Phytologia (April 4, 2016) 98(2) 146 Evidence of relictual introgression or incomplete lineage sorting in nrDNA of Juniperus excelsa and J. polycarpos in Asia Minor Robert P. Adams Biology Department, Baylor University, Box 97388, Waco, TX 76798, Robert_Adams@baylor.edu Metin Armagan Yüzüncü Yıl Üniversitesi, Eğitim Fakültesi B Blok Kampüs, Van, Turkey Adam Boratynski* Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035, Kornik, Poland Bouchra Douaihy Department of Life and Earth Sciences, Faculty of Sciences, Lebanese University, Tripoli, Lebanon 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 Central Botanical Garden, Azerbaijan National Academy of Science, Badamdar shosse 40, Baku, Azerbaijan, AZ 1073. 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 and Andrea E. Schwarzbach Department of Health and Biomedical Sciences, University of Texas - Rio Grande Valley, Brownsville, TX 78520, USA. ABSTRACT DNA analysis of Juniperus excelsa from throughout its range revealed that J. polycarpos, instead of J. excelsa occupies central and eastern Turkey. Based on nrDNA (ITS) data, it appears that relictual hybridization has occurred in southeastern Turkey between J. polycarpos and J. turcomanica. Surprisingly, evidence of incomplete lineage sorting or relictual hybridization between J. polycarpos and J. seravschanica was found in central Turkey and northwest Iran. Published on-line www.phytologia.org Phytologia 98(2): 146-155 (April 4, 2016*). ISSN 030319430 *digitally correctedAdam Boratynski, and symbols added to Fig. 3, May, 10, 2016. 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. Phytologia (April 4, 2016) 98(2) 147 Recently, Adams et al. (2016) examined J. excelsa and putative J. polycarpos from the eastern Mediterreanea, eastward into Azerbaijan. A 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 collected from Bulgaria, Cyprus, and sw Turkey, are in a clade with other J. excelsa (Fig. 1). There is some minor variation among the J. excelsa samples, mostly notably in the Afqa, Lebanon population as previously reported (Douaihy et al., 2011, 2013). 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. 1). Three other El Njass samples (Adams 14158, 14158, 14160) appear to be intermediate between J. polycarpos and J. p. var. turcomanica (Fig. 1). Figure 1. 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. 2). The newly sampled J. excelsa populations from Bulgaria, Cyprus and sw 148 Phytologia (April 4, 2016) 98(2) Turkey were identical or nearly identical to J. excelsa of Eskisehir, Turkey (Fig. 2). Both the Cyprus and southwestern Turkey populations of J. excelsa showed no differences (Fig. 2). The Bulgaria J. excelsa differed by none or one difference from Eskisehir, Turkey (Fig. 2). As previously reported (Adams et al., 2014), 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. 2). The other Lebanon populations that group with Afqa are probably J. excelsa. However, the Wadi El Njass, Lebanon (2287 m) population, although near Afqa, grouped with 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. 2). The J. excelsa, Afqa population is only about 100 - 150 km from other J. excelsa populations (Fig. 2), 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 2. 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). Adams et al. (2016) concluded that J. excelsa, as sampled in their study, was 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. However, the genetic composition of the eastern-most populations of J. excelsa in Turkey was unknown and deserved additional study. 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, 2014), utilizing DNA sequence data, recognized J. excelsa, in addition to J. polycarpos, J. p. var. Phytologia (April 4, 2016) 98(2) 149 turcomanica and J. seravschanica. Adams and 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. (2014) found that putative J. excelsa in Azerbaijan was, in fact, J. polycarpos or in one case, a putative hybrid (polycarpos x turcomanica). The distribution of J. excelsa in eastern Turkey has proved difficult to determine by modern methods of DNA sequencing, due to the lack of samples from these regions. Recently, materials were obtained of J. excelsa from central and eastern Turkey. This afforded the opportunity to further examine geographic variation of J. excelsa and J. polycarpos. 