Phytologia (Oct 1, 2014) 96(4) 247 Geographic variation in J. phoenicea var. pltoenicea from throughout its range: Analysis of nrDNA and the petN-PsbM cp region. Robert P. Adams Biology Department, Baylor University, Box 97388, Waco, TX 76798, USA [email protected] Joaquin Altarejos Departamento de Quimica Inorganica Organica, Facultad de Ciencias Experimentales, y Universidad de Jaen, E-23071 Jaen, Spain Montserrat Arista Departamento Biologia Vegetal y Ecologia, Universidad de Sevilla, Apdo. 1095, E-41080 Sevilla, Spain and Andrea E. Schwarzbach Department ofBiomedicine, University ofTexas at Brownsville, Brownsville, TX 78520, USA. ABSTRACT Juniperus phoenicea (var. phoenicea) was examined from five populations throughout its range by sequencing nrDNA and the petN-psbM cp region. In addition, data from seventeen populations ofJ. turbinata {J. phoenicea var. turbinata) were included in a Bayesian analysis. All populations of J. phoenicea were found in a well-supported clade, separate from J. turbinata. Little infra-specific variation was found in J. phoenicea, with only one substitution present in France, one in Zaragoza and one in El Penon populations. The divergence ofthe El Penon correlates with a previous report of differences in the leafvolatile oils. Published on-line www.phytologia.org Phytologia 96(4): 247-251 (Oct. 1, 2014). KEY WORDS: Juniperus phoenicea var. phoenicea, Cupressaceae nrDNA, petN-psbM, geographic , variation, J. turbinata. Adams et al. (2013) analyzed nrDNA and petN-psbM sequences for J. phoenicea L. {sensu stricto) from throughout the Mediterranean region (Fig. 1). They found J. phoenicea var. (or subsp.) phoenicea to be restricted to Spain and France, whereas J. phoenicea var. turbinata (Guss.) Pari. J turbinata Guss.) ( . was widely distributed from the Canary Islands to the Sinai. No differentiation was found between the typical Mediterranean and Canary Island populations, offering no support for the recognition of J. phoenicea subsp. canariensis (Guyot) Rivas-Martinez (Fig. 1). Juniperus turbinata is widespread, DNA ranging from Madeira - Canary Islands to the Sinai with few differences among most populations. However, some populations (Grazalema, Madeira, Sinai, central Italy) displayed (Fig. 1) moderate amounts ofdivergence (3-4 mutations). In a broad phylogenetic study of Juniperus, Adams and Schwarzbach (2013) found that J. phoenicea was not part of a clade of serrate-leafjunipers occurring in the western hemisphere, leading them to denote J. phoenicea as a 'pseudoserrate' juniper. In addition, they found J. p. var. phoenicea and DNA var. turbinata to be as different in their sequences as several other recognized species ofJuniperus. Based on these data, they recognized J. turbinata Guss., as had been proposed by Lebreton and Perez de Paz (2001) based largely on the concentration ofprodelphinidin, a polymeric tannin. The prodelphinidin . 248 Phytologia (Oct. 1, 2014) 96(4) data indicated that J. p. var. phoenicea was J. sabina outgroup , confined to the Iberian Peninsula, while var. 97.9i Grazalema, Spain J. phoenicea 99 8 | turbinata occurred throughout the var. phoenicea Mediterranean region. Lebreton and Perez de 77.3 El Penon, Spain # Paz (2001) found a clear separation between J. 99.9 Sinai phoenicea (Spain and France) and all other 99.8i— Madeira Island populations examined (J. turbinata). 92.3 97.9 fGrazalema, Spain Adams, Altarejos and Arista, 2014) southern Italy Bayesian Tree examined the volatile leaf oil of J. phoenicea based on 2131 bp " sand, Morocco coast (sensu stricto) from throughout its range from nrDNA and (Spain and France). They reported the petN-psbM Atlas Mtns, Morocco composition of the volatile leaf oils of four of Sicily the five populations varied very little except 99.4 for a chemotype (one tree) in the Zaragoza west coast, Turkey population that was high in cedrol and other ) cedarwood terpenoids. However, all five trees Setubal, Portugal sampled in the Grazalema population had the J. turbinata 99.7 (J. phoenicea -JjSabaudia, Italy cedarwood chemotype and were high in var. turbinata) 95.6 cedrol. The oil from the high cedrol plants at -[" Croatia coast Grazalema seems quite different due to the 78.8 F—Cyprus Delphi, Greece presence of cedarwood oil components, but the Cyprus oil is actually not very different, if one - Delphi, Greece removes the heartwood terpenoids and re- Corse Isl., France normalizes the remaining terpenoids (Table 1, 56.7 Tarifa sand, Spain Adams et al., 2014). Canary