Volume-6, Issue-4 Oct-Dec-2016 Coden: IJPAJX-CAS-USA, Copyrights@2015ISSN-2231-4490 Received: 10thAug-2016 Revised: 10th Sept -2016 Accepted: 11th Sept-2016 DOI: 10.21276/Ijpaes http://dx.doi.org/10.21276/ijpaes Research article MORPHOLOGICAL CHARACTERS AND RANDOM AMPLIFIED POLYMORPHIC DNA BASED GENETIC DIVERSITY ANALYSIS OF CURCUMA SPECIES (ZINGIBERACEAE) FROM INDIA Rajesh Kumar T1. Santhosh kumar R2. and Yusuf A1. Interuniveristy Center for Plant Biotechnology, Department of Botany, University of Calicut, Kerala, India-673 635 ABSTRACT: Genetic diversity of 19 Curcuma species was assessed using morphological characters and random amplified polymorphic DNA. A dichotomous key and a dendrogram for the species were developed using the selected morphological characters. The dendrogram derived from the morphological characters consists of III groups. C.bhati showed distinction from all the other species with a separate node.C.montana and C.psuedomontana shared the same node in group IIB indicating that both are synonyms and the need for reassessing their separate species status. Twenty RAPD primers produced a total of 2226 scorable bands in the 19 species of Curcuma, out of which 1025 were polymorphic. The percentage polymorphism ranged from 56.7% to 36.5% within the species. Based on UPGMA clustering, the 19 species were grouped into two main groups. The group IV contained one node for Curcuma bhatii with a similarity coefficient of 0.23 with the all the other species clarifying the questions raised against the circumscription of C.bhatii from Paracautleya bhatii. The third group contained 7 species in which C. montana and C. pseudomontana were grouped together with a similarity coefficient of 0.33 thusare synonyms. Group II contained 6 species with a similarity coefficient of 0.29 to 0.33. Group I consists of 5 species, among them,C.amadashowed distinct node justifying its special features. The results obtained show that by using morphological characters and RAPD the genetic diversity of the Curcuma species can be assessed. Key words Curcuma, Species diversity, Taxonomy, RAPD, UPGMA dendrogram *Corresponding author: Yusuf A. Interuniveristy Center for Plant Biotechnology, Department of Botany, University of Calicut, Kerala, India-673 635 Copyright: ©2016 Yusuf A. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited INTRODUCTION The family Zingiberaceae, generally known as ‘Spice family’, forms an important group with considerable economic potential, with genera such as Aframomum, K.Schum., Alpinia Roxb., Amomum Roxb., Curcuma L., Elettaria Maton., Kaempferia L. and Zingiber Boehm. Many members of this family have been used in the ayurvedic system of medicine from time immemorial and are well-known spices. The genus Curcuma L., with around 120 species distributed in tropical and subtropical Asia consists of a rather homogenous group of rhizomatous perennials [1]. International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 38 Yusuf et al Copyrights@2016 ISSN 2231-4490 Zingiberaceae is a taxonomically neglected group mainly because of thespecial floristic characters and inaccessible nature of the wet evergreen forests in which they grow. The short flowering period coincides with the monsoon season, makes their collection much difficult. Moreover, huge vegetative and massive underground parts are major constraints in herbarium preparation. Consequently, most herbarium specimens are fragmentary and the conclusions in most of the floras, based on dried specimens, are truncated accounts. The Delicate nature of flowers, loss of colour and formation of a gummy mass soon after collection makes the floral-morphology based phylogenetic evaluation much difficult for the genus Curcuma. The major problem with taxonomic descriptions is the lack of, type specimens, illustrations of old species,protologues and complete details in literature. The flowers are evanescent and even flower specimens in the herbaria cannot be of much help to a critical taxonomist. The determination of correct species identityis necessary for proper utilization and conservation of Zingiberaceous crops. Proper characterization of the taxa by using molecular and phytochemical methods is needed to solve the problem of species delimitation. The vulnerability of many of these species and imminent danger of their extinction makes it more urgent. The main identifying character of the genus is its inflorescence, a spike