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,; Crimea: Karadigski Zapovidnyk, between Kurortne and Koktebel, 44.914° N, 35.215° E, 220m, A. Boratynski, Y. Didukh, K. Romashenko, A. Romo, A. Susanna, 2001, KOR 49898, Karadag near Kolhoznoe, 44° 28' 06" N, 33° 49' 54" E, ca 530m; A. Boratynski, G. Iszkulo, A. Lewandowski, 2006, KOR 45630; Cyprus: 34° 57' 45.82" N, 33° 59' 55.33" E, 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, 550 m, Tuğrul Mataraci 2015-18, 24 May 2015, Lab Acc Adams 14569; Isparta-Eğirdir, junction of Kasımlar-Sütçüler road, 37° 28' N; 30° 59' E, 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; Sirnak, se Turkey on Turkey/Iraq border, 37° 34' 08" N, 43° 09' 45" E, 1754m, Metin Armagan ns, Lab. Acc. Adams 14709-14712, Sirnak Turkey, on Turkey/Iraq border se Anatolia, Prov., near Beytussebap, GE ca 37° 37' 18"N, 42 52' 28" E, 1420m, Metin Armagan ns, Lab. Acc. Adams14715; 3 km from Unlupinar village towards Gumushane, 40° 14' 25.14" N; 39° 27' 19.17" E. ne Turkey, 1763m, Ali Kandemir 10846, Lab Acc. Adams 14713; around Lake Ardicli, near Ergan Mountains, near Erzincan. 39° 37' 47.45" N; 39° 29' 54.3" E. ne Turkey, 1797m, Ali Kandemir 10850, Lab Acc. Adams 14714; Material from specimens at the herbarium, Yüzüncü Yıl University, Van, Turkey: Adams 14750- 1986, 38° 28' 41.7" N, 43° 31' 20.6" E, 2800m; Adams 14751- 1989, 38° 08' 37.5" N, 42° 17' 15.2" E, 1550m; Adams 14752, 1995, 38° 36' 20.2" N, 38° 47' 35.0" E, 1700m; Adams 14753- 1996, 38° 40' 6.6" N, 38° 44' 58.6" E, 1600m; Adams 14754- 2001, 38° 20' 30.7" N, 43° 37' 44.9" E, 2000m; Adams 14755- 2006, 39°13'8.08"N 43° 23' 4.11"E, 1965m; Adams 14756- 2014, 38°04'33.6"N 43° 25' 32.0"E, 2010m; Adams 14757- 2014, 39° 07' 47" N, 38° 47' 0.5" E, 1275m; Adams 14758- 2014, 39° 07' 50.6" N, 39° 07' 27.2" E, 1720m; Adams 14759- 2014, 39° 19' 42.4" N, 39° 25' 41.3" E, 1860m; Adams 14760- 2014, 39° 21' 17.6" N, 39° 28' 28.8" E, 1890m; J. polycarpos: Armenia: Lake Sevan, 1900m, Adams 8761-8763; Azerbaijan, 40° 44' 41.05" N; 47° 35' 19.14" E, 177231m, 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 1415814161; 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; Phytologia (April 4, 2016) 98(2) 150 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 FailSafe 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. R8 (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 The classification of samples on the basis of ITS and petN (cp data) is given in Table 1. First it should be noted that nrDNA does not distinguish J. excelsa (exc) from J. p. var. turcomanica (tur) (compare 1st table entry, Greece A G C C C T A exc vs. Turkmenistan (last entry) A G C C C T A tur.). Secondly, exc (or tur) is very distinct from pol in its nrDNA (exc: A G C C C T A vs. pol: C G T T T T C T). Thirdly, exc.(or tur) is very distinct from ser (exc: A G C C C T A vs. ser: C G C T T T C T). And, finally, nrDNA for pol has only one nucleotide different from ser (pol: C G T T T T C T vs ser: C G C T T T C T). Several plants had nrDNA from one taxon, but cp DNA from another taxon: El Njass (3 E,P); Metin e Turkey, 14757, (S,P); Azerbaijan 14171 (S,P); Elburz Mtn., Iran 12504 (S,P); Lushan, Iran 12798 (S,P); Hastjin, Iran 12795 (S,P); and Qushchi, Iran, 12798 (S,P). Other plants appeared to be hybrids by nrDNA: El Njass, 14161, PxE; Metin e Turkey 14753, PxS; Metin e Turkey 14754, PxS; Metin e Turkey 14758, PxS; ne Turkey 14714, PxS; se Turkey/ Iraq border 14715, PxT(or E); se Turkey/ Iraq border 14709, PxT(or E); se Turkey/ Iraq border 14710, PxT(or E); and Azerbaijan, 14165, PxT(or E). To visualize this variation, plants were mapped with their nrDNA and cp (petN) DNA coded (Fig. 3). It is sometimes difficult to determine whether a variation is due to incomplete lineage sorting or hybridization (see discussion in Naciri and Linder, 2015). In the present study, the odd occurrence of J. seravschanica nrDNA in central-eastern Turkey plants seems more likely incomplete lineage sorting than