Microsoft Word - 11. Reseda OK 4.doc Bangladesh J. Plant Taxon. 20(2): 233-238, 2013 (December) © 2013 Bangladesh Association of Plant Taxonomists STATUS OF RESEDA PENTAGYNA ABDALLAH & A.G. MILLER (RESEDACEAE) INFERRED FROM COMBINED NUCLEAR RIBOSOMAL AND CHLOROPLAST SEQUENCE DATA M. AJMAL ALI1, FAHAD M. AL-HEMAID, RITESH K. CHOUDHARY2, JOONGKU LEE2, SOO-YONG KIM2 AND M.A. RUB3 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Keywords: Reseda pentagyna; Resedaceae; Saudi Arabia; Endemic; ITS; trnL-F. Abstract The present study focuses on the status of Reseda pentagyna Abdallah & A.G. Miller (Resedaceae). The internal transcribed spacer (ITS) region of nuclear ribosomal DNA and chloroplast trnL-F gene of the questioned species were sequenced. The Basic Local Alignment Search Tool (BLAST) search showed maximum identity with R. stenostachya. The parsimony analysis of ITS, trnL-F and combined sequences data analyses revealed grouping of Reseda species consistent with established taxonomic sections of the genus, R. pentagyna showed proximity with R. stenostachya (100% bootstrap support), nested within the clade of section Reseda. Introduction The Resedaceae include six genera (i.e. Caylusea A. St.-Hil, Ochradenus Delile, Oligomeris Cambess., Randonia Coss., Reseda L. and Sesamoides Ortega) with approximately 85 species, and are widely distributed in the Old World, with a major center of species diversity in the Mediterranean basin (Martín-Bravo et al., 2007). The members of the family Resedaceae has been traditionally considered closely related to Capparaceae and Brassicaceae; however, the Angiosperm Phylogeny Group placed it under the order Brassicales (APG III, 2009). The genus Reseda consists of approximately 65 species, mostly restricted to the Mediterranean basin, while four of them (i.e. Reseda alba L., R. lutea L., R. luteola L. and R. phyteuma L.) are distributed throughout the world (Martín-Bravo et al., 2007). The genus Reseda in Saudi Arabia is represented by seven species, viz. R. alba, R. arabica Boiss., R. aucheri Boiss., R. lutea, R. muricata C. Presl, R. pentagyna Abdallah & A.G. Miller and R. sphenocleoides Deflers (Chaudhary, 1999). Among these, R. pentagyna is endemic to Saudi Arabia, and reported to occur in Northern Hijaz mountain area, Wadi Sawawin and Tabuk of north western Saudi Arabia (Miller and Nyberg, 1994; Chaudhary, 1999; Llewellyn et al., 2010). R. stenostachya is the most closely allied taxon to the endemic R. pentagyna which differs from the latter by presence of only 3-4 toothed capsules as compared to the 5-6 toothed capsules in the latter. In the last two decades, the internal transcribed spacer sequences of nuclear ribosomal DNA has gained much attention, not only because of its efficacy in carrying out phylogeny of the plants at lower taxonomic level, but also to be considered as the most trusted markers available for the DNA barcoding of the plants. Even after facing criticism of its utility, this marker stands parallel 1Corresponding author. Email: majmalali@rediffmail.com 2International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon-305 806, South Korea. 3National Herbarium & Genebank, National Agriculture & Animal Resources Research Center, Riyadh-11484, Saudi Arabia. 234 ALI et al. to the smartest genes available for the molecular phylogeny and plant DNA barcoding. Since the intrigued morphological similarities observed in between R. pentagyna and R. stenostachya (Miller and Nyberg, 1994; Chaudhary, 1999) we planned to carry out molecular phylogenetic analysis of internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA and trnL-F sequences to confirm the species status of R. pentagyna. Materials and Methods The leaf material of R. pentagyna was collected from Wadi Sirr area of Saudi Arabia, and the taxonomic identification was confirmed through consultation of Flora of Saudi Arabia (Chaudhary, 1999) and protologue (Miller and Nyberg, 1994). Total genomic DNA was extracted using the DNeasy Plant Mini kit (QIAGEN, Valencia, CA, USA). The nuclear (internal transcribed spacer sequences of nuclear ribosomal DNA), and plastid (trnL-F) genes were amplified using AccuPower HF PCR PreMix (Bioneer, Daejeon, South Korea). The standard primers ITS (White et al., 1990) and trnL-trnL-F (Taberlet et al., 1991) were used for amplification and cycle sequencing. The amplified products were purified using PCR purification kit (SolGent, Daejeon, South Korea) prior to sequencing. The purified amplified products were sequenced using ABI PRISM 3730XL (Perkin-Elmer/Applied Biosystem, USA) following manufacturer’s protocol. Each sample was sequenced in the sense and anti-sense direction. The nucleotide sequences