with prominent spiral bracts, which is laterally fused or adnate to the peduncle and form pouches, each subtending a cincinnus of flowers and a cluster of, often coloured, sterile, terminal bracts called ‘coma’. Baker [2] described 29 species in the Flora of British India and subdivided the genus into three sections - Exantha, Mesantha and Hitcheniopsis. Hitcheniopsis differed from the rest of the genus in its spurless anthers. Schumann [3] rejected the sectional classification based on spike position and suggested two subgenera [Eucurcuma and Hitcheniopsis (Baker) K. Schum.] based on the presence or absence of spur on the anthers. Fischer [4] reported eight species. But this was proved as a gross underestimate by the subsequent addition of twelve more species, bringing the total number of South West Indian species to 20 of which 12 taxa are endemic to the Indian subcontinent [5]. The species are naturally found in mixed deciduous tropical forests and tropical broad-leaved evergreen forests. The members of the genus in these regions are important resources and have great potential in terms of commercial values as a source of spices, medicines and horticultural products [6, 7,8,9,10]. Taxonomic characterization is mainly dependent on the comparative morphological characters influenced by environmental factors. Phenotype plasticity is being misunderstood as a taxonomic character in identifying species of this genus.Confusion prevails in the classification of Zingiberaceous members,due to the difficultyof conventional taxonomic toolsto discriminate the species. Studies based on the morphological, molecular, anatomical and biochemical characterization of some Curcuma species have been attempted earlier [11, 12, 13, 14]. Relying much on the morphological characters alone in species delimitation has its own disadvantages since they are not always completely representing the genetic structure. Conventional taxonomic methods in conjunction with molecular and biochemical tools will provide accurate and powerful methods for analyzing the genetic relationship among the species in the family Zingiberaceae. However, concerted efforts are lacking in the molecular characterization in Curcuma species. Molecular markers assume greater significance, as these methods detect polymorphisms by assaying subsets of the total amount of DNA sequence variation in a genome [15]. The objective of the present study was to examine the genetic diversity of selected Indian Curcuma species by using morphological and molecular markers, which has relevance in the present context of the ongoing taxonomic revision of the genus. The development of highly reliable molecular marker system for assessing the genetic diversity within the genus could help in crop improvement program through molecular breeding. MATERIALS AND METHODS Morphological characters Curcuma species selected for the study were C. aeruginosa, C. inodora, C. raktakanta, C. longa, C. amada, C. neilgherrensis, C. oligantha, C. coriacea, C. aurantiaca, C.zanthorrhiza, C. decipiens, C. vamana. C.karnatakensis, C.mutabilis,C.harita, C.aromatica, C.montana, C.psuedomontana and C. bhatii. Whole plants and their underground parts such as rhizome, roots and root tubers were collected from different parts of South India. The rhizomes collected from different regions were planted and maintained in the Calicut University Botanical Garden (CUBG). Eight morphological characters were taken into consideration inassessing the morphological similarities/dissimilarities. The characters selected were, floral (spike position, the color of calyx and color of corolla) Rhizome ( color of rhizome, aroma and taste of rhizome) and aerial ( color of leaf sheath and leaf midrib). Specimens were identified with the help of Floras, Revisions and Monographs and also referred to experts for accurate identification. International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 39 Yusuf et al Copyrights@2016 ISSN 2231-4490 The nomenclatural corrections were made according to ICBN [16] and for the abbreviation of periodicals; the BPH [17] was followed. The abbreviation of authors is according to Brumitt and Powell [18]. The types and authentic materials available in Central National Herbarium (CAL), Kolkata and Madras Herbarium (MH) at Coimbatore were studied. Voucher specimens used for this investigation are deposited in the University of Calicut Herbarium (CALI). The morphological