for 2 min, followed by 35 cycles of 94 °C (30 s), 54 °C (30 s),
and 72 °C (1 min), the final extension for 2 min at 72 °C.
Partial fragments of 360 bp mitochondrial Cyt b gene were also
amplified by using primer CytbF (5′-AAA AAG CTT CCA TCC AAC
ATC TC-3′) and CytbR (5′-AAA CTG CAG CCC CTC AGA ATG AT-3′)
[17]. The total reaction of 25 μl that consisted of 12.5 μl master mix
(Taq DNA Polymerase, dNTPs, MgCl2), 5 pmol of each primer, 20 ng
of template DNA, 9.5 μl distilled water were used. The thermal profile
initiated at 104 °C for 2 min, 95 °C for 1 min followed by 35 cycles of
94 °C (1 min), 48 °C (1 min), and 72 °C (45 s), with a final extension
of 5 min at 72 °C.
2
Ecological Genetics and Genomics 17 (2020) 100067
2.4. Purification of PCR products and sequencing
The amplified PCR products were electrophoresed using 1.5%
agarose gels and ethidium bromide staining and visualized under UV
illumination in the Gel-Doc system (BIO-RAD). The purification was
done by using an Invitrogen purification kit following the manufactur-
er's instructions. Products were labeled using the Big Dye Terminator
V.3.1 cycle sequencing kit (Applied Biosystems Inc) and sequenced
bidirectionally using ABI 3730 capillary sequencer following the man-
ufacturer's instruction.
2.5. Sequence analysis
The raw DNA sequences were edited and aligned using BioEdit se-
quence alignment editor version 7.0.5.2 [18]. The partial COI and
Cytb sequences of climbing perch of three origins were aligned to find
the final consensus size. The consensus size of the COI and Cytb gene
were ranged from 646bp to 682bp, and 356bp to 368bp respectively.
The nucleotide sequences were translated to determine the open read-
ing frame (ORF) and the translation frame with direction. The edited
sequences were submitted to the relevant GenBank databases
(MG552721-MG552726) using the BankIt submission tool. Nucleotide
sequences of the COI gene of climbing perch native to Thailand (Thai
A. testudineus, Reference: JQ661371.1), Vietnam (Vietnamese A. tes-
tudineus, Reference: KF752454.1) and India (Indian A. testudineus, Ref-
erence: JX983213.1) were retrieved from the NCBI GenBank database
(http://www.ncbi.nlm.nih.gov/) to include in the analysis as the ref-
erence sequences. The number of polymorphic sites and nucleotide di-
versity, the nucleotide composition, net base composition bias dispar-
ity between sequences, transitional/transversional bias, the test of the
homogeneity of substitution patterns between sequences, sequence di-
vergence within species using Kimura 2- parameter (K2P) model, phy-
logenetic tree (Maximum Likelihood Method), TimeTree (RelTime
method) were implemented in MEGA Version 6.01 for molecular ge-
netic analysis [19].
3. Results
In COI gene analysis, 691 sites were determined where 160
(23.15%) were conserved; 530 (76.70%) were variable; 399 (57.74%)
were parsimony informative sites and 127 (18.38%) were singleton
for three populations. The average nucleotide composition of the COI
nucleotide was for T = 31.3, C = 28.8, A = 22.3, and G = 17.6. The
GC content was lower (46.4%) than the AT content (53.6%). The dis-
parity index per site for all sequences was estimated to observe any
larger differences in base composition biases than the expected bases
on the evolutionary divergence between the COI nucleotide sequences
(according to Ref. [20]. The larger differences between the base com-
position biases than the expected bases of studied COI nucleotide se-
quences presented in Table 1. The larger differences observed be-
Table 1
Estimates of net base composition bias disparity between sequences.
