Download Phylogenetic Relationship Among Some Species of Bruchinae

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Research Article
Phylogenetic Relationship Among Some Species of
Bruchinae Based Upon 16S And 12S Ribosomal RNA
Neha Goyal and Vijay Lakshmi Sharma
Department of Zoology, Panjab University, Chandigarh - 160014
Abstract
Bruchids (pulse beetles) are cosmopolitan and the most destructive pests of stored pulses. The genus Callosobruchus
(Coleoptera:Chrysomelidae:Bruchinae) has its origin in Asia and Africa. In India, C. analis, C. maculatus and C. chinensis are the
predominant pests of this group. They cause maximum damage to the stored grains in the months of February to August when all its
developmental stages co-exist. In this study 12SrRNA and 16SrRNA gene fragments were used to analyze the phylogeneytic relationship of
two of these species i.e. Callosobruchus analis and Callosobruchus maculatus with the other Bruchid species found worldwide. The
sequences showed an A+T bias of >70% in conformation with the previous studies. The phylogenetic trees were drawn on the basis of
maximum likelihood and neighbor joining methods using MEGA 6.06 algorithm. The species of the family Cerambycidae were included in
the analysis to obtain rooted trees. Separate analysis of both the 12S and 16S genes strongly supported the monophyly of the Callosobruchus
genus. Within the major cluster, C.analis formed a common clade with the African C.chinensis species while C.maculatus showed a closer
relationship with African C.subinnotatus species. The clades were well supported by bootstrap values above 50.
Key words: Callosobruchus analis, Callosobruchus maculatus, 12SrRNA, 16SrRNA, phylogeny, MEGA 6.06.
Introduction
Pulses are a high quality source of protein and in the Indian
subcontinent where the population is predominantly vegetarian they
constitute an integral part of the daily meal. India is the largest
producer and consumer of pulses in the world. But the subcontinent's demand for pulses is rising every year as pulse
production struggles to keep pace with the country's population
growth which has doubled since 1961 whilst the pulse production
has increased by just 30% . The problem is further aggravated by
lack of proper storage practices which culminate into huge losses
that arise from insect infestation, rodent infestation or microbial
growth. About 8.5 % of annual pulse production is lost during post
harvest and storage handling (Agrawal et al., 1988). As per the latest
estimates, India achieved high pulses production record of 18.45
million tonnes (MT) in the year 2012 but still it had to import the
pulses to meet the gap between domestic demand and supply (FAO
2012)
As per reports, major damage to the pulse production is inflicted by
the insect pests both in the field before harvest and in storage
(Kusolwa, 2007). Over 200 species of insects infest various pulses
in India (Anonymous, 2007). Among the storage pests, bruchids are
of great importance. Over 1700 species of bruchids grouped under
62 genera are known all over the world (Romero et al., 2004). Of
these 108 species belonging to 11 genera of 3 subfamilies have been
reported from the Indian subcontinent (Thakur, 2012). And out of
these, 17 species are known to infest different pulses (Arora, 1977).
The edible legumes in India are attacked primarily by four species of
pulse beetles i.e. Callosobruchus maculatus (Fabricius), C. analis
(Fabricius), C. chinensis (Linn.) and Zabrotes subfasciatus (Boh.).
C. maculatus, commonly known as cowpea weevil and C. analis as
grahambean weevil, are cosmopolitan in distribution and are serious
pests of legumes mainly Vigna radiata, Vigna unguiculta, Vigna
mungo and Cicer arietinum causing heavy economic losses to the
farmers. These insects spend their entire larval and pupal stages in
individual legume seeds thereby rendering them unsuitable for
human consumption and reducing their germination potential. In
India as much as 30-40% of grain is lost because of infestations from
these pests. However during severe periods of infestation the
damage can even reach up to 100% (Pruthi and Singh, 1950).
Extensive studies have been carried out by many workers on insectplant co-evolution in the family chrysomelidae (Kergoat et al.,
2005; Funk et al., 1995) but very few phylogenetic studies have
been made for this family (Slobodchikoff and Johnson, 1973). Jei et
al. studied the genetic differentiation amongst the geographic
populations of the Callosobruchus maculatus (Fabricius) based
upon the mitochondrial COI and cytb genes.