characters were marked as presence and absence and loaded inthe binary matrix of NTSYS-pc program and computed to derive the genetic similarity index and the dendrogram was constructed by using UPGMA [19]. RAPD analysis Plant material The experimental material comprised of 19 Curcuma species, collected from different geographical locations and maintained in the field gene bank of Botanical Garden, University of Calicut (CUBG), Kerala, India. DNA extraction Genomic DNA was isolated from the young leaves by using the modified CTAB method [20]. The protocol used for the extraction of DNA worked for all the species, however, interspecific variations were observed in the quality and quantity of DNA. DNAwas extracted from uppermost first and second leaves of all the Curcuma species. The extraction buffer contained 100mM Tris - HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4 M NaCl, 2% (w/v) Cetyl trimethyl ammonium bromide (CTAB) and 0.2% (v/v) β-mercaptoethanol. Polymerase Chain Reaction Polymerase chain reactions were carried out in 25.0 µl reaction volume containing 10X PCR buffer, 0.5U Taq DNA polymerase, 200µM dNTPs, 2.0mM MgCl2 and 10pmoles of random decamer primer [21]. Amplification cycles comprised of an initial temperature of 920C for 5 min for complete denaturation.The second step consisted of 35 cycles of denaturation at 920C for 1 min., annealing at 380C for 1min., extension at 720 C for 1 min. and a final extension at 720C 10 min. The reactions were carried out in a 96 well thermal cycler with gradient block (Eppendorf mastercycler pro S, Germany). Electrophoresis of PCR products The PCR amplified products were run on an agarose gel (1%w/v) kept in 1X TAE buffer for about 1.5 hrs. at 120 Volts. The gels were stained with ethidium bromide (0.5 µg/ml) and documented using chemiluminescence imaging system (Cell-bio Science, U.S.A.). The bands were scored by using a 1Kb DNA ladder ( New England Biolabs)as a marker. Data scoring and analysis The electrophoretic patterns were visually analysed and scored as 1/0 for the presence or absence of bands for each primer. To avoid taxonomic ambiguities, the intensity of the bands was not taken into consideration, only the presence of intense and reproducible bands was considered. The data were entered into excel spreadsheet and the matrix obtained was loaded into the NTSYS-PC program [19] and Jaccard’s similarity index (JSI) between the species were calculated.A UPGMA dendrogram was constructed based on the derived JSI. The frequency of the RAPD bands detected with the 20 polymorphic RAPD primers was calculatedand estimates of genetic diversity (H) were obtained using Shannon’sinformation measure modified for RAPD by Chakraborthy and Rao [21] as H= ∑ ki=l PilogePi where Piis the frequency of the ith RAPD bands. RESULTS AND DISCUSSION Morphological evaluation Based on the morphological characters a dichotomous taxonomic key was developed for all the 19 Curcuma species studied. 1. Anther lobes ecalcarate ...................................................................................... C. aurantiaca 1. Anther lobes spurred ............................................................................................................... 2 2. Leafy shoot 15-60 cm high...................................................................................................... 3 2. Leafy shoot 65-125 cm high .................................................................................................. 11 3. Inflorescence with or without inconspicuous coma ................................................................ 4 3. Inflorescence with well-developed coma ................................................................................ 8 4. Rhizome stoloniferous; flowers shorter than the bracts ..................................... 18. C. vamana International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 40 Yusuf et al Copyrights@2016 ISSN 2231-4490 4. Rhizome ovoid; flowers longer than the bracts ....................................................................... 5 5. Leaves 0.7-1.5 cm broad; inflorescence only central ............................................... 5 C. bhatii 5. Leaves 7-14 cm broad; inflorescence both lateral and central ................................................ 6 6. Leafy shoot upto 35 cm tall; flowers 5-5.7 cm long ........................................ 