DNA BARCODING, PHYLOGENETIC DIVERSITY STUDIES OF ETROPLUS SURATENSIS FISH FROM POORANANKUPPAM BRACKISH WATER, PUDUCHERRY

Sachithanandam V.1, Mohan P.M.1, Muruganandam N.2, Chaaithanya I.K.2, Arun Kumar P3, Siva Sankar R3

ijcrr 1Department of Ocean Studies and Marine Biology, Pondicherry University, Vol 04 issue 08 Andaman 2Regional Medical Research Centre (ICMR), Andaman Category: Research 3Department of Ecology and Environmental Sciences, Pondicherry University, Received on:29/01/12 Puducherry Revised on:16/02/12 E-mail of Corresponding Author: [email protected] Accepted on:03/03/12

ABSTRACT Etroplus suratensis is known for the high commercial value fish available in South India. The identification of the species of this fish cumbersome and inaccurate in different life stages of the fish. Therefore, DNA sequence of cytochrome Oxidase subunit I gene was analysed for the species identification and phylogenetic relationship of the species. The average genetic distance of conspecifics species value was found to be 0.005%. The present work suggests that COI sequence provides sufficient information on phylogenetic and evolutionary relationship to distinguish the Etroplus suratensis species, the brackish waters species of pearl spots, unambiguously. Further, this work revealed that every species having individual genetic distances depended upon the environmental stress and water quality, which play an important role for its minor morphometric variations. Therefore, it was concluded that a DNA COI barcoding tool can be used for fish identification by non technical personnel (other than taxonomist). ______Keywords: DNA barcoding, COI, brackish of more than US$ 3/kg2. These fish is available water, Pooranankuppam and Etroplus suratensis throughout the year. The average production is about 1000 kg/ha/year over 8-10 month grow- INTRODUCTION out period. The chromids or the pearl-spots (Family: Morphometric studies are not only essential to Cichlidae) form an important group among the understand the taxonomy but also the health of a brackish water fishes of the tropics. One of the species (including reproduction) in an genus Etroplus contains E. suratensis fish is environment. The morphometric features of the inhabitant in fresh water and brackish water in fish are unique to the species whereas the southern India. E. suratensis has many desirable variations in its feature are probably related to features which make them ideal fishes for the habit and habitat3. aquaculture like wide salinity tolerance, ability Morphometric measurements have been widely to breed in confined waters, fast rate of growth, used to discriminate populations of various fish good body weight, tasty flesh, highly adaptable species4-6. Fishes are considered to be feeding habits, robust, sturdy body1. phenotypically more variable than most other Experimental cultures of this species show its vertebrates, having relatively higher within- potential for polyculture and integrated farming population coefficients of variation of with poultry. In addition to export, it has high phenotypic characters. Genetic polymorphism or demand in the local market and fetches a price environmental factors may induce 33 International Journal of Current Research and Review www.ijcrr.com Vol. 04 issue 08 April 2012