Islands It is of interest to examine variation in Crete DNA sequence data from the same populations (Adams et al., 2014) in France and Spain. The purpose ofthe present study is to Figure 1. Bayesian tree ofJ. phoenicea and J. turbinata present analyses ofnrDNA and petN-psbM (J. p. var. turbinata) from throughout the species ranges, sequences from populations of J. phoenicea (from Adams et al. 2013). , var. phoenicea throughout its range. MATERIALS AND METHODS Figure 2 shows the distributions ofJ. phoenicea var. phoenicea and populations sampled in this study. Specimens used in this study: J. p. var.phoenicea: France, Narbonne, near St. Pierre sur Mer, 43° 10’ 0.2" N, 3° 09’ 57.6" E, 23 m, J. Altarejos 1-5 Baylor , specs. Adams 14123-14127. Andorra, Coll de Jou near Sant Julia de Loria, 42° 26’ 56.8" N, 1° 28’ 04.6" E, 1426 m, J. Altarejos 6-10, Baylor specs. Adams 14128-14132. W Spain, Zaragoza, Montes de la Retuerta de Pina ofBujaraloz, 41° 28’ 59"N, 0° 19’ 31.2"W, 317 m, J. Altarejos 11-15 Baylor specs. Adams 14133-14137 , Spain, El Penon, approx. 25 km west ofGuadahortuna, 37 ° 35' 38" N, 3 ° 31' 22" W, 760 m, Adams 7077-7079 , Spain, Cadiz, Sierra de Grazalema, 36° 47' 51.5" N, 5°24' 43.7"W, 835 m; M. Arista 1-5, Baylor specs. Adams 13813-13817. Phytologia (Oct 1, 2014) 96(4) 249 Locations ofpopulations ofJ. turbinata are listed in Adams et al. (2014). Voucher specimens BAYLU are deposited at herbarium Baylor University. One gram (fresh weight) ofthe foliage was placed in 20 g of activated silica gel and transported DNA DNA to the lab, thence stored at -20° C until the was extracted. was extracted from juniper leaves by use of a Qiagen mini-plant kit (Qiagen, Valencia, CA) as per manufacturer's instructions. Amplifications were performed in 30 pi reactions using 6 ng of genomic DNA, 1.5 units Epi-Centre Fail- K Safe Taq polymerase, 15 pi 2x buffer E (petN, trnD-T, trnL-F, trnS-G) or (nrDNA) (final mM mM pM concentration: 50 KC1, 50 Tris-HCl (pH 8.3), 200 each dNTP, plus Epi-Centre proprietary enhancers with 1.5 - 3.5 mM MgCl2 according to the buffer used) 1.8 pM each primer. See Adams, Bartel and Price (2009) for the ITS and petN-psbM primers utilized. PCR The reaction was subjected to purification by agarose gel electrophoresis. In each case, the band was excised and purified using a Qiagen QIAquick gel extraction kit (Qiagen, Valencia, CA). The DNA gel purified band with the appropriate sequencing primer was sent to McLab Inc. (San Francisco) for sequencing. Sequences for both strands were edited and a consensus sequence was produced using Chromas, version 2.31 (Technelysium Pty Ltd.) or Sequencher v. 5 (genecodes.com). Sequence datasets were analyzed using Geneious v. R6-1 (Biomatters. Available from http://www.geneious.com/) and the MAFFT alignment program. Analyses utilized the Bayesian analysis software Mr. Bayes v.3.1 (Ronquist and Huelsenbeck, 2003). For phylogenetic analyses, appropriate nucleotide substitution models were selected using Modeltest v3.7 (Posada and Crandall, 1998) and Akaike's information criterion. Figure 2. Distributions ofJ. phoenicea and J. turbinata (adapted from Adams, 2014; Lebreton and Perez de Paz, 2001). Gray shaded area shows the general distribution of J. phoenicea (var. phoenicea and ) solid circles inside squares show the five populations ofJ. phoenicea sampled in the present study. Figure 3. Distribution of J. phoenicea var. phoenicea showing populations sampled in this study (closed circles) 250 Phytologia (Oct. 1, 2014) 96(4) RESULTS AND DISCUSSION c 0.90 El Penon, Spain phoenicea J. — El Penon, Spain Sequencing nrDNA and petN-psbM 0.96 — Grazalema, Spain resulted in 2131 bp of data. The Bayesian — Grazalema, Spain — Andorra 0.85 tree shows (Fig. 4) all populations of J. Andorr—a phoenicea (var. phoenicea in a well- i.o — France ) — |' France supported clade, separated from J. turbinata Q-99 — Zaragoza, Spain (= J. phoenicea var. turbinata). [ Zaragoza, Spain The El Penon population shows the 1.0 largest differentiation (Fig. 4). France and - Turkey Zaragoza populations show small differences - Turkey (Fig. 4). No substitutional differences were - Turkey - Essaouria sand, Morocco found between the Andorra and Grazalema - Essaouria sand, Morocco populations (Fig. 4). - Portugal - Portugal - Sicily To examine this geographical 0.99 - Sicily pattern, the order of linkage in the Bayesian - Sinai J. turbinata - Sinai tree (Fig. 4, J. phoenicea clade) was contour- (J. p. var. turbinata) - Oukaimeden