15 C. oligantha 6. Leafy shoot upto 60 cm tall; flowers 4.5-6 cm long ................................................................ 7 7. Labellum white with a median bright yellow band; anther 4.5 mm long......10. C. karnatakensis 7. Labellum yellow or white with yellow center; anther 3.5-4 mm long ............ 13. C. mutabilis 8. Root tubers cylindrical, 10-18 cm long; leaves coriaceous, densely pubescent on both sides6. C. coriacea 8. Root tuber small, spherical, ovoid or oblong; leaves not coriaceous, glabrous or sparsely pubescent 9 9. Flowers equal to the bracts; lip purple .................................................................................. 10 9. Flowers longer than bracts; lip yellow ..................................................... 14. C. neilgherrensis 10. Leaves broadly ovate, sub cordate at base; fertile bracts recurved; lip purple towards base7. C. decipiens 10. Leaves elliptic, base oblique; fertile bracts not recurved; lip deep purple with a bright yellow band 9. C. inodora 11. Leaves with purple patch along the midrib ........................................................................... 12 11. Leaves without a purple patch along midrib ......................................................................... 13 12. Rhizome blue within; leaves with a purple patch on the distal half on the upper side only1. C. aeruginosa 12. Rhizome yellow to deep yellow within; leaves with purple patch on both sides along the whole length of the midrib .................................................................................................................... 19. C. zanthorrhiza 13. Lateral staminodes with a patch of glandular hairs at centre ................................................. 14 13. Lateral staminoides without glandular hairs .......................................................................... 15 14. Pseudostem reddish purple; leaves spreading, oblong-lanceolate, glabrous below 17. C. raktakanta 14. Pseudostem green with a few light pink dots; leaves erect, semiplicate, ovate-elliptic, densely pubescent on the lower surface .................................................................................................................... 8 C. haritha 15. Rhizome with the smell of green mango ................................................................ 2. C. amada 15. Rhizome without the smell of green mango .......................................................................... 16 16. Rhizome with sessile tubers .................................................................................................. 17 16. Rhizome without sessile tubers ............................................................. 16. C. pseudomontana 17. Rhizome deep orange-yellow within; lip light yellow with a median dark yellow band4. C. longa 17. Rhizome yellow or light orange yellow within; lip deep yellow ........................................... 18 18. Rhizome light orange-yellow within; corolla white; calyx 8 mm long ...............12C. montana 18. Rhizome greyish-yellow within; corolla pinkish white; calyx 2 cm long…………..3. C. aromatica Taxonomic classifications based onmorphological characters in Curcuma species rely on the position of the spike, presence or absence of root tubers and the colour of coma. Roxburgh [22] pointed out that the positional difference of spikes is affected by the flowering season; the early spikes develop laterally and later spikes develop terminaly. Santapau [23,24] added that in C. pseudomontana a large lateral spike emerges from the sides of the leaves in the beginning of the rainy season (June-July). Gradually by the beginning of August, this spike decays and a central spike appear surrounded by leaves, resulting in a central and lateral spikes in the same plant. International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 41 Yusuf et al Copyrights@2016 ISSN 2231-4490 C. aurantiaca shows diverse floral plasticity, which is the distinguishing feature of this species. The plant is medium sized with small rhizomes and tubers. The inflorescence is central in position and the main feature is the exceptionally coloured inflorescence. The bract colour varies from green, white, brown, purple to rose, justifying its specific epithet (Fig.1). The distinguishing feature of C.inodora is its beautiful and attractive spike. The inflorescence has variations in the shape of comma bracts,the labellum has colour variations from dark