morphological variability among spatially vulnerability to exploitation of tropical reef separated fish populations7, and phenotypic fishes19. plasticity in fish morphology has been As the morphometric measurements could lead documented for various species, including to misidentification of the species in different cichlids8,9. E. suratensis is known to have life stages of fish especially E. suratensis, which variations in various morphological features would affect the conservation strategy and the which are dependent on the geographical market value of the same. Molecular taxonomy partition. Further environmental comparisons of appears to be the best tool for the species these estuaries would be worthwhile in identification and advantageous over the other understanding the evolution of such variations. method of taxonomy, so the effort was taken to In addition, genetic investigations of the identify the E. suratensis through molecular variation and differentiation involving more taxonomy. estuarine samples of E. suratensis will be useful in substantiating the conclusions. The genotypic METHODS and phenotypic variation of species is a pre- Study Area, Sample collection and requisite in conserving them. DNA barcoding is preservation: highly efficient method in the analysis of genetic Fishes were collected from local fish landings at divergences among species as well as for intra Pondicherry brackish area of Pooranankuppam species-level identifications10. (Fig.1). The identification of fishes was done as Among the marine living organisms of the Indo- described in FAO14. A piece of muscle in the West Pacific, Teleosts are among the best- lateral line was collected and stored in 95% described, even though their systematics and ethyl alcohol at -20 C for DNA extraction. The taxonomy still need considerable research DNA Isolation and PCR condition work was effort11,12. Southeast Asia has been identified as carried out in Department of Ocean studies and one of the world‘s biodiversity hotspots based Marine Biology centre at Port Blair, Andaman on both plant and animal diversity13. Many time and Nicobar Islands. taxonomic ambiguities exist due to Molecular Taxonomy: morphological and meristic similarities. Modern Total DNA extracted from 0.25g of muscle by taxonomic work includes analysis of a host of the standard proteinase-K/ phenol-chloroform- other traits, including anatomy, physiology, isoamyl alcohol-ethanol method20. PCR behaviour, genes, and geography, yet amplification of a 650bp DNA fragment coding morphological traits remain cornerstone14. COI gene of the mitochondrial (mt) DNA In such circumstances, DNA barcodes has genome was amplified using published primer revealed that this could be helpful even for set11. PCR components and conditions for 50 l 15 larval stage fish taxonomical identification . To reaction were as described in our previous facilitate DNA barcode identification of fishes, work21. The PCR product was resolved in 1% regional working groups are conjoining under Agarose gel and visualized using Gel Doc the Fish Barcode of Life (FISH-BOL) System, to confirm the presence of amplified 16 initiative , which seeks to establish a barcode product sized 650 bp. Nucleotide sequencing 17 reference sequence library for all fishes . The was performed using BigDye Terminator Cycle phylogenetic systems, in combination with Sequencing kit, following manufacturer‘s conservation genetics, provide a critical frame instructions (Applied Bio-systems, Foster City, 18 work for understanding diversity and predict CA, USA). 34 International Journal of Current Research and Review www.ijcrr.com Vol. 04 issue 08 April 2012

Sequence Analysis: The NJ and K2P genetic distances were created The DNA sequences of phenotypically identified to provide a graphic representation of the fishes were assembled using the SeqMan II patterns of divergences. Two distinct clad with version 5.03 (DNASTAR). The sequence of two sub clad of the same species were Etroplus suratensis reference sequences recognized with more than 90% bootstrap value. retrieved from the NCBI GenBank were aligned These two sub clad formation was identified using Clustal W pair-wise and multiple based on the independent assemblages of close alignment of MEGA version 4.122. Sequence related species with differences in region and divergence was calculated using the Kimura 2- environmental closeness. The results clearly parameter (K2P) model23 and the mid-point shows that every species having individual rooted Neighbour-joining (NJ) tree of K2P genetic distances depended upon survival of any distances was created to provide a graphic species adaptation of the environmental stress representation of the species divergence24 (Fig. and its water quality, which play an important 2). role of significant values of the genetic distances internally and morphometric minor variations RESULTS externally. DNA barcoding is a unique concept with many innovative attributes undertakes continuous DISCUSSION improvement in taxonomy. The estimated Fishes are largest group of vertebrates, which Etroplus suratensis species DNA sequences exhibits remarkable diversity of morphological were submitted to GenBank under the attributes and biological adaptations25. In these mentioned accession number in Table 1. NCBI circumstances fish taxonomist facing a large BLASTn result revealed that 21 reference problems while the identification of fishes. To sequences were matched with maximum identity overcome this problem, a morphology- based of Etroplus suratensis of Puducherry as identification combined with molecular based described in Table 2. The average genetic approach for the species identification using distance within the species (K2P) is 0.005. DNA barcoding would be an ideal tool26. This The species genetic evolutionary pair wise tool is an efficient method for species-level distance proximity was calculated by the species identification of the mitochondrial Cytochrome c similarity of genetic base pair. The Etroplus Oxidase I (COI) gene27. Mitochondrial DNA suratensis DOSMB species closely related to the (mtDNA) has been widely employed in species (FJ237544 – 0.006: GU5666028 – phylogenetic studies of animals because it 0.006: FJ347966 – 0.006 and AY263870 – evolves much more rapidly than nuclear DNA, 0.009) in the Indian waters and USA resulting in the accumulation of differences respectively (Table 3). The nucleotide between closely related species 28-30. composition of Etroplus suratensis from the This study provides the interspecific present studied species is A = 23.4%, T = heterogeneity which enhanced the efficiency of 29.9%, G = 18.6% and C = 28.1%. The average species identification through bar coding. This nucleotide composition of E. suratensis among was proved in Australian marine fishes11, the species level is noted as A = 23.48%, T = freshwater fish barcoding from Canadian31 and 30.08%, G = 18.1% and C = 28.36% (Table 4, carangid fishes from Indian waters32. The Fig.3). variations of phenotypic character of species are unique and it is probably related to the habit and 35 International Journal of Current Research and Review www.ijcrr.com Vol. 04 issue 08 April 2012

