Download DNA Barcoding: Glyptothorax Species TAXONOMY IN ACTION

DNA Barcoding and Molecular Phylogeny
of Glyptothorax Species (Siluriformes,
sisoridae)
Mahender Singh1*, W S Lakra2, Y Ph
Kartavtsev3, A K Singh1, S N Bahuguna4
1National
Bureau of Fish Genetic Resources, Lucknow, India;
Central Institute of Fisheries Education, Mumbai, India
3A.V. Zhirmunsky Institute of Marine Biology, Vladivostok,
Russia
4Hemwati Nandan Bahuguna Garhwal University, Srinagar,
Uttrakhand, India;
ISO: 9001: 2008
http://www.nbfgr.res.in/ 2
DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
Glyptothorax
G. garhwali
http://www.nbfgr.res.in/
Siluriformes, sisoridae

Attached to rocks, boulders, stones at
the bottom of the water bodies where
they live by means of a thoracic
adhesive apparatus : a sort of
sucking disk as an adaptive structure.
G. dakpathari
G. brevipinnis
G. ngapang
 several new species
have been reported from India in
the past few years (Vishwanath &
Linthiongambi 2006, 2007).
 The debate over the criteria to
recognize the species and
subspecies boundaries has
received considerable attention in
recent years
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
Difficult to identify !
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 (Siluriformes, sisoridae) : with
89 species world over and
29 species from India alone is
highly difficult
at species level
to resolve
 Identification in Glyptothorax
has also been a matter
debate
of
 Identification generally based on
dichotomous keys: ratios
© Author of image | affiliation
of various lengths
Adhesive Apparatus of seven investigated Glyptothorax species. A- G. garhwali (G3), B- G. garhwali (GG), C- G. garhwali (G4), D- G. dakpathri,
E- G. brevipinnis, F- G. ngapang, G- G. granulus, H- G. ventrolineatus, I- G. davissinghi
Impact of geo-climatic factors: affect of boulders and water current on dimensions of
adhesive apparatus, size of cavity, which are the key identification characters ?
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
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recognition based on small
DNA barcoding: for taxon  Taxon
sequence fragment (655 base
recognition and classification
pairs) near the 5’end of
mitochondrial gene cytochrome
c oxidase I (COI) with universal
primers is proved to be effective
(Hebert et al., 2003).
 This barcode is also suitable
marker for discriminating between
closely related species of marine
and freshwater fishes
© Arthur D. Chapman | Australian Biodiversity Information Services
DNA barcoding is also being used for detection of species substitution in
processed food products, illegal products from regulated species control of
invasive species, pest management, water quality control etc.
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
Present Study: Resolving ambiguities
in Glyptothorax Species
Voucher specimens of Glyptothorax
http://www.nbfgr.res.in/
(i) Use of well recognized DNA
barcoding methodology vis-àvis morphological characterbased criteria to fix the
molecular signature for species
ii) To determine if COI based
barcoding is suitable for
species identification as well
as molecular phylogeny in
Glyptothorax catfishes with
overlapping range of
morphometric characters
and
Antonio
G. Valdecasas
|MNCN,
CSIC | Spain
iii)© To
prove
whether
Glyptothorax
genus of catfish is monophyletic based on
currently analyzed COI dataset as well as concatamer with earlier dataset of
Cyt b.
