Communications in Plant Sciences (January-June 2015), Volume 5, Issues 1-2, Pages 1-8 Commun. Plant Sci. (ISSN 2237-4027) www.complantsci.wordpress.com Manuscript received on July 29, 2014, and published on October 16, 2014 Species delimitation in congenerics of Genus Daemonorops from India using DNA barcodes 1,2 1 3 4 Senthilkumar Umapathy , Narasimhan Duvuru , Sanjappa Munivenkatappa , Uma Shaanker Ramanan , 2* and Ravikanth Gudasalamani 1 Center for Floristic Research, Department of Botany, Madras Christian College, East Tambaram, Chennai, Tamil Nadu, INDIA-600 059. Conservation Genetics Lab, Ashoka Trust for Research in Ecology and the Environment (ATREE), Srirampura, Jakkur PO, Bangalore, Karnataka, INDIA-560 064. 3 Department of Botany, University of Agricultural Sciences, GKVK, Bangalore, Karnataka, INDIA-560 065. 4 School of Ecology and Conservation, Department of Crop physiology, University of Agricultural Sciences, GKVK, Bangalore, Karnataka, INDIA-560 065. 2 ABSTRACT Daemonorops is one of the largest genus of palms, widely distributed in tropical evergreen forests of South and Southeast Asia and especially abundant in peninsular Malaysia. It shares the phylogenetic clade with genus Calamus, but distinctly characterized by the presence of the prophyllar bracts in subtribe Calaminae of family Arecaceae (Palmae). However, the species of Daemonorops are highly variable and homoplasious in their morphological characters. Indian Daemonorops has six species, of which five distributed in Andaman group of islands and one in Indo-China. The study mainly focused on the species of Indo-Myanmar (Andaman Islands) because they have showed uncertainty in their stand as distinct species. Hence it was attempted to test the species delimitation in congenerics using phylogenetic and character analysis of the nuclear genes. The study indicates three species of both Griffith and Beccari (D. jenkinsiana, D. manii, and D. kurziana) in India, and another three species of Renuka et al. (D. wrightmyoensis, D. aurea, and D. rarispinosa) from Andaman group of islands could be the variabilities of D. manii that is colonized throughout the archipelago. Keywords: Daemonorops, Species delimitation, DNA barcodes, Andaman and Nicobar Islands. ______________________________ *Corresponding author E-mail: gravikanth@gmail.com 2 Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India using DNA barcodes. INTRODUCTION Rattans (otherwise called ‘canes’) are spiny, climbing palms, widely distributed in the evergreen rain forests of the paleotropics in Africa, Madagascar, India, Myanmar, Peninsular Malaysia, Sumatra and occasionally in Papua-New Guinea and Queensland (Dransfield et al. 2008). The subfamily Calamoideae, accounts for the greatest diversity of climbing palms in South and South East Asia (Baker et al. 1998). Of the different genera, the genus Calamus and Daemonorops are abundant with highest species richness in the oriental landmass (Baker et al. 2000a). They have also colonized most of the volcanic islands of Indian and east Pacific oceans apart from the continental lands (Baker et al. 1998). The phylogenetic studies on calamoideaes indicate that Calamus is not monophyletic and at the same time Daemonorops is paraphyletic (they will have common ancestor but they are not monophyletic to each other and largely mixed at the tips from the node of origin) within the sub-tribe Calaminae based on ITS2 and 5S rRNA (Baker et al. 2000a, 2000b). However, selection of DNA regions and taxon sampling were limited in these studies, so paraphyletic relationship was not highly intended (Baker et al. 2000b). The pleisomorphic transition of Plectocomiinae (considered to be the basal clade for sub-tribe Calaminae) to both Calamus and Daemonorops was distinguished by the presence of prophyllar bracts (Baker et al. 2000b). Moreover, the genus Daemonorops was further divided into two subgenera i.e. Piptospatha and Daemonorops based on the nature of prophyllar bracts (Beccari 1911, Furtado 1953). Daemonorops is distributed over 101 species throughout South and South East Asia (Dransfield et al. 2008). In India, out of 6 species, five are present in Indo-Myanmar (Andaman Islands) and one in IndoChina (Arunachal Pradesh and Nagaland) (Renuka et al. 2010, Senthilkumar et al. 2014). Among