dna barcoding of indian ant species based on cox1 gene

Indian Journal of Biotechnology

Vol 13, April 2014, pp 165-171

DNA barcoding of Indian ant species based on cox1 gene

Rakshit Ojha1, S K Jalali

1*, T M Mushtak Ali

2, T Venkatesan

1, Sean W Prosser

3 and N K Krishna Kumar

1

1National Bureau of Agriculturally Important Insects, Hebbal, Bangalore 560 024, India 2Department of Entomology, University of Agricultural Sciences, GKVK Campus, Bangalore 560 065, India

3Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph

Guelph, Ontario, Canada N1G 2W1

Received 17 January 2013; revised 6 May 2013; accepted 14 June 2013

Sixteen species of ants collected from Karnataka, India, were sequenced and barcoded for a 658 bp region of the

mitochondrial cytochrome c oxidase subunit 1 gene (cox1). This gene is frequently termed as CO1 in barcoding approach

and serves as the core of a global bioidentification marker for insects and other animals. The AT content in DNA of 16 ant

species was estimated as 68.55%, which is in accordance with invertebrates. The variance of AT content among species was

much higher for the 1st base (AT1) compared to the 2nd and 3rd bases, which were nearly invariant. The distance within

species was calculated using Kimura 2-parameter (K2P) and it was 0.166, 0.000 and 0.333% for mean, minimum and

maximum, respectively. Moreover, our data also showed some phylogenetic signal. In a neighbor-joining tree for all

sequences, two clusters were obtained, the first cluster consisted of subfamilies Formicinae and Myrmicinae, while other

clade showed relationship between subfamily Myrmicinae and Ponerinae clustered with subfamily Dolichodrinae, which is

not in contradiction with cladistics analysis of morphological data for ants and is consistent with traditional phylogeny of

ants. The present results thus favour DNA barcoding as a decisive tool in quick and reliable identifications of ants.

Keywords: Barcodes, cox1, cytochrome c oxidase subunit 1, Indian ants, molecular characterization

Introduction

The science behind classifying living species based

on shared features, i.e., taxonomy, has been a part of

human society for centuries. The quantification of

biodiversity, which is increasingly decimated,

presents a daunting challenge to taxonomists because

it requires discovery and analysis to proceed at a

greatly accelerated pace1. Recognition of subtle

anatomical differences between closely related

species requires subjective judgment of a specialist

and also demands great deal of taxonomic

knowledge2. Hebert et al

3 observed that, for critical

identification of 10-15 million species based on

morphological diagnosis, a community of 15,000

taxonomists will be required. In order to identify these

species at the rate of expertise available, it may

require centuries to even complete a preliminary

‘Encyclopedia of life’4, signaling the need for a new

approach to taxon recognition5.

Utilization of DNA sequence diversity to identify

organisms assessed directly or indirectly through

protein analysis has been gaining importance6,7

.

About five decades ago, starch gel electrophoresis

of proteins was first used to identify species8.

Tautz et al9 advocated the case for a DNA-based

taxonomic system, whereas Hebert et al3 believed

that a single gene sequence would be sufficient to

differentiate vast majority of animal species, and

proposed the use of the mitochondrial DNA gene

cytochrome c oxidase subunit 1 (cox1) as a global

identification marker for animals. The cox1 region is

useful for inferring phylogenetic relationships among

populations and it is also used as the primary DNA

barcode throughout the world3. Although the cox1

region is highly conserved, differences do exist in the

length and sequence of the regions flanking cox1.

Previous phylogenetic studies have shown the utility

of cox1 for the identification of genetic variability1.

The sequence obtained was equated to a barcode with

species being distinguished by their particular

sequence10

. Just as the unique pattern of bars in a

Universal Product Code (UPC) identifies each

consumer product, a “DNA barcode” identifies each

organism. Short DNA barcodes, about 700 bp in

length, can be quickly processed from thousands of

specimens and unambiguously analyzed by computer

———————

*Author for correspondence:

Mobile: +91-9449673949

Email: [email protected]

INDIAN J BIOTECHNOL, APRIL 2014

166

programs3. Therefore, DNA barcoding could

revolutionize taxonomy and diversity by linking

established museum collections to unknown species

from the field, facilitating the description of new

species, revealing cryptic species, and linking adult

with juvenile or male with female11,12

.

Concerns have been raised and reported regarding

the efficacy of the cox1 approach to DNA

barcoding13,14

. However, large scale sequencing of

short cox1 fragments for biodiversity inventories

indicates that sequence variation is highly structured

and partitioned with discrete genetic clusters

that correspond broadly to species level entities15

.

