Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 Biomolecular Approach To Oligochaete Taxonomy Dr. Jaya. M 1* ,Dr. Aja. M2 and Dr. K. Vijayakumaran Nair3 1* Assistant Professor, Department of Zoology, Sree keralavarma College, Thrissur, Email: [email protected] 2Senior Research Fellow, Department of Zoology, University of Kerala, Kariavatom, Email: [email protected] 3Associate Professor, Department of Zoology, Mar Ivanios College, Thiruvananthapuram, Email: [email protected]

ABSTRACT This paper comprises the molecular approach for the identification of earthworm along with the traditional taxonomic method. The mitochondrial CO 1 gene of the Pontoscolex corethrurus (Glossoscolecidae), Travoscolides chengannures, Amynthas corticis, Perionyx sansibaricus (Megascolecidae), Progizzardus varadiamensis and Glyphidrilus annandalei (Almidae) were sequenced. The cytochrome-c oxidase I (CO1) exhibited a unique barcode to a particular species. The further exploration of mitochondrial diversity in earthworms will lead to major improvements in our understanding of the evolutionary pathways and rates of the mitochondrial genome Key words: Barcoding, cytochrome c oxidase 1 (COI), 16S ribosomal DNA INTRODUCTION DNA barcoding is a taxonomic method that uses a short genetic marker in the DNA to identify an organism. It differs from molecular phylogeny in that the main goal is to identify an unknown sample in terms of a known classification [26]. DNA sequence can be used to identify different species, in the same way as the supermarket scanner uses the black stripes of the UPC barcode to identify the items. This database will rapidly link a specimen to a Binomial Linnaean name and through that link it will provide all available information and studies on the species. Moreover by allowing easy access to species identity, these tags would greatly enhance the rate of species discovery and further supplement and strengthen the traditional taxonomy. Most eukaryote cells contain mitochondria, and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variation in mtDNA sequences. This evolves much more rapidly than nuclear DNA, thereby resulting in the accumulation of differences between closely related species [6], [30] and [31]. This is more pronounced interspecifically (between species) than intraspecifically (within species). The DNA barcode itself consists of a 648 bp region, 58-705 from the 5’-end of the cytochrome c oxidase 1 (COI) gene using the mouse mitochondrial genome as a reference. It is based on the postulate that every species will most likely have a unique DNA barcode. There are 4650 possible ATGC-combinations compared to an estimated 10 million species remaining to be discovered [53]. It is also on the assumption that interspecific genetic variation exceeds intraspecific genetic variation [19], [21]. [37] reported that COI barcodes are also a good proxy for estimating intrageneric (within genus) phylogenetic diversity and relationships in earthworms. Early history of DNA barcode DNA barcode approach has become increasingly popular and the idea of a standardized molecular identification system emerged progressively during the 1990s with the development of PCR-based approaches for species identification. Molecular identification has largely been applied to bacterial studies, microbial diversity surveys [56] and [57] and routine pathogenic strains diagnoses [47] and [55] due to a need for culture-independent identification systems. PCR-based methods have also been frequently used in fields related to taxonomy, food and forensic molecular identification [50] and for identification of eukaryotic pathogens and vectors [28]. The Barcode of Life project aimed to create a universal system for a eukaryotic species inventory based on a standard molecular approach. It