habitat among the variants of this species33. also informed that every species having Genetic variation of the green chromid has not individual genetic distances depended upon the been studied previously of the genus E. environmental stress and water quality which suratensis in Indian waters. play an important role for its minor In the studies of bar coding, it has been reported morphometric variations. Moreover, the mtDNA that K2P values between two species should be COI gene based identification provides high greater than 0.0227,34 and in e.g. Indian mosquito resolution in species identification in fishes. DNA barcoding average K2P values is 0.032935. The present bar code exhibited K2P pair wise ACKNOWLEDGMENTS genetic distances variation among the species The Authors express their sincere acknowledges level is 0.005. However, such variation has to Prof. J. A. K. Tareen, Vice-Chancellor of greater impact on the survival of the haplotypes Pondicherry University and the constant help and its evolution. and encouragement of Dr. P. Vijayachari, The efficiency of species identification by Director, Regional Medical Research Centre molecular method was enhanced by the (ICMR), Port Blair for the extension of facility interspecific heterogenetic relationship during this study. The manuscript valuable displayed36. Based on the above investigation it review and commands supported by Chandal Lal is clearly evident that the E. suratensis identified and Sayi Dev. We express thanks to the in Puducherry waters represented the haplotypes Pondicherry University and Central Marine species morphometrically, the other part of the Living Resources and Ecology (CMLRE) for India and USA waters. However, bar code funding this work. Authors acknowledge the results suggested that they are genetically varied. immense help received from the scholars whose Since, this species identified as haplotypes it articles are cited and included in references of may be of low level differences in this manuscript. The authors are also grateful to morphometrically because of low genetic authors/ editors/ publishers of all those articles, distances. Results of phylogenetic and journals and books from where the literature for evolutionary relationship of present study were this article has been reviewed and discussed. supported by earlier studies11. Further, it has also confirmed that the DNA barcoding help to REFERENCES recover phylogenetic information and to 1. Hora, S. L and PilIay, T. V. R. (1962). understand the relationship with the species as Hand·book on the fish culture in the well as Order level36. The present study also Indo·Pacific region. FAO Fish. BioI. Tech. supported that mtDNA COI barcode region pp. 14:124. offers best species identification, which is most 2. James, C. M. (2000). Potential of marine applicable and comparable with the mtDNA 12S fish farming in India: - 16S rRNA region sequences37. www\Grp\Grouper\Research\Economics\20 00\2103.htm pp. 1-3. CONCLUSION 3. Mauro José Cavalcanti., Leandro Rabello The study concludes that the fish which Monteiro and Paulo Roberto Duarte Lopes. identified morphometrically and DNA barcoding (1999). Landmarkbased Morphometric method using COI gene sequence are one and Analysis in Selected Species of Serranid same species of E. suratensis, the brackish Fishes (Perciformes: Teleostei) Zool.stud. waters species of pearl spots. Further, this result 38: pp. 287294. 36 International Journal of Current Research and Review www.ijcrr.com Vol. 04 issue 08 April 2012

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Table 1 Etroplus suratensis species and their mtDNA COI Sequences GenBank accession Number

Sl No Name of the species Common Name NCBI GenBank Number 1 Family:Cichlidae - Pearl spots - chromids JN228382 Etroplus suratensis

Table 2 BALSTn SEARCH REQUEST AND RESULTS COI PUBLIC RECORDS DATABASE:

Table 2a Identification Summary: Taxonomic Level Taxon Assignment Probability of Placement (%) phylum Chordata 100 class Actinopterygii 100 order Perciformes 100 family Cichlidae 100 genus Etroplus 100 species Etroplus suratensis 99.1