of both the DNA strands (sense and anti-sense) were obtained and analyzed using Sequence Navigator (Perkin-Elmer/Applied Biosystems) to ensure accuracy of the base pair sequence. For the molecular phylogenetic analysis, ITS and trnL-F sequences of a total of 36 related species of Reseda (comprising representative from all six sections i.e. Glaucoreseda, Leucoreseda, Luteola, Neoreseda, Phyteuma and Reseda as recognized by Martín-Bravo et al., 2007) were retrieved from GenBank (Table 1). According to Martín-Bravo et al. (2007) Oligomeris arose within the ranks of Reseda; hence, sequences of Oligomeris were retrieved from GenBank, and were used as outgroup in the phylogenetic analyses (Table 1). Sequence alignments were performed using Clustal X, version 1.81 (Thompson et al., 1997). Sequence alignments were subsequently adjusted manually using BioEdit (Hall, 1999). Gaps were treated as missing data in phylogenetic analyses. The voucher specimen (Chaudhary et al. 13704) of sequenced plant accession deposited at National Herbarium (RIY) of Saudi Arabia; and the generated sequences submitted in GenBank (Table 1). Maximum parsimony (MP) analysis was performed using PAUP* 4.0b10 (Swofford, 2002). Results and Discussion The combined length of ITS region (ITS1-5.8S-ITS2) in Reseda pentagyna was 634 bp. The ITS1 region was 261 bp (GC content 61%), the 5.8S gene was 162 bp (GC content 56%), and the ITS2 region was 211 bp (GC content 63%). The trnL-F sequence in R. pentagyna was 777 bp (GC content 33%). BLAST search of ITS sequence of R. pentagyna showed maximum identity (99%) with R. stenostachya followed by R. aucheri and R. ellenbeckii (95%), while trnL-F sequence showed maximum identity (100%) with R. stenostachya followed by R. alphonsi, R. buhseana, R. gilgiana and R. sessilifolia (97%). ITS sequence of R. pentagyna differs from R. stenostachya at position 67 and 75 in alignment, however, in trnL-F sequences, no base pair difference was observed in between sequence of R. pentagyna and R. stenostachya. Sequence characteristics and statistics of maximum parsimony trees derived from analyses of ITS, trnL-F and combined data are summarized in Table 2. The maximum parsimony tree derived from analysis of ITS and trnL-F sequence revealed comparatively week bootstrap support than combined analysis; and therefore, only the maximum parsimony trees topology derived from analysis of combined sequence data is discussed here. STATUS OF RESEDA PENTAGYNA ABDALLAH & A.G. MILLER 235 Table 1. Plant accessions used for the molecular phylogenetic analysis of Reseda pentagyna. Taxa GenBank Accession No. ITS trnL-F Ingroup Sect. Glaucoreseda 1. Reseda battandieri Pit. DQ987183 DQ987045 2. R. complicata Bory DQ987172 DQ987046 3. R. glauca L. DQ987182 DQ987040 4. R. gredensis (Cutanda & Willk.) Müll.-Arg. DQ987174 DQ987047 5. R. virgata Boiss. & Reut. DQ987177 DQ987048 Sect. Luteola 6. R. luteola L. DQ987187 DQ987050 Sect. Leucoreseda Subsect. Leucoreseda 7. R. alba L. DQ987198 DQ987053 8. R. attenuata Ball DQ987201 DQ987057 9. R. gayana Boiss. DQ987205 DQ987055 10. R. undata L. DQ987203 DQ987056 11. R.valentina Pau DQ987207 DQ987059 Sect. Leucoreseda Subsect. Erythroreseda 12. R. suffruticosa Loefl. DQ987210 DQ987062 Sect. Neoreseda 13. R. ellenbeckii Perkins DQ987110 DQ986998 14. R. telephiifolia (Chiov.) Abdallah & de Wit DQ987128 DQ986994 Sect. Phyteuma 15. R. alopecuros Boiss. DQ987139 DQ987028 16. R. arabica Boiss. DQ987132 DQ987029 17. R. collina Müll.-Arg. DQ987136 DQ987031 18. R. diffusa Ball DQ987141 DQ987033 19. R. inodora Rchb. DQ987142 DQ987030 20. R. odorata L. DQ987133 DQ987026 21. R. orientalis (Müll.-Arg.) Boiss. DQ987137 DQ987025 22. R. phyteuma L. DQ987146 DQ987032 Sect. Reseda 23. R. alphonsi Müll.-Arg. DQ987108 DQ987005 24. R. amblycarpa Fresen. DQ987125 DQ987001 25. R. aucheri Boiss. DQ987123 DQ986989 26. R. buhseana Müll.-Arg. DQ987119 DQ987004 27. R. crystallina Webb & Berthel. DQ987088 DQ987021 28. R. gilgiana Perkins DQ987114 DQ986999 29. R. lanceolata Lag. DQ987099 DQ987015 30. R. lutea L. DQ987094 DQ987018 31. R. pentagyna Abdallah & Miller JX867260 JX867261 32. R. sessilifolia Thulin DQ987127 DQ986995 33. R. sphenocleoides Deflers DQ987117 DQ986993 34. R. stenostachya Boiss. DQ987156 DQ987007 35. R. stricta Pers. DQ987103 DQ987013 36. R. urnigera Webb DQ987098 DQ987014 37. R. viridis Balf. f. DQ987130 DQ986996 Outgroup 38. Oligomeris dipetala (Aiton) Turcz. DQ987168 DQ987037 39. O. dregeana (Müll. Arg.) Müll.