SI·NO. Populations 1 2 3 4 5 6
1 Thai A. testudineus (Reference) –
2 Vietnamese A. testudineus 0.000
(Reference)
3 Indian A. testudineus (Reference) 0.056 0.000
4 Bangladeshi A. testudineus 0.084 0.007 0.000
(Sequenced)
5 Thai A. testudineus (Sequenced) 0.000 0.000 0.056 0.084
6 Vietnamese A. testudineus 0.000 0.000 0.000 0.007 0.000 –
(Sequenced)
The values (>0) indicated any larger differences in base composition biases
than the expected bases on the evolutionary divergence between the studied
COI nucleotide sequences (according to Ref. [20].
I. Parvez et al. Ecological Genetics and Genomics 17 (2020) 100067

tween the sequence pair of reference Thai and reference Indian A. tes- of A. testudineus (0.056),which indicated that the sequences COI gene
tudineus (0.056), sequenced native and reference Thai A. testudineus in Bangladeshi A. testudineus evolved with the same pattern of substi-
(0.084), sequenced Thai and reference Indian A. testudineus (0.056), tution (Table 3).
and sequenced Bangladeshi and sequenced exotic Thai A. testudineus The pairwise genetic distance values (Nucleotide sequence diver-
(0.084). gences) using the Kimura 2- parameter (K2P) model are shown below
The transition/transversion biases of COI gene sequences substitu- the diagonal in Table 4. There was no genetic distance (0.00) between
tion were estimated using a Kimura 2-parameter (K2P) model (Kimura reference vs sequenced Thai and reference vs sequenced Vietnamese
1980). The estimated transition/transversion bias (R) among the COI A. testudineus COI gene sequences. Very little genetic distance (0.02)
gene sequences of A. testudineus was 8.44. The rates of different tran- was found between sequenced Bangladeshi and the reference Indian
sitional substitutions are shown in bold and transversionsal substitu- A. testudineus COI gene sequences. The highest genetic distance
tions are shown in italics (Table 2). The nucleotide frequencies were (0.094) was found between sequenced Bangladeshi and sequenced
A = 22.98%, T/U = 31.09%, C = 29.29% and G = 16.64%. The Vietnamese A. testudineus, followed by sequenced Bangladeshi and se-
rate of transitional substitution from A to G, T to C, C to T, G to A quenced Thai A. testudineus. The lowest genetic distance (0.026) was
were 24.8955, 23.7226, 22.3527, and 18.022, respectively. On the found between sequenced Vietnamese and sequenced Thai A. tes-
other hand, the rate of transversional substitution from A to T, A to C, tudineus (Table 4).
T to A, T to G, C to A, C to G, G to T, G to C were 1.2646, 1.2646, The phylogenetic tree was constructed based on the maximum
1.7108, 1.7108, 1.6120, 1.6120, 0.9155 and 0.9155 respectively likelihood method using COI nucleotide sequences showed that both
(Table 2). The rate of transitional substitution among the COI nu- reference and sequenced Thai and Vietnamese A. testudineus formed
cleotide sequences was found higher (88.9941) than the transver- one clade. The reference Thai and sequenced Thai A. testudineus
sional substitution (11.0050). formed one sister clade, and Vietnamese reference and sequenced A.
The probability of rejecting the null hypothesis that sequences testudineus formed another sister clade and the Bangladeshi and refer-
have evolved with the same pattern of substitution, based on the ex- ence Indian A. testudineus formed another clade (Fig. 1). From the
tent of differences in base composition biases between sequences, was time tree, the highest times of divergence (0.04 MYA) was found be-
estimated according to Ref. [20]. A Monte Carlo test (500 replicates) tween sequenced Bangladeshi and sequenced Thai A. testudineus, fol-
was used to estimate the p values [20], that is presented above the di-
agonal in Table 3. The p values smaller than 0.05 were considered sig- Table 4
nificant (marked with asterisk symbol) and the estimates of the dis- Pair wise genetic distance values using Kimura 2- parameter (K2P) model.
parity index per site were shown for each sequence pair that is pre-
SI. Population 1 2 3 4 5 6
sented below the diagonal in Table 3. It was found that the p values
No.