To that end, we describe in this communication how

cox1 DNA barcoding enables rapid identification of

molecular operational taxonomic units as described

by,

Floyd et al16

and Blaxter17

for the assessment

of diversity of some ant species in Karnataka state

of India.

India is one of the world’s mega diversity regions

for ants, where they occur frequently throughout the

country in various ecosystems including forest,

grassland and human habitat. Ants are important

components of ecosystems not only because they

constitute a great part of animal biomass, but also as

ecosystem engineers. It is estimated that there are

more species of ants in a square kilometer of Brazilian

forest than all the lions and elephants in Africa18

.

Although ants dominate the biomass of most

terrestrial communities, act as pollinators and seed

dispersers, and are critical to nutrient cycling and

ecosystem function, there is a global lack of studies of

ant diversity or community structure19

. This may be

largely because of the difficulty of species-level

identifications. The vast diversity of ant fauna

includes approx 660 species from 87 genera in India

and will continue to increase in numbers as more and

more systematic explorations in diverse habitats are

undertaken by taxonomists. Myrmicines family forms

the bulk of Indian ant diversity with 45% of total

Indian ants, while family Formicines is the second

leading ant group (25% of species), with Camponotus

and Polyrachis constituting the majority of the

diversity19

. Ants on the Andaman and Nicobar

Islands, India were surveyed and it resulted in

doubling the number of ant species recorded from

these islands. Records include five endemic species,

but no endemic genera. However, the surveys were

fairly superficial and it is likely that many species

remain to be discovered on these islands20

. Five

Indian ant species are on the International Union for

Conservation of Nature (IUCN) red list20

.

In the present study, we examined the cox1, a

protein coding mitochondrial gene, for barcoding

approach to assemble ants of India in a global

library of DNA barcodes at BOLDSYSTEMS

(http://www.barcodinglife.com/). Species clusters

were identified using ClustalW tool and a tree based

approach was used to study phylogeny. This approach

can be used to barcode the total diversity of Indian ant

species.

Materials and Methods Collection and Identification

Ants were collected during the month of September

to December 2011 in Karnataka state of India from

the following locations: i) Bangalore (12°58' N;

77°38' E; 3018 ft), ii) Mandya (12°13'N; 76°20' E;

2224 ft); iii) Medikeri (12°19' N; 75°53' E; 5000 ft);

and iv) Kolar (13°01' N; 77°71' E; 4850 ft). At each

of these sites, ants were collected by brush and cotton

wool pad and transferred to collection tubes

containing 95% alcohol. The specimens were

identified to species level and distributed into their

respective subfamily to obtain a clear phylogenetic

signal (Table 1) immediately upon their collection by

Table 1—Distribution of ant species into their respective

subfamilies and tribes on the basis of classification

Species Tribe Collection site

Subfamily: Formicinae

Camponotus irritance Camponotini Bangalore

C. parius Camponotini Bangalore

C. compressus GR-17 Camponotini Mandya

C. compressus Camponotini Bangalore

Anoplolepis gracilipes Lasiini Medikeri

Oecophylla samaragdina Oecophyllini Mandya

Paratrechina longicornis Plagiolepidini Bangalore

Plagiolepis sp. Plagiolepidini Bangalore

Subfamily: Myrmicinae

Aphaenogaster beccarii Pheidolini Bangalore

Pheidologeton diversus Pheidolini Medikeri

Solenopsis geminata Solinopsidni Kolar

Monomorium scabriceps Solinopsidni Mandya

Myrmicaria brunnea Myrmicarini Kolar

Subfamily: Ponerinae

Leptogenys chinensis Ponerini Bangalore

Subfamily: Dolichoderinae

Tapinoma melanocephalum Dolichoderini Bangalore

Technomyrmex albipes Dolichoderini Bangalore

OJHA et al: DNA BARCODING OF INDIAN ANT SPECIES.

167

one of the authors. The specimens, thus, collected

and morphologically identified were used for cox1

barcoding at the National Bureau of Agriculturally

Important Insects (NBAII) Bangalore, India. Genetic Analysis

DNA was extracted from somatic tissues (thorax,

abdomen) rich in mitochondria using Qiagen

DNeasy® kit, following the manufacturer’s protocols.

