Geographic variation in the volatile leaf oils J. phoenicea var. phoenicea from throughout its range

Phytologia (April 1, 2014) 96(2) 110 Geographic variation in the volatile leaf oils J. phoenicea var. phoenicea from throughout its range Robert P. Adams Biology Department, Baylor University, Box 97388, Waco, TX 76798, USA [email protected] Joaquín Altarejos Departamento de Química Inorgánica y Orgánica, Facultad de Ciencias Experimentales, Universidad de Jaén, E-23071 Jaén, Spain Montserrat Arista Departamento Biología Vegetal y Ecología, Universidad de Sevilla, Apdo. 1095, E-41080 Sevilla, Spain ABSTRACT The compositions of the volatile leaf oils from 5 populations from throughout the range of Juniperus phoenicea var. phoenicea were analyzed. Two chemotypes were found: normal leaf oils and leaf oils containing cedarwood oil components. Except for the chemotypes (hi cedrol), the leaf oils of J. phoenicea are high in α-pinene (41.2 - 51.9%) and manoyl oxide (14.0 - 28.0%) with moderate amounts of α-pinene, myrcene, β-phellandrene and (E)-caryophyllene. Little geographic variation was found in the major components from Narbonne to Andorra, Zaragoza thence to El Peñón. The oil from the high cedrol plants at Grazalema seems quite different due to the presence of cedarwood oil components, but it is actually not very different, if one removes the heartwood terpenoids and re-normalizes the remaining terpenoids. Trees with high cedarwood oil had 16.4 - 31.9% cedrol and moderate amounts of other cedarwood oil components (eg., α- & β-cedrene, 2-epi-funebrene, cis-thujopsene, α- & β-alaskene, (E)-β- bisabolene, liguloxide, allo-cedrol). Published on-line www.phytologia.org Phytologia 96(2): 110-116 (April 1, 2014). KEY WORDS: Juniperus phoenicea var. phoenicea, Cupressaceae, terpenes, leaf essential oil, geographic variation. Recently, Adams et al. (2013) analyzed nrDNA and petN sequences for J. phoenicea L. (sensu stricto) from throughout the Mediterranean region (Fig. 1). They found J. phoenicea var. (or subsp.) phoenicea was restricted to Spain and France, whereas J. phoenicea var. turbinata (Guss.) Parl. (J. turbinata Guss.) were widely distributed from the Canary Islands to the Sinai (Fig. 4). 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-Martínez (Fig. 1). Juniperus turbinata appears to be widespread from Madeira - Canary Islands to the Sinai with few DNA differences among most populations. However, some populations (Grazalema, Madeira, Sinai, central Italy) displayed (Fig. 1) moderate amounts of divergence (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-leaf junipers occurring in the western hemisphere, leading them to denote J. phoenicea as a 'pseudoserrate' juniper. In addition, they found J. p. var. phoenicea and var. turbinata to be as different in their DNA sequences as several other recognized species of Juniperus. This lends support for the recognition of J. turbinata Guss., as proposed by Lebreton and Pérez de Paz (2001) based largely on the concentration of prodelphinidin, a polymeric tannin. The prodelphinidin data suggested that J. phoenicea var. phoenicea was confined to the Iberian Peninsula with var. turbinata widespread throughout the Mediterranean region. Lebreton and Pérez de Paz (2001) found a clear Phytologia (April 1, 2014) 96(2) 111 separation between J. phoenicea (Spain and France) and all other populations examined (J. turbinata). Several