were higher than 0.05 between sequence pair of Bangladeshi se-
quenced vs both Thai reference and sequenced COI nucleotide se- 1 Thai A. testudineus – 0.007 0.013 0.013 0.000 0.007
(Reference)
quences of A. testudineus (0.084), and slightly higher between
2 Vietnamese A. testudineus 0.026 – 0.013 0.014 0.007 0.000
Bangladeshi sequenced vs Indian reference COI nucleotide sequences (Reference)
3 Indian A. testudineus 0.085 0.092 – 0.002 0.013 0.013
(Reference)
Table 2
4 Bangladeshi A. testudineus 0.087 0.094 0.002 – 0.013 0.014
Maximum likelihood estimate of the pattern of nucleotide substitution. (Sequenced)
5 Thai A. testudineus 0.000 0.026 0.085 0.087 – 0.007
A T C G
(Sequenced)
A – 1.7108 1.6120 18.0233 6 Vietnamese A. testudineus 0.026 0.000 0.092 0.094 0.026 –
T 1.2646 – 22.3527 0.9155 (Sequenced)
C 1.2646 23.7226 – 0.9155 The pairwise genetic distance values (Nucleotide sequence divergences) using
G 24.8955 1.7108 1.6120 – the Kimura 2- parameter (K2P) model were shown below the diagonal.

The rates of different transitional substitutions are shown in bold and trans-
versionsal substitutions are shown in italics.

Table 3
Test of the homogeneity of substitution patterns between COI sequences of A.
testudineus.

Populations 1 2 3 4 5 6

1 Thai A. testudineus – 0.000* 0.056 0.084 0.000* 0.000*

(Reference)
2 Vietnamese A. testudineus 1.000 – 0.000* 0.007* 0.000* 0.000*
(Reference)
3 Indian A. testudineus 0.152 1.000 – 0.000* 0.056 0.000*
(Reference)
4 Bangladeshi A. testudineus 0.116 0.332 1.000 – 0.084 0.007*
(Sequenced)
5 Thai A. testudineus 1.000 1.000 0.116 0.110 – 0.000*
(Sequenced)
6 Vietnamese A. testudineus 1.000 1.000 1.000 0.334 1.000 –
(Sequenced)

The p values smaller than 0.05 were shown above the diagonal, considered
significant (marked with asterisk symbol), which rejecting the null hypothesis
of sequences have evolved with the same pattern of substitution. The esti-
mates of the disparity index per site were shown for each sequence pair that is Fig. 1. Phylogenetic tree inferred from COI nucleotide sequences using ML
presented below the diagonal. method.

3
I. Parvez et al.
lowed by sequenced Bangladeshi and sequenced Vietnamese A. tes-
tudineus. The divergence time of evolution was zero (0) between se-
quenced Bangladeshi and reference Indian A. testudineus (Fig. 2).
The phylogenetic tree was also constructed using the Cytb nu-
cleotide sequences to validate the findings of the phylogenetic tree
based on the COI nucleotide sequence. The results showed that se-
quenced Thai and Vietnamese A. testudineus were closely related but
sequenced Bangladeshi A. testudineus was distantly related from them
(Fig. 3). The time tree revealed that the highest divergence time was
0.03 million years ago (MYA) between sequenced Bangladeshi and se-
quenced Thai A. testudineus followed by sequenced Bangladeshi A. tes-
tudineus and sequenced Vietnamese A. testudineus (Fig. 4).
4. Discussion
In this study, significant genetic variations were found between
native and exotic A. testudineus based on DNA barcoding of the COI
gene. DNA barcoding using the COI gene has been used as an effective
tool for fish species identification [21], for example, identification of
three Ompok species [10] and Serranidae [8]. [22] Barcoded more
than 5000 species by using a 648 bp region of the COI (mitochondr-
Fig. 2. Time tree inferred from COI nucleotide sequences using RelTime
method.
Fig. 3. Phylogenetic tree inferred from Cytb nucleotide sequences using ML
method.
