Journal of Experimental Biology and Agricultural Sciences, April - 2015; Volume – 3(2)

Journal of Experimental Biology and Agricultural Sciences

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ISSN No. 2320 – 8694

PHYLOGENETIC ANALYSIS OF FRESHWATER Corbicula regularis BY 18S rRNA GENE SEQUENCING

Magare V N1, Kulkarnii C P2, Maurya C B3 , Patil R C4,* and Upadhye M V5

1Principal, Department of Zoology, Kirti M. Doongursee College of Arts, Science and Commerce, Dadar (W), Mumbai- 400028, INDIA 2Department of Chemistry, Kirti M. Doongursee College of Arts, Science and Commerce, Dadar (W), Mumbai - 400028, INDIA 3Department of Chemistry, G.N. Khalsa College of Arts, Science & Commerce, Matunga (E), Mumbai - 400019, INDIA 4Department of Microbiology, Bhavan’s College, Andheri (W), Mumbai - 400058, INDIA 5Department of Life Sciences, University of Mumbai, Santacruz (E), Mumbai - 400098, INDIA

Received – April 05, 2015; Revision – April 15, 2015; Accepted – April 25, 2015 Available Online – April 25, 2015

DOI: http://dx.doi.org10.18006/2015.3(2).213.219

KEYWORDS ABSTRACT Phylogenetics Corbicula regularis is a freshwater mussel found in the Indian sub-continent. In the present study, 18S ribosomal gene phylogenetic characterization of this important bivalve was attempted using 18S ribosomal RNA gene markers. Genomic DNA was extracted and 18S rRNA gene was amplified by universal primers. The Corbicula regularis amplification product was sequenced and compared with the nucleotide databases available online to evaluate phylogenetic relationship of the under study. Results indicated that 18S rRNA gene Freshwater sequences of C. regularis showed high degree of similarity to another freshwater mussel, C. fluminea. This work constitutes the first ever sequence deposition of the C. regularis in the nucleotide databases State of Maharashtra highlighting the usefulness of 18S ribosomal gene markers for phylogenetic analysis.

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* Corresponding author E-mail: [email protected] (Patil R C)