studies have been made on the leaf terpenoids of J. phoenicea. San Feliciano and workers examined acidic diterpenes (San Feliciano et al., 1988; 1993). Incomplete analyses have been published on the volatile leaf oils of J. phoenicea from Egypt (Afifi et al., 1992), Saudi Arabia (Dawidar et al., 1991) and France (Tabacik and La Porte, 1971). See Adams, Barrero and Lara (1996) for a review of the early literature. Adams, Barrero and Lara (1996) presented the first comprehensive analyses of the volatile leaf oils of J. phoenicea, J. p. subsp. eu-mediterranea and J. p. var. turbinata; they concluded that J. p. subsp. eu-mediterranea and var. turbinata were conspecific as their oils were nearly identical. More recently, Adams et al. (2009) presented complete analysis of the leaf oils of J. phoenicea (var. turbinata) from the Canary Islands and Madeira and compared these with oils from Morocco and Spain. The purpose of the present study is to Figure 1. Bayesian tree of J. phoenicea and J. turbinata present a detailed analyses of the volatile leaf (J. p. var. turbinata) from throughout the species ranges. oils from populations of J. phoenicea var. (from Adams and Schwarzbach, 2013). phoenicea from throughout its ranges. MATERIALS AND METHODS Figure 2 shows the distributions of J. phoenicea var. phoenicea and populations sampled in this study. Specimens used in this study: J. phoenicea var. phoenicea: France, Narbonne, near St. Pierre sur Mere, 43º 10’ 0.2" N, 3º 09’ 57.6" E, 23 m, J. Altarejos 1-5, Baylor specs. Adams 14123-14127. Andorra, Coll de Jou near Sant Julià de Lòria, 42º 26’ 56.8" N, 1º 28’ 04.6" E, 1426 m, J. Altarejos 6-10, Baylor specs. Adams 14128-14132. Spain, Zaragoza, Montes de la Retuerta de Pina W of Bujaraloz, 41º 28’ 59"N, 0º 19’ 31.2"W, 317 m, J. Altarejos 11-15, Baylor specs. Adams 14133-14137. Spain, El Peñón, 37 º 35' 38" N, 3 º 31' 22" W, 760 m, Adams 7077-7079, Spain, Cádiz, Sierra de Grazalema, 36º 47' 51.5'' N, 5º24' 43.7''W, 835 m; M. Arista 1-5, Baylor specs. Adams 13813-13817. Phytologia (April 1, 2014) 96(2) 112 Figure 2. Distribution of J. phoenicea (adapted from Lebreton and Pérez de Paz, 2001, and Adams et al. 2010). Squares show the five populations of J. phoenicea sampled in the present terpene study. Fresh, air dried leaves (50-100 g) were steam distilled for 2 h using a circulatory Clevenger-type apparatus (Adams, 1991). The oil samples were concentrated (ether trap removed) with nitrogen and the samples stored at 20 ºC until analyzed. The extracted leaves were oven dried (100 ºC, 48 h) for determination of oil yields. Oils from 4 - 5 trees of each taxon were analyzed and average values reported. The oils were analyzed on a HP 5971 MSD mass spectrometer, scan time 1/ sec., directly coupled to a HP 5890 gas chromatograph, using a J & W DB-5, 0.26 mm x 30 m, 0.25 micron coating thickness, fused silica capillary column (see Adams, 2007 for operating details). Identifications were made by library searches of our volatile oil library (Adams, 2007), using the HP Chemstation library search routines, coupled with retention time data of authentic reference compounds. Quantitation was by FID on an HP 5890 gas chromatograph using a J & W DB-5, 0.26 mm x 30 m, 0.25 micron coating thickness, fused silica capillary column using the HP Chemstation software. Terpenoids (as per cent total oil) were coded and compared among the species by the Gower metric (1971). Principal