Sequencing of nrDNA, plus four cp DNA regions: petN-psbM, trnS-trnG, trnD-trnT and trnL-trnF of newly acquired samples of J. excelsa from Bulgaria, Cyprus and Turkey showed little variation in J. excelsa (sensu stricto), except for the unusual situation in Lebanon, where J. excelsa and J. polycarpos (and likely J. p. var. turcomanica) grow near each other and may be hybridizing. The genetic composition of the eastern-most populations of J. excelsa in Turkey is unknown and deserves further study

Adams, Robert P.

Dagher-Kharrat, Magda Dou

Douaihy, Bouchra

Farzaliyev, Vahid

Gucel, Salih

Mataraci, Tuğrul

Schwarzbach, Andrea E.

Tashev, Alexander N.

English

Scholarworks@UTRGV Univ. of Texas RioGrande Valley

University of Texas Rio Grande Valley ScholarWorks @ UTRGV Health and Biomedical Sciences Faculty Publications and Presentations College of Health Professions 1-5-2016 Geographic variation in nrDNA and four cpDNA regions of Juniperus excelsa: Analysis of new records from Bulgaria, Cyprus and southwestern Turkey Robert P. Adams Baylor University Salih Gucel Tuğrul Mataraci Alexander N. Tashev Bouchra Douaihy See next page for additional authors Follow this and additional works at: https://scholarworks.utrgv.edu/hbs_fac  Part of the Plant Sciences Commons Recommended Citation Adams, R., Gucel, S., Mataraci, T., Müz, E., Vadi, S., İstanbul, Y.-B., Tashev, A., Douaihy, B., Bou Dagher Kharrat, M., Farzaliyev, V., & Schwarzbach, A. E. (2016). Geographic variation in nrDNA and four cpDNA regions of Juniperus excelsa: Analysis of new records from Bulgaria, Cyprus and southwestern Turkey. Phytologia, 98(1). This Article is brought to you for free and open access by the College of Health Professions at ScholarWorks @ UTRGV. It has been accepted for inclusion in Health and Biomedical Sciences Faculty Publications and Presentations by an authorized administrator of ScholarWorks @ UTRGV. For more information, please contact justin.white@utrgv.edu, william.flores01@utrgv.edu. Authors Robert P. Adams, Salih Gucel, Tuğrul Mataraci, Alexander N. Tashev, Bouchra Douaihy, Magda Dou Dagher-Kharrat, Vahid Farzaliyev, and Andrea E. Schwarzbach This article is available at ScholarWorks @ UTRGV: https://scholarworks.utrgv.edu/hbs_fac/39 Phytologia (Jan 5, 2016) 98(1)  1Geographic variation in nrDNA and four cpDNA regions of Juniperus excelsa: Analysis of  new records from Bulgaria, Cyprus and southwestern Turkey  Robert P. Adams Biology Department, Baylor University, Box 97388, Waco, TX 76798, Robert_Adams@baylor.edu  Salih Gucel Environmental Research Institute, Near East University, North Nicosia, Cyprus  Tuğrul Mataraci Eskidji Müz. AŞ., Sanayi Cad. Vadi Sokak No:2, Yenibosna-Bahçelievler, İstanbul, Turkey  Alexander N. Tashev University of Forestry, Dept. of Dendrology, 10, Kliment Ochridsky Blvd., 1756 Sofia, Bulgaria  Bouchra Douaihy and Magda Dou Dagher-Kharrat Laboratoire Caractérisation Genomique des Plantes, Université Saint-Joseph, Campus Sciences et Technologies, Mar Roukos, Mkalles, BP 1514, Riad el Solh, Beirut 1107 2050, Lebanon  Vahid Farzaliyev Deputy Director for Science, Central Botanical Garden, Azerbaijan National Academy of Science, Badamdar shosse 40, Baku, Azerbaijan, AZ 1073.  and  Andrea E. Schwarzbach Department of Health and Biomedical Sciences, University of Texas - Rio Grande Valley,  Brownsville, TX 78520, USA.  ABSTRACT   Sequencing of nrDNA, plus four cp DNA regions: petN-psbM, trnS-trnG, trnD-trnT and trnL-trnF of newly acquired samples of J. excelsa from Bulgaria, Cyprus and Turkey showed little variation in J. excelsa (sensu stricto), except for the unusual situation in Lebanon, where J. excelsa and J. polycarpos (and likely J. p. var. turcomanica) grow near each other and may be hybridizing.  