Remaining parts of each ants and respective

individuals were kept as voucher specimens at

NBAII. The extracts were subjected to PCR

amplification of a 658 bp region near the 5' terminus

of the cox1 gene following standard protocol (Hebert

et al)3. Primers used were: forward primer (LCO

1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'),

and reverse primer (HCO 2198: 5'-TAAACTTCA

GGGTGACCAAAAAATCA-3').

PCR reactions were carried out in 96-well plates,

50 µL reaction volume containing: 5 µL GeNeiTM

Taq

buffer, 1 µL GeNeiTM

10mM dNTP mix, 2.5 µL

(20 pmol/µL) forward primer, 2.5 µL (20 pmol/µL)

reverse primer, 1 µL GeNeiTM

Taq DNA polymerase

(1 U/µL), 2 µL DNA (50 ng/µL), and 36 µL sterile

water. Thermo cycling consisted of an initial

denaturation of 94°C for 5 min, followed by 30 cycles

of denaturation at 94°C for 1 min, annealing at 55°C

for 1 min and extension at 72°C for 1 min. PCR

was performed using a C1000™ Thermal Cycler. The

amplified products were analyzed on a 1.5% agarose

gel electrophoresis as described by Sambrook and

Russell21

. The amplified products were sent to two

commercial sequencing companies, SciGenome Pvt.

Ltd., India and Ocimum Biosolutions Pvt. Ltd., India.

Each ant species was bi-directionally sequenced and

checked for homology, insertions and deletions,

stop codons, and frame shifts by using NCBI BLAST.

All sequences were uploaded to GenBank and

the Barcode of Life Database (BOLD,

http://www.boldsystems.org).

Data Analysis

The pairwise analysis of 16 sequences was

conducted using Kimura 2-parameter (K2P) method

in MEGA4. The number of base substitutions per site

was analyzed between all sequences. Codon positions

included were 1st+2

nd+3

rd+non-coding. All positions

containing gaps and missing data were eliminated

from the dataset. The A, T, G, C, AT and GC content

of all 16 sequences was obtained using a computer

program designed in the Bioinformatics Lab at

NBAII, Bangalore, India. The AT% at three codon

positions was calculated using the same program.

Sequences were aligned using the MegAlign tool

of the DNASTAR software package to highlight the

conserved gene regions using Boxshade option.

Residue distances were estimated using the ClustalW

program of MEGA 4.0.2 software with default

settings of gap opening penalty 10 and a

gap-extension 0.1 in pairwise and 0.05 in multiple

alignments. Sequence divergences were calculated

and a NJ tree of distances was created to provide a

graphic representation of the among-species

divergences22

. Sequencing neutrality, rate of

substitutions and per cent identities were calculated

using DNASTAR Lasergene 8. Sequences and

other specimen information are available in the

project “Ants of India” in the campaign section

and “Ants of the World” in BOLD Systems

at the website (http://www.boldsystems.org). Five

sequences (Anoplolepis gracilipes Smith, Oecophylla

smaragdina Fabricius, Plagiolepis sp., Solenopsis

geminata Fabricius & Technomyrmex albipes Smith)

were examined for homology by downloading each

sequence from GenBank since these were the only

available sequences present in GenBank when we

checked all the 16 species reported by us. Sequences

and other specimen informations are available at

BOLD Systems (http://www.boldsystems.org).

Results and Discussion All cox1 sequences were submitted to the NCBI-

GenBank under accession numbers: Camponotus

compressus Fabricius-JN886027, S. geminate

Fabricius-JN886028, Myrmicaria brunnea Saunders-

JN886029, Leptogenys chinensis Mayr-JN886030,

Aphaenogaster beccarii Emery-JN886031, C. parius

Emery-JN886032, C. irritans Smith-JN886033,

Paratrechina longicornis Latreille-JN886034,

O. smaragdina Fabricius-JN886035, Tapinoma

melanocephalum Fabricius-JN886036, Plagiolepis

sp.-JN886037, T. albipes Smith-JN886038,

C. compressus Fabricius-JN987857, Monomorium

scabriceps Mayr-JN987858, Pheidologeton diversus

Jerdon-JN987859 and A. gracilipes Smith-JN987860.