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214 Patil et al

1 Introduction and different body parts were carefully dissected and separated. Freshly dissected body parts were subjected to Freshwater mussels are one of the most important components isolation of genomic DNA. of freshwater aquatic ecosystem. These are filter feeders that siphon phytoplankton and organic detritus 2.2 Isolation of Genomic DNA suspended or dissolved in the water column. In this manner, they serve as indicators of ecosystem health and help to sustain Genomic DNA from mantle tissue of the bivalve was extracted the water quality (Goudreau et al., 1993). Apart from this role, by following the protocol standardized by Upadhye et al. like other bivalves, they also serve as food for benthic (2011). Briefly, 100 mg fresh mantle tissue was ground in 2 ml invertebrates, fish, birds and some mammals (Langdon & of extraction buffer by adding buffer in parts to obtain a Newell, 1996). They have also been used for making buttons, homogenous suspension. This suspension was centrifuged at as food, source material for tools and ornamental objects and 10000 rpm for 5 min and supernatant was discarded. To the recently for cultured pearl production (Upadhye et al., 2011). pellet, 700 µl of suspension buffer and 80 µl of SDS were added and incubated at 65°C for 30 min. To this mixture 200 Though India has a rich diversity of freshwater pearly mussels µl ammonium acetate was added and incubated for another 30 with a widespread distribution of around 52 different species of min. After incubation, 500 µl chloroform - isoamyl alcohol freshwater mussels; the marine molluscs have received more (24:1) mixture was added and mixed it by several inversions. attention because of their aesthetic and gastronomic appeals This mixture was centrifuged at 10000 rpm for 8 min. Top and the use of freshwater mussels for commercial applications aqueous layer was collected in a new microfuge tube and equal is in infancy (Subba Rao, 1993). In India, in comparison to volume of absolute ethanol was added. This mixture was kept marine mussels, freshwater molluscs are drab coloured and in ice cold condition for 1 hour and after incubation centrifuge have attracted less attention. Notwithstanding their importance at 10000 rpm for 15 min. The pellet was dissolved in 200 µl in the ecosystem, freshwater molluscs are less explored and elution buffer and stored at 4°C. Agarose gel electrophoresis studied. (1.0 % agarose) was carried out to check the presence and quality of DNA. C. regularis is an important member of this Indian freshwater mussel fauna. This bivalve belongs to family Corbiculidae and 2.3 18S rRNA Gene Amplification using PCR was earlier reported to be quite common all over the country (Subba Rao, 1989). However, due to increasing anthropogenic Isolated bivalve DNA was used for phylogenetic analysis. PCR activities and pollution resulting in habitat destruction, this amplification was carried out with reaction mixture of 50 µl mussel has disappeared from many locations where it was containing was 25 µl of PCR master mix, 1 µl of template earlier used to be abundantly present. Work done on this DNA (diluted to 10-50 ng/µl), 1.2 µl of forward primer UnivF- freshwater bivalve has been restricted to studying its 15 (5’ CTG CCA GTA GTC ATA TGC) (10 µM), 1.2 µl of physiological, biochemical and toxicological aspects reverse primer Univ R-1765 (5’ ACC TTG TTA CGA CTT (Mudkhede & Nagabhushanam, 1977; Lomte & Jadhav, 1982; TAC) (10 µM) and 8.1 µl of nuclease free water in a PCR tube Mali & Afsar, 2010) and there are absolutely no reports in Biometra thermal cycler. PCR conditions were as follows: available on any scientific study on this mussel with the aid of initial denaturation at 95°C for 2 min, followed by 30 cycles of modern sophisticated molecular biological tools. each 95°C for 1 min, primer annealing at 52°C for 1 min and final primer extension at 72°C for 5 min. Amplified gene Considering the fact that its resources are getting depleted products were visualized on 1% agarose gel under UV rapidly and this important mussel could soon be on the verge visibility @ 254 nm. PCR products were purified with a kit of getting endangered, an attempt was made in the present and sequenced by using forward and reverse sequencing study to characterize C. regularis using amplified sequence of primers. DNA sequencing was performed on automatic DNA its 18S ribosomal RNA gene and study its phylogenetic sequencer supplied by Applied Biosystems using BigDye position with respect to available gene sequences of other Terminator v 3.1 Cycle Sequencing Kit. mussels. 2.4 BLAST and Phylogenetic analysis 2 Materials and Methods The amplified PCR product was partially sequenced and the 2.1 Sample collection gene sequences were analyzed against the DNA database of Japan (DDBJ) database using BLAST N (nucleotide- Thirty healthy specimens of C. regularis were hand collected nucleotide BLAST) program packages (Zhang et al., 2000) and from Darna river near Nashik city in the State of Maharashtra. matched to known 18S rRNA gene sequences. After Collected samples were brought alive in plastic containers to identifying the phylogenetic neighbors of the study animal, the the Department of Zoology, Kirti M. Doongursee College, sequences were also analyzed by using CLUSTAL W and Mumbai and acclimatized for 24 hours in separate glass Phylodraw softwares (Choi et al., 2000). Distance between the aquaria. After acclimatization, 15 live mussels were sacrificed genes for each pair was found out and phylogenetic tree was

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Phylogenetic analysis of freshwater mussel corbicula regularis by 18s rRNA gene sequencing., 215 constructed. Sequence of C. regularis was submitted to DNA morphological data matrices. Since last couple of decades, Database of Japan (DDBJ). phylogeny studies of various molluscan taxa with the aid of ribosomal marker gene have been a reliable tool in elucidating 2.5 Comparison of sequence with other freshwater mussels of the phyletic relationships below the class level (Taylor et al., Maharashtra 2007a; Taylor et al.,2007b). However, with only couple of report by Jadhav and Jamkhedkar (2009) who studied The 18S rRNA gene sequence of C. regularis was analyzed by phylogeny of freshwater mussel, L. corrianus, and that of Clustal W software to find out the pair distance between 18S Upadhye et al., (2011) who studied phylogeny of freshwater rRNA gene sequence of other freshwater mussels present in the mussel, P. corrugata there are absolutely no other reports Maharashtra state such as Parreysia corrugata (accession available on DNA sequencing of freshwater mussels from the number AB533279) and Lamellidens corrianus (accession state of Maharashtra so the present study was undertaken. number EU394233). Phylogenetic tree was constructed for these three species using Phylodraw software. The 18S rRNA gene of freshwater mussel C. regularis collected from Darana River in the state of Maharashtra when 3 Results amplified using universal 18S rDNA targeted oligonucleotide primers UnivF-15 and UnivR-1765 produced good quality Phylogenetics is the study of evolutionary relatedness among PCR amplification product. This amplified PCR products was various groups of organisms (e.g. species, populations), which partially sequenced to obtain a sequence of 687 nucleotide is discovered through molecular sequencing data and bases.

Figure 1 Phylogenetic tree of freshwater mussel C. regularis with respect to its Phylogenetic neighbours as indicated by a rectangle cladogram.

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216 Patil et al

Table 1 BLAST results for Corbicula regularis 18S rRNA gene sequence from DDBJ database.