Mtns, Morocco mapped (Fig. 5). This shows the Andorra Ouk—aimeden Mtns, Morocco (AN) population linked to the Grazalema o,95 |— Croatia —' Croatia (GR) population (no substitutional 0.83 northern Italy j differences found). Next, the Zaragoza (ZA) northern Italy 0.90 - Grazalema, Spain population enters the tree; then, the France — Grazalema, Spain (FA) population enters (Fig. 5). The most Q.72 — Cyprus Cyprus divergent population (El Penon, EP) enters Delphi, the tree as the last member of the J. Greece phoenicea clade (Fig. 5). — Delphi, Greece Crete — The uniformity of the J. phoenicea Bayesian Tree Crete Corse populations is shown in their petN-psbM based on 2131 bp - Corse sequences that had only one substitution (in from nrDNA and 0.55 —- Tarifa sand, Spain the France population) and two indels. One petN-psbM —Tarifa sand, Spain —Tenerife, Canary 1st 19 bp indel was present in both samples from Tencerife, Canary 1st Grazalema and one sample from El Penon. 0.96 Corse 1 A Corse The other indel, a poly 10-mer, was in all — °-99 — Madeira samples, except for an 11-mer in one sample '| Madeira from France and El Penon and found as a 12- mer in the second sample from France. Figure 4. Bayesian tree for J. phoenicea populations (upper clade). Juniperus turbinata samples included for reference. Numbers at the branch points are posterior probabilities (1.0 - 0.0). The J. phoenicea populations were a little less uniform in their nrDNA with 5 substitutions and one indel. One substitution event was found in both Zaragoza individuals. Another substitution was present in both El Penon samples and the third substitution event was present in both trees from France. The final two substitution events were present in only one tree: 7078 from El Penon. The 3-bp indel was absent in both trees from El Penon and one sample from Grazalema. Phytologia (Oct 1, 2014) 96(4) 251 Figure 5. Contoured Bayesian analysis of J. phoenicea populations based on nrDNA and petN- psbM. AN = Andorra, FR = France, ZA = Zaragoza, GR = Grazalema, EP = El Penon. In our study of geographic variation in the leaf essential oil from these same five populations of J. phoenicea (Adams et al., 2014), the leaf oils of one individual from Zaragoza and all the trees from Grazalema contained large amounts of typical heartwood oil components (a-cedrene, cis-thujopsene, a- alaskene, allo-cedrol, cedrol, etc.). When we corrected the leaf constituents (by removing the heartwood components and re-normalizing the percentages), there was little variation among the five populations, except for four compounds in the El Penon population: myrcene, p-phellandrene, a-terpineol were in higher concentrations, whereas, (E)-caryophyllene was present in a lower concentration. The divergence DNA of the El Penon trees is congruent with the divergence in the data presented in this study. Additional research is needed to understand the divergence ofthe El Penon population. ACKNOWLEDGEMENTS Thanks to Tonnie Yanke for lab assistance. This research was supported in part with funds from Baylor University. Thanks Dr. Carlos Fernandez (University of Jaen, Spain) for providing assistance with the herbarium work. LITERATURE CITED Adams, R. P. 2014. The junipers ofthe world: The genus Juniperus. 4th ed. Trafford Publ., Victoria, BC. Adams, R. P., J. Altarejos and M. Arista. 2014. Geographic variation in the volatile leafoils J. phoenicea var. phoenicea from throughout its range. Phytologia 96: 110-116. A Adams, R. P., J. A. Bartel and R. A. Price. 2009. new genus, Hesperocyparis, for the cypresses ofthe new world. Phytologia 91: 160-185. Adams, R. P., A. Boratynski, M. Arista, A. E. Schwarzbach, H. Leschner, Z. Liber, P. Minissale, T. Mataraci and A. Manolis. 2013. Analysis ofJuniperusphoenicea from throughout its range in the Mediterranean using DNA sequence data from nrDNA and petN-psbM: The case for the recognition ofJ. turbinata Guss. Phytologia 95: 202-209. Adams, R. P. and A. E. Schwarzbach. 2013. Phylogeny ofJuniperus using nrDNA and four cpDNA regions. Phytologia 95: 179-187. Lebreton, P. and P. L. Perez de Paz. 2001. Definition du Genevrier de Phenicie Juniperus aggr. ( phoenicea reconsidere a ses limites biogeographiques Mediterranee orientale (Crete et Chypre) et ), : Atlantique (lies Canaries). Bull. Mens. Soc. Linn. Lyon, 70(4) 73-92. : MODEL DNA Posada, D. and K. A. Crandall. 1998. TEST: testing the model of substitution. Bioinformatics 14: 817-818. Ronquist, F. and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.