Bangladesh J. Plant Taxon. 22(2): 111-118, 2015 (December) MOLECULAR EVOLUTIONARY RELATIONSHIPS OF EUPHORBIA SCORDIFOLIA JACQ. WITHIN THE GENUS INFERRED FROM ANALYSIS OF INTERNAL TRANSCRIBED SPACER SEQUENCES FAHAD M.A. AL-HEMAID, M. AJMAL ALI1, JOONGKU LEE2, SOO-YONG KIM3 AND M. OLIUR RAHMAN4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Keywords: Euphorbia scordifolia; Euphorbiaceae; ITS; Genotyping. Abstract The present study explored molecular phylogenetic analysis of 28 species of Euphorbia L. for the identification and establishment of molecular evolutionary relationships of Euphorbia scordifolia Jacq. within the genus based on the internal transcribed spacers (ITS) sequences (ITS1-5.8S-ITS2) of nuclear ribosomal DNA (nrDNA). The sequence similarity search using Basic Local Alignment Search Tool (BLAST) of the ITS sequence of E. scordifolia showed the closest sequence similarity to E. supina Raf. The analysis of ITS sequence data revealed four major clades consistent with subgeneric classifications of the genus. Molecular data support placement of E. scordifolia in the subgenus Chamaesyce. Introduction The genus Euphorbia L. (Euphorbiaceae) comprising ca. 2000 species, which is one of the largest genera of the flowering plants (Frodin, 2004; Riina et al., 2013). The main molecular phylogenetic studies of Euphorbia species have addressed the overall phylogeny of the genus, with its four subgeneric clades of Rhizanthium, Esula, Euphorbia, and Chamaesyce (Steinmann and Porter, 2002; Bruyns et al., 2006; Park and Jansen, 2007; Zimmermann et al., 2010). In Saudi Arabia, the genus Euphorbia is represented by 38 species. Of them, E. scordifolia Jacq. is distributed in Cape Verde Island, Ethiopia, Somalia, Sudan, Yemen and also in western region of Saudi Arabia (Abedin et al., 2001). The morphological characters of E. scordifolia overlap with E. supina Raf. (Abedin et al., 2001). The internal transcribed spacers (ITS) sequence of nuclear ribosomal DNA region including the 5.8S gene is the most widely used molecular marker to infer phylogenetic relationships among plant species (Baldwin et al., 1995; Ali et al., 2014). Although reliance on nrDNA ITS sequence as the sole source of phylogenetic evidence has come under criticism because of certain features of its evolution; however, it remains the most efficient locus for generating species-specific phylogenetic inferences and genotyping in most groups of plants (Ali et al., 2013, 2014, 2015). While searching for DNA sequences of E. scordifolia in GenBank as a part of a research for genotyping of unresolved taxonomic status of flowering plants of Saudi Arabia, it was found that E. scordifolia have not previously been sequenced. A perusal of taxonomic literature revealed that 1Corresponding author. Email: majmalali@rediffmail.com 2Department of Environment and Forest Resources, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, South Korea. 3International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806, South Korea. 4Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh. mailto:majmalali@rediffmail.com 112 AL-HEMAID et al. the molecular evolutionary relationships of E. scordifolia distributed in Saudi Arabia is also unknown. Therefore, the present study aims at molecular genotyping of E. scordifolia based on ITS sequence of nrDNA. Materials and Methods Taxon sampling: Leaf materials of E. scordifolia were collected from the herbarium specimens [voucher- Al- Rawshan, altitude 1122 m, 19.08.1978, Don Bermant 146] housed at National Herbaium & Genebank, National Agriculture & Animal Resources Research Center, Ministry of Agriculture, Riyadh, Saudi Arabia (RIY); and the taxonomic identification was confirmed through consultation of Flora of Saudi Arabia (Abedin et al., 2001). DNA extraction, amplification and sequencing: Total genomic DNA was extracted using Qiagen DNeasy Plant Mini Kit (Valencia, CA, USA). ITS sequences of nuclear ribosomal DNA were amplified using AccuPower HF PCR PreMix (Bioneer, Daejeon, South Korea) and primer ITS1 (5/-GTCCACTGAACCTTATCATTT