purple, white, yellow or golden (Fig.2). The plant produces two inflorescences in a year; one lateral and one central. The plant undergoes a dormant phase during the summer; new sprouts with fresh leaves emerge by the end of April hence called as hidden purple ginger. The species shares morphological features with C. decipiensbut differs in the number of flowers equaling the bracts, 3-4 flowered cincinni, purple corolla and staminodes and labellum with a dark yellow band in the center. Identification of the species to some extent is possible by the qualitative observation of leaf morphology, rhizome colour, size,etc. Highest leaf length and breadth in the descending order was observed from C. zanthorrhiza, C. aromatica, C. aeruginosa, C. longa, C.amada, C.coriacea, C.haritha, C.raktakanta and C.pseudomontana. In C. zanthorrhiza,the midrib has a purple colour throughout the midrib of the leaf whereas in C. aeruginosa the purple colour is present only towards the upper half of the leaves. C.raktakanta closely resembles C.aeruginosa however, differs in the yellowish to gray colour of the rhizomes instead of blue colour in the center, purple coloured pseudostem and peduncle and absence of a purple patch on the leaves. C. amada is closely related to C.longa, but the characteristic smell and pale yellow colour of the rhizome, pale yellow flowers and light violet coma in C. amada displays some degree of differentiation. The characteristic camphor smell of the rhizome and the densely pubescent lower surface of the leaf are differentiating characters of C.aromatica. C.coriacea is endemic to Kerala, with coriaceous dense pubescent leaves, lateral inflorescence and bright yellow corolla and C. decipiens can be distinguished from other species of Curcuma by the deep purple corolla and coma and two flowered cincinni. From its close ally, C.inodora, the differences arein flowers being longer than the bracts, 2-3 flowered cincinni, labellum purple with deep yellow bands. C. vamana has the smallest flowers among the different Curcuma species reported so far from India. It has a sub equal leaf base, much shorter, condensed spike and bracts, few flowered cincinni, absence of anther crest and presence of spurs on fertile anther. C. karnatakensis closely resembles C.oligantha but differs from it in the large size of the plants and large white flowers with a median yellow band [25]. C. oligantha was described as a new species under the name C. cannanorensis [26]. This plant is closely akin to C. albiflora but differs from it in the smaller size of leaves with acuminate or apiculate apex; the petiole shorter than the lamina, shorter spike and larger size of the flowers. It is also related to C. neilgherrensis but differs mainly in the absence of a coma and smaller bracteoles. Bhat [27] studied this species in detail and confirmed it as C. oligantha. Velayudhan et al., [28] retained it as C. cannanorensis but the type specimen and protologue of C.oligantha were not taken into account and rejected. C. mutabilis is the most interesting species due to the variations in flowercolour (Fig.3). Corolla color varies from whitish pink, pink-red, reddish orange, dark pink to dark violet; labellum and lateral staminodes are pure white, white with yellow or reddish streaks in the throat of labellum or base of the lateral staminodes, different shades ranging from creamy, light yellow to deep yellow. Since there is no correlation between the color of the corolla and staminodes, the combination of these two characters makes each individual a slightly different look. Velayudhan et al.[29] described this species as C. nilamburensis based on a collection from Nilambur, Kerala, India. Unfortunately, the species was not validly published according to the St. Louis code [30] as the description lacked a Latin diagnosis and a type was not designated. In addition, the publication by Velayudhan et al., [29] is of limited circulation. Hence, the species was validly published under C. mutabilis [31]. C. haritha is closely related to C. aromaticaSalisb., but differs in the yellowish-gray, non-aromatic rhizome, leathery, semiplicate, erect leaves, white corolla, and light yellow lip with a median dark yellow band. It also resembled C. raktakanta but differs in having green pseudostem with light pink spots, white corolla lobes and swollen placenta. C.bhatii showed variations from all other species which was originally described as another