Available online @ www.ijntse.com 74 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 was initiated in 2003 by researchers at the University of Guelph in Ontario, Canada (http://www.barcoding.si.edu) and promoted in 2004 by the international initiative ‘Consortium for the Barcode of Life’ (CBOL). By then, it had more than 150 member organizations from 45 countries including natural history museums, zoos, herbaria, botanical gardens, university departments as well as private companies and governmental organizations. The DNA barcode project does not have the ambition to build the tree of life nor to perform molecular taxonomy [12] and [17], but rather to produce a simple diagnostic tool based on strong taxonomic knowledge that is collated in the DNA barcode reference library [44]. The DNA Barcode of Life Data System (BOLD, http://www.boldsystems.org) has progressively been developed since 2004 and was officially established in 2007 [41]. This data system enables the acquisition, storage, analysis and publication of DNA barcode records. During the year 2008, the total available DNA Barcode records were 363,584 sequences for 50,039 species. The efficiency of DNA barcoding has also been reported in the detection and description of new cryptic species [2], [7], [16], [24], [38], [49] and of sibling species [1]. DNA barcodes in biological specimens Until now, biological specimens were identified using morphological and anatomical features. If a specimen is damaged or is in an immature stage of development, even experienced taxonomist may be unable to make identifications. Barcoding solves these problems, because non-specialists can obtain barcodes from tiny amounts of tissue. This is not to say that traditional taxonomy has become less important, but rather that DNA barcoding can serve a dual purpose as a new tool in the taxonomists toolbox supplementing his knowledge as well as being an innovative device for researchers who need to make a quick (CBOL) identification [8]. DNA barcode is a powerful tool for identifying species of earthworms and provides a useful complement to traditional morphological taxonomy [23]. DNA barcode used in earthworm identification DNA barcoding promises great advances in species discovery and broad dissemination of taxonomic knowledge. This provides a framework for a first-stage taxonomic screening of earthworm diversity using a single standardised gene marker- a 648 base pair fragment of the mitochondrial cytochrome c oxidase subunit 1 gene. Earthworm barcode of life is a recently launched campaign under the broad umbrella of barcode of life initiative. The earthworm barcoding campaign objective is to build an extensive library of genetic tags (CO 1 barcode region- 648bp) for 5000 species. This also helps to discriminate morphologically similar and genetically different species termed cryptic species [9], [10], [11] and [35] by using the DNA sequences of the mitochondrial cytochrome c oxidase subunit1 (COI). However, reports are available that several genes have been chosen for identifying the earthworm species through DNA barcoding such as mitochondrial cytochrome-c oxidase I, 18S, 28S and 16S ribosomal DNA [5], [27], [33], [36], [39], [40], and [42]. Among these, DNA barcodes based on a fragment of the COI and 16S rDNA genes have been demonstrated to work well for species identification for earthworms. The aim of the study is to determine whether the mtCOI barcode is able to discriminate species under consideration and also check whether these are in agreement with those obtained through traditional approaches. Materials and Methods Collection of specimens The study area comprises the five districts of Kerala state (Thiruvananthapuram, Idukki, Thrissur, Palakad and Wyanad). The study was carried out during the period of September 2010 to October 2011. The earthworms were collected from the field by digging and hand sorting and either brought to the laboratory for preservation or washed and preserved in absolute alcohol (100%) on the spot. A total of 27 earthworm samples were collected from different habitats such as grassy land, river bank, forest, banana plantation, vegetation sites, residential area, coconut plantation, hill region, and cultivated land. All species were serially numbered and assigned a unique code. The adult worms were

