Jisha Krishnan and Sebastian, IJALS, Volume (8) Issue (2) May RESEARCH ARTICLE

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1 Analysis of evolutionary divergence of Neurothemis tullia (Odonata:Libellulidae) using cytochrome oxidase subunit I gene E.K. Jisha Krishnan and C.D. Sebastian Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala , India, Corresponding Author C.D. Sebastian Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala , India drcdsebastian@gmail.com Article History Received on 19 March, 2015; Received in revised form 15 April, 2015; Accepted 01 May, drcdsebastian@gmail.com Abstract Neurothemis tullia is an andromorphic libellulidae member commonly found in ponds, marshes, paddy fields, swamps and tanks. Since this species exhibits female polymorphism and sexual dimorphism, the present study analyzed the nucleotide sequence of mitochondrial cytochrome oxidase I (COI) gene to predict any genetic changes that had occurred in this highly conserved region. Results showed that male, female and andromorphic female members showed the same 351 bp length COI gene ( GenBank Accession: KP ). Phylogenetic tree constructed by the neighbour joining method showed that it is having a sister clade relationship of the same species found in Mizoram with a maximum bootstrap value of 100. Even though sexual dimorphism in the body colouration, body patterns, wing spot, etc may cause misidentification, the present DNA sequence analysis leads to the absolute identification of this species. The male, female and andromorphic female have same base pair length DNA, which confirmed them to belong to the same species. Phylogenetic tree depicted that different species of Neurothemis have a sequence divergence in the range of 1-12% and all showed a monophyletic ancestry representing splitting from a single clade and thereby confirmed genus level taxonomy. The study concludes that a vicariance may be the probable reason for the splitting up of this genus into different geographical areas and caused the reproductive isolation of the same genus which in turn leads to the formation of different species. Keywords : Odonata, Neurothemis tullia, sexual dimorphism, mitochondrial COI gene, predators and evolutionary divergence Introduction Neurothemis tullia is a dragonfly species, common species, commonly called as Pied Paddy Skimmer, widely distributed in south and southeast Asia. It is a small black dragonfly with black and white (male) or brown and black (female) wings with abdomen mm and hind wing with mm long. They abundantly found as large colonies in swamps and heavily-weeded tanks, aquatic weeds usually flies more than five feet above the ground with a weak and slow fluttering flight. They usually have two or three generations per year and the nymphal stage are found in stagnant water in and around rice fields. They are important predators of rice pests such as plant hoppers, leafhoppers, and rice-stem borers (Che-salmah et al., 1999). Odonate larvae found in rice fields can be used to control mosquito larvae. Most adult Odonates exhibits sexual dimorphism and have an important role in the courtship behaviour and it is restricted to certain geographical areas. In the females 2 or more clearly different phenotypes existing in the same population of a species, namely polymorphism and it is based on colour not on morphology. Female members of certain Anisopteran libellulidae Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 110

2 especially certain genera such as Crocothemis, Neurothemis and Sympatrum show a different colour pattern on their wings which mimics the colour pattern of males known as andromorphic (Corbet, 1999 and Prasad, 2000). Neurothemis tullia shows such a kind of female polymorphism. Molecular technique is a powerful tool for the study of insect population ecology and insect systematics. Analysis of mitochondrial DNA (mt DNA) is particularly useful as molecular marker to discriminating between closely related species and to monitor a specific population in the field. Mitochondrial genes have a high copy number allowing them to be recovered from trace samples compared to nuclear DNA. These genes typically lack recombination and promoted to the fixation of mtdna halotypes for species identification (Avise et al., 1984). Molecular phylogeny analysis using mitochondrial COI gene sequences were extensively conducted in various insect groups ranging Heteroptera (Sreejith and Sebastian, 2014), Lepidoptera (Akhilesh and Sebastian 2014 and Sebastian, 2014) Diptera (Bindu and Sebastian, 2014; Pavana and Sebastian, 2014), and Hymenoptera (Rukhsana and Sebastian, 2014). The present study analyses the nucleotide sequence of cytochrome oxidase I gene among male, female and andromorphic female of Neurothemis tullia species in order to check any variation can be found in this sexually dimorphic species and also to elucidate its phylogenetic status. Methodology Male, female and andromorphic female members of Neurothemis tullia were collected from the field by hand sweep netting method. The morphological and taxonomical characters were cross checked with standard references, museum specimens, expert consultation and field guides of Odonates (Fraser, 1957). The identified specimens were stored in separate vials in 70% ethanol at 20 C as voucher specimens for future reference. Extraction of DNA was made from the thoracic legs of male, female and andromorphic female members of N. tullia species. The tissue was homogenized and genomic DNA in the homogenate was isolated using Genei Ultrapure DNA Pre Kit. About 2 ng of genomic DNA was amplified for mitochondrial cytochrome oxidase subunit Ι (CO Ι) gen e using the forward primer, 5'-CATTGGAGATGACCAAATT TA-3' and reverse primer with nucleotide sequence 5'-TCAGTTAATAGTATAGTAATAGC-3'. The PCR product was column purified using Mo Bio Ultra Clean PCR Cleanup Kit (Mo Bio Laboratories, Inc. California). The purified PCR product was sequenced using Sanger s technique (Sanger and Coulson, 1975). Sequences obtained were assembled using ClustalW and the consensus was taken for the analysis. The final sequence was searched for its similarity using BLAST of NCBI ( Phylo - genetic tree was plotted with a Neighbour Joining method using MEGA6 software (Tamura et al., 2013). Results and Discussion The present study revealed the partial cytochrome oxidase I gene of male, female, and andromorphic female species of N. tullia, (Fig.-1) which yielded a product of 351 bp DNA (GenBank Accession: KP ). The female polymorphism is one of the mechanisms exhibited by this species. Andromorphic female (Polymorphic female) of N. tullia has been reported from the Northeastern states of India (Kumar, 1988 and Mitra, 1991). Most of the hypothesis states that andromorphic females have the advantage that are not recognized by the mate searching males and can avoid unnecessary long copulations and thereby dedicating this time to feeding and egg Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 111