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
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Glyptothorax
Sampling Sites [
India
] in
 Khanda Gad Near Srinagar in
Garhwal, Uttarakhand
 Maldevta, Dehradun,
Uttarakhand
 Yamuna Barrage, Dakpathar,
Dehradun, Uttarakhand
 Tatapani, Shimla, Himachal
Pradesh
 Downstream of Poringalkuthu
Dam, Trishur, Kerala
 Serou,
Manipur
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
Species Name
http://www.nbfgr.res.in/
Lat/ Long/ Alt
Accession Numbers
(1) G. garhwali
Sample
Size
30.13/ 078.47/ 1780 31
(2) G. dakpathri
31.14/ 077.05/ 2087 13
EU637835-EU637839,
EU637785,EU637786,
EU637788,
EU637791-802, EU637804-812
EU637830-34, EU637777-84
0.9103
(3) G. brevipinnis
30.20/ 078.07/ 2382 23
-“-
30.30/ 077.47/ 1476 7
-“-
30.12/ 078.44/ 2083 6
(4) G. ngapang
24.16/ 093.52/ 2521 15
(5) G. granulus
24.16/ 093.52/ 2521 14
FJ347806, FJ347807, FJ347815-17, 0.7917
FJ347820,
FJ347827,FJ347830,
FJ347831,
FJ347834,
FJ347835,
FJ347837-41
FJ347842-47, FJ347857-65
0.3956
(6) G. ventrolineatus
24.16/ 093.52/ 2521 9
FJ347797-805
0.8056
(7) G. davissinghi
Total
10.16/076.26/221
FJ347789-96
0.7500
8
126
Haplotype
Diversity
0.2968
FJ423570-75, FJ208927-33,EU637840- 0.5261
44, EU637813-29,
Samples were collected from different regions across India. Muscle and fin
tissues —were
preserved in 95 % v/v ethanol.
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
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 Approximately 50 mg of caudal or anal fin or muscular tissue was used for
DNA isolation following standard phenol/chloroform method (Sambrook et
al., 1989) with partial modifications.
 The partial COI gene was amplified using the universal primers Fish F1
5’TCAACCAACCACAAAGACATTGGCAC3
& FishR1-5’TAGACTTCTGGGTGGCCAAA GAATCA3 (Ward et al. 2005).
 DNA sequencing was performed following the dideoxynucleotide chain
termination method (Sanger et al., 1977), using an automated ABI 3730
sequencer
 The COI sequences were aligned and primer sequences were trimmed, to
get uniform length sequences of 655 base.
 Sequences were aligned using CLUSTALW (Thompson et al. 1994), refereed
against electropherogram and submitted to GenBank.
The phylogenetic trees were visualized and edited where necessary with FigTree
(http://tree.bio.ed.ac.uk/software/figtree/) and Dendroscope (http://ab.inf.uni— the leader in establishing and operating partnerships
for taxonomy
developing2010)
countries software
| www.bionet-intl.org | [email protected]
tuebingen.de/software/dendroscope/)
(Huson
et al.,in2007;
TAXONOMY IN ACTION
Sr. No.
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Species
Voucher Code
G. burmanicus
KIZ:0329
Accession
COI
HQ593567
G. deqinensis
KIZ:200515001
HQ593564
G. f. fokiensis
Both COI & Cyt b sequences taken from same
NC018769
mitochondrial genome
G. hainanensis
KIZ:WWY20080124
HQ593584
HQ593597
G. honghensis
KIZ:2007004027
HQ593583
HQ593596
G. interspinalus
KIZ:200101023
HQ593563
HQ593589
G. longicauda
KIZ:05488
HQ593578
HQ593593
G. longinema
KIZ:05219
HQ593573
HQ322509
G. macromaculatus
KIZ:20040929016
HQ593568
HQ593592
G. minimaculatus
KIZ 05698
HQ593579
HQ322547
G. obliquimaculatus
KIZ:20050415021
HQ593576
HQ322553
G. quadriocellatus
KIZ:200101022
HQ593562
HQ593588
G. trilineatus
KIZ:20050417014
HQ593572
HQ322512
G. zanaensis
KIZ1818
HQ593574
HQ322507
Silurus lanzhouensis
Both COI & Cyt b sequences taken from same
NC015650
mitochondrial genome
Silurus asotus
Both COI & Cyt b sequences taken from same
AP012022
mitochondrial genome
Bagarius yarrelli
INHS:93673
EU490904
EU490855
No.