Available online @ www.ijntse.com 75 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 identified by using traditional taxonomic keys. The preserved samples (in absolute alcohol, 100%, three-in-one AR grade) were stored at 4oC until processing for DNA extraction. The samples (posterior-most portion of the preserved specimen) were then processed for DNA sequencing. A total of 27 samples were used for DNA barcoding. The voucher specimens are deposited in the Zoological Museum of the Department of Zoology, Mar Ivanios College, Thiruvananthapuram, Kerala. Extraction of DNA DNA sequencing was done at ‘Rajiv Gandhi Centre for Biotechnology’, Department of Biotechnology, Government of India, Thiruvanathapuram. About 25 mg tissue of the each worm was used to extract the DNA. DNA was extracted from the sample using Qiagen® Dneasy Kit. The extraction protocol involves the following steps: 1. 25 mg of tissue cut into small pieces was placed in a 1.5 ml microcentrifuge tube. 2. 180 μl of AL buffer and 20 μl proteinase K were added. It was then mixed by vortexing and was incubated at 55◦C in a water bath until the tissue was completely lysed. 3. 4 μl of RNAase A was added to this and mixed by vortexing. It was incubated at room temperature for 2 minutes. 4. After vortexing for 15 seconds 200 μl of AL buffer was added to the sample. It was again mixed thoroughly by vortexing and was incubated for 70◦C for 10 minutes. 5. 200 μl of ethanol (96 - 100%) was added to the sample and mixed thoroughly by vortexing. 6. The mixture was pipetted to the DNeasy spin column and placed in a 2ml tube. 7. It was centrifuged at 8000 rpm for one minute. The flow through and collection tube was discarded. 8. The DNeasy spin column was placed in a new 2 ml collection tube and 500 μl AW1 buffer was added. 9. The mixture was centrifuged at 8000 rpm for one minute. The flow through was again discarded. 10. It was placed in a new collection tube and 500 μl of AW2 buffer was added. 11. The mixture was centrifuged at 8000 rpm for three minutes. The flow through was again discarded. 12. The DNeasy spin column was placed in a clean 1.5 ml microcentrifuge tube and 200 μl of AE buffer was pipetted into it and centrifuged for one minute. 13. Elution was repeated with 100 μl of AE buffer to increase the final DNA concentration. DNA Sequence acquisition Total genomic DNA was isolated from one to six individuals per species. A fragment of the mitochondrial cytochrome c oxidase subunit I sequence (COI) was amplified using the universal primers F - 5’-GGTCAACAAATCATAAAGATATTG-3’ and R - 5’- TAAACTTCAGGGTGACCAAAAAATCA-3’ [13]. The reaction mix consisted of 10 μl of Taq PCR Master mix (Qiagen, New Delhi, India), 2 μl of MgCl2 (25 mM), 0.5 μl of each primer (10 μM), 4 μl of dH2O and 3 μl of template DNA (10–20 ng), and the thermocycling conditions for the amplification of CO1 gene fragment were as follows: 95°C for 5 min, followed by 10 cycles of 95°C for 30 s, 45°C for 40 s, 72°C for 90 s, followed by 30 cycles of 95°C for 30 s, 51°C for 40 s, 72°C for 90 s, and a final extension step at 72°C for 5 min. PCR products were purified by using ExoSap IT (USB, USA). The sequencing was performed for both forward and reverse strands using the same PCR primers — the products were labelled using the BigDye Terminator V.3.1 Cycle sequencing Kit (Applied Biosystems) and sequenced using an ABI 3730 capillary sequencer following manufacturer’s instructions. Sequences were manually checked and aligned using the default parameters with ClustalW, built into BIOEDIT.

Available online @ www.ijntse.com 76 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 RESULTS The topology of the maximum likelihood tree inferred from the dataset clearly shows very strong signal of COI at the species level in earthworms (Fig. 1). A total of 27 samples were processed in the present study. Morphological examination of the adult worms revealed 9 species — Pontoscolex corethrurus (Glossoscolecidae), Travoscolides chengannures, Amynthas corticis, Perionyx sansibaricus, P. foveatus var. nairii, nov., Amynthas sp (Megascolecidae), Progizzardus varadiamensis, Glyphidrilus annandalei (Almidae) and Dichogaster bolaui var. vijai, nov. (Octochaetidae) (Table1). It was noted that CO1 exhibited a unique barcode to a particular species. The DNA sequences from CO1 were deposited in GenBank under the accession numbers listed in Table 1. Table 5.1.1: Accession numbers of sequences submitted to GenBank Family Genus Species Labels Accession Glossoscolecidae Pontoscolex Pontoscolex corethrurus E8- CO1 JN185607 Megascolecidae Travoscolides Travoscolides chengannures E4- CO1 JN185606 Megascolecidae Amynthas Amynthas corticis E 13-CO1 JX535189 Megascolecidae Amynthas Amynthas sp E14-CO1 KC248377 Megascolecidae Perionyx Perionyx sansibaricus E19- CO1 JX535190 Perionyx foveatus var. nairii, Megascolecidae Perionyx nov. E-15-CO1 KC248378 Almidae Progizzardus Progizzardus varadiamensis E12-CO1 JX535188 Almidae Glyphidrilus Glyphidrilus annandalei E32- CO1 JX535191 Dichogaster bolaui var. Octochaetidae Dichogaster vijai,nov E9-CO1 KC248376