The genetic composition of the eastern-most populations of J. excelsa in Turkey is unknown and deserves further study.  Published on-line www.phytologia.org Phytologia 98(1): 1-7 (Jan. 5, 2016). ISSN 030319430  KEY WORDS:  Juniperus excelsa, J. polycarpos var. polycarpos, J. polycarpos var. turcomanica, J. seravschanica, DNA sequencing, nrDNA, petN-psbM, trnS-trnG, trnD-trnT, trnL-trnF.     Recently, Adams et al. (2014a) examined J. excelsa and putative J. polycarpos from the eastern Mediterreanea eastward into Azerbaijan.  They reported that putative J. excelsa from Azerbaijan is J. polycarpos (= J. excelsa subsp. polycarpos).  Two Lebanon Juniperus populations from Afqa (1306 m) and Wadi El Njass (2287 m), previously shown to be divergent in their microsatellites, were shown to be J. excelsa and J. polycarpos, respectively.  This was the first report of the occurrence of J. polycarpos in Lebanon.                                                                                                                         Phytologia (Jan 5, 2016) 98(1) 2  Analyses of the volatile leaf oils of J. excelsa (Adams et al. 2014b) revealed that the oils from Bulgaria and Greece were higher in α-pinene, limonene and β-phellandrene than populations from Turkey, Cyprus and Lebanon.  Otherwise, there was little variation in the oils between these populations.  Cedrol was a major component in each of the populations, ranging from 22.6 to 29.3%.  Analysei of J. polycarpos var. polycarpos from Azerbaijan revealed the presence of high cedrol and zero cedrol chemotypes.  The high cedrol chemotype was similar to the oil from Armenia.  The Azerbaijan zero cedrol chemotype was similar to the oil from El Njass, Lebanon.    The aforementioned reports were preceded by the Douaihy et al. (2011) study in which 3 microsatellites of putative J. excelsa, reported that the Nei genetic distance separated their 12 populations into 3 groups: Lebanon (Leb 1, 2, 4, 5), Lebanon (Leb 3, 6) and the Crimea, Cyprus, Greece and Turkey populations.  PCO of the data removed 38.8% and 27.5% on the first two axes.  Ordination clearly showed: Lebanon (Leb 1, 2, 4, 5), Lebanon (Leb 3, 6) and the Crimea, Cyprus, Greece and Turkey populations.  El Njass (Leb 3, 2287 m) and Aarsal (Leb 6, 2180 m) are from higher elevations in Lebanon.     Examination of plants (RPA) from Afqa, 1300 m and El Njass, 2287 m, found that Afqa plants had very fine, small leaves.  The  Afqa  leaves   were Figure 1.  Bayesian tree based on nrDNA (ITS) and four cp regions:  bluish green similar to J. excelsa petN-psbM, trnS-trnG, trnD-trnT and trnL-trnF (4430 bp).  Numbers  from Greece.   The  leaves of the at the branch points are posterior probabilities.    El  Njass  plants  were larger,  coarse and yellowish green similar to J. polycarpos from Armenia and J. p. var. turcomanica from Turkmenistan.     In a comprehensive study of the reproductive ecology of Juniperus in Lebanon, Douaihy et al. (2013) reported differences between the higher (El Njass, Aarsal) and lower (Afqa, etc.) populations in cones density classes, frequencies of adult and juvenile trees, and dioecious (El Njass, Aarsal) vs. monecious (Afqa, etc.) individuals.  Interestingly, Adams (2014) describes J. excelsa as monoecious or dioecious and J. polycarpos as dioecious.   Juniperus excelsa M.-Bieb. grows from Greece  (Fig. 2).  Farjon (2005, 2010) treated J. polycarpos, J. p. var. seravschanica and J. p. var. turcomanica as J. excelsa subsp. polycarpos.  However, Adams et al. (2008), Adams and Schwarzbach (2012) and Adams (2013), utilizing DNA sequence data, recognized J. excelsa, in addition to J. polycarpos, J. p. var. turcomanica and J. seravschanica.  