DNA based phylogenetic analysis of aquatic beetle, Dytiscus marginalis isolated from North Kerala, using mitochondrial COI marker

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DNA BASED PHYLOGENETIC ANALYSIS OF AQUATIC BEETLE DYTISCUS MARGINALIS ISOLATED
FROM NORTH KERALA, USING MITOCHONDRIAL COI MARKER
Akhilesh, V. P. and *Sebastian, C. D.
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala 673 635 India
ARTICLE INFO
ABSTRACT
Dytiscus marginalis
elytra. It is the largest order including more species known to science than any other order not only in
the class insect, but also in the entire animal kingdom. The order is huge
of groups. In the present study we have isolated, PCR amplified and deciphered the partial nucleotide
sequence of the mitochondrial cytochrome oxidase subunit I gene (COI) of
and its phylogenetic statu
(GenBankAcession:
813688) and Cybister cognatus (DQ 813672)
of the COI sequences among the morphologically distinct and geographically isolated
Copyright © 2015 Akhilesh, V. P. and Sebastian, C. D.
This
unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
INTRODUCTION
Dytiscus marginalis
, the diving beetles are insects belonging to
the Order Coleoptera which means ‘sheathed wings’ as the
wings are covered by hard wing elytra. The order is huge and
includes a wide variety of groups, some of which have a clo
association with water, living on or in it for most of their adult
life. It is frequently encountered in wet tropical and subtropical
forests (Michael et al
., 2004). Water beetles prefer shallower
areas of water such as streams, ditches, river bottoms a
margins, lake margins, ponds, pools, marshes and puddles.
marginalis
has a beautifully streamlined body shape and is
dark brown to blackish in colour with yellow legs and a
yellow border around both the head and the
are ridged in females but smooth in males. Males can also be
distinguished from females by the presence of suction pads on
the front legs; two of wh
ich are very large.
air supply closed in under the elytra gives the tip a silver seam.
Like most water insects, the diving beetle needs to come up for
a new supply of fresh air. This air is taken in by bringing the tip
of the abdomen to t
he water surface and then lowering it.
routine identification of known species can be difficult, often
requiring highly specialized knowledge and representing a
limiting factor in ecological studies and biodiversity
inventories.
*Corresponding author: Sebastian, C. D.,
Molecular Biology Laboratory, Department of Zoology, University of
Calicut, Kerala 673 635 India.
ISSN: 0975-833X
Article History:
Received 17th February, 2015
Received in revised form
23rd March, 2015
Accepted 17h April, 2015
Published online 31st May, 2015
Key words:
Molecular Systematics, Dytiscus
Marginalis, Mitochondrial DNA,
COI gene sequence.
Citation
:
Akhilesh, V. P. and Sebastian, C. D,
2015.
North Kerala, using mitochondrial coi marker”, Internation
RESEARCH ARTICLE
DNA BASED PHYLOGENETIC ANALYSIS OF AQUATIC BEETLE DYTISCUS MARGINALIS ISOLATED
FROM NORTH KERALA, USING MITOCHONDRIAL COI MARKER
Akhilesh, V. P. and *Sebastian, C. D.
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala 673 635 India
ABSTRACT
Dytiscus marginalis
are the diving beetles with their wings covered by hard wing cases known as
elytra. It is the largest order including more species known to science than any other order not only in
the class insect, but also in the entire animal kingdom. The order is huge
of groups. In the present study we have isolated, PCR amplified and deciphered the partial nucleotide
sequence of the mitochondrial cytochrome oxidase subunit I gene (COI) of
and its phylogenetic status. DNA sequence similarity searches of COI gene of
(GenBankAcession:
KM 230115
) revealed that it is genetically identical to
813688) and Cybister cognatus (DQ 813672)
isolated from USA. The results indicate slow evolution
of the COI sequences among the morphologically distinct and geographically isolated
This
is an open access article distributed
under the Creative Commons Att
use, distribution, and reproduction in any medium, provided the original work is properly cited.
, the diving beetles are insects belonging to
the Order Coleoptera which means ‘sheathed wings’ as the
wings are covered by hard wing elytra. The order is huge and
includes a wide variety of groups, some of which have a clo
se
association with water, living on or in it for most of their adult
life. It is frequently encountered in wet tropical and subtropical
., 2004). Water beetles prefer shallower
areas of water such as streams, ditches, river bottoms a
nd
margins, lake margins, ponds, pools, marshes and puddles.
D.
has a beautifully streamlined body shape and is
dark brown to blackish in colour with yellow legs and a
yellow border around both the head and the
thorax. The elytra
are ridged in females but smooth in males. Males can also be
distinguished from females by the presence of suction pads on
ich are very large.
The border of the
air supply closed in under the elytra gives the tip a silver seam.
Like most water insects, the diving beetle needs to come up for
a new supply of fresh air. This air is taken in by bringing the tip
he water surface and then lowering it.