them, Daemonorops jenkinsianus belongs to the section Piptospatha and all other species belong to section Daemonorops (Basu 1992). Particularly, the species distributed in the Andaman Islands are morphologically highly variable in their quantitative measures (Senthilkumar et al. 2014).Though they have colonized most of islets of Andaman groups, but few species are highly circumscribed into smaller forests such as D. kurzianus, D.wrightmyoensis, and D. aurea (Renuka et al. 2010). However the taxonomic relatedness is quite uncertain among them. The morphological uncertainty plays a pivot role in this particular genus due to the variance at intraspecific level (within populations) and also the less dissimilarities within genera (inter-specific). So, here, Communications in Plant Sciences (ISSN 2237-4027) based on the molecular studies, the congeneric species of genus Daemonorops were tested based on phylogenetic and character analysis using nuclear DNA barcodes. MATERIAL AND METHODS Sampling. For the present study, all the six species of Daemonorops were collected from Arunachal Pradesh and Andaman group of islands during the survey on these climbing palms. Comparative analyses of the morphological characters of the species are given in Table 1. Herbarium specimens were prepared with replicates and deposited at School of Ecology and Conservation, UAS, GKVK, Bangalore (Table 2). The Genbank accession numbers and voucher data of the species are presented in Table 2. Out-groups were included from sub tribe Plectocomiinae, Korthalsiinae and also a species from Calamus to construct both ITS2 and RPB2 trees. DNA barcodes. The ribosomal internal transcribed spacer 2-ITS2 (ITS3 F and 28R – Hamzeh and Dayanandan 2004) and RNA polymerase II (RPB2Palm INT23 – Loo et al. 2007) of the nuclear genome were selected based on their divergence at both inter and intra-specific level and also based on the earlier studies in these climbing palms (Baker et al. 2000). PCR amplification, sequencing. Genomic DNA was extracted using a modified CTAB method (Doyle and Doyle 1987). DNA was amplified for both the markers ITS2 and RPB2. PCR conditions were followed based on Senthilkumar et al. (2013). The purified PCR product was sequenced using an ABI310 automated genetic analyzer. Sequence editing, character analysis and datasets. Sequences were edited in Geneious Pro 5.4.6 (Drummond 2011) and aligned using the program Muscle (Edgar 2004) inbuilt in MEGA 6.0 (Tamura et al. 2013). The alignment matrix was imported into the program Mesquite 2.7.4 (Maddison and Maddison 2010) for the manual alignments when required. Based on the final alignment, the datasets were designed into two for each barcode as multiple (using multiple individuals of 6 congeneric species) and consensus (using only consensus sequence of multiple individuals from each species) (Table 3). Further, the distance analysis and character analysis were performed simultaneously to check the divergence at both inter and intra-specific level and also the evolutionary models (Table 3). The aligned matrix was tested for models using the program jModeltest v. 2.1.4 (Darriba et al. 2012). Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015) Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India Using DNA barcodes. Table 1. Details of voucher specimens and nucleotide submissions of Daemonorops and out groups used in this study. Species Name Calamus viminalis Wild. Locality Voucher ID KFRI-Peechi [Kerala] Senthilkumar 216 ITS2 RPB2 KJ532894 KJ501055 KJ532903 KJ532904 KJ532905 KJ532906 KJ532907 KJ532908 KJ532909 KJ532910 KJ532911 KJ532912 KJ532913 KJ532914 KJ532915 KJ501059 KJ501060 KJ501061 D. aurea Renuka & Vijayak. Chidiyatapu, Tharmughali Island [South Andaman (SA)] Senthilkumar 689, 794, 802 D. jenkinsiana (Griff.) Mart. IBG [West Bengal]; SFRIItanagar [Arunachal Pradesh] Senthilkumar 318RA, 605, 643 Nayasahar, Carbyn Chowk, Kalatang RF [SA] Senthilkumar 702, 714, 721 Indian Botanical GardenHowrah [West Bengal]; Tharmughali Island [SA] Senthilkumar 604, 800, 803 Krishna Nallah 12KM, Little Andaman [SA] Senthilkumar 851, 870, 871 KJ532916 KJ532917 KJ501067 KJ501068 KJ501069 KJ501070 Papeeta Pahad, Wrightmyo, SA Senthilkumar 847 KJ532918 KJ501071 D. fissa Blume South East Asia Baker et al. (2000) AJ242073, 74, 75 D. didymophylla