3 Jisha Krishnan and Sebastian, IJALS, Volume (8) Issue (2) May RESEARCH ARTICLE (a) Female (b) Male (c) Anandromorphic female Fig.- 1. Sexually dimorphic members of Neurothemis tullia Table - 1. Percentage of sequence divergence of Neurothemis tullia from Kerala with other odonate species Species Name Neurothemis tullia (Kerala) Neurothemis tullia (Mizoram) Neurothemis ramburii (Japan) Neurothemisintermedia (Mizoram) Neurothemis fluctans (Japan) Neurothemis terminate (Japan) Orthetrum juliafalsum (Germany) Acisoma panorpoides (Mizoram) Trithemis aurora (Japan) GenBank Accession KP KC AB KC AB AB KC KC AB % of Divergence 0 1% 8% 10% 10% 12% 13% 14% Trithemis aurora (Japan) AB % Fig.- 2. Phylogenetic tree of Neurothmis tullia constructed by Neighbour-joining method Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 112

4 maturation. The study revealed that gene sequences were found to be similar in male, female and andromorphic female. Hence it is clear that this kind of female polymorphism is contributed mainly by the environment. Sexual dimorphism in the body colouration, body patterns, the wing spot etc, of male and female species of most of the dragonflies causes the misidentification of species. This is also a character exhibited by this species. The male, female and andromorphic female has same base pair length DNA, which confirmed they belong to the same species. Phylogenetic tree constructed by the neighbour joining method showed the relationship of different species of Neurothemis and other Libellulidae members. N. tullia found in both Kerala and Tamilnadu showed sister clade relationships with a maximum bootstrap value of 100 and thereby confirmed both genus and species level taxonomy (Fig.- 2). Also the other two members like N. fluctans and N. intermedia were found very close together than other members and also share a sister clade relation (Fig.- 2). Even though different species of Neurothemis have a sequence divergence in the range of 1-12% they all showed a monophyletic ancestry which is originating from the clade having the bootstrap value of 74 (Table - 1). All the different species of Neurothemis found in different geographical areas may be the result of vicariance speciation. Due to continental shift and differences in climate, predation, food sources, and other factors in the newly established habitat causes the splitting of the ancestral species into many species and leads to their reproductive isolation. Thus, it can be concluded that molecular identification tools like DNA barcoding can be used as an effective tool for confirming taxonomy and phylogenetic relationships. Council for Science Technology and Environment, Thiruvananthapuram under Science Research Scheme is gratefully acknowledged. References Akhilesh, V. P. and Sebastian, C.D Molecular barcoding and phylogeny analysis of Herpetogramma stultalis (Lepidoptera: Crambidae) using cytochrome oxidase subunit I gene sequence. Int. J. Adv. Lif. Sci., 7: Avise, J.C., Neigel, J.E. and Arnold, E Demographic influences on the mitochondrial DNA lineage survivorship in animal population. J. Mol. Evol., 20: Bindu, P. U. and Sebastian, C. D Genetic structure of mitochondrial cytochrome oxidase subunit I gene of the mosquito, Armigeres subalbatus. International Journal of Research., 1(10): Che-salmah, M.R., Hassan, S.T.S., Ali, A. and Abu Hassan Life history of Neurothemis tullia in a tropical rainfed rice field. Notule Odonatologicae, 28: Corbet, P.S Dragonfly behaviour and Ecology of Odonata. Harley Books, Coichester, England Fraser, F.C A reclassification of the order Odonata. Royal Zoological Society, New South Wales. pp Kumar, A On the Andromorphic female of Neurothemis tullia (Anisoptera: Libellulidae). Notule Odonatologicae., 3: Mitra, T.R Another record of an andromorphic female member of Neurothemis tullia. Notule Odonatologicae., 3: Acknowledgement The financial assistance from Kerala State Pavana, E. and Sebastian, C.D Genetic diversity and phylogenetic analysis of lepidopteran species Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 113

5 by molecular barcoding using CO I gene sequences. Int. J. Sc. Res., 3(5): Prasad, M., Kulkarni, P.P., Talimale, S.S New record of andromorphic females in 2 species of Neurothemis dragonflies (Odonata : Libellulidae) from central India. Bionotes., 2: Priya Bhaskaran, K.P. and Sebastian, C.D Molecular barcoding of green bottle fly, Lucilia sericata (Diptera: Calliphoridae) using COI gene sequences. J. Entomol. Zool. Studies., 3(1): Rukhsana, K. and Sebastian, C.D Deciphering the molecular phylogenetics of the Asian honey bee, Apis cerana (Hymenoptera: Apidae) and inferring the phylogeographical relationships using DNA barcoding. J. Entomol. Zool. Studies., 2(4): Sanger, F. and Coulson, A.R A rapid method for determining the sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol., 94 (3): Sreejith, K. and Sebastian, C.D Phylogenetic analysis and sequencing of the mitochondrial cytochrome oxidase subunit I (COI) of white backed plant hopper, Sogatella furcifera (Horvath). Int. Res. J. Pharmacy, 5 (12): Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution., 30: Corresponding Author : C.D. Sebastian, Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala , India, drcdsebastian@gmail.com 2015, IJALS. All Rights Reserved. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 114