4
Ecological Genetics and Genomics 17 (2020) 100067
Fig. 4. Time tree inferred from Cytb nucleotide sequences using RelTime
method.
ial) gene. In this study, we also used the 648 bp barcode COI gene to
determine the level of genetic variation among the climbing perch
currently available in Bangladesh. The sequence analysis identified a
total of 691sites, where 160 (23.15%) were conserved; 530 (76.70%)
were variable; 399 (57.74%) were parsimony informative and 127
(18.38%) were singleton. Among Clarias gariepinus, Coptodon zillii and
Sarotherodon melanotheron from Southwestern Nigeria, among 696
sites - 347 (49.86%) were conserved; 346 (49.71%) were variable;
225 (32.33%) were parsimony informative sites and 121 (17.39%)
were singleton sites [23]. The nucleotide frequency of COI gene of se-
quenced and reference A. testudineus were 22.84% for A, 31.03% for
T, 28.57% for C, and 17.56% for G which was similar to Ref. [23]
that described nucleotide frequencies of A, 22.5%; T, 31.2.0%; C,
22.3% and G, 24.0%. The base composition bias was estimated
through the disparity index between the studied COI sequences of A.
testudineus. Larger differences were observed in base composition bi-
ases than the expected bases among the sequences. The transition/
transversion bias (R) was 8.44 in the COI sequences of the present
study, transition/transversion bias in perciforms was 1.46 [24–26].
The rate of transitional substitution was higher (89%) than the rate of
transversional substitution (11%). The substitution pattern of se-
quences was tested according to Ref. [20] to test whether the COI
gene sequences of A. testudineus evolved in the same pattern or not.
We found that the sequences of the COI gene in A. testudineus were
evolved with the same pattern of substitution with p-value <0.00
(Table 3).
The K2P pair-wise genetic distance between sequenced vs refer-
ence Thai A. testudineus, and sequenced vs reference Vietnamese A.
testudineus was 0.00%. The inter-specific genetic distance of S. richard-
sonii was 0.00% (ranged from 0.00040 to 0.00080%) and S. progastus
was 0.00% (ranged from 0.00036 to 0.000206) [9]. The genetic dis-
tance between sequenced Bangladeshi vs sequenced Vietnamese A.
testudineus was 0.094 (9.40%), and sequenced Bangladeshi vs se-
quenced Thai A. testudineus was 0.087 (8.7%), and sequenced
Bangladeshi vs reference Indian A. testudineus was 0.02 (2%). The in-
terspecies genetic distance ranged from 0.000 to 0.012 in the genus
Rastrelliger using COI, whereas genetic distance ranged from 0.039 to
0.086 for inter-species variation [27]. [28] showed that the mean
Kimura2-parameter (K2P) genetic divergence for intra-species ranged
from 0.000 (0%) to 0.087 (8.7%), and from 0.000 (0%) to 0.245
(24.5%) for congeneric comparisons. Accordingly, the intraspecific ge-
netic distances of the indigenous and exotic A. testudineus in
Bangladesh is considered high genetic distance, which could become
sub-species or separate species over time if the genetic distance con-
tinues to increase.
In phylogenetic tree analysis (ML), sister group formation was
found between sequenced and reference Thai A. testudineus, se-
quenced and reference Vietnamese A. testudineus and sequenced
I. Parvez et al.
Bangladeshi and reference Indian A. testudineus. The results of sister
group formation are aligned with the pairwise genetic distance values.
Formation of sister group by the closely related individual/species
was found in Plicofolli stenuispinis and P. polystaphylodon; Arius venosus
and A. venosus; Netuma thalasinus and N. thalasinus [29]. To validate
the results of COI gene sequence analyses, we constructed a phyloge-
netic tree based on the 360 bp barcode region of the Cytb gene [17].
used the 360 bp barcode region of Tenualosa ilisha populations of the
Ganga and the Hooghly rivers. The cluster formation using the Cytb
gene was congruent with the cluster formation by using the COI gene.