of Accession No. of Cyt b
HQ593591
HQ593590
For phylogenetic tree making, COI and Cyt b sequences of 14 Glyptothorax species submitted in NCBI by other
researchers —
were
alsoin downloaded
to present
a comprehensive
overview
elucidate phylogenetic
relationship
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establishing and operating
partnerships
for taxonomy in developing
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| [email protected]
DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
Result highlights:
http://www.nbfgr.res.in/
Analysis of COI :
Out of 655 positions,




G. garhwali
514 were conserved (82.6%)
114 (17.4%) were variable
10 were singleton (1.5%)
104 (15.9%) were
parsimoniously informative (at
least two of nucleotides
occurring with a minimum
frequency of two).
As the adhesive apparatus is key character, it was photographed in mature
specimens of all seven species. By adhesive apparatus and morphological data,
two populations of G. garhwali considered as different species
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
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Divergence within each group
Number of base substitutions per site (K2P model in MEGA) were calculated and it is
found to be highest in G. dakpathri and G. davissinghi (K2P = 0.0040 in both)
whereas the least divergence is shown within G. garhwali (K2P = 0.0005)
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DNA Barcoding: Glyptothorax
TAXONOMY IN ACTION
Species
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Divergence between
and within species
G. garhwali
G. dakpathri
G. brevipinnis
G. ngapang
G. granulus
G.
ventrolineatus
G. davissinghi
G.
garhwali
G.
dakpathri
G.
brevipin
nis
G.
ngapan
g
0.0005
0.0851
0.0818
0.0991
0.0930
0.0806
0.0761
G.
granulus
G.
G.
ventroline davissin
atus
ghi
0.0040
0.0345
0.0735
0.0774
0.0505
0.0014
0.0677
0.0723
0.0402
0.0024
0.0628
0.0712
0.0009
0.0689
0.0022
0.0409
0.0303
0.0495
0.0611
0.0497
0.0040
Estimates of evolutionary divergence in K2P over sequence pairs
within and between 7 species of genus Glyptothorax
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DNA Barcoding: Glyptothorax
Species
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Average genetic
divergence: over sequence pairs
between species
 The calculation of
average genetic
divergence over
sequence pairs between
groups was found to be
highest between
G. ngapang and
G. garhwali (K2P =
0.0991)
 while the least divergence
is shown between
G. davissinghi and
G. brevippinisi
(K2P = 0.0303) species
0,69
1
1
0,54
COI gene
phylogram
G_f_fokiensis
G_hainanensis
G_honghensis
G_deqinensis
G_quadriocellatus
G_garhwali_G3_2
G_garhwali_G3_3
G_garhwali_G4_3
0,99
G_garhwali_G4_4
G_garhwali_GG1
0,9
G_garhwali_GG2
G_burmanicus
0,8
1
G_macromaculatus
The phylogenetic
and evolutionary
history :
 G. dakpathari,
 59 sequences for
G. davissinghi,
our and GenBank
G. brevipinnis and
representatives of
G. ventrolineatus
Glyptothorax
G. Garhwali
catfish jointly with 3 G. Ngapang
G. Granulus
outgroup
with 14 GenBank
specimens
species-sequences
with well supported
nodes, as proved by
two genes data
 Slight difference in
topology may be due
Bayesian (BA) algorithm
to mutation rate
G_obliquimaculatus
G_granulus_GR8413A
G_granulus_GR8413B
G_granulus_GR8413C
1
G_granulus_GR8413D
G_granulus_GR8413E
G_granulus_GR8413F
G_ngapang_GN8205A
G_ngapang_GN8412A
0,75
G_ngapang_GN8412I
1
G_ngapang_GN8412J
1 0,89
G_ngapang_GN8412K
G_ngapang_GN8412N
G_minimaculatus
G_longinema
G_zanaensis
G_davissinghi_GD8318A
0,81
G_davissinghi_GD8318C
G_davissinghi_GD8318F
0,97 0,66
G_davissinghi_GD8318B
0,95
G_davissinghi_GD8318E