genus Paracautleya in 1977 by R. M. Smith. Skornickova and Sabu [31] observed the characters were not sufficient to provide a separate genus status, hence merged Paracautleya bhatii R. M. Smith with Curcuma bhatii on the basis of type specimen collected by K.G. Bhat from Udupi, Karnataka, South India. The genus Paracautleya was established by Smith [32] based on the same specimen. International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 42 Yusuf et al Copyrights@2016 ISSN 2231-4490 The affinity of Paracautleya congruent with Curcuma, but pointed out that Curcuma flowers are borne in cincinni within pouches formed by adnate bracts and confirmed the close affinity of Paracautleya with Cautleya and Roscoea mainly based on the singly borne ebracteolate flowers and leafy stems. However, the assigned generic status of this taxon is mainly based on its stemless habit, elongated naked peduncle and ovules attached at the base of the ovary. The dendrogram constructed by computing the selected morphological characters of the 19 species using UPGMA clustering produced a phylogenetic tree with three prominent groups (Fig.4). The groups diverged at a JSI of 0.37 and C. bhati showed distinctiveness from the rest of the species. The group I comprised of a distinct node for C. aeruginosa with 0.45 JSI from the other species. C.inodora and C.raktakanta shared a common node with a JSI of 0.53 with all the other species. C.longa formed a separate node with a JSI of 0.474 justifying its amphidiploid nature.Group IIA comprised of C. amada with 0.53 JSI from the rest of the species indicating its special biochemical features. C.aurantiaca and C.neilgherensis shared a common node with a JSI of 0.84 with all the other species. C.oligantha showed a JSI of 0.68 and C.coriacea with 0.65 JSI with the other species. C.aromatica and C.mutabilis showed the same JSI as they shared the same node. Group IIB comprised of C.montana, C. pseudomontana, C.haritha, C.karnatakensis and C. zanthorrhiza. C.montana, C. pseudomontana showed maximum similarity among all the species studied hence can be considered as synonyms. Group IIC comprised of C.decipiens and C.vamana which showed a JSI of 0.47. Group III comprised of C.bhatiwith 0.37 JSI, which raises the question in the circumscription of P. bhati. Fig.1. Inflorescence bract colour variations in C.aurantiaca. International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 43 Yusuf et al Copyrights@2016 ISSN 2231-4490 Fig.2. Phenotypic plasticity in the flower colors of C.inodora International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 44 Yusuf et al Copyrights@2016 ISSN 2231-4490 International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 45 Yusuf et al Copyrights@2016 ISSN 2231-4490 Fig-4: Dendogram of 19 Curcuma Species from India resulting from a UPGMA cluster analysis obtained from various morphological characters International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 46 Yusuf et al Copyrights@2016 ISSN 2231-4490 International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 47 Yusuf et al Copyrights@2016 ISSN 2231-4490 Fig-6: Dendrogram of 19 Curcuma Species from India resulting from a UPGMA cluster analysis obtained from 20 RAPD primers Molecular characterization Polymorphic bands were produced by 20 random decamer primers in PCR for all the 19 species of Curcuma (Fig.5). The remaining 22primers produced monomorphic bands and were not considered for phylogenetic analysis. The sequence of the RAPD primers used for the molecular characterization, total number of bands produced by each primer, number of polymorphic bands, percentage polymorphism and genetic diversity are presented (Table 2). The scoring was done by running the PCR products on an agarose gel, where the bands were clearly visible and reproducible in three replicated amplification reactions. Amplified fragments were manually scored for presence (1) and absence (0) and the binary matrices were subjected to statistical analyses using NTSYS-pc [Numerical Taxonomic Multivariate Analysis System version 2.1 [33]. The 20 random decamer RAPD primers produced a total of 2226 scorable bands in all the species of Curcuma studied, out of which 1025 were polymorphic. The percentage polymorphism ranged from a maximum of 56.7% to a minimum of 36.5%. Highest genetic diversity was demonstrated by the OPA 14 primer (Table 2). International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 48 Yusuf et al Copyrights@2016 ISSN 2231-4490 Table-2: Total number of bands generated, number of polymorphic bands and percentage polymorphism exhibited by the RAPD primers in 19Curcuma Sp. S.No. 1. 