Table 2b A species level match has been made Sl. Order Family Genus Species Specimen No‘s Similarity (%) 1 Perciformes Cichlidae Etroplus suratensis 99.07 2 Perciformes Cichlidae Etroplus suratensis 98.92 3 Perciformes Cichlidae Etroplus suratensis 98.77 4 Characiformes Characidae Pseudocorynopoma doriae 85.23 5 Perciformes Haemulidae Pomadasys hasta 82.33 6 Perciformes Haemulidae Pomadasys hasta 82.33 7 Perciformes Haemulidae Pomadasys hasta 82.33 8 Perciformes Haemulidae Pomadasys hasta 82.33 9 Perciformes Haemulidae Pomadasys hasta 82.33 10 Characiformes Alestidae longipinnis longipinnis 82.31 11 Characiformes Alestidae longipinnis longipinnis 82.27 12 Perciformes Cichlidae Paretroplus damii 82.25 13 Perciformes Cichlidae Etroplus maculatus 82.22 14 Characiformes Alestidae longipinnis 82.19 15 Beloniformes Hemiramphidae Hyporhamphus quoyi 82.18 16 Perciformes Lutjanidae Lutjanus johnii 82.18 17 Beloniformes Hemiramphidae Hyporhamphus limbatus 82.18 18 Beloniformes Hemiramphidae Hyporhamphus limbatus 82.18 19 Beloniformes Hemiramphidae Hyporhamphus limbatus 82.18 20 Beloniformes Hemiramphidae Hyporhamphus limbatus 82.18

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Table 3 Kimura two parameter Genetic distances Sl.No Species Name/ GenBank No‟s K2P Country 1 Etroplus suratensis DOSMB 0.000 Pondicherry 2 FJ237544-Etroplus suratensis 0.006 India" 10.02 N 76.13 E 3 FJ347966 Etroplus suratensis 0.006 India" 10.02 N 76.13 E 4 GU566028-Etroplus suratensis 0.006 Kerala, India" 5 AY263870 Etroplus suratensis 0.007 USA Average Interspecific K2P Distances is 0.005

Table 4 Nucleotide composition: Sl. No Species Names and GenBank nos. T % C% A% G% 1 Etroplus suratensis DOSMB 29.9 28.1 23.4 18.6 2 FJ237544 Etroplus suratensis 30.4 28.4 23.4 17.9 3 AY263870 Etroplus suratensis 30.2 28.0 23.6 18.2 4 FJ347966 Etroplus suratensis 30.5 28.2 23.4 17.9 5 GU566028 Etroplus suratensis 29.4 29.1 23.6 17.9 Average base pair composition 30.08 28.36 23.48 18.1

Fig 1 Study Area

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Fig 2 Kimura two parameter (K2P) Genetic distances Values

Fig 3 Nucleotide composition of E. suratensis with references sequences species.

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69 Etroplus suratensisDOSMB AY263870-Etroplus suratensis

100 FJ237544-Etroplus suratensis

GU566028-Etroplus suratensis

FJ347966-Etroplus suratensis

DQ119195-Herotilapia multispinosa

100 HQ654752-Parachromis managuensis 100 HQ654751-Parachromis managuensis

GU817266-Australoheros facetus ssp

HQ956111-Scorpaeniformes sp. 54 FJ583406-Forcipiger flavissimus

FJ583412-Forcipiger flavissimus 100 FJ583407-Forcipiger flavissimus 74 FJ583411-Forcipiger flavissimus

70 HM882986-Hepsetus odoe

81 HM882988-Hepsetus odoe HM882985-Hepsetus odoe 100 63 HM882987-Hepsetus odoe

HM882978-Hepsetus odoe

64 HM882979-Hepsetus odoe

HQ573341-Perciformes sp.

Fig 4 Neighbour-Joining (NJ) Method for Phylogenetic analysis and Evolutionary relationships of E. suratensis with NCBI references sequence The bootstrap test (1000 replicates) is shown next to the branches length is = 0.41765812 [Felsenstein 1985]. The evolutionary distances were computed using the Maximum Composite Likelihood method. Codon positions included were 1st+2nd+3rd. Phylogenetic analyses were conducted in MEGA4 [Tamura, et al 2007].

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