In the present study, a comparison of the mitochondrial 12S and
16SrRNA gene sequences of two species of Callosobruchus i.e.
Callosobruchus analis and Callosobruchus maculatus with the
members of Bruchinae has been carried out to understand their
phylogenetic relationship.
Material and Methods
Sample culture: Sedentary individuals of the two species of
Bruchids i.e. Callosobruchus analis (Fabricus) and Callosobruchus
maculatus (Fabricus) constituted the material for the present
investigation. Both these species are serious pests of the legumes of
the family fabeaccae. Small populations of the species were reared
Received:December 2014
Accepted: December 2014
*Corresponding Author
Email: [email protected]
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under laboratory conditions on Vigna radiata seeds in glass jars
inside a growth chamber at 30 ± 2°C and 70% relative humidity.
Muslin cloth was used to close the mouth of the jars. The specimens
were identified on the basis of the color pattern on elytra and the
color and distribution of pubescence on the pygidium. (Fig.1).
DNA isolation: The voucher specimens were preserved in absolute
ethanol mixed with a few drops of glycerol and maintained at
Department of Zoology, Panjab University, Chandigarh (UT), India,
till the extraction of genomic DNA. DNA was extracted from whole
insect using modified phenol:chloroform method (Sambrook et al.,
1989).
Isolated DNA was suspended in 100µl of TE buffer and stored at 20°C. The integrity of the genomic DNA was checked on 0.8%
agarose gel by horizontal gel electrophoresis. Quantification of
DNA was done by nanodrop-spectrophotometer.
PCR amplification and sequencing: Partial fragments of 12S
rRNA and 16S rRNA gene sequences given by Simon et al., 1994
were used for the amplification of both gene fragments: 12S-f
5'tactatgttacgacttat3'; 12S-r 5'aaactaggattagataccc3'; 16S-f
5'ccggtttgaactcagatcatgt-3' & 16S-r 5'cgcctgtttaacaaaaacat3'. PCR
amplifications were carried out in 25 µl reaction mixture containing
2/2.5 µl of 10 × Taq DNA polymerase buffer, 2-2.5 mM MgCl2, 0.1
mM dNTPs, 5 pM each primer and 0.5 U Taq DNA polymerase.
Amplifications were carried out in a thermal cycler (Biometra,
Germany) and the cycling profile was as follows: Initial denaturion
at 95°C for 5 min, 30 cycles of 30s denaturion at 95°C, 45s annealing
at 49°C (12SrRNA)/ 52 °C (16SrRNA), 1 min extension at 72°C,
followed by a final extension of 7 min at 72°C. The length of the
amplified products was checked by horizontal gel electrophoresis
by running on 2% agarose gel (stained with ethidium bromide) with
100bp DNA ladder.
The amplified products of 12SrRNA and 16SrRNA gene were got
sequenced from Chromous Biotech Pvt. Ltd. (Bangalore, India).
The data was retrieved in the form of chromatograms.
Data Analysis
The sequences were first edited manually for discarding the
ambiguous and skipped bases and files were converted into FASTA
format. The edited sequences were compared with related
sequences from the nucleotide database of the National Centre for
Biotechnology Information (NCBI) using Basic Local Alignment
Search Tool (blastn) algorithm (Altschul et al., 1997).
The accession numbers of the sequences selected for the present
analysis are shown in Table 1 (12SrRNA) and Table 2 (16SrRNA).
Consensus sequences were aligned using the Clustal omega
software (Sievers et al., 2011).
The sequence ambiguities were corrected manually. The sequence
data was then imported into the MEGA 6.06 software package and
analysed to generate neighbor-joining and maximum likelihood
trees using Kimura 2-Parameter (K2P) method (Kimura, 1980)
Analysis of nucleotide composition, overall transition: transversion
ratio (ts:tv), and pairwise nucleotide distances were calculated using
MEGA 6 (Tamura et al., 2013). Bootstrap analysis using 1000
pseudoreplications (Felsenstein, 1985) was included to test the
reliability of inferred trees and all codon positions (1st, 2nd & 3rd) were
included to verify the robustness of the internal nodes.
