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 Jalali1*, T M Mushtak Ali2, T Venkatesan1, Sean W Prosser3 and N K Krishna Kumar1 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 st nd rd much higher for the 1 base (AT1) compared to the 2 and 3 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 protein analysis has been gaining importance6,7. The science behind classifying living species based About five decades ago, starch gel electrophoresis on shared features, i.e., taxonomy, has been a part of of proteins was first used to identify species8. human society for centuries. The quantification of Tautz et al9 advocated the case for a DNA-based biodiversity, which is increasingly decimated, taxonomic system, whereas Hebert et al3 believed presents a daunting challenge to taxonomists because that a single gene sequence would be sufficient to it requires discovery and analysis to proceed at a differentiate vast majority of animal species, and greatly accelerated pace1. Recognition of subtle proposed the use of the mitochondrial DNA gene anatomical differences between closely related cytochrome c oxidase subunit 1 (cox1) as a global species requires subjective judgment of a specialist identification marker for animals. The cox1 region is and also demands great deal of taxonomic useful for inferring phylogenetic relationships among knowledge2. Hebert et al3 observed that, for critical populations and it is also used as the primary DNA identification of 10-15 million species based on barcode throughout the world3. Although the cox1 morphological diagnosis, a community of 15,000 region is highly conserved, differences do exist in the taxonomists will be required. In order to identify these length and sequence of the regions flanking cox1. species at the rate of expertise available, it may Previous phylogenetic studies have shown the utility require centuries to even complete a preliminary of cox1 for the identification of genetic variability1. ‘Encyclopedia of life’4, signaling the need for a new The sequence obtained was equated to a barcode with approach to taxon recognition5. species being distinguished by their particular Utilization of DNA sequence diversity to identify sequence10. Just as the unique pattern of bars in a organisms assessed directly or indirectly through Universal Product Code (UPC) identifies each consumer product, a “DNA barcode” identifies each ——————— organism. Short DNA barcodes, about 700 bp in *Author for correspondence: Mobile: +91-9449673949 length, can be quickly processed from thousands of Email: [email protected] specimens and unambiguously analyzed by computer 166 INDIAN J BIOTECHNOL, APRIL 2014 programs3. Therefore, DNA barcoding could Indian ant species are on the International Union for revolutionize taxonomy and diversity by linking Conservation of Nature (IUCN) red list20. established museum collections to unknown species In the present study, we examined the cox1, a from the field, facilitating the description of new protein coding mitochondrial gene, for barcoding species, revealing cryptic species, and linking adult approach to assemble ants of India in a global with juvenile or male with female11,12. library of DNA barcodes at BOLDSYSTEMS Concerns have been raised and reported regarding (http://www.barcodinglife.com/). Species clusters the efficacy of the cox1 approach to DNA were identified using ClustalW tool and a tree based barcoding13,14. However, large scale sequencing of approach was used to study phylogeny. This approach short cox1 fragments for biodiversity inventories can be used to barcode the total diversity of Indian ant indicates that sequence variation is highly structured species. and partitioned with discrete genetic clusters 15 Materials and Methods that correspond broadly to species level entities . Collection and Identification To that end, we describe in this communication how Ants were collected during the month of September cox1 DNA barcoding enables rapid identification of to December 2011 in Karnataka state of India from molecular operational taxonomic units as described , 16 17 the following locations: i) Bangalore (12°58' N; by Floyd et al and Blaxter for the assessment 77°38' E; 3018 ft), ii) Mandya (12°13'N; 76°20' E; of diversity of some ant species in Karnataka state 2224 ft); iii) Medikeri (12°19' N; 75°53' E; 5000 ft); of India. and iv) Kolar (13°01' N; 77°71' E; 4850 ft). At each India is one of the world’s mega diversity regions of these sites, ants were collected by brush and cotton for ants, where they occur frequently throughout the wool pad and transferred to collection tubes country in various ecosystems including forest, containing 95% alcohol. The specimens were grassland and human habitat. Ants are important identified to species level and distributed into their components of ecosystems not only because they respective subfamily to obtain a clear phylogenetic constitute a great part of animal biomass, but also as signal (Table 1) immediately upon their collection by ecosystem engineers. It is estimated that there are Table 1—Distribution of ant species into their respective more species of ants in a square kilometer of Brazilian subfamilies and tribes on the basis of classification forest than all the lions and elephants in Africa18. Although ants dominate the biomass of most Species Tribe Collection site terrestrial communities, act as pollinators and seed Subfamily: Formicinae dispersers, and are critical to nutrient cycling and Camponotus irritance Camponotini Bangalore ecosystem function, there is a global lack of studies of C. parius Camponotini Bangalore 19 ant diversity or community structure . This may be C. compressus GR-17 Camponotini Mandya largely because of the difficulty of species-level C. compressus Camponotini Bangalore identifications. The vast diversity of ant fauna Anoplolepis gracilipes Lasiini Medikeri includes approx 660 species from 87 genera in India Oecophylla samaragdina Oecophyllini Mandya and will continue to increase in numbers as more and Paratrechina longicornis Plagiolepidini Bangalore more systematic explorations in diverse habitats are Plagiolepis sp. Plagiolepidini Bangalore undertaken by taxonomists. Myrmicines family forms Subfamily: Myrmicinae the bulk of Indian ant diversity with 45% of total Aphaenogaster beccarii Pheidolini Bangalore Indian ants, while family Formicines is the second Pheidologeton diversus Pheidolini Medikeri leading ant group (25% of species), with Camponotus Solenopsis geminata Solinopsidni Kolar and Polyrachis constituting the majority of the 19 Monomorium scabriceps Solinopsidni Mandya diversity . Ants on the Andaman and Nicobar Myrmicaria brunnea Myrmicarini Kolar Islands, India were surveyed and it resulted in doubling the number of ant species recorded from Subfamily: Ponerinae these islands. Records include five endemic species, Leptogenys chinensis Ponerini Bangalore but no endemic genera. However, the surveys were Subfamily: Dolichoderinae fairly superficial and it is likely that many species Tapinoma melanocephalum Dolichoderini Bangalore 20 remain to be discovered on these islands . Five Technomyrmex albipes Dolichoderini Bangalore OJHA et al: DNA BARCODING OF INDIAN ANT SPECIES. 167 one of the authors. The specimens, thus, collected NBAII, Bangalore, India. The AT% at three codon and morphologically identified were used for cox1 positions was calculated using the same program. barcoding at the National Bureau of Agriculturally Sequences were aligned using the MegAlign tool Important Insects (NBAII) Bangalore, India. of the DNASTAR software package to highlight the conserved gene regions using Boxshade option. Genetic Analysis Residue distances were estimated using the ClustalW DNA was extracted from somatic tissues (thorax, program of MEGA 4.0.2 software with default abdomen)