Genetic diversity and phylogenetic analysis of lepidopteran species by molecular barcoding using CO I gene sequences

Abstract

Lepidoptera is one of the largest orders of insects that include moths and butterflies. Most of the Lepidopterans are morphologically similar, therefore the identification of these insects is tedious using morphotaxonomy and that is detrimental in designing a better strategy to control and conserve them. These are extremely important group of 'model' organisms and are used to investigate many areas of biological research, including such diverse fields as navigation, pest control, embryology, mimicry, evolution, genetics, population dynamics and biodiversity conservation. The knowledge of Lepidopteran genomics will create new methods of insect pest management and will contribute to sustainable agriculture, protection of the environment and the maintenance of biodiversity. In this study we amplified cytochrome oxidase I gene of Junonia atlites for species identification and constructed phylogenetic tree for recognizing evolutionary relationship.

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International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Impact Factor (2012): 3.358
Volume 3 Issue 5, May 2014
www.ijsr.net
Genetic Diversity and Phylogenetic Analysis of
Lepidopteran Species by Molecular Barcoding
Using Co I Gene Sequences
Pavana E.1, Sebastian C. D2
1Anna Univerity, Rajalakshmi Engineering College, Chennai-600 025, India
2Molecular Biology Laboratory, Department of Zoology, Calicut University, Kerala-673 635, India
Abstract: Lepidoptera is one of the largest orders of insects that include moths and butterflies. Most of the Lepidopterans are
morphologically similar, therefore the identification of these insects is tedious using morphotaxonomy and that is detrimental in
designing a better strategy to control and conserve them. These are extremely important group of ‘model’ organisms and are used to
investigate many areas of biological research, including such diverse fields as navigation, pest control, embryology, mimicry, evolution,
genetics, population dynamics and biodiversity conservation. The knowledge of Lepidopteran genomics will create new methods of insect
pest management and will contribute to sustainable agriculture, protection of the environment and the maintenance of biodiversity. In
this study we amplified cytochrome oxidase I gene of Junonia atlites for species identification and constructed phylogenetic tree for
recognizing evolutionary relationship.
Keywords: Molecular systematics, Lepidoptera, DNA barcoding, CO I gene sequences, Mitochondrial DNA, Junonia atlites.
1. Introduction
Identification and conservation of genetic diversity is
important for Lepidoptera since it is one of the widespread
and widely recognizable insect orders in the world. The
strength of Lepidopteran genomics lies in the diversity of
the group as a whole. Although there are clear insect
models for genetic analysis (Drosophila) and disease
vectors (Anopheles), the Lepidoptera are rich in diverse
model systems for a variety of biological processes. Much
of our knowledge of endocrinology, reproduction,
behavior and immunity is derived from the studies in
Lepidoptera. They are important test case for the use of
mitochondrial DNA in species identification. This is the
important order among class insecta that have appeared on
endangered species list.
The genome of lepidoptera is characterized by larger size
and higher chromosome number, typically about 30.
Genetic crosses are routinely accomplished in Lepidoptera.
The GC content of Lepidopteran DNA is about 35-40%.
The large body size, accessible genetics, and extreme
diversity of Lepidopteran species are important
experimental advantages. Identification at the molecular
level is important since phenotypic variability and
convergent evolution causes misidentification of many
cryptic species, in addition sexual dimorphism causes
more confusion to species identification. To solve these
problems in the taxonomy, recently a short nucleotide
sequence of mitochondrial DNA (CO I) is widely accepted
as a marker for the accurate and easy identification of
species. DNA sequences of the mitochondrial cytochrome
oxidase I (CO I) gene can serve as a DNA barcode for
identifying all kinds of animals [1]. It is an important
advancement in molecular biology for rapidly and cost-
efficiently catalog fauna using a short reference sequence
of DNA. The present work reveals the partial
mitochondrial CO I gene sequences of Junonia atlites
(Figure 1) their genetic divergence and phylogenetic status
and also the effectiveness of DNA barcoding in
identification of cryptic species.
2. Materials and Methods
2.1 Collection and Preservation
The samples were collected from Calicut University
Botanical Garden (CUBG), Kerala using hand sweeping
net. The sample was identified as Junonia atlites and
placed in separate glassine envelope with 70% ethanol,
assigned code number and stored at -20°C as voucher
specimen until further use.
Figure 1: a) Junonia atlites
2.2 DNA Extraction, Amplification and Sequencing
DNA was extracted from the leg piece of the specimen
using phenol chloroform method [7]. The obtained DNA
was amplified for CO I gene using forward primer (5'-
GGTCAACAAATCATAAAGATATTGG-3') and reverse
primer (5'-TAAACTTCAGGGTGACCAAAAAATCA-
3'). PCR reaction was carried out in total volume of 50 µl
containing 2 ng of genomic DNA (1 µl), 1 µl of each
forward and reverse primer at a concentration of 10 µM, 1
µl of dNTPs (2 Mm), 5 µl of MgCl2, 5 µl of 10X reaction
Paper ID: 020131820
450