Adams Phytologia (Jan 5, 2016) 98(1)  3and Hojjati (2012) and Adams, Hojjati and Schwarzbach (2014), using sequences from 4 gene regions, did not find J. excelsa in Iran, but did confirm J. polycarpos, J. p. var. turcomanica and J. seravschanica in Iran.  Putative J. excelsa from Qushchi, in extreme northwest Iran, had none or only one SNP difference compared with J. polycarpos var. polycarpos from Armenia and was concluded to be J. polycarpos (Adams and Hojjati, 2012).  Adams et al. (2014a) found that putative J. excelsa in Azerbaijan was, in fact, J. polycarpos or in one case, a putative hybrid.   The distribution of J. excelsa in Bulgaria, Cyprus and southwestern Turkey has proved difficult to determine by modern methods of DNA sequencing and leaf essential oil data, due to the lack of samples from these regions.  Recently, materials were obtained of J. excelsa from Bulgaria, Cyprus and southwestern Turkey.  This afforded the opportunity to further examine geographic variation in the DNA sequences of J. excelsa.     The purpose of the paper is to examine nrDNA, and 4 cp DNA regions: petN-psbM, trnS-trnG, trnD-trnT and trnL-trnF and report on variation in J. excelsa.                       Figure 2. Distribution of J. excelsa, J. polycarpos var. polycarpos (P) and J. p. var. turcomanica (T) .  Questionable locations of J. excelsa and J. polycarpos are indicated by E? and P? (modified from Adams, et al., 2014a).  MATERIALS AND METHODS  Plant material -  J. excelsa:  Bulgaria, Central Rhodopes, above the town of Kritchim, Reserve “Izgorialoto Gune”, 42° 01' 22.0" N; 24° 28' 03.1" E, 356 m, Alex Tashev, 2012-1-JE -5-JE, 1 Sep 2012, Lab Acc. Adams 13720-13724,; Cyprus: 34° 57' 45.82" N, 33° 59' 55.33" E, elev. 1461m, Salih Gucel ns, 3 July  2015, Lab Acc. Adams 14570-14574; Greece: Lemos, ca 40° 49' N, 21° 03' E, 1100m, Adams 5983-5985, 5987; Lebanon: Afqa,  34° 04' 58.12"N, 35° 53' 08.52" E, 1306 m,  Bouchra Douaihy 1-3, 4 Nov 2013, Lab Acc. Adams 14155-14157; Turkey: Antalya-Manavgat, Köprülü Canyon National Park, 37° 20' N; 31° 16' E, elev. 550 m,                                                                                                                          Phytologia (Jan 5, 2016) 98(1) 4 Tuğrul Mataraci 2015-18, 24 May 2015, Lab Acc Adams 14569; Isparta-Eğirdir, junction of Kasımlar-Sütçüler road, 37° 28' N; 30° 59' E, elev. 1180 m, Tugrul Mataraci, 2015-7, 24 May 2015, Lab Acc. Adams 14596; ~40 km north of Eskişehir, with Oaks, 39 58.307’N; 30 41.045’ E, Turkey, 820m, Adams 9433-9435;  J. polycarpos:  Armenia: Lake Sevan, 1900m, Adams 8761-8763; Azerbaijan, 40° 44' 41.05" N; 47° 35' 19.14" E, 177-231m, Vahid Farzaliyev 1-10, Dec 2013, Lab Acc. Adams 14162-14171; Lebanon:, Wadi El Njass, 34° 20' 47.79"N, 36° 05' 45.54"E, 2287m, Bouchra Douaihy 4-7,  14 Nov 2013, Lab Acc. Adams 14158-14161;  J. polycarpos var. turcomanica: Turkmenistan:  Kopet Mtns., 38 25.12'N, 56 58.80'E, 1535 m, 22 May 1999, Adams 8758-8790;  J. procera: Ethiopia: on the road to Guder, ca. 40 km w of Addis Abba, ca. 9° 02'N, 38° 24' W, 2400 m, Adams 6184-6188;  J. seravschanica: Pakistan: near Quetta, Baluchistan, A. Hafeez Buzdar ns, 6 Apr 1998, Lab Acc. Adams 8483-8485; Kazakhstan: west end of Talasskiy Ala-Tau Range, ca. 2 km S. of Dzhabagly, 420 24.53’N, 700 28.50’E, 1770m, 12 Sept 1997, Adams 8224-8226. Voucher specimens deposited in the Herbarium, Baylor University (BAYLU).   One gram (fresh weight) of the foliage was placed in 20 g of activated silica gel and transported to the lab, thence stored at -20o C until the DNA was extracted.  