Figure 1. Photographs pf the normal female forms of two
species of Callosobruchus
Figure 2. Amplification product of 12SrRNA gene
Callosobruchus species under study
Lane M: 100 bp DNAladder
in
Figure 3. Amplification product of 16SrRNA gene in
Callosobruchus species under study
Lane L1-L3: C. maculatus (2 populations)
Lane L4-L5: C.analis(2 populations)
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Results
The amplified 12SrRNA and 16SrRNA gene fragments in the present
study were 430 bp (Fig. 2) and 419 bp long (Fig. 3) respectively, for
both the two species of Callosobruchus under study. However after
final editing 404bp of 12SrRNA gene and 332bp of the 16SrRNA
gene sequence were considered for sequence analysis. The
sequences correspond to the positions 15342-15744 and 1425014576 respectively, of complete mitogenome of Diabrotica
virgifera virgifera (Coleoptera: Chrysomelidae); Accession No.
KF658070 (Coates, 2014).
12S phylogeny
The 12SrRNA gene fragment revealed the occurrence of 175
variable sites of which 122 were found to be parsimony informative.
The average nucleotide composition across the populations of both
the species was found to be T=38, A=39, C=15 and G=8. The
transition:transvertion ratio (R) was 0.86. As shown in Table. 3
within the genus Callosobruchus the minimum interspecies
distance of 0.005 was observed between two populations of
Callosobruchus maculatus under study and the Callosobruchus
chinensis showed maximum divergence of 0.098 - 0.106 from other
species of the same genus. Overall the interspecific distance ranged
from 0.089 - 0.274. Amongst the outgroup members of the family
cerambicydae Psacothea hilaris revealed maximum sequence
divergence from the members of Bruchinae (0.216-0.274). The 12S
trees drawn on the basis of neighbor joining ( Fig. 4) and maximum
likelihood (Fig. 5) methods revealed almost identical topologies
with minor differences. However the neighbor joining tree exhibited
a better bootstrap support for most of the nodes. The
Callosobruchus genus formed a separate monophylectic group with
a high bootstrap value of 81% in NJ tree and 74% in ML tree. Within
the major cluster Callosobruchus analis formed a common clade
with the Callosobruchus chinensis species (AY625319). The
Callosobruchus maculatus species (AY625320) grouped closely
with its counterparts collected from the Indian subcontinent. The
Callosobruchus subinnotatus species (AY625322) showed a close
relationship to C.maculatus and hence formed a common clade. All
the nodes were well supported by bootstrap values above 50. In both
the trees the outgroup genera occupied similar positions but with
minor intrageneric configurational variations.
Figure 4.12S Neighbor Joining tree
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Figure 5. 12S Maximum likelihood tree
Figure 6. 16S Neighbor Joining tree
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Figure 7. 16S Maximum Likelihood tree
Discussion
gene and 73% in 16S gene sequences of our species is in confirmation
with the previous studies reported on coleopterans (Liu and
Beckenbach, 1992; Forgie et al., 2006). A probable hypothesis for the
favourable selection of A+T nucleotides in insect mtDNAs, is that,
during long term evolution the DNA became rich in AT and the
transcription enzymes started functioning optimally with this
composition and therefore less optimally with GC-rich DNA (Clary
and Wolstenholme, 1985). As the sequences for both the genes of the
Callosobruchus species were rarely available in GenBank so
somewhat separate species of the related genera were used to draw
the phylogeny based upon on the 12S and 16S genes. We consider our
study to be a robust estimate of the phylogeny of these sequences
because the trees drawn on the basis of maximum likelihood and
neighbor joining methods revealed similar configuration with minor
alterations and the nodes were well supported by high bootstrap. The
trees in both the cases showed two major groupings - the species of
the family Cerambicydae occupying the basal position and the
species of the family Chrysomelidae forming one large cluster. The
family Cerambycidae commonly known as long horned beetles is a
diverse group of phytophagous insects. Owing to their immense
importance in the ecosystem as pests, decomposers and pollinators it is one of the most extensively studied group of the order coleoptera
(Marvaldi et al., 2009).