2. 3. 4. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Primer OPA 05 OPA 07 OPA 08 OPA 10 OPA 11 OPA 12 OPA 14 OPA 15 OPA 17 OPA 18 OPA 19 OPB09 OPB 12 OPB 16 OPC 07 OPC 08 OPC 09 OPC 10 OPC 11 OPC 12 Sequence(5’ 3’) AGGGGTCTTG GAAACGGGTG GTGACGTAGG GTGATCGCAG CAATCGCCGT TCGGCGATAG TCTGTGCTGG TTCCGAACCC GACCGCTTGT AGGTGACCGT CAAACGTCGG TGGGGGACTC CCTTGACGCA TTTGCCCGGA GTCCCGACGA TGGACCGGTG CTCACCGTCC TGTCTGGGTG AAAGCTGCGG TGTCATCCCC Total Total no. of bands 97 116 116 109 97 111 134 132 133 116 80 120 137 89 117 80 76 133 122 111 2226 No. of polymorphic bands 46 43 48 58 47 60 76 57 59 60 41 59 50 34 44 32 40 61 54 56 1025 Polymor phism (%) 47.42 37.06 41.37 53.21 48.45 54.05 56.71 43.18 44.36 51.72 51.25 49.16 36.50 38.20 37.61 40 52.63 45.86 44.26 50.45 46.04 Genetic Diversity 0.0448 0.0419 0.0468 0.0565 0.0458 0.0585 0.0741 0.0556 0.0575 0.0585 0.0400 0.0575 0.0487 0.0331 0.0429 0.0312 0.0390 0.0595 0.0526 0.0546 To compute pairwise genetic similarities, Jaccard’s similarity coefficients (J ij) were calculated by using the formula, Jij= a/(n-d) where a is the number of RAPD bands present in both i and j accessions, d is the number of bands absent in both I and j accessions, and n is the total number of RAPD bands. The similarity matrices were computed and the corresponding dendrogram of genetic relatedness was constructed by applying Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering algorithm (Fig.-6 and Table-3). Table-3.Jaccard’ssimilarity coefficient among 19 species of Curcuma for 20 RAPD polymorphic primers International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 49 Yusuf et al Copyrights@2016 ISSN 2231-4490 The species were grouped by subjecting the similarity values to UPGMA clustering. Amongst the Curcuma species the lowest genetic similarity was observed between C. aurantiaca and C. oligantha (0.184) and the highest in C. montana and C. pseudomontana (0.4). Based on the dendrogram developed, all the 19 species were grouped into four groups. Group IV had only one node representing Curcuma bhatii exhibiting a JSI of 0.23 with all the other species, indicating the conversion of Paracautleyabhatii to C. bhatii is not correct and deserves a separate genus status.Group I was formed with five species of which C. karnatakensis and C. mutabilis shared 0.37 JSI between them. C. neilgherrensis and C.oligantha showed a distinct node with a JSI of 0.36. C. amada shared a 0.28 Jaccard’ssimilarity with the C. neilgherrensis/ C. oligantha cluster and formed a distinct node justifying its special morphological characters and biochemical features. Group II formed of C. haritha and C. aromatica with ashared JSI of 0.32 among them. C. aeruginosa and C. zanthorrhizashowed 0.33Jaccard’s similarity coefficient. C. coriacea and C. longa were grouped together with a similarity coefficient of 0.24. The group III contained 7 species in which C.montana and C. pseudomontana were grouped together with maximum similarity (JSI 0.4) between them and can be considered as synonyms. C. decipiens and C.inodora showed 0.29 JSI among them and C. aurantiaca grouped along with C. decipiens/C.inodora cluster with 0.368 similarity. C. vamana showed 0.29 JSI with all the other species in the cluster, and form a separate node. The highest JSI was observed for C. karnatakensis and C. mutabilis indicating that these two species have the maximum similarity revealed by the RAPD banding pattern. The two species have diverse geographical existence, C. karnatakensis is reported from Uttar kannad districts of Karnataka and C. mutabilis was collected from Nilambur of Kerala state. However, the genetic similarity between these two species revealed by RAPD suggests that the genetic closeness exhibited by the two species may not have an environmental impact as suggested by [15]. C. amada shared Jaccard’s similarity coefficient of 0.28 with the C. neilgherrensis/C. oligantha cluster suggesting that C. amada has a divergent character from the other members of this cluster. Morphological and biochemical features of C. amada bestow it with a separate species status and the specific aromatic ‘mango’ flavor and secondary metabolites are characters unique to the species justifying its specific