0,72
G_davissinghi_GD8318D
G_dakpathari_GP1
0,64
G_dakpathari_GP2
G_dakpathari_GP4
1 G_dakpathari_GP3
0,99
G_dakpathari_GP5
G_dakpathari_GP6
G_ventrolineatus_GV8417A
G_ventrolineatus_GV8417B
G_ventrolineatus_GV8417C
10,99
G_ventrolineatus_GV8417F
G_ventrolineatus_GV8417D
1
G_ventrolineatus_GV8417E
0,88
G_interspinalus
0,65
G_trilineatus
G_longicauda
G_brevipinnis_GT1
G_brevipinnis_GT2
1G_brevipinnis_8204GK1
G_brevipinnis_8204GK2
G_brevipinnis_GB1
0,93
G_brevipinnis_GB2
variation among
genes and lineages
1
1
Bagarius_yarrelli
0.2
Silurus_lanzhouensis
Silurus_asotus
Combined
phylogram of COI Results
and Cyt-b genes revealed
 59 sequences for our that COI, as well
and GenBank
as Cyt-b
representatives of
Glyptothorax
catfishes jointly with 3
outgroup specimens
 In the nodes the
support levels (%) are:
BA, ML, MP, and NJ.
(Singh et al., 2012
and this study), are
the effective
markers for
identifications of
seven species of
Glyptothorax
from India
 Abbreviation 100 x 4
denotes:
100,100,100,100 and
100 x 3 denotes:
100,100,100
Bayesian (BA) algorithm
The lowest stable
ancestor (LSA)
consensus tree :
A
B
COI and Cyt-b
 At A the consensus tree
made by COI gene trees is
represented
 At B the consensus tree
built by BA algorithm from
Cyt-b
 Judging on both trees
we may conclude that some
intragenus lineages as was firstly
noted on main COI data and
combined two genes data are
Tanglegram built for COI vs. Cyt-b genes as previously for 59
retained with this more
sequences of our and GenBank representatives jointly with 3
sophisticated approach
outgroup specimens in two approaches depicted at A and B
DNA Barcoding: Glyptothorax
Species
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Findings:
 These seven species of Sisoridae from the Indian rivers were found
genetically distinct from each other.
 The adhesive apparatus, considered as the key character for species
identification, is not supported by barcode data as G. garhwali has two types
of adhesive apparatus
 Pairwise comparisons, sequence alignment, intra-specific and inter-specific
sequence divergence the pattern of nucleotide substitution found in COI gene
of Glyptothorax species, is quite expected for a functionally conserved protein
coding region, which has evolved over recent evolutionary time.
 Glyptothorax exhibited more nucleotide change at third codon position, with
most mutations being synonymous. We also observed anti G bias in this
position in the seven species, and the G is mostly substituted with A (both
purines with double ring nitrogenous base).
DNA Barcoding: Glyptothorax
Species
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Findings:
 Estimate of genetic divergence with COI gene was sufficient enough to
discriminate individuals of different Glyptothorx species within Sisoridae.
 In all seven species, levels of intra-specific variation (K2P sequence
divergence) were varied from G. garhwali (0.05%), G. dakpathri (0.40%), G.
ngapang (0.24%), G. davissinghi (0.40% ), G. brevipinnis (0.14%), G. granulus
(0.09%), G. ventrolineatus (0.22%).
 High inter-specific K2P sequence divergence is estimated between
G. brevipinnis & G. garhwali (0.129) despite small geographical distance
between them and low inter-specific K2P sequence divergence is estimated
between G. davissinghi and G. dakpathri (0.002) despite a distance of over
2000 km.
DNA Barcoding: Glyptothorax
Species
Authors
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are thankful to Prof. W. Vishwanath for helping in
identification of Glyptothorax species from North East India, National
Bureau of Fish Genetic Resources of India for supporting this work.
THANK YOU!