hybridization, because no pure J. seravschanica grows sympatric with J. polycarpos in the area. Long distance cross-pollination is possible but unlikely as the nearest known J. seravschanica is quite distant (Fig. 4). In northwestern Iran one P,P and three S,P plants were found. This may be due to either hybridization or incomplete lineage sorting. Additional samples are needed to better understand that region. Phytologia (April 4, 2016) 98(2) 151 Table 1. Classification of samples, on ITS and cp (petN) sequence data. exe = excelsa, pol = polycarpos, tur = turcomanica, ser = seravschanica. PxE = hybrid pol x exc; PxS = hybrid pol x ser; PxT(E) = pol x tur (or exc, as ITS for exe = tur). source n Greece, 1010m n Greece, 1010m n Greece, 903m Bulgaria, 365m Bulgaria, 365m Bulgaria, 365m Cyprus, 1461m Cyprus, 1461m Cyprus, 1461m Crimea, 220m Crimea, 530m sw Turkey, 550m sw Turkey, 1461m nw Turkey, Eskisehir, 820m nw Turkey, Eskisehir, 820m Afqa Leb M1, 1306m Afqa Leb M2, 1306m Afqa Leb M3, 1306m El Njass Leb M4, 2287m El Njass Leb M5, 2287m El Njass Leb M5, 2287m El Njass Leb M7, 2287m Metin e Turkey, 2800m Metin e Turkey, 1550m Metin e Turkey, 1700m Metin e Turkey, 1600m Metin e Turkey, 2000m Metin e Turkey, 1965m Metin e Turkey, 2010m Metin e Turkey, 1275m Metin e Turkey, 1720m Metin e Turkey, 1860m Metin e Turkey, 1890m ne Turkey , 1,753m ne Turkey, 1,783m se Turkey/ n Iraq, 1,420m se Turkey/ n Iraq, 1,743m se Turkey/ n Iraq, 1,743m se Turkey/ n Iraq, 1,743m se Turkey/ n Iraq, 1,743m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Azerbaijan, 200m Armenia, 1900m Armenia, 1900m Elburz Mtn., Iran, 2033m Elburz Mtn., Iran, 2033m Lushan, Iran, 1120m Hastjin, Iran,1610m Qushchi, Iran, 1760m Pakistan, seravschanica Pakistan, seravschanica Kazakhstan, seravschanica Kazakhstan, seravschanica Turkmenistan, turcomanica Turkmenistan, turcomanica acc # 8785 8786 14742 13720 13721 13722 14570 14571 14572 14906 14907 14569 14596 9433 9434 14155 14156 14157 14158 14159 14160 14161 14750 14751 14752 14753 14754 14755 14756 14757 14758 14759 14760 14713 14714 14715 14709 14710 14711 14712 14162 14163 14164 14165 14166 14167 14168 14169 14170 14171 8761 8762 12603 12604 12789 12795 12798 8483 8484 8224 8225 8757 8758 230 A A A A A A A A A A A A A A A A A A A A A Y-C/T na C C C C C C C C C C C C Y-C/T Y-C/T Y-C/T C C C C C Y-C/T C C C C C C C C C C C C C C C C C A A 232 G G G G G G G G G G G G G G G G G G G G G G na G G G G A G G G G G G R-A/G G G G G G G R-A/G G G G G G G G G G A G G G G G G G G G G G 238 C C C C C C C C C C C C C C C C C C C C C Y-C/T na T T T T T T T T T T T T Y-C/T Y-C/T Y-C/T T T T T T Y-C/T T T T T T T T T T T T T T T T T T C C 354 C C C C C C C C C C C C C C C C C C C C C C na T T Y-C/T Y-C/T T T C Y-C/T T T T Y-C/T C C C T T T T T C T T T T T C T T T C C C C C C C C C C 427 C C C C C C C C C C C C C C C C C C C C C Y-C/T T T T T T T T T T T T T T Y-C/T Y-C/T Y-C/T T T T T T Y-C/T T T T T T T T T T T T T T T T T T C C 732 T T T T T T T T T T T T T T T T T T T T T Y-C/T C C C C C C C C C C C C C Y-C/T Y-C/T Y-C/T C C C C C Y-C/T C C C C C C C C C C C C C C C C C T T 8952 A A A A A A A A A A A A A A A A A A A A A W-A/T T T T T T T T T T T T T T W-A/T W-A/T W-A/T T T T T T W-A/T T T T T T T T T T T T T T T T T T A A ITS exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc PxE poly poly poly PxS PxS poly poly serav PxS poly poly poly PxSa PxT(E) PxT(E) PxT(E) poly poly poly polya poly PxT(E) poly poly poly poly poly serav poly poly poly serav serav serav serav serav serav serav serav turc=exc turc=exc cp exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc exc poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly poly serav serav serav serav turco turco 152 Phytologia (April 4, 2016) 98(2) Figure 3. Distribution of J. excelsa, J. polycarpos, putative hybrids and introgressants based on ITS and cp sequences. The pair of capital letters (eg., E,E) gives the sample classification based on ITS (1st letter) and cp (2nd letter). The situation in Lebanon seems to favor hybridization between J. excelsa and J. polycarpos, even though no pure J. polycarpos was found (Adams et al., 2014). Note the four plants in the