Out of the 27 samples studied, 4 species were identified namely, Progizzardus varadiamensis, Perionyx foveatus var. nairii, nov., Dichogaster bolaui var. vijai, nov. and Travoscolides chengannures which are endemic to Kerala and Western Ghats and 2 species, Pontoscolex corethrurus and Amynthas corticis are exotic peregrines and Perionyx sansibaricus is a native peregrine. Discussion DNA barcodes were obtained from all specimens studied. After alignment there were 558 common sites in the partial sequences of CO1 genes including inserted gaps used for this analysis. No areas of uncertain alignment were identified.The results of the present study strongly support the usefulness of DNA barcodes in species identification of earthworms. Phylogenetic analyses were performed using MEGA ver. 5 inference, maximum likelihood methods and the branch support was evaluated using 1000 bootstrap replicates. The analyses were performed using different methods to check the consistency of the topology of the resulting tree. Quidnipagus palatum (a bivalve) was used as the outgroup for the analysis. Pair wise genetic distances between the monophyletic clades were determined using the Kimura 2-parameter method [25], [58] in the program MEGA ver. 5. Sequences from 27 individuals were used in the analyses, and after final alignment. Among the 558 nucleotide sites utilized for the phylogenetic analyses, 384 sites were variable and 232 sites were parsimony-informative (a site is considered to be parsimony-informative if there were at least two different nucleotides at that site, each of which occur at least twice). The GTR + I + G nuclear substitution model was selected. The phylogenetic trees obtained with maximum likelihood revealed 15 distinct monophyletic and high bootstrap support values (Fig.1).

Available online @ www.ijntse.com 77 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 The individuals belonging to ‘clade g’ represent the species Pontoscolex corethrurus (Fig. 1). This clade was highly divergent from all other clades but the individuals of the clade (E18, EW6, E7, E8, E6, S29, E3 and EW2) have 0% divergence between themselves indicating individuals of the clade belong to the same species Pontoscolex corethrurus. We identify clade f, clade e, clade i and clade 1 as a possible new candidate species from a possibly new genus based on the extent of genetic divergence (Table 1) from the other species; however, the validation of these earthworms as new species and genera would require the designation of a type specimen and relevant comparisons with other type specimens. FiG. 1: Maximum likelihood (ML) phylogenetic tree of earthworm inferred from DNA sequences of mitochondrial gene CO1. 10 monophyletic clades (Clade a- Pontoscolex corethrurus; clade b— unidentified (possibly a new species/genus ); clade c – Glyphidrilus annandalei ; clade d unidentified (possibly a new species / genus); clade e – Travoscolides chengannures; clade f— unidentified (possibly a new species/ genus); clade g— Amynthas corticis; clade h— Perionyx sp.; clade h— Dichogaster bolaui var. vijai, nov and Progizzardus varadiamensis; clade 1— Unidentified (possibly a new genus) are highlighted together with pictures of representative specimens. The numbers at the nodes indicate the posterior probabilities.