coordinate analysis was performed by factoring the associational matrix using the formulation of Gower (1966) and Veldman (1967). RESULTS AND DISCUSSION The composition of the volatile leaf oils of four of the five populations varied very little except for a chemotype (one tree) in the Zaragoza population that was high in cedrol and other cedarwood terpenoids (Table 1). However, all five trees sampled in the Grazalema population had the cedarwood chemotype and were high in cedrol (Table 1). The activation of the cedarwood oil pathway (in the leaf glands) reduces the concentrations of the non-heartwood components, as the terpene pool is siphoned off to produce cedarwood components in the leaf oil. There appears to be a single gene ('cedarwood synthase') that is turned on in the heartwood (or some associated tissue) that activates the cedarwood oil pathway (α- & β-cedrene, 2-epi-funebrene, cis-thujopsene, α- & β-alaskene, (E)-β-bisabolene, liguloxide, allo-cedrol, cedrol, widdrol, epi-cedrol, etc. (see Adams, 2014). Normally, this gene ('cedarwood synthase') is not active in Juniperus (and Cupressaceae) leaf oil glands. Most Juniperus species produce Phytologia (April 1, 2014) 96(2) 113 two kinds of essential oils: leaf oils and heartwood oils and these oils have few components in common (Adams, 1991). Juniperus phoenicea, J. excelsa, J. foetidissima, J. polycarpos, and J. seravschanica have leaf oils that may contain significant amounts of the heartwood oil components (Adams and Hojjati, 2013). For example, Adams (1990) reported 4.4, 0.2, trace and 8.3% cedrol in the leaf oils from four trees of J. foetidissima from Greece. Whereas, Tunalier et al. (2004) reported 13.0 and 12.2% of cedrol and widdrol in the stem heartwood of J. foetidissima from Turkey. Ucar and Balaban (2002) analyzed the sapwood (white wood) of J. excelsa, Turkey, and reported the oil to contain 22.5% widdrol and 9.0% cedrol (these components are difficult to separate on non-polar columns and the mass spectra are nearly identical, so their identification is often problematic). When Juniperus species contain heartwood components in the leaf oils, it is common to find chemical polymorphisms in cedrol (and associated heartwood terpenes) between trees. That is the case for trees from Zaragoza. Four trees had only the typical leaf oil components (Table 1) and their oil is very similar to nearby populations at Andorra and Narbonne, France (Table 1). However, one of 5 trees in the Zaragoza population had 31.9% cedrol and related compounds (Table 1) and thus, only 33.4% αpinene. The oil of this tree, is quite similar to the hi cedrol Grazalema population that has 16.4% cedrol and 29.7% α-pinene (Table 1). It is interesting to compare cedrol + manoyl oxide for hi cedrol Zaragoza (31.9+13.3 = 45.2) vs. hi cedrol Grazalema (16.4+32.9 = 49.3%). Except for the cedarwood oil chemotypes (hi cedrol), the leaf oils of J. phoenicea are high in αpinene and manoyl oxide with moderate amounts of α-pinene, myrcene, β-phellandrene and (E)- caryophyllene. Little geographic variation was found in the major components from Narbonne, Andorra, Zaragoza thence to El Peñón. The oil from the high cedrol plants at Grazalema seems quite different due to the presence of cedarwood oil components, but the oil is actually not very different, if one removes the heartwood terpenoids and re-normalizes the remaining terpenoids (Table 1). ACKNOWLEDGEMENTS Thanks to Tonnie Yanke for lab assistance. This research was supported in part with funds from Baylor University. Thanks Dr. Carlos Fernández (University of Jaén, Spain) for providing assistance with the herbarium work. LITERATURE CITED Adams, R. P., A. Boratynski, M. Arista, A. E. Schwarzbach, H. Leschner, Z. Liber, P. Minissale, T. Mataraci and A. Manolis. 