Accession Organism Description Length Identities and % Number similarity EF613239 Corbicula fluminea 18S ribosomal RNA gene, Freshwater mussel 1723 684/687 (99%) partial sequence. AM774558 Corbicula fluminea partial 18S rRNA gene Freshwater mussel 1776 684/687 (99%) AF120557 Corbicula fluminea 18S ribosomal RNA gene, Freshwater mussel 1777 684/687 (99%) complete sequence FM999789 Cyrenoida floridana partial 18S rRNA gene Florida Marsh marine 1772 682/687 (99%) AM774559 Glauconome virens partial 18S rRNA gene Greenish glauconomya 1776 679/687 (98%) Marine clam AM774567 disrupta partial 18S rRNA gene Matine clam 1778 679/687 (98%) EF426294 Saxidomus purpuratus 18S ribosomal RNA gene, Marine clam 1829 678/687 (98%) complete sequence AM774563 partial 18S rRNA gene Mahogany clam 1774 677/687 (98%) Marine EF426289 Cyclina sinensis 18S ribosomal RNA gene, Oriental cylcina 1838 676/687 (98%) complete sequence. Marine clam AM774564 Calyptogena pacifica partial 18S rRNA gene Marine clam 1777 676/687 (98%) AF120556 Calyptogena magnifica 18S ribosomal RNA Marine clam 1777 676/687 (98%) gene, complete sequence. U93555 Arctica islandica 18S ribosomal RNA gene, Mahogany clam 1771 676/687 (98%) partial sequence. Marine DQ279943 Chamelea striatula 18S ribosomal RNA gene, Marine clam 1778 677/687 (98%) partial sequence AJ007613 18S rRNA gene Smooth clam Marine 1780 677/687 (98%)

Figure 2 Phylogenetic position of C. regularis with respect to other freshwater mussels in the state of Maharashtra as indicated by slanted cladogram.

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Phylogenetic analysis of freshwater mussel corbicula regularis by 18s rRNA gene sequencing., 217 3.1 BLAST analysis of sequences of C. regularis Asia. This species has also been introduced into many parts of the world, including North America and Europe. However, BLASTN tool of DDBJ database indicated that the studied other phylogenetic neighobours were all found to be marine species C. regularis was showing 99% identical sequencing in . Next closest phylogenetic neighbour was C. floridana terms of its 18S rRNA gene sequence of freshwater mussel which was studied by Taylor et al. (2009). C. floridana (family Corbicula fluminea. These two species were showing 684 out Cyrenoididae) is commonly called as Florida marsh clam and it of the 687 similar nucleotides. The 18S rRNA gene sequence is commonly distributed in the West Atlantic sea region. also showed close homology with sequences of couple of more Species under present study was also showed similarity with C. fluminea species present in the GenBank / EMBL / DDBJ the sequence of G. virens which was also studied by Taylor et nucleotide database. It also showed 99% similarity with al. (2007b). G. virens commonly called as Greenish Florida marsh marine clam Cyrenoida floridana but in this glauconomya and it is a marine clam belonging to case the match was 682 out of 687 of C. regularis (Table 1). family Glauconomidae. This clam is found in seas of Indo- Many other marine clams such as Glauconome virens, Callista West Pacific; from Thailand and Philippines to northern disrupta, Saxidomus purpuratus, Arctica islandica, Cyclina Australia. sinensis, Calyptogena pacifica, Calyptogena magnifica, Chamelea striatula and Callista chione were also found to be Sequence alignment with BLAST tool further indicated that Phylogenetic neighbor of the study animal as their 18S rRNA partial sequence of C. regularis showed resemblance with gene sequence showed 98% similarity with the partial 18S different marine clams belonging to family , rRNA gene sequence of C. regularis (Table 1). Vesicomyidae, Arcticidae which is comprised of commercially important clams (Taylor et al., 2007b). 3.2 Construction of Phylogenetic tree by using Phylodraw software Therefore, results in the present study clearly indicate that C. regularis is 99% phylogenetically closer to the freshwater After preliminary analysis with CLUSTAL W software, data bivalve C. fluminea. Jadhav & Jamkhedkar (2009) also was subjected to Phylodraw software for constructing reported that the partial sequences of freshwater mussel, L. phylogenetic tree. Tree drawn in phylogram format (Figure 1) corrianus from Maharashtra showed approximately 99.2% indicates the Phylogenetic position of C. regularis. similarity with the fresh water bivalves such as Inversidens japanesis, Unio pictorum, Unio douglasiaei and Anodonta 3.3 Comparison of sequence with other freshwater mussels of cygnea suggesting that freshwater mussels should be Maharashtra phylogenetically closest to other freshwater mussels. However, sequences of C. regularis in the present study also showed Results indicated that distance between C. regularis and P. high percentage of similarity with sequences of a number of corrugata was 0.72772 whereas pair distance between C. other marine bivalve mussels suggesting that there is an regularis and Lamellidens corrianus was found out to be evolutionary phylogenetic link between freshwater and marine 0.68317. This indicated that C. regularis was phylogenetically mussels. Upadhye et al. (2011) had also made similar more closer to Lamellidens corrianus than P. corrugata. Based observation in case of 18S rRNA gene sequences of freshwater on this initial analysis, a slanted cladogram for these bivalve P. corrugata. freshwater mussels from Maharashtra state was constructed (Figure 2). Reliable species identification is basic to all ecological, genetic and conservation studies. Identification of bivalve species is 3.4 Submission of 18S rRNA gene sequences to DDBJ mostly done by the keys based on their morphological characters and in many instances these characters tend to The partial sequence of amplified 18S rRNA gene of C. overlap making the species distinction difficult. Some regularis from Maharashtra state has been submitted to researchers suggest the importance of both anatomical and collaborative nucleotide databases of GenBank / EMBL / reproductive features to classify freshwater mussels. However, DDBJ, where the submitted sequence was checked, approved these different schemes have led to different and inconsistent and provided with accession number. The information on the classification of unionids (Hoeh et al., 2001) and there are still same is available on accession number AB562350. certain taxonomic confusions and uncertainties in taxonomical classification of freshwater malacofauna. Identification of 4 Discussion some species within the genus Corbicula remains a challenging exercise due to the wide range of shell form and Therefore the current study clearly indicates that partial 18S shell color variations displayed by individuals in different rRNA gene sequence of C. regularis is closely related to 18S habitats. Species belonging to genus Corbicula can easily be ribosomal RNA gene sequences of freshwater Asiatic clam, subjected to this kind of identification problem since both C. Corbicula fluminea and this fact is clearly suggesting that this regularis and C. fluminea closely resemble each other in terms species is a phylogenetic neighbour of C. fluminea. Species C. of morphological characters and can be found in the same fluminea also belongs to family Corbiculidae and is found both location leading to confusions. Failure to resolve freshwater in lottic and lentic habitats over its native range in southeastern mussel classification inconsistencies could hamper the creation