Becc. South East Asia Baker et al. (2000) AJ242070, 71, 72 Korthalsia laciniosa(Griff.) Mart. Mannarghat [SA] Senthilkumar 694 KJ532919 KJ501072 TBGRI-Palod [Kerala] Senthilkumar 457A KJ532921 KJ501077 Tippi-Subansiri [Arunachal Pradesh] Senthilkumar 631 KJ532926 KJ501080 D. kurziana Hook.f. ex Becc. D. manii Becc. D. rarispinosa Renuka & Vijayak. D. wrightmyoensis Renuka & Vijayak. Korthalsia rogersii Becc. Plectocomia himalayana Griff. KJ501062 KJ501063 KJ501064 KJ501065 KJ501066 Table 2. Morphometric analysis of congeneric species of Daemonorops. Character/ Species Stem size (diameter) Sheath armature D. aurea D. jenkinsiana D. kurziana D. manii D. rarispinosa D. wrightmyoensis 3.5 cm 3 cm 5 cm 3 2.5 cm 4.5 cm Sparingly armed with group of spines below the knee Densely armed with needle like spines Armed with distinct papery spines at mouth Armed with dimorphic spines Unarmed or rarely armed Armed with dimorphic spines Sheath indementum Sheath colour Absent Reddish brown Brown scurfy Brown tomentum Absent Dusty tomentum Yellowish green Reddish brown Pale brown Brownish green Straw yellow Pale green Inflor. Length 35 cm 80 cm 45 cm 30 cm 40 cm 30 cm Prophyllar bracts Greenish brown Reddish brown Straw yellow Yellowish brown Pale brown Pale brown Female Rachillae Distichus Sinuous Sinuous Sinuous Distichus Distichus Fruit shape Globose Globose Spherical Spherical Globose Oblongoid 2 x 1.5 cm 2 x 1.8 cm Yellow; tightly imbricated 2 cm across 1.5 cm across 1.5 cm across 1 x 1.5 cm Orange yellow Brown Pale yellow Brown Fruit size Fruit scales Golden Yellow Table 3. Datasets and its character analysis. Dataset No. of Taxa Alignment length (bps) Pi Pui C Evolutionary Model* ITS2_multiple 12 (25 inds) 303 81 (26.7) 144 (47.5) 158 (52.2) TPM1uf+G 12 303 49 (16.2) 66 (21.9) 236 (77.9) TPM1uf+G 10 (17 inds) 679 50 (7.4) 74 (10.9) 604 (90) HKY+G RPB2_consensus 10 680 45 (6.6) 78 (11.5) 600 (88.2) HKY Combined 12 983 95 (9.7) 50 (5) 838 (85.3) HKY+G ITS2_consensus RPB2_multi Pi-Parsimony informative; Pui- Parsimony uninformative; C-Conserved sites; * Model selected based on BIC values. The percentage is in parentheses. Phylogenetic analysis. Bayesian analysis was perfomed for all the four datasets using mixed models for the posterior analysis in MrBayes v3.1.2. (Ronquist and Huelsenbeck 2003). MCMC (Markov Chain Monte Communications in Plant Sciences (ISSN 2237-4027) Carlo) was run for 1M generations with 4 chains (of hot and cold). The run was not continued further once it reached the split frequency of standard deviation <0.01. The trees were summarized as 50% majority Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015) 3 4 Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India using DNA barcodes. rule consensus tree and viewed in FigTree v1.3.1 (Rambaut 2009). The related attributes of the tree such as posterior values and substitution changes per sites were drawn for each tree. Maximum parsimony analysis was carried out for only combined dataset to derive the entire parsimonious information from both the barcodes using PAUP* v.4.0b10 (Swofford 2002). Bootstrap analysis was conducted for 1000 replicates using default settings except for the ‘maxtrees’ option that was left unchanged. UPGMA (Unweighted Pair Group Method with Arithmatic mean) analysis was done in MEGA 6.0 for RPB2 consenus dataset with Indian Daemonorops. high between these two species (0.165) when they are analyzed separately (Figure 3). RESULTS AND DISCUSSION Morphometric analysis. Both discrete and continuous trait analysis have shown relatively sufficient information on congeneric species whether they are distinct or not (Table 2). Moreover, the comparative character analysis distinguishes the section Piptospatha (D. jenkinsiana) and Daemonorops (other 5 species from Andaman Islands). The following characters such as stem size, sheath armature, size of the prophyllar bract, fruit shape are considered as diagnostic keys among these species. Similarly, D. jenkinsiana is distinguished by its long prophyllar bract that enfolds the inner bracts within and forms the beak (Figure 1). D. kurziana and D. manii differ based on the large sized stem and densely covered brown scurfy tomentum respectively (Figures 1 and 2). Mouth of the sheath covered with papery spines is distinct in D. kurziana (Figure 2). But, the other 3 species of Daemonorops are more or less similar to each other and D. wrightmyoensis differs only in fruit shape (oblongoid) (Figure 1). D. aurea and D. rarispinoa stand out as distinct species based on their sheath armature (rarely or sparingly armed) but the other quantitative traits are variable within populations (Figures 1 and 2). Phylogenetic analysis Distance analysis. The aligned matrix of all the five datasets for the distance analysis was carried out to obtain the pairwise distance (p-distance) using neighbor joining (NJ) method for both the barcodes. The region ITS2 shows higher inter-generic mean distance (0.126) than the RPB2 (0.06) (Figure 2). Similarly, the distance at inter-specific is relatively high (0.053) when South East Asian species was included in the analysis (0.0053) (Figure 3). But, when D. fissa and D. didymophylla were excluded from the matrix, the distance is none (0). The distance is significantly Communications in Plant Sciences (ISSN 2237-4027) Figure 1. Genus Daemonorops Bl. (1) Sheath sparsely armed-Daemonorops aurea Renuka & Vijayak. [insectGlobose fruits]; (2) Sheath densely armed & covered by brown indumentum-D. manii Becc.; (3) Sheath armed with half whorled spines-D. jenkinsiana (Griff.) Mart. [insect=Sheath covered by reddish tomentum]; (4) D. wrightmyoensis Renuda & Vijayak. [insect=Oblongoid fruits; copyright to Renuda & Sreekumar, 2012]. Further, both the regions failed to differentiate the species (the inter-specific of RPB2 was 0.002, Figure 3). Among them, RPB2 is comparatively better to distinguish the congeneric species. So, the distance tree is generated based on the UPGMA method here. The UPGMA tree shows two separate clades i.e. Piptospatha and Daemonorops, which are distinctly clustered here (Figure 4). The clade 1 collapsed because they are identical to each other at inter-specific level. The phylogenetic distance is weighed as 0.025 from tips to the root which indicate inter-generic distance at substitutions level (Figure 4). The evolutionary models were tested for all the datasets based on their substitution schemes of invariable sites (I) and gamma distribution (G) (Table 3). The models were selected based on the BIC (Bayesian information Criterion) from computed likelihood scores. Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015) Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India Using DNA barcodes. Figure 2. Daemonorops Bl. (1) Sheath armed with papery spines-D. Kurziana Hook.f. ex Becc. [insect=rachillae with yellow fruits]; (2) Sheath sparingly armed-D. rarispinosa Renuka & Vijayak. [insect=Shiny yellow fruits with strongly grooved scales]. clade, which is due to the contribution of RPB2 intron 23 (Figure 6). So here, the genetic distance is minimized to 0.02 in ITS2 dataset but again the distance is solely represented by south East Asian species (Figure 6). Daemonorops is one among the largest genus in calamoideaes (scaly fruited palms) widely distributed in Asian tropics. It showed paraphyletic relationship with genus Calamus (Baker et al. 2000b, Baker et al. 2009). Further, the congeneric species of this genus is highly variable in their morphology and has been challenged for several decades (Beccari 1911, Furtado 1953, Dransfield et al. 2008, Renuka et al. 2010). The problem of misidentification is due to higher variability in their visible morphological characters even at the population level. So the study was carried out to contribute an information on species delimitation in these congeneric species based on the measure of both inter and intraspecific divergence. However, the three species namely D. jenkinsiana, D. manii, and D. kurziana being distinct and another three species that are highly versatile to be designated as distinct species based on their described characters that are quantitatively highly variable. Molecular analysis carried out in the present study indicates a very low genetic distance (0) in the Indian Daemonorops compared to the Daemonorops distributed in the South East Asia (0.165). The phylogenetic analysis based on both Bayesian and parsimony reveals that there is relative increase of node support at the deeper nodes of Daemonorops when multi loci analysis was carried out using the combined dataset. RPB2 intron region