2013. Analysis of Juniperus phoenicea from throughout its range in the Mediterranean using DNA sequence data from nrDNA and petN-psbM: The case for the recognition of J. turbinata Guss. Phytologia 95(2): 202-209. Adams, R. P., A. F. Barrero and A. Lara. 1996. Comparisons of the leaf essential oils of Juniperus phoenicea, J. phoenicea subsp. eu-mediterranea Lebr. & Thiv. and J. phoenicea var. turbinata (Guss.) Parl. J. Essent. Oil Res. 8: 367-371 Adams, R. P. and F. Hojjati. 2013. Leaf essential oils of Juniperus in central and southern Iran. Phytologia 95: 288-295. Adams, R. P., N. Pandy, S. Rezzi and J. Casanova. 2002. Geographic variation in the random amplified polymorphic DNAs (RAPDs) of Juniperus phoenicea, J. p. var. canariensis, J. p. subsp. eumediterreanea, and J. p. var. turbinata. Biochem. Syst. Ecol. 30: 223-229. Adams, R. P. and A. E. Schwarzbach. 2013. Phylogeny of Juniperus using nrDNA and four cpDNA regions. Phytologia 95: 179-187. Afifi, M. S., S. H. El-Sharkawy, G. T. Maatoog, M. el-Sohly and J. P. N. Rosazza. 1992. Essential oils of Thuja occidentalis, Thuja orientalis, Cupressus sempervirens, and Juniperus phoenicea. Mansoura J. Pharm. Sci. 8: 37-46. Phytologia (April 1, 2014) 96(2) 114 Dawidar,A. M., S. T. Ezmirly and M. Abdel-Mogib. 1991. Sesquiterpenes and diterpenes from Juniperus phoenicea L. Pharmazie 46: 472-473. Lebreton, P. and P. L. Pérez de Paz. 2001. Définition du Genévrier de Phénicie (Juniperus aggr. phoenicea), reconsidéré à ses limites biogéographiques : Méditerranée orientale (Crète et Chypre) et Atlantique (Iles Canaries). Bull. Mens. Soc. linn. Lyon, 70(4) : 73-92. San Feliciano, A., A. Medarde, F. Tomé, J. M. Miguel del Corral and A. F. Barrero. 1988. Sandaracopimaric and isopimaric acids in Juniperus phoenicea L from different areas. Ann. Quím. 84: 360-363. San Feliciano, A., J. M. Miguel del Corral, M. Gordaliza, M. A. Salinero and B. Del Rey. 1993. Acid constituents of Juniperus phoenicea subsp. turbinata leaves. Fitoterapia 64: 185-186. Tabacki, C. and Y. LaPorte. 1971. Diterpenes of Juniperus phoenicea. Major constituents. Phytochemistry 10: 2147-2153. Tunalier, Z., N. Kirimer and K. H. C. Baser. 2004. A potential new source of cedarwood oil: Juniperus foeditissima Willd. J. Essent. Oil Res. 16: 233-235. Ucar, G. and M. Balaban. 2002. The composition of volatile extractives from the wood of Juniperus excelsa, Juniperus foetidissima and Juniperus oxycedrus. Holz als Roh- und Werkstoff 60: 356-362. Phytologia (April 1, 2014) 96(2) 115 Table 1. Composition of the leaf oils of J. phoenicea (var. phoenicea): Narbonne, France; Andorra; El Peñón, Spain; Zaragoza (lo and hi cedrol), Spain; and hi cedrol, Grazalema, Spain. Those compounds that appear to distinguish taxa are in boldface. Cedarwood oil components are in italics. Values in parenthesis ( ) for larger components of hi cedrol Zaragoza and hi cedrol Grazalema columns are corrected values, computed by correcting for cedarwood oil components. AI Compound lo cedrol France lo cedrol Andorra lo cedrol El Peñón lo cedrol