PERIODICUM BIOLOGORUM UDC 57:61 VOL. 118, No 4, 417–424, 2016 CODEN PDBIAD DOI: 10.18054/pb.v118i4.3856 ISSN 0031-5362 original research article Contribution to Globularia phylogeny based on nuclear ribosomal spacer and two chloroplast DNA regions Abstract K. HAZLER PILEPIĆ1 M. FRIŠČIĆ1 Background and Purpose: Molecular approach has a major impact A. DURAN2 on phylogenetic studies of plants, considering that it gives useful information S. MASLO3 about evolutionary events and relations on all taxonomic levels. The sequ- 4 R. GARIĆ ence data of the nuclear ITS and of two chloroplast regions, trnL-trnF S. ČULJAK1 spacer and rbcL gene, obtained from thirteen Globularia L. taxa, including K. ŠUTALO1 five Anatolian endemics, representing six sections altogether, were analyzed 1 Department of Pharmaceutical Botany in order to determine the relations between the European and the Anatoli- Faculty of Pharmacy and Biochemistry an species and get a better insight into the phylogeny of several closely related University of Zagreb Schrottova 39, 10 000 Zagreb, Croatia Globularia taxa. 2 Department of Biology Materials and Methods: Total cellular DNA was extracted from fre- Faculty of Science sh or frozen leaf tissue of thirteen Globularia samples. The ITS regions of Selçuk University nuclear DNA and two chloroplast DNA regions were amplified and sequ- 42 075 Selçuklu, Konya, Turkey enced. Obtained nuclear and combined plastid data matrices were subjected 3 Lundåkerskolan to Maximum Parsimony analyses. Södra Storgatan 45, 332 33 Gislaved, Sweden Results and Conclusions: Molecular data that were obtained in this 4 Institute for Marine and Coastal Research study indicate the existence of separate centers of diversification for the Eu- University of Dubrovnik ropean and the Anatolian Globularia. The results provide support for rela- Kneza Damjana Jude 12, 20 000 Dubrovnik, Croatia tionships among the studied Anatolian endemic species and indications for a redefinition of affinities of some of the European species. The results pre- sented herein are discussed along with available morphological, karyological, Correspondence: Kroata Hazler Pilepi} phytogeographical and molecular data. Department of Pharmaceutical Botany Faculty of Pharmacy and Biochemistry University of Zagreb Schrottova 39, 10 000 Zagreb, Croatia INTRODUCTION E-mail: [email protected] lobularia L. is a small Angiosperm genus mostly comprised of ev- Gergreen perennials and small shrubs, recognizable by blue flowers Keywords: Globularia, ITS, trnL-trnF, rbcL, Maximum assembled in globular capitula, which it was named after. The first -ex Parsimony, Quaternary tensive classifications of Globularia were proposed by Schwarz (1, 2). They comprised 22-25 taxa, which were divided into nine sections. Sub- sequently, Schwarz’s studies regarding this genus have undergone some modifications by several authors(3, 4, 5). The finally accepted classifica- tion of Globularia separates the genus into eight sections based on mor- phological characters, cytological data, ecology and distribution. These include Lytanthus, Polycephalium, Carradoria, Hellenion, Globularia, Alypum, Empetron and Gymnocladium (5). Since most of the taxa are localized in Central and Southern Europe, Anatolia, Northern Africa and Macaronesia, the Mediterranean basin Received January 07, 2016. Revised June 23, 2016. is viewed as their primary center of diversification, which has started Accepted September 01, 2016. approximately 7.57 million years ago in the Miocene (6, 7). Globularia vulgaris L. is the only taxon distributed more northward, reaching the K. Hazler Pilepić et al. Contribution to Globularia phylogeny based on nuclear ribosomal spacer and two chloroplast DNA regions Baltic region. A total of 15 Globularia species are includ- Pharmacy and Biochemistry, University of Zagreb, Cro- ed in the European flora (8) and 11 taxa in the flora of atia. Table 1 lists all the data about plant samples, in- Turkey (9, 10). Among and above these taxa there is a cluding the geographical distribution of investigated considerable number of endemic species, particularly in taxa and GenBank accession numbers of the analyzed Macaronesia (11), Morocco (12), Italy (13), Greece (14) sequences. Total cellular DNA was extracted from 100– and Turkey (9, 15). Taxa of Globularia are known as out- 140g dry, fresh or frozen leaf tissue following the proce- crossing and mainly exhibit two ploidy levels, diploids dure of Doyle and Doyle (27), modified as reported in (2n=2x=16 chromosomes) and tetraploids (2n=4x=32), Petit et al (28). whereby autopolyploidy is a proposed mode of genome duplication (1, 3, 4, 16). Cytogeographical relations indi- Amplification and Sequencing cate that endemic taxa and taxa which grow in moderate The