El Njass population all have J. polycarpos cp, and one appears to be a ExP hybrid by nrDNA (Fig. 3). It seems likely that pure (P,P) J. polycarpos grows in the region, although these plants may have become extinct, in which case, this may be relictual hybridization. Liao (1999), in a seminal paper on concerted evolution, defined it as "The molecular process that leads to homogenization of DNA sequences belonging to a given repetitive family". Liao reasoned that because rRNA functions only when assembled into large complexes, homogeneity of rRNAs is critical if all the steps of ribosome assembly and translation are to proceed normally. Liao (1999) says "One can therefore envision that a possible biological function of concerted evolution is to maintain homogeneous gene copies in a family so that homogeneous transcripts can be produced". However, it seems possible that there could be "silent" base substitutions that do not impact the shape or function of a rRNA. If so, these "silent" changes might persist indefinitely in a derived taxon. Naciri and Linder (2015) estimated that typical tree species with Ne of 1 million individuals and a generation time of 10 yrs would require 50 M yr after speciation to reach full monophyly. Syring et al. (2007) concluded that the presence of shared nrDNA haplotypes among Pinus species was due to incomplete lineage sorting. They estimated that reciprocal monophyly will be more likely than paraphyly in 1.7 to 2.4 M yr, but complete genome-wide coalescence in species could take up to 76 M yr. Phytologia (April 4, 2016) 98(2) 153 However, Bouillé and Bousquet (2005) examined trans-specific allelic polymorphism in three low-copy nuclear genes in different Picea species and they estimated that allelic coalescence time between randomly selected alleles in Picea was 10 to 18 million years ago. The effective population size can greatly effect coalescence times (Naciri and Linder, 2015), such that species with smaller effective population sizes would coalescence faster than species with larger effective population sizes. Mao et al. (2010) published an ancestral reconstruction of Juniperus based on all three (3) known Juniperus fossils. They showed J. excelsa and J. polycarpos joined in a clade at approximately 7 M yr. If that result is correct, then the amount of time available for complete nrDNA coalescence in the J. excelsa - J. polycarpos clade seems insufficient, compared with the 10 to 18 M yr required in Picea species (Bouillé and Bousquet, 2005). The ca. 7 M yr in J. excelsa/ polycarpos is far less than the 76 M yr that Syring et al. (2007) suggested was needed for coalescence of nrDNA in Pinus. It is difficult to know how accurate the dates are in Mao et al. (2010) due to the very small number (3) of Juniperus fossils used. But, it does appear that there has been insufficient time for complete coalescence of nrDNA in the present day J. excelsa/ J. polycarpos. Incomplete lineage sorting would explain the presence of J. seravschanica (S) nrDNA in otherwise, typical J. polycarpos in central-eastern Turkey, Azerbaijan and northwest Iran (Fig. 3). The currently understood distributions of J. excelsa, J. polycarpos, J. seravschanica and J. p. var. turcomanica are depicted in Figure 4. The dashed line in central Turkey indicates the boundary between J. excelsa and J. polycarpos is unknown at present. The population of Juniperus on the north coast of the Black Sea may be J. excelsa or J. polycarpos. Recent events have made it impossible to collect in that area. Figure 4. Distributions of J. excelsa, J. polycarpos, J. p. var. turcomanica and J. seravschanica as understood at present. The dashed line indicates the uncertain limits of J. excelsa and J. polycarpos in central Turkey. See text for discussion. Phytologia (April 4, 2016) 98(2) 154 ACKNOWLEDGEMENTS Thanks to Amy Tebeest for lab assistance and A. Kandemir for providing specimens. research was supported in part with funds from Baylor University. This LITERATURE CITED Adams, R. P. 1975. Statistical character weighting and similarity stability. Brittonia 27: 305-316. Adams, R. P. 2013. 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