DNA 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 µl reactions using 6 ng of genomic DNA, 1.5 units Epi-Centre Fail-Safe Taq polymerase, 15 µl 2x buffer E (petN, trnD-T, trnL-F, trnS-G) or K (nrDNA) (final concentration: 50 mM KCl, 50 mM Tris-HCl (pH 8.3), 200 µM each dNTP, plus Epi-Centre proprietary enhancers with 1.5 - 3.5 mM MgCl2 according to the buffer used) 1.8 µM each primer.  See Adams, Bartel and Price (2009) for the ITS and petN-psbM primers utilized.  The primers for trnD-trnT, trnL-trnF and trnS-trnG regions have been previously reported (Adams and Kauffmann, 2010).    The PCR 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 gel purified DNA 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. R7 (Biomatters. Available from http://www.geneious.com/), the MAFFT alignment program.  Further 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.  Minimum spanning networks were constructed from mutational events (ME) data using PCODNA software (Adams et al., 2009; Adams, 1975; Veldman, 1967).   RESULTS AND DISCUSSION   Sequencing nrDNA (ITS) and four cp regions petN-psbM, trnS-trnG, trnD-trnT and trnL-trnF yielded 4430 bp of data.  The Bayesian consensus tree shows Juniperus seravschanica, J. polycarpos, J. p. var. turcomanica, J. procera and J. excelsa in well-supported clades.   J. excelsa samples, newly Phytologia (Jan 5, 2016) 98(1)  5collected from Bulgaria, Cyprus, and sw Turkey, are in a clade with other J. excelsa (Fig. 3).  There is some minor variation among the J. excelsa samples, mostly notably in the Afqa, Lebanon population.   All of the J. polycarpos samples from Azerbaijan are closely related with J. polycarpos, Armenia along with the El Njass, Lebanon (Adams 14161) sample (Fig. 3).  Three other El Njass samples (Adams 14158, 14158, 14160) appear to be intermediate between J. polycarpos and J. p. var. turcomanica (Fig. 3).                                   Figure 3. Bayesian analysis based on nrDNA, petN-psbM, trnSG, trnDT and trnLF.  Numbers at the branch points are posterior probabilities.     Overlaying a minimum spanning network onto a distribution map gives one a perspective of the geographic trends (Fig. 4).  The newly sampled J. excelsa populations from Bulgaria, Cyprus and sw Turkey are identical or nearly identical to J. excelsa of Eskisehir, Turkey (Fig. 4).  Both the Cyprus and southwestern Turkey populations of J. excelsa showed no differences (Fig. 4).  The Bulgaria J. excelsa differed by none or one difference from Eskisehir, Turkey (Fig. 4).                                                                                                                           Phytologia (Jan 5, 2016) 98(1) 6  As previously reported (Adams et al., 2014a), the Afqa, Lebanon J. excelsa population differs by 2 MEs from Eskisehir, Turkey, which in turn, differs by only 1 ME from the Lemos, Greece population (Fig. 4).  The other Lebanon populations that group with Afqa are probably J. excelsa.     However, the Wadi El Njass, Lebanon (2287 m) population, although near Afqa, is J. polycarpos and differs by 1 to 3 MEs from J. p. var. turcomanica, Turkmenistan and by 1 to 2 MEs from J. polycarpos, Armenia (Fig. 4).  The J. excelsa, Afqa population is only about 100 - 150 km from other J. excelsa populations (Fig. 4), but the Wadi El Njass, J. polycarpos population is 700 to 1000 km from the nearest J. polycarpos population, still, it differs by only 1 to 3 MEs.    Figure 4. Minimum spanning network mapped onto the distributions of J. excelsa and J. polycarpos.  Numbers next to lines are the number of MEs (Mutational Events = base substitutions plus indels).    Clearly, DNA sequencing shows that J. excelsa, as sampled in this study, is a fairly uniform species, except for the unusual situation in Lebanon, where J. excelsa and J. polycarpos (and likely J. p. var. turcomanica) grow near each other and may be hybridizing.  