To analyze the phylogeny of the Indian Callosobruchus species
sequences of the species of the same genus from other regions of the
world and of the outgroup species of the related genera were obtained
from GenBank, to draw a better picture. The species of the family
cerambicydae were included in the final analysis to obtain rooted
trees. The predictably high A+T content of 77% in sequences of 12S
The genus Callosobruchus of the tribe bruchini occupied a central
position in all the trees. In the 16S tree the genera Bruchidius and
Callosobruchus formed sister grouping in conformation with the fact
that both belong to the subtribe Bruchidina. In case of the 12S gene
phylogeny the Callosobruchus species shows up as a sister group of
the acanthoscelides in the maximum likelihood tree. However, the
16S phylogeny
Of the 332 bp included in the final analysis of the 16SrRNA amplicon
209bp were found to be conserved while 119bp were variable with 96
parsimony informative sites. The sequences of both the species
revealed an average A:T:G:C ratio of 34:39:18:9. The ts:tv ratio came
out to be 0.38. The interspecies distance between Callosobruchus
species under study ranged from 0.003 to 0.010. The species
Monochamus alternatus showed maximum divergence from
Callosobruchus maculatus with an intraspecific distance of 0.257.
Similarly the species of the genus Bruchidius showed less divergence
from the Callosobruchus species under study with the intraspecific
distance ranging from 0.131- 0.175 (Table 4). The phylogenetic trees
drawn on the basis of neighbor joining and method maximum
likelihood are shown in Fig. 6 and Fig. 7 respectively. Both the trees
revealed similar topology with minor variations. The species
grouped according to their respective genera. The outgroup species
of the family Cerambicydae included in the present study formed an
entirely separate group and occupied the bottom position of the tree.
The 16SrRNA trees also support the monophyly of the
Callosobruchus genus. The nodes were well supported by high
bootstrap values.
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Table1. List of the species whose 12S gene sequences were retrieved from GenBank public database and included in analysis
Sl No
Taxon
Family
Subfamily
Reared on
Locality
Accession number
1
Callosobruchus maculatus
Chrysomelidae
Bruchinae
Vigna radiata
Kenya
AY625320
2
Callosobruchus
subinnotatus
Chrysomelidae
Bruchinae
Vigna subterranea
Senegal
AY625322
3
Callosobruchus chinensis
Chrysomelidae
Bruchinae
Cajanus cajan
Egypt
AY625319
4
Bruchidius murinus
Chrysomelidae
Bruchinae
Trifolium
subterraneum
Greece
HQ178008
5
Bruchidius tibialis
Chrysomelidae
Bruchinae
Medicago polymorpha
Greece
HQ178011
6
Bruchidius dispar
Chrysomelidae
Bruchinae
Trifolium medium
France
AY390643
7
Bruchidius fulvicornis
Chrysomelidae
Bruchinae
Trifolium vesiculosum
France
AY390644
8
Bruchidius pusillus
Chrysomelidae
Bruchinae
Hippocrepis emerus
France
AY390650
9
Bruchidius seminarius
Chrysomelidae
Bruchinae
Tetragonobolus
maritimus
France
AY390652
10
Bruchus brachialis
Chrysomelidae
Bruchinae
Vicia villosa
France
AY390660
11
Bruchus luteicornis
Chrysomelidae
Bruchinae
Vicia sativa
France
AY390662
12
Bruchus atomarius
Chrysomelidae
Bruchinae
Lathyrus macrorhyzus
France
DQ307623
13
Tuberculobruchus babaulti
Chrysomelidae
Bruchinae
Acacia
amythethophylla
Kenya
AY625326
14
Tuberculobruchus natalensis Chrysomelidae
Bruchinae
Acacia sieberiana
Senegal
AY625327
15
Tuberculobruchus sinaitus
Chrysomelidae
Bruchinae
Acacia tortilis
Senegal
AY625329
16
Acanthoscelides flavescens
Chrysomelidae
Bruchinae
Rhynchosia minima
Mexico
AY945973
17
Acanthoscelides guazumae
Chrysomelidae