ITS regions of nuclear DNA and two chloroplast climate areas are predominantly diploids, while taxa with DNA regions were amplified via the polymerase chain broader distributions and growing in rigorous habitat reaction (PCR) using the primer pairs designed by White conditions are mainly tetraploids (1, 3). All Globularia et al. (29) for ITS, Taberlet et al. (30) for trnL-trnF and examined by now have the same pollen morphology (17). Hasebe et al. (31) for rbcL. Amplification reactions were From an economic point of view, several Globularia spe- performed in volumes of 50mL containing 0.5mM of each cies are valuable herbal plants, for example G. alypum L. m primer, 200 M of each nucleotide, 2mM MgCl2 and and G. trichosantha Fisch & C.A. Mey., used in tradi- 1.25 units of TaKaRa Taq HS polymerase (Takara Bio tional medicine of countries such as Spain, Italy, Tunisia Inc, Japan). Amplifications were performed using a Biorad and Turkey (18, 19, 20). Several recent investigations MyCycler 1065 under the following conditions: first de- showed that some widespread European taxa also exhib- naturation at 93°C for 3 min, followed by 35 cycles of it medicinal potential (21, 22). template denaturation at 93°C for 1 min, primer anneal- In the past few decades, molecular studies have great- ing at 50°C for 1 min, elongation at 72°C for 1 min and ly contributed to a better understanding of relations and a final extension at 72°C for 10 min. The PCR products plant phylogeny at all taxonomic levels. Accordingly, the were verified by electrophoresis on 0.7% agarose gels con- genus Globularia that had traditionally been included in taining ethidium bromide in Tris-acetate EDTA (TAE) the homonymous family of Globulariaceae, was mean- buffer and detected under UV light. The size of fragments while proposed for inclusion into the Plantaginaceae, was estimated by comparison with a molecular size stan- based on molecular evidence (23, 24). Moreover, the ITS dard (GeneRulerTM DNA Ladder Mix, Fermentas). The molecular data obtained from 23 Globularia taxa pro- PCR products were purified by using the GFX PCR DNA posed interesting conclusions about their Miocene origin and Gel Band Purification Kit (GE Healthcare, USA) in and Pleistocene independent development of three Euro- accordance with the protocol of the manufacturer. DNA pean Alpine/montane and two Mediterranean Globu- sequencing was performed by Macrogen (Seoul, South laria lineages with proposed rate of 0.33 net speciation Korea) on an ABI3730XL DNA sequencer, using the events per million years (6, 7, 25, 26). same primer set as for PCR. Double-stranded sequencing In order to provide better understanding of intrage- was performed for at least 75% of their total length. All neric relationships among Globularia, Maximum Parsi- sequences have been deposited in the GenBank Sequence mony analyses were conducted on three DNA regions Database (Table 1). from seven European, five Anatolian and one Mediter- ranean Globularia taxon. Taking into account the above Sequence Alignment and Phylogenetic mentioned studies that comprise most of the Globularia Reconstruction species, including Macaronesian endemic species, our Nucleotide sequence fragments were manually edited study is focused on the comparison of several European and aligned using ClustalX.2.1 (32). The alignment is and Anatolian species, among which five are endemics. available from the author upon request. The data set con- Phylogenetic relationships were reconstructed using the tained a total of 13 Globularia taxa sequenced in this internal transcribed spacer (ITS) of the nuclear rDNA work, and the sequences of Plantago major L. (trnL-trnF, repeats and two chloroplast regions, nontranscribed trnL- rbcL), P. lanceolata L. (ITS), Veronica officinalis L. (ITS, trnF spacer and plastid gene rbcL. trnL-trnF, rbcL), Digitalis lanata Ehrh. (ITS, trnL-trnF) and D. lutea L. (rbcL), which were retrieved from Gen- MATERIALS AND METHODS Bank. GenBank accession numbers of outgroup taxa are listed in Table 2. We analyzed the nuclear and combined Plant Material and DNA Extraction plastid data sets independently and thus two sequence Samples of 13 Globularia taxa belonging to six sec- data matrices were generated: (1) an ITS data matrix and tions were studied. The plants were collected from their (2) a trnL-trnF combined with a rbcL data matrix, all natural habitats or obtained from botanical gardens. comprised of 13 Globularia taxa and 3 outgroup taxa. Voucher specimens were deposited in the Herbarium of Sequence data matrices were subjected to phylogenetic the Department of Pharmaceutical Botany, Faculty of analyses using the Maximum Parsimony Method in 418 Period biol, Vol 118, No 4, 2016. Contribution to Globularia phylogeny based on nuclear