AG-3/) and ITS4 (5/-TCCTCCGCTTATTGATATGC-3/) of White et al. (1990) via polymerase chain reaction (PCR). Each 20 μl volumes of PCR premix contained 2 μl of 10x buffer, 300 μM dNTPs, 1 μl of a 10 pM solution of each primer and 1 unit of HF DNA polymerase. One round of amplification consisted of denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 49 °C for 1 min and extension at 72 °C for 1 min, and a final extension for 5 min at 72 °C. PCR products were purified with the SolGent PCR Purification Kit- Ultra (SolGent, Daejeon, South Korea) prior to sequencing. The sequencing reaction was performed in a 10 µl final volume with the BigDye Terminator cycle sequencing kit (Perkin- Elmer, Applied Biosystems). Cycling conditions included an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. The sequenced products were precipitated with 17 µl of deionized sterile water, 3 µl of 3 M NaOAc, and 70 µl of 95% EtOH. The capillary gel electrophoresis was conducted with Long Ranger Single Packs (FMC BioProducts) by an ABI 3100 automated DNA sequencer (Perkin-Elmer, Applied Biosystems). The sequences were analyzed by ABI Sequence Navigator (Perkin-Elmer/Applied Biosystems). Nucleotide sequences of both DNA strands were analyzed to ensure accuracy. The sequences were subjected to BLAST-searched (Altschul et al., 1990) by NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis: ITS sequences of nrDNA of 28 species of Euphorbia (Table 1) were retrieved from GenBank database of National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Neoguillauminia cleopatra and Dichostemma glaucescens were chosen as outgroup taxa according to previous work (Barres et al., 2011) and were retrieved from GenBank (Table 1). Sequence alignment was performed using CLUSTAL X version 1.81 (Thompson et al., 1997). Sequence alignment was subsequently adjusted manually using BioEdit (Hall, 1999). Gaps were treated as missing data in phylogenetic analyses. The generated sequences were submitted to GenBank (Table 1). The boundaries between the ITS1, 5.8S and ITS2 gene for E. scordifolia were determined in the aligned data matrix, and were exported as a Nexus file and subsequently analysed using Maximum Parsimony (MP) and Maximum Likelihood (ML) methods by MEGA5 (Tamura et al., 2011). The distribution and pattern of nucleotide substitution in all sequences was investigated using HYPERMUT (Rose and Korber, 2000). http://blast.ncbi.nlm.nih.gov/Blast.cgi). http://www.ncbi.nlm.nih.gov). MOLECULAR EVOLUTIONARY RELATIONSHIPS OF EUPHORBIA SCORDIFOLIA 113 Table 1. Plant accessions used for the molecular phylogenetic analysis of Euphorbia scordifolia. Group Subgenus Taxon GenBank Accession No. Ingroup Rhizanthium Euphorbia antso Denis AF537579 Euphorbia atrispina N.E. Br. AF537568 Euphorbia balsamifera Ait. AF537571 Euphorbia clava Jacq. AF537569 Euphorbia namuskluftensis L.C. Leach AF537562 Euphorbia obesa Hook. f. AF537566 Esula Euphorbia aphylla Brouss. AF537540 Euphorbia dendroides L. AF537539 Euphorbia peplus L. AF537532 Euphorbia schimperi C. Presl AF537537 Euphorbia schimperiana Hochst. ex A. Rich. JN207816 Euphorbia Euphorbia abdelkuri Balf. f. AF537458 Euphorbia beharensis Leandri AJ508983 Euphorbia cylindrifolia Marn.-Lap. & Rauh AJ508955 Euphorbia drupifera Thonn. AF537480 Euphorbia epiphylloides Kurz AF537484 Euphorbia milii Des Moul. AJ508974 Euphorbia ramipressa Croizat AF537481 Euphorbia supina Raf. EU659773 Euphorbia teke Schweinf. ex Pax AF537485 Chamaesyce Euphorbia fulgens Karw. ex Klotzsch AF537404 Euphorbia graminea Jacq. AF537410 Euphorbia heterophylla L. GU214931 Euphorbia ipecacuanhae L. AF537397 Euphorbia leucocephala Lotsy GU214932 Euphorbia misera Benth. AF537383 Euphorbia pulcherrima Willd. ex Klotzsch GU214943 Euphorbia scordifolia Jacq. KR704890 Euphorbia sphaerorhiza Benth. AF537412 Outgroup Neoguillauminia cleopatra (Baill.) Croizat AF537581 Dichostemma glaucescens Pierre AF537584 Results and Discussion The combined length of ITS region (ITS1-5.8S-ITS2) in E. scordifolia was 642 bp. The ITS1 region was 266 bp (GC