-Arg. DQ987166 DQ987038 40. O. linifolia (Vahl) J.F. Macbr. DQ987165 DQ987039 236 ALI et al. Table 2. Summary of sequence characteristics and MP trees derived from analyses of ITS, trnL-F and combined data. Characters ITS trnL-F Combined data Number of taxa included in analysis (including outgroup) 40 40 40 Sequence characteristics Length of sequenced 627-639 698-785 1325-1424 Aligned length 644 955 1622 Parsimony informative 92 146 433 Tree characteristics Number of trees 334 323 1299 Length 339 327 1305 CI (Consistency Index) 0.643 0.832 0.656 RI (Retention Index) 0.885 0.926 0.860 RC (Rescaled Consistency Index) 0.569 0.770 0.564 HI (Homoplasy Index) 0.478 0.318 0.446 The bootstrap strict consensus tree resulted from combined sequence data analysis has been shown in Fig. 1. The study revealed the grouping of Reseda species according to previously recognized taxonomic sections, which is consistent with earlier report (Martín-Bravo et al., 2007). Moreover, R. pentagyna nested within the clade of the section Reseda, and showed proximity (100% bootstrap support) with morphologically similar R. stenostachya. The ITS sequence of R. pentagyna (which was described based on 5-6 toothed capsule characters) differs from morphologically allied R. stenostachya (3-4 toothed capsule) at aligned position 67 (C in R. pentagyna but missing nucleotide in R. stenostachya) and 75 (C in R. pentagyna, T in R. stenostachya) possibly due to nucleotide polymorphism, a known features of ITS sequences of nrDNA. Bentham and Hooker (1862) reported Reseda as a polymorphic genus with not more than 30 existing species. Latter, Abdallah and de Wit (1978) updated the list with some addition, and emphasized the need of experimental taxonomical research to get a strong support for the delimitation of species. Muller (1864) also described the variations in the morphology of leaf blades of Reseda that might be arranged in various manners and could be entire, crenate to ternately or pinnately (or rarely bi-pinnately) lobed. The occurrence of brachycarpous or macrocarpous capsules in Reseda is a known feature (Muller, 1864). Under various ecological conditions, plants may show certain morphological changes, viz. R. lutea shows change in the proximity of the veins in the lamina (Abdallah and de Wit, 1978). Further, the emergence of indumentums depends more or less on the moisture content present in the plant. In dry condition, these hairs can shrink, flatten or curl; while in wet conditions, they appear as blisters, or a scabrid, or muricated surface. As variations in fruit size within the same species usually do occur, therefore it cannot be taken as a strong taxonomic character for species level delimitation (Donald, 1988); and thus, the wide degree of variation in quantitative fruit-spine characters limits their use taxonomically. The proximity of questioned sequenced material with R. stenostachya in the MPTs indicates the quantitative differences of tooth characters or the variable trait which limits its use in species delimitation; therefore, we herein propose the merger of R. pentagyna into R. stenostachya. STATUS OF RESEDA PENTAGYNA ABDALLAH & A.G. MILLER 237 Reseda complicata Reseda gredensis Reseda virgata Reseda battandieri Reseda glauca Reseda ellenbeckii Reseda buhseana Reseda sphenocleoides Reseda aucheri Reseda sessilifolia Reseda telephiifolia Reseda amblycarpa Reseda viridis Reseda gilgiana Reseda alphonsi Reseda pentagyna Reseda stenostachya Reseda stricta Reseda lanceolata Reseda urnigera Reseda crystallina Reseda lutea Reseda odorata Reseda arabica Reseda collina Reseda orientalis Reseda diffusa Reseda inodora Reseda alopecuros Reseda phyteuma Reseda gayana Reseda suffruticosa Reseda valentina Reseda attenuata Reseda alba Reseda undata Reseda luteola Oligomeris linifolia Oligomeris dipetala Oligomeris dregeana 84 100 59 66 97 100 100 67 84 88 92 59 55 100 97 86 92 100 98 92 56 72 94 100 86 79 89 Fig. 1. Bootstrap strict consensus tree inferred from combined sequence data analysis of internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA and trnL-F region. The Bootstrap strict consensus tree of 1299 maximally parsimonious trees (MPTs) with a total length of 1305 steps, a consistency index (CI) of 0.656, a homoplasy index (HI) of 0.446, rescaled consistency index (RC) of 0.564 and a retention index (RI) of 0.860. Bootstrap values greater than 50% in 1000 bootstrap replicates are shown above lines. Acknowledgement The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. RGP-VPP-195. 238 ALI et al. References Abdallah, M.S. and de Wit, H.C.D. 1978. The Resedaceae: a taxonomical revision of the family. Meded. Landbouwhoogeschool, Wageningen, p. 78. APG III 2009. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants. Bot. J. Linn. Soc. 161: 105-121. Bentham, G., and Hooker, J.D. 1862. Genera Plantarum. Reeve, Williams & Norgate, London. 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