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Journal of Plant Biology Research 2015, 4(2): 73-82 eISSN: 2233-0275 pISSN: 2233-1980 http://www.inast.org/jpbr.html REGULAR ARTICLE Molecular Systematics of some Bulbophyllum species in Peninsular Malaysia based on ITS sequences Shahla Hosseini1* and Kourosh Dadkhah2 1 Department of Biological Science & Biotechnology University of Kurdistan, Sanandaj, Iran. 2 Department of Statistics, Faculty of Science, University of Kurdistan, Sanandaj, Iran. ABSTRACT In this article, ITS sequences of 34 species in Bulbophyllum genus were researched. Alignment between ITS sequences from the species was performed, and discovered that average percentage sequence divergence (uncorrected p distance) within Bulbophyllum species was 9.87% and maximum in-group ITS divergence was 13.68% (between B. membranifolium with B. sulcatum). Molecular systematic analysis of ITS was revealed that, 34 species of Bulbophyllum were monophyletic and they were divided into six groups, which is partly inconsistent with the viewpoint of morphological classification of Bulbophyllum in Peninsular Malaysia. Keywords: Bulbophyllum, Classification, ITS Sequence, Orchidaceae, Peninsular Malaysia INTRODUCTION epiphytic and they are found in different habitats Bulbophyllum Thou. is the largest genus of ranging from (sub) tropical dry forests to wet subtribe Bulbophyllinae (Schlechter, 1926). More montane cloud forests and most of them are fly than 95% of the species in the subtribe belong in pollinated (Bartareau, 1994; Borba & Semir, 1998; the large genus Bulbophyllum, with more than Nishida et al., 2004; Tan et al., 2002; Teixeira et 2000 species found mostly in Asia (Seidenfaden & al., 2004). Orchids of the genus Bulbophyllum are Wood, 1992). Latest taxonomy (Seidenfaden & one of the important plants in Malaysia in terms of Wood, 1992) described the Bulbophyllum in 17 their abundance, but identification of sections with more than 100 species. However Bulbophyllum at species level still remains a based on this study, the members of this large problem for practising taxonomists. In recent genus have undergone extreme reduction in years, there have been several attempts to delimit number of species in the natural habitat and have orchid species boundaries based on phylogenetic acquired an efficient adaptation to the canopy reconstructions, using chloroplast and nuclear environment. Nevertheless, areas of earlier DNA sequences (Bellstedt et al., 2001; Fisher et collection site were visited for possible sampling. al., 2007; Van Den Berg et al., 2009; Hosseini et However not all the previous collection sites are al., 2012). Although some of these studies have forested areas. At least 50% of the recorded revealed concordance with species defined by collection sites are now palm oil plantation area. classical taxonomy, others have not. To date it has Therefore there is possibility to overthrow of not reported that ITS sequence was applied in the several Bulbophyllum species in Peninsular systematic evolution and sibship of Malaysian Malaysia. Bulbophyllum species are mostly Bulbophyllum. ITS sequence is made up of ITS-1 *Corresponding author: Shahla Hosseini. Corresponding author e-mail: [email protected] J. Plant Bio. Res. 2015, 4(2): 73-82 and ITS-2, which are respectively genetic area CLUSTAL W (Thompson et al., 1994). The primer between ribosome 18S and 5.8S, ribosome 5.8S part trimmed from first of the each sequences, and 26S (Tian & Li, 2002; Wang & Liu, 2004). because the character base sequence should not be The nucleotide sequences in ITS sequence take on include primer. Then the end of sequences high variability, which can provide rich matched together and the unmatched sequence information for systematic classification, and then trimmed. All sequences have been deposited in conveniently allow people to study genetic Genbank (Table 1). diversity and sibship among population or species. Data Analysis: As a heuristic tool to explore the Recently, ITS is generally used in studies about degree of saturation present in the datasets, we systematic evolution and sibship of plant plotted raw sequence divergence (uncorrected p population or species (Chanderbali et al., 2001; distance) vs. number of transition and transversion Roalson & Friar, 2004; Kazempour Osaloo et al., substitutions for all pairwise comparisons among 2005 ) which all proved that it is feasible to study taxa, for each position. the systematic evolution and sibship of species in Afterward, ITS sequences were analyzed with dicotyledon by ITS sequence. In this article, ITS PAUP* 4.0b10 (Swofford, 2002), and the was used to study and discuss the sibship among Dendrochilum pallideflavens and Dendrobium species of Bulbophyllum in order to provide more rosellum was