Fig. 1 :Pontoscolex corethrurus

The individuals (E4, E5, EW5, E17 and EW4) of the clade d belongs to the genus Travoscolides chengannures. The individuals of this clade show 0% divergence among themselves indicating them to be the same species Travoscolides chengannures. The taxonomic identification and phylogenetic position of the species like S32-Glyphidrilus annandalei-JX535191, E14-Amynthas corticis, E13-Amynthas corticis -JX535189, E15-Perionyx

Available online @ www.ijntse.com 78 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 foveatus var. nairii- KC248378, E12- Progizzardus varadiamensis- JX535188, E9- Dichogaster bolaui var. vijai, nov - KC248376 , E14- Amynthas sp- KC248377, E8- Pontoscolex corethrurus- JN185607 and E19-Perionyx sansibaricus-JX535190 were fixed (Fig.1) and their closely related species identified. Dichogaster bolaui var. vijai shows more than 18.8% divergence from all other species of earthworms compared in the present study and Progizzardus varadiamensis shows 15.5% divergence from E1, an unidentified species and more than 19.6% divergence with all other species of earthworms compared in the present study. This high degree of divergence for the two species supports their position as a distinct genus. The unresolved cases in the present study may represent the parthenogenetically degraded morphs that do not reproduce by normal meiosis and cross-fertilization to produce diploid offspring. These are outside the conventional species concept. The question whether such parthenogenetic morphs should be distinguished by scientific names is debatable. However, in the present study, such cases are reported as unidentified morphs. Two different species of Amynthas (A. corticis and Amynthas sp.) were sequenced. By morphotaxonomy it was found that, Amynthas sp. is closely related to Amynthas corticis except in the number of spermathecae (= 2 pair). In addition, Amynthas sp. carry numerous papillae like projections in the segment between 12-16 covering the clitellar segment. These two specimens, E13 and E14, however show 0% divergence. In a similar situation, [11] reported three different species of Amynthas sharing a common CO1 haplotype. Therefore, a careful re-examination of these specimens is required to resolve this matter. The cytochrome c oxidase I (CO1) gene is present in all animals, and sequence comparisons are straightforward because insertions and deletions are rare. Thus, the CO1 gene, defined as the DNA barcode by [19], has been used to identify species of birds [20], whale [34], spiders [4], springtails [22], tropical Lepidoptera [18], [54], and insect pests [3], [45], including invasive leaf miners [43]. DNA barcoding can be performed at any life stage and reliable identifications of juvenile [42], [48] or even partial specimens are possible. [23] analyzed partial CO1 sequences from 86 specimens of 28 Chinese earthworm species. They tested a 566 bp portion of the gene and obtained positive results. However, [11] re-evaluated the use of DNA barcodes in earthworm species identification by re- analyzing sequence data for the CO1 gene. They discussed the problems in the light of results presented by [23] as well as previous systematic studies of earthworms based on CO1 sequences and morphology. The present findings are unique in four species (Travoscolides chengannures (JN185606), Progizzardus varadiamensis (JX535188), Perionyx foveatus var nairii, nov. (KC 248378) and Dichogaster bolaui var. vijai, nov (KC248376), among those studied currently are endemic to Kerala and the Western Ghats region and the barcodes made available are new to science. The DNA barcoded specimens also include a newly created genus and two new sub species. Ideally these results should be confirmed with more markers and specimens, and so potential sampling and systematic error could be evaluated. Since the earthworms are key organisms of soil ecosystems and are important indicators of soil health and quality it is crucial to improve the resolution and the reliability of species identification [14] and to enable identifications for all life stages. Molecular markers can highly supplement the conventional taxonomy especially in the light of large portion of cryptic diversity in the group. As the Earthworm Barcode of Life campaign envisage, DNA barcoding promises great advances in species discovery and broad dissemination of taxonomic knowledge by providing a framework for a first-pass taxonomic screening of earthworm diversity using a single standardized gene marker - a 648 base pair fragment of the mitochondrial cytochrome oxidase subunit I gene. The DNA barcoding should be further refined to address the problems facing the conventional taxonomy of terrestrial oligochaetes. It is also expected that a comprehensive Indian earthworm barcode database can be a powerful taxonomic tool that merits further development.

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