International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Impact Factor (2012): 3.358
Volume 3 Issue 5, May 2014
www.ijsr.net
buffer, 0.5 µl Taq polymerase (5U/µl) and 36.5 µl of
water. The PCR profile consisted of an initial denaturation
step of 5 min at 95°C followed by 30 cycles of 10 sec at
95°C, 10 sec at 50°C and 45 sec at 72°C and ending with a
final phase of 72°C for 30 min. The obtained PCR product
was checked on 2% agarose gel and was column purified
using Gene JETTM PCR Purification kit (Fermentas Life
science). The purified PCR product was sequenced at Sci
Genome Laboratories Ltd., Cochin, Kerala. The obtained
sequence was checked for its quality by examining
chromatograms and the forward and reverse sequence
were assembled using Clustal W. Sequence analysis and
sample identification were done by the trimmed sequence
in NCBI’s BLAST tool. Phylogenetic tree was then
constructed using MEGA 6 software [8].
3. Result and Discussion
DNA Sequences of good quality and length of 543 bp
were generated in the present study. The forward and
reverse sequences obtained were trimmed for primer
sequence and assembled using Clustal W (Figure 2). The
nucleotide BLAST against the nucleotide redundant
database revealed that the CO I gene sequence obtained is
showing 100% similarity to Junonia atlites. The CO I
hyper variable region of Junonia atlites DNA is 100%
similar to that of Junonia atlites CO I gene reported from
Western Ghats (GenBank accession number
GU0126131.1). Here geographical barrier may act as
evolutionary tool for the sequence divergence.
The evolutionary history of Junonia atlites is inferred
using Neighobour joining method of MEGA 6 software
which shows clearly the inter and intra species divergence
(Figure 3). NJ clustering analysis showed that Junonia
atlites from Calicut, Kerala and Western Ghats belong to
same clade without any overlap while Junonia atlites from
Africa (GenBank accession number EU053290.1) belongs
to another clade with 100% similarity. The above
phylogenetic tree shows that the Junonia atlites from
Calicut, Kerala, Western Ghats and Africa have same
ancestors. DNA barcoding provide rapid and automatable
species identification by short standardized DNA fragment
as species tag and its makes the Linnaean classification
system more accessible [5].
Figure 2: The DNA sequence of the mitochondrial cytochrome oxidase I (CO I) gene of Junonia atlites
EU053290.1 Junonia ansorgei
AY788644.1 Junonia erigone
EU053318.1 Junonia terea
EU053322.1 Junonia villida
EU053291.1Junonia atlites
GU012613.1 Junonia atlit es
Calicut Kerala Junonia atlites KJ636444
KC158400.1 Junonia orithya
HQ962223.1 Junonia iphita
KF398346.1 Junonia hedonia
0.5
Figure 3: Phylogenetic Status of Junonia Atlites Using Neighbor Joining Method
There is no intra species nucleotide divergence between
Junonia atlites since the nucleotide BLAST against the
nucleotide redundant database is showing 100% similarity
to Junonia atlites. The intra species divergence is found to
be between 2-9%. The average nucleotide composition
proportions for these twelve sequences were G, 15.3%; A,
Paper ID: 020131820
451

International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Impact Factor (2012): 3.358
Volume 3 Issue 5, May 2014
www.ijsr.net
31.7%; T, 37.9%; and C, 15.1%. The present results
indicate that an identification system for insect life based
on the COI gene will be highly effective.
Transition/Transversion bias (R) calculated for selected
species of Junonia atlites is 0.80. Maximum transitional
substitutions are C to T and transversional substitutions are
A to T. Variation in the nucleotide sequence is a
fundamental property of all living organisms which can be
used for their identification and phylogenetic status.
The conceptual peptide translation yielded 180
aminoacids. The peptide blast of nucleotide sequence
showed 100% similarity to Junonia atlites which again
confirmes the sample as Junonia atlites. The molecular
barcode generated for Junonia atlites in the present study
can be used for its accurate taxonomic identification.
Sequences generated in this study were submitted to
GenBank with accession number KJ636444 (Junonia
atlites) which can be used as molecular barcode of this
species and can be used for its accurate taxonomic
identification.
The present study on molecular evolutionary analysis
using partial mitochondrial cytochrome oxidase subunit I
(CO I) gene sequence explicates phylogenetic
relationships of Junonia atlites. The CO I gene is generally
effective as a barcode sequence, delivering more than 95%
species level resolution [1]. Thus present study reveals the
importance of DNA barcoding in the identification of
species. The molecular database will act as molecular field
guide facilitating the identification of cryptic species [4].
4. Conclusion
The study concludes the efficacy of using COI gene for
identification of species at molecular level and applying
this molecular taxonomy for identification of cryptic
biodiversity. The study also concludes that DNA
barcoding is most effective tool for identification of
species at molecular level than the morphotaxonomy that
are ordinarily employed for identification.
Morphotaxonomy often generates errors due to
intraspecific variability between the species. The study
developed a comprehensive DNA barcode database for
Lepidoptera Junonia atlites (GenBank accession number
KJ636444).
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