designated as outgroup. The MP and information for classification of Bulbophyllum on ML analysis included a default heuristic search for molecule level. the most parsimonious and most likelihood trees: starting trees were obtained via stepwise addition. MATERIALS AND METHODS Sequences were added via simple addition with In this article, the experimental materials were one tree held at each step. Branch swapping was fresh leaves of 34 species in Bulbophyllum were performed via tree-bisection-reconnection (TBR). stored at -80 º C for DNA extraction. Origin of Bootstrap support (BP) was calculated by materials and other information were clearly listed bootstrap analysis from 1000 bootstrap replicates in Table 1. and 500 replications in maximum parsimony and DNA Extraction: Total genomic DNA was maximum likelihood analysis respectively. isolated from leaves of Bulbophyllum and two outgroup species of Dendrobium using Wizard ® RESULTS Genomic DNA Purification Kit (Promega) with ITS Gene Sequences and Variations modifications. Yield and purity of genomic DNA Complete ITS sequences (including the 5.8 S was estimated by spectrophotometry at 260nm and coding region) were analysed for 34 Bulbophyllum the integrity of genomic DNA was determined by species plus Dendrobium rosellum and denaturing agarose gel electrophoresis. Dendrochilum pallideflavens as outgroups. PCR Amplification: Two pairs of primers were Lengths of the ITS 1, 5.8 S and ITS 2 regions were used in this study, The Primers 26SE and 17SE of within the range recorded for other flowering Sun et al. (1994) where used for the amplification plants (Baldwin et al., 1995). The lengths of the of the nrITS regions with the following PCR ITS regions (including the 5.8 S coding region) program: an initial 2 min premelt at 94º C and 34 varied from 677 to 678 bp. For all species, ITS 1 cycles of 1 min denaturation at 94º C, 1 min (261 to 262 bp) was longer than ITS 2 (254 to 255) annealing at 50º C and 1 min extension at 72º C bp). In this study the 5.8 S coding region was 160 followed by a final extension for 7 min. For bp long in all species which was slightly shorter sequencing, the primers ITS4 than that in other angiosperms (161 to 164 bp). (TCCTCCGCTTATTGATATGC) and ITS5 The aligned sequences consisted of 678 nucleotide (GGAAGT AAAAGTCGTAACAAGG) (White et sites which 81 characters are constant among all al., 1990) were used. In addition, PCR products taxa, 603 sites were variable and 86 variable were separated by 1.5% agarose gel characters are parsimony-uninformative, and 517 electrophoresis. sites were parsimony informative. The number and Sequence Alignment: The purified DNA was sent percentage of information sites and mutation sites to First Base Laboratory for sequencing. Multiple in ITS sequence of Bulbophyllum are shown in alignments of sequences were performed using Table 2. Average percentage sequence divergence 74 J. Plant Bio. Res. 2015, 4(2): 73-82 Table 1: Plant materials used in this study. Genbank No. Section Taxon Location Herbarium/ Voucher accession No. 1. Hirtula B. dayanum Gunung Jerai, Malaysia UPM/ B0014 JF428119 2. Hirtula B. hirtulum Fraser Hill, Malaysia UPM/ FAN. FH- 314 JF428126 3. Hirtula B. hirtulum Gunung Belumut, Malaysia UPM/ SH.K-100 JF428127 4. Hirtula B. limbatum Gunung Belumut, Malaysia UPM/ B0054 JF428122 5. Hirtula B. dayanum Gunung Jerai, Malaysia UPM/ SH.K-101 JF428120 6. Cirrhopetalum B. acuminatum Gunung Belumut, Malaysia UPM/ RG 2291 JF428117 7. Cirrhopetalum B. gracillimum Gunung Panti, Malaysia UPM/ B0053 JF428125 8. Cirrhopetalum B. sp1 Genting Highland, Malaysia UPM/ SH.K-102 JF428114 9. Cirrhopetalum B. biflorum Malaysia SZU/OR5791999 EF195919 10. Aphanobulbon B. flavescens Fraser Hill, Malaysia UPM/ FAN. FH- 062 JF428102 11. Aphanobulbon B. mutabile Fraser Hill, Malaysia UPM/ FAN. FH- 105 JF428101 12. Aphanobulbon B. linearifolium Fraser Hill, Malaysia UPM/ FAN. FH- 258 JF428103 13. Aphanobulbon B. odoratum Pahang, Malaysia UPM/ B0056 JF428113 14. Aphanobulbon B. apodum Cameron Highlands, Malaysia UPM/ FAN. FH- 276 JF428121 15. Aphanobulbon B. armeniacum Fraser Hill, Malaysia UPM/ SH.K-105 JF428112 16. Aphanobulbon B. caudatisepalum Fraser Hill, Malaysia UPM/ SH.K-106 JF428108 17. Desmosanthes B. sulcatum Gunung Jerai, Malaysia UPM/ FAN. FH- 304 JF428106 18. Desmosanthes B. sp9 Penang, Malaysia UPM/ SH.K-113 JF428118 19. Sestochilus B. macranthum Cameron Highlands, Malaysia UPM/ FAN. FH- 153 JF428097 20. Sestochilus B. inunctum Gunung Jerai, Malaysia UPM/ SH.K-109 JF428110 21. Sestochilus B. lobbii Cameron Highlands, Malaysia UPM/ FAN. FH- 426 JF428099 22. Sestochilus B. uniflorum Genting Highland, Malaysia UPM/ FAN. FH- 107 JF428098 23. Sestochilus B. patens Gunung Jerai, Malaysia UPM/ B005 JF428100 24. Sestochilus B. pileatum Gunung Belumut,