The
routine identification of known species can be difficult, often
requiring highly specialized knowledge and representing a
limiting factor in ecological studies and biodiversity
Molecular Biology Laboratory, Department of Zoology, University of
In response, recent proposals have called for a more prominent
role of efficient DNA based methods in the delineation and
identification of species (Blaxter, 2004; Floyed
Hebert et al
., 2003). The objective of any method of species
delineation, including DNA-
based approaches, is to identify
reproductively isolated groups of organisms that warrant
classification as distinct species (Michael
Mitochondrial DNA is one of the most widely used and
informative molecular markers due to t
maternal inheritance (Avise, 2004). Mitochondrial genes have
been used extensively in population genetic and
phylogeographical analyses; in part due to a high rate of
nucleotide substitution in animal mt DNA. DNA barcoding is
the well
adopted method for accurate taxonomic identification
of animals. The main purpose attributed for DNA barcoding
are the assignment of specimens to known species and
discovery of new species. Identification by DNA barcoding is
based on matching an unknown s
to one or more sequences from specimens that have been
positively identified by other sequences.
analysis using mitochondrial COI gene sequences were
extensively conducted in various insect groups ranging
Odonata (Jisha et al
., 2015), Hymenoptera (Rukhsana
2014) Lepidoptera (Akhilesh
(Sreejith et al
., 2014) and Diptera (Bindu
et al., 2014).
The present study reveals the partial DNA
sequence of t
he mitochondrial cytochrome oxidase subunit I
(COI) gene of the
D. marginalis
molecular phylogenetic status with related members.
Available online at http://www.journalcra.com
International Journal of Current Research
Vol. 7, Issue, 05, pp.16426-16429, May, 2015
INTERNATIONAL
2015.
“DNA based phylogenetic analysis of aquatic beetle
dytiscus marginalis
International Journal of Current Research, 7, (5), 16426-16429
z
DNA BASED PHYLOGENETIC ANALYSIS OF AQUATIC BEETLE DYTISCUS MARGINALIS ISOLATED
FROM NORTH KERALA, USING MITOCHONDRIAL COI MARKER
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala 673 635 India
are the diving beetles with their wings covered by hard wing cases known as
elytra. It is the largest order including more species known to science than any other order not only in
the class insect, but also in the entire animal kingdom. The order is huge
and includes a wide variety
of groups. In the present study we have isolated, PCR amplified and deciphered the partial nucleotide
sequence of the mitochondrial cytochrome oxidase subunit I gene (COI) of
D. marginalis from Kerala
s. DNA sequence similarity searches of COI gene of
D. marginalis
) revealed that it is genetically identical to
Cybister ventralis (DQ
isolated from USA. The results indicate slow evolution
of the COI sequences among the morphologically distinct and geographically isolated
D. marginalis.
under the Creative Commons Att
ribution License, which permits
In response, recent proposals have called for a more prominent
role of efficient DNA based methods in the delineation and
identification of species (Blaxter, 2004; Floyed
et al., 2002;
., 2003). The objective of any method of species
based approaches, is to identify
reproductively isolated groups of organisms that warrant
classification as distinct species (Michael
et al. 2005)
Mitochondrial DNA is one of the most widely used and
informative molecular markers due to t
heir precise size and
maternal inheritance (Avise, 2004). Mitochondrial genes have
been used extensively in population genetic and
phylogeographical analyses; in part due to a high rate of
nucleotide substitution in animal mt DNA. DNA barcoding is
adopted method for accurate taxonomic identification
of animals. The main purpose attributed for DNA barcoding
are the assignment of specimens to known species and
discovery of new species. Identification by DNA barcoding is
based on matching an unknown s
pecimen’s barcode sequence
to one or more sequences from specimens that have been
positively identified by other sequences.
Molecular phylogeny
analysis using mitochondrial COI gene sequences were
extensively conducted in various insect groups ranging
., 2015), Hymenoptera (Rukhsana
et al.,
2014) Lepidoptera (Akhilesh
et al., 2014), Heteroptera
., 2014) and Diptera (Bindu
et al., 2014; Priya
The present study reveals the partial DNA
he mitochondrial cytochrome oxidase subunit I
D. marginalis
isolated from Kerala and its
molecular phylogenetic status with related members.
INTERNATIONAL
JOURNAL
OF CURRENT RESEARCH
dytiscus marginalis
isolated from
16429.

MATERIALS AND METHODS
Genomic DNA from D. marginalis was isolated using genomic
DNA extraction kit of NucleoSpin XS (Takara). About 2 ng of
genomic DNA was amplified for mitochondrial COI gene
using the forward primer (5'-
GGTCAACAAATCATAAGATATTGG-3') and reverse
primer (5'-TAAACTTCAGGGTGACCAAAAAATCA-3').