PCR products from different ant species were easily

produced and aligned as no insertions, deletions or

stop codons were observed as 1st frame of DNA

sequences were chosen from ORF finder for

submission. The visualized PCR product contained

only discrete single bands (Fig. 1), thus indicating that

sequences obtained were mitochondrial DNA and not


168

Fig. 1—PCR amplification of cox1 region (M, Mol marker;

1, Camponotus compressus; 2, Solenopsis geminate; 3, Myrmicaria

brunnea; 4, Leptogenys chinensis; 5, Aphaenogaster beccarii;

6, C. parius; 7, Paratrechina longicornis; 8, Monomorium

scabriceps; 9, Oecophylla smaragdina, 10, Tapinoma

melanocephalum; 11, Technomyrmex albipes; 12, Pheidologeton

diversus; 13, Anoplolepis gracilipes; 14, Plagiolepis sp.;

15. C. compressus GR-17; & 16, C. irritans)

nuclear pseudogenes24

. The cox1 region in almost all

the samples was in the range of 648-744 bp.

A total of 16 species were studied, giving

120 comparisons out of a total of 240 datasets

(software generated) according to the full K2P/NJ

tree, constructed as electronic supplementary material

(Table 2). All 16 species could be differentiated by

cox1 barcoding. Most of the amplified sequences

were more than 600 bp in length, except for

L. chinensis (560 bp). In the present study, the lengths

of sequences suggest that nuclear DNA sequences of

mitochondrial origin (NUMTs) were not sequenced

because NUMTs does not code for protein, leading

to incorrect sequence and misidentification25

. The

average K2P distance of individuals within species

Table 2—Estimates of evolutionary divergence between sequences, the numbers of base substitutions per site is shown

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 Aphaenogaster

beccarii

2 Leptogenys

chinensis

0.322

3 Pheidologeton

diversus

0.281 0.308

4 Myrmicaria

brunnea

0.282 0.255 0.255

5 Tepinoma

melanocephalum

0.328 0.333 0.295 0.295

6 Technomyrmex

albipes

0.291 0.272 0.259 0.227 0.234

7 Solenopsis

geminate

0.282 0.279 0.243 0.266 0.323 0.286

8 Monomorium

scabriceps

0.284 0.302 0.279 0.271 0.312 0.258 0.196

9 Camponotus

irritance

0.291 0.296 0.306 0.270 0.295 0.293 0.272 0.313

10 C. parius 0.291 0.296 0.306 0.270 0.295 0.293 0.272 0.313 0.000

11 C. compressus

GR-17

0.301 0.247 0.288 0.270 0.311 0.284 0.277 0.298 0.201 0.201

12 C. compressus 0.308 0.258 0.290 0.267 0.321 0.293 0.274 0.304 0.214 0.214 0.022

13 Anoplolepis

gracilipes

0.264 0.295 0.240 0.243 0.274 0.253 0.298 0.300 0.243 0.243 0.253 0.258

14 Paratrechina

longicorins

0.259 0.264 0.235 0.249 0.296 0.257 0.233 0.269 0.252 0.252 0.229 0.237 0.202

15 Oecophylla

samaragdina

0.273 0.270 0.248 0.258 0.262 0.253 0.271 0.309 0.259 0.259 0.210 0.215 0.223 0.210

16 Plagiolepis sp. 0.292 0.264 0.245 0.266 0.257 0.260 0.255 0.275 0.284 0.284 0.226 0.229 0.212 0.197 0.180

Note: Analyses were conducted using the Kimura 2-parameter (K2P) method in MEGA41,2. Codon positions included were

1st+2nd+3rd+non-coding. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option)

Column no: 1-15 consists of 120 comparisons from a total of 240 datasets including 15 blank columns (software generated). All results

are based on the pair-wise analysis of 16 sequences.


169

was 0.166%, with minimum 0.000% and maximum

0.333% (Table 2). Our analysis is the first step

towards assembling the ants of India into a global

library of DNA barcodes. Comparative analysis of A,

T, G, C content was found to have a relatively high

AT content of 68.55% (64.5%-76.3%) compared to

31.44% of GC content. This difference was attributed

to the AT content of the 1st codon (AT1), which

ranged from 20.44-33.13%. The AT content at

2nd

and 3rd

codon positions was nearly invariant

(Table 3). As expected, closely allied species, which

grouped closely in the N-J tree, showed high

percentage of identity and low divergence. For

example, C. irritance and C. parius showed 91.9%

identity and 0.0% divergence, while L. chinensis and

P. diversus showed 58.4% identity and 30.8%

divergence. An interesting observation was that

higher genetic distance was observed in the 1st codon

position compared to the 2nd

and 3rd

codon positions.

Average genetic distances among the different groups

of ants used in this study showed higher values at

the1st codon position, indicating that detailed study on

the 1st codon position for ants might reveal possible

evolutionary information among this closely related

group of organisms. Sequences were heavily AT

biased due to this 1st codon position, which is

expected in insect mtDNA1.