content 53%), the 5.8S gene was 162 bp long (GC content 56%), and the ITS2 region was 213 bp (GC content 58%). The BLAST search of ITS sequence of E. scordifolia showed high identity level (95%) with E. humifusa Willd. followed by E. glyptosperma Engelm., E. maculata L., E. tettensis Klotzsch and E. meganaesos Featherm. Parsimony analysis of the entire ITS region resulted in five maximally parsimonious trees, the consistency index was 0.491, the retention index was 0.709, and the composite index was 0.367 (0.348) for all sites and parsimony-informative sites (in parentheses). There were a total of 499 114 AL-HEMAID et al. positions in the final dataset, of which 223 were parsimony informative. The phylogenetic tree recovered by the analyses provided a clear resolution of taxon included in the analysis at the subgeneric level. Eupphorbia scordifolia nested within the clade of the subgenus Chamaesyce. The ML analyses recovered tree topology similar to MPT; and therefore, only the ML topology is presented here (Fig. 1). A total of 36 specific nucleotide differences, i.e. 19 in ITS1 and 17 in ITS2 region were detected between E. scordifolia and E. supina (Table 2). Table 2. Differences of DNA base pairs between the ITS sequences of Euphorbia supina and E. scordifolia. Specific nucleotide differences ITS1 ITS2 Position in sequence alignment E. supina E. scordifolia Position in sequence alignment E. supina E. scordifolia 18 G A 3 T C 41 T T 22 C T 45 C G 25 T C 56 G T 37 - G 93 C T 49 C T 106 T - 56 A R 113 C T 74 T C 135 A C 94 T C 136 A T 126 T C 137 A T 146 A G 147 T C 151 C A 148 G T 163 C T 149 C T 170 T A 208 C T 173 G A 212 C T 174 A T 215 C T 191 T C 232 T C 192 G A 254 G A 258 G A The Tandem Repeats Finder (Benson, 1999) was used to detect repeats in the ITS sequences. Differences in substitution rates can discriminate functional forms of pseudogenes (Buckler and Holtsford, 1996a,b). The analysis using the program HYPERMUT showed excessive levels of G =>A mutations which indicates that all differences arose from a single substitution sequence. The result was compared to the reference sequences and their physical locations along the sequences were graphically illustrated (Fig. 2). The use of DNA sequences to identify organisms has been proposed as a more efficient approach than traditional and morphological taxonomic parameters (Tautz et al., 2003). In fact, the recent development in DNA molecular systematic techniques including molecular hybridization, cloning, restriction endonuclease digestions and DNA sequencing and phylogenetic theory have changed the epitome of species identification as well as our understanding of the relationships among organisms at various levels in the tree of life which has been advanced greatly MOLECULAR EVOLUTIONARY RELATIONSHIPS OF EUPHORBIA SCORDIFOLIA 115 Fig.1. A maximum likelihood (ML) tree inferred from analysis of sequence data of internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Bootstrap values (1000 × replicates) are indicated. 116 AL-HEMAID et al. Fig. 2. Schematic illustration of the distribution of substitution sites across the ITS region obtained from 29 species of Euphorbia, using Dichostemma glaucescens as reference (red = GG > AG, cyan = GA > AA, green = GC > AC, magenta = GT > AT, black = not G > A transition, yellow = gap). (Ali et al., 2014). From the first report of the utility of the nrDNA ITS sequence in plants (Baldwin, 1992), it has been extensively used to distinguish even very closely related species (Chen et al., 2010; Yao et al., 2010). Moreover, during the last two decades, the nrDNA ITS sequence has gained much attention as smartest gene available for the molecular signature of a taxon (Ali et al., 2013). The present study is the first report of inferring the nrDNA ITS based molecular genotyping of the E. scordifolia. Since, the majority of the species of the genus Euphorbia have to be sequenced; the present study will nevertheless help in DNA barcoding / molecular identification of E. 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