The PCR profile consisted of an initial denaturation step of 5
minutes at 950C followed by 30 cycles in 5 sec at 950C, 30 sec
at 500C and 45 sec at 720C and ending with a final phase 720C
for 3 minutes.
The PCR products were resolved on a 2% TAE agarose gel,
stained with Ethidium bromide and photographed using a gel
documentation system. After ascertaining the amplification of
the corresponding COI fragment, the PCR product was column
purified using Mo Bio Ultra PCR Clean-up Kit as per the
manufacturer’s instructions. The purified PCR product is
sequenced from both ends using forward and reverse primers
used for the PCR using the Sanger’s sequencing method
(Sanger, 1975). The forward and reverse sequences obtained
were trimmed off the primer sequences and assembled by using
Clustal W and was searched for its similarity using BLAST
Programme of NCBI. A phylogenetic tree of D. marginalis was
constructed using MEGA6 software (Tamura et al., 2013).
Figure 1. The chromatogram showing PCR amplified COI gene sequences of D. marginalis (Kerala), GenBank Accession: KM 230115
Figure 3. The phylogenetic tree plotted for D. marginalis was inferred using cytochrome oxidase subunit I (COI) gene
partial sequence by Neighbor joining method.
16427 Akhilesh and Sebastian, DNA based Phylogenetic analysis of aquatic beetle Dytiscus marginalis isolated from north Kerala,
using mitochondrial COI marker

RESULTS AND DISCUSSION
The PCR amplified sequences of mitochondrial cytochrome
oxidase subunit I gene fragment of D. marginalis yielded a
single product of 626 bp. The sequence has been deposited in
the NCBI GenBank with Acession No. KM 230115 (Figure 1).
The phylogenetic tree plotted using neighbor joining method in
rectangle format presented in Figure 2.
Genetic diversity is central to the breeding success of most
populations. Reduced genetic variation can greatly impair a
population growth and can jeopardize the recovery of
endangered species. The DNA sequences in organisms are
maintained from generation to generation with very little
change. Although such genetic stability is crucial for the
survival of individuals, the survival of organisms may depend
on genetic variation through which they can adapt to a
changing environment. DNA sequence based identification
technique has revealed the morphological and ecological traits
of many species during larval stages (Rukhsana et al., 2014).
Thus an important property of the DNA in cells is its ability to
undergo rearrangements that can vary the particular
combination of genes present in any individual genome as well
as the timing and the level of expression of these genes.
Cytochrome oxidase subunit 1 is the most widely used gene for
molecular barcoding and phylogeny analysis of organisms
especially higher eukaryotes for its high level of sequence
variations compared to the other region of mitochondrial DNA.
The partial DNA sequence of cytochrome oxidase subunit I
gene of D. marginalis collected from Kerala is genetically
more similar to Cybister ventralis (DQ 813688) and Cybister
cognatus (DQ 813672) isolated from USA. D. marginalis
isolated from Finland having 12% and 13% sequence
divergence against D. marginalis isolated from Kerala. D.
marginalis isolated from USA having 13% sequence
divergence against D. marginalis isolated from Kerala. The
present study finds clear genetic breaks and deep divergence
between many con-generic species, which is likely to have
resulted from taxonomic sampling across a comparatively
broad range of evolutionary divergence, and geographic
regions. This study represents important progress towards
understanding the evolution and biodiversity of aquatic beetles
and provides a foundation for similar future work. The D.
marginalis provides an excellent study system as the order is
huge and includes a wide variety of groups, some of which
have a close association with aquatic habitat, living in or on for
most of their lifespan.
Acknowledgement
The financial assistance from University Grants Commission,
New Delhi under Major Research Project is gratefully
acknowledged.
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Table 1. Percentage of evolutionary divergence between D. marginalis (Kerala) and other sequences from GenBank
Sl. No. GenBank Accession No. Species Name % of Divergence
KM 230115 Dytiscus marginalis 0%
DQ 813688 Cybister ventralis 3%
DQ 813672 Cybister cognatus 3%
DQ 813691 Dytiscus sp. 12%
KJ 963783 Dytiscus sp. 13%
JX 535701 Pterostichus ganesh_ganesh 15%
JX 260148 Pterostichus luctuosus 16%
JX 260039 Harpalus caliginosus 16%
AB 095262 Carabus japonicus 16%
KJ 204080 Platynus m 16%
HQ 978622 Carabidae sp. 16%
KC 016848 Gyrinus opacus 17%
KC 016846 Gyrinus opacus 17%
AB 095242 Carabus yamato 17%
HM 909088 Carabus clathratus 17%
KC 016843 Gyrinus opacus 17%
JX 260149 Pterostichus sp. 17%
16428 International Journal of Current Research, Vol. 7, Issue, 05, pp.16426-16429, May,
2015

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16429 Akhilesh and Sebastian, DNA based Phylogenetic analysis of aquatic beetle Dytiscus marginalis isolated from north Kerala,
using mitochondrial COI marker