shows comparatively high mean distance than ITS2 for Indian Daemonorops. Thus, species delimitation cannot be derived alone with single gene or marker. Moreover, for such studies, the marker selection should be robust and highly informative. Besides, the sampling of the study system also should be complete and dense for robust analysis. CONCLUSION Figure 3. Distance analysis between the gene trees of ITS2 and RPB2 in MEGA 6.0. The histogram represented the mean distance with standard error bars. Character based analysis. Bayesian analysis result the majority rule consensus tree which shows higher posterior density values at each node. But the clade Daemonorops is not resolved in data sets of multiple individuals of both ITS2 and RPB2 or otherwise it shows that they are identical (Figure 5). Hence, when the genetic distance is compared, there is large gap (0.04-0.009) between the gene trees. Moreover, the combined dataset shows the relative increase of the posterior values at deeper nodes in Daemonorops Communications in Plant Sciences (ISSN 2237-4027) The study indicates three species (D. jenkinsiana, D. manii, and D. kurziana) in India, and another three species (D. wrightmyoensis, D. aurea, and D. rarispinosa) from Andaman group of islands could be the variabilities of D. manii that is colonized throughout the archipelago. The study however has not been able to resolve the distance between D. kurziana and D. manii due to the limited markers that were used. The study suggests a complete revision of the genus Daemonorops using a number of DNA regions as well as species to resolve the species delimitation not only in the Indian subcontinent but also for other South East Asian species. Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015) 5 6 Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India using DNA barcodes. Figure 4. UPGMA tree of congeneric species of genus Daemonorops using RPB2 palm Intron 23; tree shows both clade 1 and 2 of Indo-China and Indo-Myanmarese, respectively. Figure 5. Bayesian posterior analysis using multiple individuals of congeneric species of Daemonorops using nuclear ribosomal spacer (ITS2) and intron (RPB2) regions. Asterisk indicates deeper nodes with relatively high posterior values in RPB2 than ITS2. Communications in Plant Sciences (ISSN 2237-4027) Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015) Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India Using DNA barcodes. Figure 6. Bayesian posterior tree based on combined analysis (ITS2+RPB2). Tree represented by consensus of multiples of each congeneric species (RPB2 sequences are not available for South East Asian species). Values at right side of the node indicate the posterior probabilities in percent and the values above the bootsrap support derived from parsimony analysis. Acknowledgements We are thankful to the Principal Chief Conservator of Forests, Haddo, Port Blair, and Chief Wildlife warden, Haddo, Port Blair, for permitting to explore the forests throughout the A and N islands. We thank Dr. C. Murugan, Scientist ‘C’, Ms. Lakra, Botanist of the Andaman and Nicobar regional centre, BSI, for their helpful guidance to visit different localities. We also acknowledge the Department of Biotechnology, Government of India, for its financial support to carry out this study. References Baker WJ, Coode MJE, Dransfield J, Dransfield S, Harley MM, Hoffmann P, Johns RJ. 1998. Patterns of distribution of Malesian vascular plants. In: Hall, R., Holloway, J.D. (Eds.), Biogeography and Geological Evolution of SE Asia. Backhuys Publishers, Leiden, The Netherlands. pp. 243–258. Baker WJ, Hedderson TA, Dransfield J. 2000a. Molecular phylogenetics of subfamily Calamoideae (Palmae) based on nrDNA ITS and cpDNA rps16 intron sequence data. Molecular Phylogenetics and Evolution 14: 195–217. Baker WJ, Dransfield J, Hedderson TA. 2000b. Phylogeny, character evolution, and a new classification of the calamoid palms. Systematic Botany 25: 297–322. Baker WJ, Savolainen V, Asmussen-Lange CB. 2009. Complete generic-level phylogenetic analyses of palms (Arecaceae) with comparisons of supertree and supermatrix approaches. Systematic Biology 58: 240–256. Basu SK. 1992. Rattans (Canes) in India: A monographic revision. Rattan Information Centre, Kepong, Kuala Lumpur, pp. 37-45. Beccari O. 1911. Asiatic Palms–Lepidocaryeae, Part II, The species of Daemonorops. Annals of Royal Botanical Garden 12: 1–237. Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bull 19: 11–15. Dransfield J, Uhl NW, Asmussen CB, Baker JW, Harley MM, Lewis CE. 2008. Genera Palmarum: the evolution and classification of palms. Richmond, UK: Kew Publishing. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Communications in Plant Sciences (ISSN 2237-4027) Sturrock S, Thierer T, Wilson A. 2011. Geneious v5.4. Available from http://www.geneious.com. Edgar RC. 2004. MUSCLE: Multiple sequence alignment with high accuracy and throughput. Nucleic Acids Research 32: 1792-1797. Furtado CX. 1953. The genus Daemonorops in Malaya. The Gardens Bulletin 14: 49–147. Hamzeh M, Dayanandan S. 2004. Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast trnTtrnF region and nuclear rDNA. American Journal of Botany 91(9: 1398–1408. Loo AH, Dransfield J, Chase MW, Baker WJ. 2006. Low-copy nuclear DNA, phylogeny and the evolution of dichogamy in the betel nut palms and their relatives (Arecinae; Arecaceae). Molecular Phylogenetics and Evolution 39: 598–618. Maddison WP, Maddison DR. 2010. Mesquite: A modular system for evolutionary analysis, version 2.7.3. Available from http://mesquiteproject.org. Rambaut A. 2009. FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software /figtree). Renuka C, Bhat KV, Pandalai RC. 2010. Rattans of India: Taxonomy, biology and utilization. Kerala Forest Research Institute, Peechi, India. pp. 172. Renuka C, Sreekumar VB. 2012. A field guide to the palms of India. KFRI, Kerala, pp.165 (inset Figure 1a). Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Senthilkumar U, Shivaprakash N, Srirama R, Ravikanth G, Narasimhan D, Ullasa K, Sanjappa M, Ganeshaiah K, Uma Shaanker R, Dayanandan S. 2013. Early Pliocene diversification and dispersal of rattans (Tribe Calamae, Family Arecaceae) in Asian tropical forests. Proceedings of the 49th Annual Meeting of the Canadian Botanical Association, Thompson Rivers University, Kamloops, Canada, June 1-5. Senthilkumar U, Choudhary RK, Sanjappa M, Narasimhan D, Uma Shaanker R, Ravikanth G. 2014. Livelihood and Revenue: Role of rattans among Mongoloid tribes and settlers of Andaman and Nicobar islands, India. Ethnobotany Research and Applications 12: 141–154. Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sinauer Associates, Sunderland. Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015) 7 8 Umapathy et al. 2015. Species delimitation in congenerics of Genus Daemonorops from India using DNA barcodes. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA 6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30: 2725–2729. RESUMO Delimitação de espécies congêneres do gênero Daemonorops da Índia, usando código de barras de DNA. Daemonorops é um dos gêneros mais ricos de palmeiras, amplamente distribuído em florestas perenifólias do Sul e Sudeste da Ásia, e especialmente abundante na Malásia peninsular. O gênero compartilha o clado filogenético com o gênero Calamus, mas distintamente caracterizado pela presença de brácteas profilares na subtribo Calaminae da família Areraceae (Palmae). Contudo, as espécies do gênero Daemonorops apresentam grande variabilidade e homoplasia em suas características morfológicas. Plantas do gênero Daemonorops de origem na Índia contemplam seis espécies, das quais cinco estão distribuídas no grupo de ilhas Andaman e uma espécie na Indo-China. Os objetos de estudo são as espécies da Indo-Myanmar (Ilhas Andamar), pois há dúvidas quanto à separação em espécies distintas. Portanto, o objetivo da pesquisa foi testar a delimitação de espécies congêneres usando análises filogenéticas e de caráter dos genes nucleares. O estudo indica a ocorrência de três espécies (D. jenkinsiana, D. manii, and D. kurziana) na Índia; enquanto outras três espécies (D. wrightmyoensis, D. aurea, and D. rarispinosa) que ocorrem no grupo de ilhas Andaman podem ser variedades de D. manii que colonizaram o arquipélago. Palavras-chave: Daemonorops, Delimitação de espécies, Código de barras de DNA, Ilhas Andaman e Nicobar. Quality of English writing and reference information are of totally responsibility of authors. Communications in Plant Sciences (ISSN 2237-4027) Commun. Plant Sci. 5(1-2): 1-8 (Jan-Jun 2015)