The results of the phylogenetic tree using COI and Cytb genes were
further tested by time tree construction using the molecular clock hy-
pothesis. The time tree showed the same cluster formation as with
phylogenetic trees of COI and Cytb genes. The time tree estimated the
times of divergence among the sequences, the time tree was congru-
ent with the phylogenetic tree based on COI and Cty b gene. Among
the studied A. testudineus, the highest times of divergence (0.04 MYA)
found between sequenced Bangladeshi A. testudineus and sequenced
Thai A. testudineus. The divergence time was 0.00 MYA between se-
quenced vs reference Thai A. testudineus, sequenced vs reference Viet-
namese A. testudineus, and native sequenced vs reference Indian A. tes-
tudineus. Divergence times among model organisms in fishes were esti-
mated using partitioned Bayesian approaches. The resultant trees
from the two datasets were consistent in this study and also congruent
with previous studies. The approximate divergence times between
spotted green pufferfish and model organisms in fishes were 85 mil-
lion years ago (MYA) vs. torafugu, 183 MYA vs. three-spined stickle-
back, 191 MYA vs. medaka, and 324 MYA vs. zebrafish, all of which
were about twice as old as the divergence times estimated by their
earliest occurrences in fossil records [30]. Ray-finned fish phylogeny
and timing of diversification were estimated using multiple nuclear
gene sequences in conjunction with 36 fossil age constraints [31].
They constructed a well-supported phylogeny of all major ray-finned
fish lineages and estimated the times of divergence that was consis-
tent with the previous fossil records. The molecular divergence times
were estimated in other groups of organisms. To resolve the taxo-
nomic conflicts in Malaysian Bactroceran species, times of divergence
were estimated based on combined data of COI and ND1 marker gene
analyses. The results of the time tree were congruent with the results
of phylogenetic tree which were highly important in species identifi-
cation. Particularly the complex taxonomical status of the sister-
species, B. papayae and B. carambolae has been resolved using the di-
vergence tree where they separated from each other approximately
2.743 MYA, while B. papayae and B. carambolae distinctly diverged
between 1.9619 and 2.2884 MYA [32]. In a separate study, we also
estimated the times of divergence using retrieved Cytb gene sequences
to study the evolution of cyprinid fishes in Bangladesh. We found that
the divergence time was very low among closely related species such
as C. mrigala and L. gonius and, the highest times of divergence (4.89
MYA) observed between D. rerio and outgroup taxa A. testudineus
[33].
5. Conclusion
Nucleotide sequence divergence, phylogenetic tree, and time tree
analysis showed that the native Bangladeshi A. testudineus are dis-
tinctly separate from both Thai and Vietnamese origin. The level of
genetic variation and cluster formation predicted the chance of speci-
ation among them in the future. The purity of native A. testudineus
should be maintained during breeding and aquaculture to protect na-
tive origin from genetic loss. Further studies involving a higher sam-
ple number characterized by multiple molecular markers should vali-
date the possibility of speciation among them.
5
Ecological Genetics and Genomics 17 (2020) 100067
Ethical approval
“All applicable international, national, and/or institutional guide-
lines for the care and use of animals were followed by the authors.”
CRediT authorship contribution statement
Imran Parvez: Conceptualization, Funding acquisition, Data cura-
tion, Formal analysis, and analyses, Writing - original draft, Original
draft, . Tanjiba Mahajebin: Methodology, Methodology, Experimen-
tal Work, Data curation, Writing - original draft, Original draft prepa-
ration. Michèle L. Clarke: Writing - review & editing, Review and
editing manuscript, English Improvement. Mousumi Sarker
Chhanda: Data curation, Writing - original draft, Original draft
preparation. Shirin Sultana: Methodology, Writing - original draft,
Experimental Work, Original draft preparation.
Declaration of competing interest
The authors have no conflict of interest. This research was funded
to the first author and corresponding author Imran Parvez as Principal
Investigator from the University Grants Commission of Bangladesh
(UGC), Bangladesh, FY 2017-2018.
Acknowledgment
The authors acknowledge the National Institute of Biotechnology,
National Science & Information and Communication Technology for
supporting us with DNA sequencing. We are also grateful to the Fresh-
water Sub-station at Saidpur, Bangladesh Fisheries Research Institute,
and Sharnalata Agro-Fishery for providing samples of Thai and Viet-
namese Anabas testudineus, respectively.
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