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218 Patil et al of conservation and/or management measures, which is usually Maharashtra by 28s rRNA and 18s rRNA sequences. Research impacted by residential, commercial and municipal Journal of Biotechnology 4: 37-44. development. Thus, freshwater mussel biologists continue to look for ways to resolve inconsistencies in species Langdon CJ, Newell RIE (1996) Digestion and nutrition in identification and develop a single uniform scheme for unionid larvae and adults. In: Kennedy VS, Newell RIE, Eble AF classification (Molina, 2004). Appropriate identification of (Eds.) The Eastern , virginica. Maryland species is also essential for the selection of correct candidate Sea Grant College, College Park, USA, Pp. 231-260. species of freshwater mussel for aquaculture and pearl culture operations and for other biological studies. Lomte VS, Jadhav ML (1982) Effects of toxic compounds on oxygen consumption in the freshwater bivalve, Corbicula So, the results of the present study clearly indicate usefulness regularis (Prime, 1860). Comparative Physiology and Ecology of 18S rRNA gene sequencing to confirm the phylogenetic 7: 37-38. position of freshwater bivalve C. regularis. Rice et al. (1993), Adamkewicz et al. (1997), Maruyama et al. (1998); Canapa et Mali RP, Afsar SK (2010) Regulation of nitrogenous excretion al. (1999) and Jadhav & Jamkhedkar (2009) and Upadhye et al. under influence of temperature variations in freshwater clam, (2011) have already suggested that the 18S rRNA comparative Corbicula regularis. Asian Journal of Animal Sciences 5: 209- studies of various molluscan taxa have been reliable in 211.n elucidating the phyletic relationships below the class level. Maruyama T, Ishikura M, Yamazaki S, Kanai S (1998) Conflict of interest Molecular phylogeny of zooxanthellate bivalves. The Biological Bulletin 195: 70-77. Authors would hereby like to declare that there is no conflict of interests that could possibly arise. Molina RE (2004) Morphological and Genetic Description of the Freshwater Mussel, Elliptio Complanata (Lightfoot, 1786) Acknowledgements in the Cape Fear River System, N.C. PhD. Thesis submitted to the North Carolina State University, USA. Authors would like to express their sincere thanks to Dr. P. R. Bhamre, KTHM College, Nashik, Dr. U. A. Jadhav of Mudkhede LM, Nagabhushanam R (1977) Heat tolerance of University of Mumbai and Dr. Archana Thakur, Goa for their the fresh water clam, Corbicula regularis. Marathwada valuable help and support during the course of this study. University Journal of Science, Natural History 16:151-154.

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