A phylogeny tree constructed using the N-J method

revealed two clusters (Fig. 2). The first cluster

consisted of two sub-clades representing two

subfamilies of ant species: Formicinae (8 species) and

Myrmicinae (4 species), which were originated from

similar ancestors. The second main cluster consisted

of the subfamilies Ponerinae (1 species), Myrmicinae

(1 species), and Dolichoderinae (2 species), showing

sharing of similar characters from their ancestors.

L. chinensis (Ponerinae) is a primitive species of ant

related to M. brunnea (Myrmicinae), which is

consistent with cladistics analysis of morphological

data for ants by Baroni Urbani et al26

and also favours

traditional phylogeny, since all the 16 species were

morphologically identified using traditionally

morphological keys27

. The sequences obtained in the present study

[C. compressus (680 bp), S. geminata (678 bp), M. brunnea (689 bp), L. chinensis (560 bp), A. beccarii

(683 bp), C. parius (687 bp), P. longicornis (648 bp), M. scabriceps (687bp), O. smaragdina (685 bp), T. melanocephalum (690 bp), T. albipes (688 bp), P. diversus (685 bp), A. gracilipes (691 bp), Plagiolepis sp. (744 bp), C. compressus GR-17. (686 bp) and C. irritans (689 bp)] were compared to homologous sequences available in GenBank (Fig. 3). Of all the sequences reported in the study, five were found similar with their respective sequences in GenBank (identities ranged from 89% to 100%), while the remaining 11 sequences were reported for the first time.

Table 3—Per cent AT at the 1st, 2nd, and 3rd codon positions of

16 different ant species of the family Formicidae

Family: Formicidae 1st 2nd 3rd

Anoplolepis gracilipes 20.96 27.58 19.51

Aphaenogaster beccarii 20.44 24.91 20.27

Camponotus compressus 30.74 22.26 20.49

C. compressus GR-17 30.39 21.58 20.51

C. irritans 20.49 24.95 19.83

C. parius 25.22 20.51 20.36

Leptogenys chinensis 21.32 30.90 20.82

Monomorium scabriceps 25.07 19.90 20.51

Myrmicaria brunnea 20.66 26.11 20.00

Oecophylla smaragdina 33.13 22.18 20.97

Paratrechina longicornis 28.57 20.66 21.12

Pheidologeton diversus 24.20 19.78 20.49

Plagiolepis sp. 28.11 20.82 21.12

Solenopsis geminata 25.22 19.75 20.66

Tapinoma melanocephalum 24.46 20.82 20.51

Technomyrmex albipes 26.89 20.51 20.82

Mean 25.36 22.70 20.49

Fig. 2—Cluster analysis based dendrogram depicting genetic

relationships among 16 different ant species, generated by

Bootstrap Test Phylogeny using N-J (Neighbour-joining) method

of MEGA 4 Software. All species are from 4 subfamilies, which

distribute into two main clades (subfamily Myrmicinae clustered

in both clades) that are 12% similar.


170

Fig. 3—Nucleotide alignment of all 16 ant barcodes obtained in the study. Sequences were first assembled using the SeqBuilder tool and

then aligned using the MegAlign tool of the DNASTAR software package. Matching residues are highlighted in grey.


171

The present study clearly shows that availability of

DNA tools for diversity assessment will greatly

facilitate and complement taxonomic studies. The

combination of DNA sequencing data with traditional

taxonomy will serve as a model that can be applied

across disciplines. It will increase the rate of species

identification, which will eventually help to deal with

current biodiversity crisis1. In situation where species

identification is difficult, the potential utility of DNA

barcoding is immense. Our results reveal that

cox1 barcoding will permit the unambiguous

identification of ant species of India, thus there is

need to look for integrated approach in taxonomy for

quick identification of insect biodiversity.

Acknowledgements

The authors wish to express their sincere thanks to

NAIP Project of National Agricultural Bioinformatics

Grid, New Delhi, granted to National Bureau of

Agriculturally Important Insects, Bangalore, under

which necessary facilities were provided for the

present study. Authors are also thankful to Dr P D N

Hebert of Biodiversity Institute of Ontario, Canada

for his useful suggestions and advice and Dr C A

Viraktamath of GKVK, Bangalore, for his useful

suggestions, corrections and proper classification of

ant species.

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dna barcoding of indian ant species based on cox1 gene

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