The genetic composition of the eastern-most populations of J. excelsa in Turkey is unknown and deserves study.   ACKNOWLEDGEMENTS   Thanks to Amy Tebeest for lab assistance.  This research was supported in part with funds from Baylor University. Phytologia (Jan 5, 2016) 98(1)  7LITERAT URE CITED  Adams, R. P. 1975.  Statistical character weighting and similarity stability.  Brittonia 27: 305-316. Adams, R. P.  2013.  Correction to Adams and Schwarzbach, Taxonomy of the multi-seeded, entire leaf taxa of the Juniperus, section Sabina: sequence analysis of nrDNA and four cp DNA regions.  Phytologia 95: 226-227. Adams, R. P.  2014. The junipers of the world: The genus Juniperus. 4th ed. Trafford Publ., Victoria, BC.  Adams, R. P., J. A. Bartel and R. A. Price. 2009. A new genus, Hesperocyparis, for the cypresses of the new world.  Phytologia 91: 160-185. Adams, R. P., B. Douaihy, M. D. Dagher-Kharrat, A. E. Schwarzbach and V. Farzaliyev.  2014a. Geographic variation in nrDNA and four cpDNAs sequences of Juniperus excelsa and J. polycarpos in Greece, Turkey. Lebanon and Azerbaijan.  Phytologia 96: 89-95. Adams, R. P., B. Douaihy, M. D. Dagher-Kharrat, V. Farzaliyev, Alexander N. Tashev, K. H. C. Baser and A. K. Christou.  2014b. Geographic variation in the volatile leaf oils of Juniperus excelsa and J. polycarpos.  Phytologia 96: 96-106. Adams, R. P. and F. Hojjati.  2012.  Taxonomy of Juniperus in Iran: Insight from DNA sequencing. Phytologia 94: 219-227. Adams, R. P., F. Hojjati and A. E. Schwarzbach.  2014.  Taxonomy of Juniperus in Iran: DNA sequences of nrDNA plus three cpDNAs reveal Juniperus polycarpos var. turcomanica and J. seravschanica in southern Iran.  Phytologia 96: 19-25. Adams, R. P. and M. E. Kauffmann.  2010.  Geographic variation in nrDNA and cp DNA of Juniperus californica, J. grandis, J. occidentalis and J. osteosperma (Cupressaceae).  Phytologia 92: 266-276. Adams, R. P., J. A. Morris and A. E. Schwarzbach. 2008. Taxonomic study of Juniperus excelsa and J. polycarpos using SNPs from nrDNA and cp trnC-trnD plus essential oils and RAPD data. Phytologia 90: 208-225. Adams, R. P. and A. E. Schwarzbach.  2012. Taxonomy of the multi-seeded, entire leaf taxa of Juniperus section Sabina: Sequence analysis of nrDNA and four cpDNA regions.  Phytologia 94: 350-368. Douaihy, B. G., G. Restoux, N. Machon and M. B. Dagher-Karrat.  2013.  Ecological characterization of the Juniperus excelsa stands in Lebanon.  Ecologia Mediterranea 39: 169-180. Douaihy, B. G., G. Vendramin, A. Boratynski, N. Machon and M. B. Dagher-Karrat.  2011.  High genetic diversity with moderate differentiation in Juniperus excelsa from Lebanon and the eastern Mediterrean region.  AoB PLANTS 2011 pir003 doi:10.1093/aobpla/pir003. Farjon, A. 2005. A monograph of Cupressaceae and Sciadopitys.  Royal Botanic Garderns, Kew Press, London. Farjon, A. 2010. A handbook of the world's conifers. Vol. I. Koninklijke Brill NV, Leiden, The Netherlands. Posada, D. and K. A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817-818. Ronquist, F. and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. Veldman, D. J., 1967. Fortran programming for the behavioral sciences. Holt, Rinehart and Winston Publ., NY.    

Geographic variation in nrDNA and four cpDNA regions of Juniperus excelsa: Analysis of new records from Bulgaria, Cyprus and southwestern Turkey

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Geographic variation in nrDNA and four cpDNA regions of Juniperus excelsa: Analysis of new records from Bulgaria, Cyprus and southwestern Turkey

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