position in cluster I A. Thus the five species which are geographically close and taxonomically grouped at similar level showed similarities with the members of the same group. C. amada showed distinctiveness from the other members of the group as described before [11] Cluster IB formed with 6 species and cluster IC with 7 species. These two sub clusters showed 0.27 Jaccard’s similarity among themselves. C. aromatica and C. haritha have a common ecological niche in the mid land and base of Western Ghats of India. C. aromatica is a seed setting species with somatic chromosome number 2n = 42 [34] even though C. haritha has the same somatic chromosome number this species but never sets seeds [35].The geographical niche of the two species may also affect the RAPD profile of the species. The group formed together by C. aeruginosa and C. zanthorrizha has 0.347 JSI, have the implication on the genetic constitution of the species. Both the species have 2n = 63, 64 chromosomes and this ploidy level may be the basis for the existence of the two species in the same group. The most cultivated species C. longa is grouped along with C. coriacea and both of them showed 0.33 Jaccard’s similarity with each other. The ecological niche of these two species is same and this may be contributing to the identical banding pattern and similarity of the two species. Group III comprised of 7 species which shared Jaccard’s similarity index of 0.27. C. montana and C. pseudomontana shared equal distance among themselves. They were treated as two different species in earlier taxonomic investigations [36]. However, they showed the maximum JSI (0.4) among the species taken for the study and can be considered as synonyms. C. decipiens and C. inodora showed 0.38 Jaccard’s similarity between them and form a common node, however, the morphological character based classification illustrated the highest level of dissimilarity. C. aurantiaca showed a JSI of 0.37 between the species is indicative of its relatedness with the other species geographically located in the same ecological niche. Of all the species C. bhatii forms a clear node, without exhibiting many similarities with the other species. In earlier classification based on morphological characters, this species was treated as a separate genus, Paracautleya [32]. However, studies by Skornickova and Sabu [31] shown that depending on the bracts which forms pouches as in Curcuma circumscribed it as Curcuma bhatii. But RAPD data showed that C. bhatii exists as a separate entity, showing very less similarity with all the other Curcuma species, indicating its genetic identity as a separate genus. International Journal of Plant, Animal and Environmental Sciences Available online at www.ijpaes.com Page: 50 Yusuf et al Copyrights@2016 ISSN 2231-4490 CONCLUSION This study was aimed at establishing genetic diversity of 19 Curcuma species which shared the morphological similarity.Though, there wasa dialectical situation of species of un-identical morphology falling in the same group or vice versa. The study proved that information on the morphological characters alone in species delimitation has its own limitations for the genus Curcuma. Molecular biology techniques like RAPD markers in conjunction with conventional taxonomic tools, thus assume significance and can be used for diversity studies of this important genus and can be used for future breeding programs. ACKNOWLEDGMENTS We sincerely thank Dr. Nirmal Babu, Principal Scientist, Indian Institute of Spices Research, Calicut, for providing the facilities for the data analysis and Director, Interuniversity Centre for Plant Biotechnology, University of Calicut for the lab facilities provided and extending his support during this work. REFERENCES [1] Skornickova, J; Sabu, M. and Prasanth kumar, M.G. 2004. Curcuma mutabilis (Zingiberaceae): A new species from South India. Gardens Bull Singapore 56: 43-54. [2] Baker, J.G. 1890. Scitamineae. in. J.D. Hooker ed., Flora of British India 6: 198-264 London. [3] Schumann, K. 1904. Zingiberaceae. In: A. Engler, Das Pflanzenreich 4: 46 (Heft 20): 1-458 Leip Zig, Berlin. [4] Fischer, C.E.C. 1928. Zingiberaceae. in: J.S. Gamble. Flora of the presidency of Madras, Pt. 8: 1478-1493. London. [5] Sabu, M. 2006. Zingiberaceae and Costaceae of South India. Indian Association for Angiosperm Taxonomy, Calicut University, India. 1-282. 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