Bruchinae
Guazuma tomentosa
Mexico
AY945974
18
Acanthoscelides sanfordi
Chrysomelidae
Bruchinae
Pachyrhizus erosus
Mexico
Ay945987
19
Gaurotes tuberculicolis
Cerambicydae
Lepturinae
-
China
KF737658
20
Monochamus alternatus
Cerambicydae
Lamiinae
-
China
KJ809086
21
Moechotypa diphysis
Cerambicydae
Lamiinae
-
China
KF737697
22
Lamiinae
Cerambicydae
Lamiinae
-
Korea
FJ424074
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Table 2. List of the species whose 16S gene sequences were retrieved from GenBank public database and included in analysis
Sl No
Taxon
Family
Subfamily
Reared on
Locality
Accession
1
Bruchidius seminarius
Chrysomelidae
Bruchinae
Tetragonobolus maritimus
France
Hq178231
2
Bruchidius cinerascens
Chrysomelidae
Bruchinae
Eryngium campestre
France
HQ178221
3
Bruchidius villosus
Chrysomelidae
Bruchinae
Laburnum anagyroides
France
HQ178236
4
Bruchidius murinus
Chrysomelidae
Bruchinae
Trifolium subterraneum
Greece
HQ178227
5
Bruchidius tibialis
Chrysomelidae
Bruchinae
Medicago polymorpha
Greece
HQ178233
6
Bruchidius varius
Chrysomelidae
Bruchinae
Trifolium angustifolium
France
HQ178235
7
Bruchidius pygmaeus
Chrysomelidae
Bruchinae
Trifolium angustifolium
France
HQ178230
8
Bruchidius bimaculatus
Chrysomelidae
Bruchinae
Bruchinae
France
HQ178219
9
Mimosestes anomalus
Chrysomelidae
Bruchinae
Acacia pennatula
Mexico
AB499927
10
Mimosestes cinerifer
Chrysomelidae
Bruchinae
Acacia sphaerocephala
Mexico
AB499928
11
Mimosestes viduatus
Chrysomelidae
Bruchinae
Acacia chiapensis
Mexico
AB499940
12
Mimosestes nubigens
Chrysomelidae
Bruchinae
Acacia schaffneri
Mexico
AB499937
13
Mimosestes amicus
Chrysomelidae
Bruchinae
Prosopis pallida
Hawaii
AB499925
14
Gaurotes tuberculicolis
Cerambicydae
Cerambicyd
Lepturinae
China
KF737721
15
Monochamus alternatus
Cerambicydae
Cerambicyd
Lamiinae
China
KF737765
16
.Pachyta bicuneata
Cerambicydae
Cerambicyd
Pachyrhizus erosus
China
DQ861334
17
Psacothea hilaris
Cerambicydae
Cerambicyd
Lepturinae
Korea
KF737766
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Table 3. Percentage divergence matrix of 12S sequences
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Table 4. Percentage Divergence matrix of 16S sequences
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grouping is not supported by relevant internal bootstrap node value
29. In the neighbor joining tree, the genus Callosobruchus clearly
appears as a monophyletic group with a bootstrap support of 81%.
Similarly the gene sequence data also firmly supports the
monophyly of this group with a bootstrap value of 100%.
Conclusion
The results clearly point towards the monophyly of one of the most
destructive pulse pests of India. This study reveals the usefulness of
the mitochondrial ribosomal genes as potent molecular markers for
unravelling the deeper phylogenetic relationship of the family
chrysomelidae. The sequence data generated will be helpful for
further studies on phylogenetic origins and biogeographical
evolution of the group.
Acknowledgments
We acknowledge the Department of Zoology, Panjab University,
Chandigarh for providing all the necessary facilities for this work.
We are thankful to the University Grants Commission (UGC), New
Delhi for financial support under CAS programme. We are grateful
to Chromous Biotech Pvt. Ltd., Bangalore, India, for providing the
services of sequencing.
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To cite this article: Neha Goyal and Vijay Lakshmi Sharma 2015 Phylogenetic Relationship among Some Species of Bruchinae
Based Upon 16S and 12S Ribosomal RNA Gene Sequences. The Scitech Journal 02(01): 16-25
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