Zaragoza hi cedrol Zaragoza hi cedrol Grazalema 921 tricyclene 0.1 0.1 0.1 0.1 0.1 t 932 α-pinene 42.4 42.9 41.2 51.9 33.4(45.7) 29.7(35.4) 945 α-fenchene 0.2 0.1 0.1 0.1 0.1 t 946 camphene 0.3 0.3 0.1 0.4 0.3 0.3 953 thuja-2,4-diene t 0.1 0.1 0.1 0.1 t 961 verbenene 0.1 0.1 0.3 0.1 0.1 t 969 sabinene t t 0.1 t t t 974 β-pinene 1.7 1.7 2.1 2.0 1.2(1.6) 1.2(1.4) 988 myrcene 2.7 2.6 3.2 2.8 1.9(2.6) 2.3(2.7) 1001 δ-2-carene t t 0.1 t t t 1002 α-phellandrene 0.3 0.2 0.7 0.2 0.2 t 1008 δ-3-carene 2.0 1.3 1.5 t t t 1014 α-terpinene t t 0.1 t t t 1020 p-cymene 0.3 0.3 0.4 0.3 0.2 0.6 1024 limonene 0.6 0.6 0.6 0.5 0.4 0.4 1025 β-phellandrene 2.0 1.9 4.9 1.8 1.1(1.5) 0.6(0.7) 1054 γ-terpinene 0.2 0.2 0.2 0.2 0.2 0.9 1069 cis-linalool oxide 0.2 0.2 t 0.1 t t 1086 terpinolene 0.7 0.6 0.7 0.6 0.4 0.4 1095 linalool 0.7 0.3 1.0 0.6 0.5 0.2 1118 cis-p-menth-2-en-1-ol t t 0.2 t t - 1122 α-campholenal 0.1 0.2 0.2 0.2 0.1 t 1135 trans-pinocarveol 0.1 0.2 0.3 0.4 0.2 t 1139 C10OH, 41,55,81,95,152 - - 1.4 - - - 1140 trans-verbenol - - - - - 0.2 1141 camphor 0.4 0.5 - 0.5 0.3 - 1144 neo-isopulegol 0.3 0.4 t 0.4 0.3 t 1158 trans-pinocamphone t t 0.1 t t - 1165 borneol 0.2 0.3 0.6 0.3 0.1 - 1172 cis-pinocamphone 0.1 0.1 0.2 0.2 0.1 - 1174 terpinen-4-ol t t 0.1 t t t 1178 naphthalene 0.3 0.1 t t t - 1179 p-cymen-8-ol t t 0.1 t t - 1186 α-terpineol 0.8 0.5 2.3 0.5 0.4 t 1195 myrtenal/ myrtenol t 0.1 0.1 0.2 0.1 - 1204 verbenone t 0.2 0.2 0.2 0.1 - 1215 trans-carveol 0.1 0.1 0.1 0.2 t - 1223 citronellol 0.2 0.2 0.5 0.4 0.1 - 1249 piperitone t t 0.2 t t - 1255 (4Z)-decenol 0.1 0.1 0.2 0.1 t t 1315 (E,E)-2,4-decadienal t 0.5 0.3 0.2 t - 1335 δ-elemene 0.1 0.1 t 0.2 0.1 t 1387 β-bourbonene 0.1 t - 0.1 t - 1389 β-elemene 0.4 0.1 0.1 0.3 0.1 - 1400 β-longipinene t 0.4 t 0.4 0.1 t 1410 α-cedrene - - - - 0.9 1.0 Phytologia (April 1, 2014) 96(2) 116 AI Compound lo cedrol France lo cedrol Andorra lo cedrol El Peñón lo cedrol Zaragoza hi cedrol Zaragoza hi cedrol Grazalema 1411 2-epi-funebrene - - - t 0.9 - 1417 (E)-caryophyllene 2.9 2.6 1.2 2.7 - 1.3(1.5) 1429 cis-thujopsene - - - - 0.4 0.3 1434 γ-elemene 0.1 t t 0.1 - - 1452 α-humulene 0.2 0.1 - 0.2 - - 1454 (E)-β-farnesene - - - - 0.3 - 1478 γ-muurolene t t - 0.1 0.6 - 1484 germacrene D 2.1 1.1 0.5 1.7 t 0.3 1484 allo-aromadendr-9-ene t t - 0.1 - - 1498 β-alaskene - - - - 0.1 - 1500 β-himachalene - - - - 0.1 - 1505 β-bisabolene - - - - - 0.4 1509 C15OH,41,55,81,161,220 - - 0.3 - - - 1512 α-alaskene - - - - 0.6 0.4 1513 γ-cadinene 0.1 0.1 0.1 0.1 t t 1521 β-sesquiphellandrene - - - - 0.3 - 1522 δ-cadinene 0.3 0.2 0.2 0.3 t 0.2 1529 (E)-γ-bisabolene - - - - 0.2 - 1534 liguloxide - - - - 0.2 - 1535 C15OH,41,69,105,161,204 - - 1.0 - - - 1541 C15OH,43,95,207,222 0.7 1.1 - 0.7 - - 1548 elemol 0.5 0.9 1.8 1.8 0.5 0.5 1559 germacrene B 1.5 1.1 0.6 1.9 0.9 0.2 1561 (E)-nerolidol 0.1 0.1 t 0.1 t - 1574 germacrene-D-4-ol 0.1 0.1 0.2 0.1 t - 1582 caryophyllene oxide 0.7 1.1 1.0 1.2 0.5 - 1589 allo-cedrol - - - - 1.4 1.1 1600 cedrol 0.2 0.1 - 0.7 31.9 16.4 1625 C15OH, 43,119,161,220 0.3 0.5 0.4 0.6 t - 1630 γ-eudesmol t t 0.2 t t t 1632 α-acorenol - - - - 0.4 - 1638 epi-α-cadinol t t 0.2 t t t 1638 epi-α-muurolol t t 0.1 t t t 1649 β-eudesmol 0.2 0.4 0.4 0.6 0.2 - 1652 α-eudesmol 0.4 0.6 0.3 0.7 0.3 t 1652 α-cadinol - - 0.3 - - t 1687 eudesma-4(15),7-dien-1-β-ol - - 0.1 - - - 1688 shyobunol 0.8 1.4 1.5 1.1 0.5 0.5 1715 (2Z,6E)-farnesol t t 1.2 0.2 t - 1968 sandaracopimara-8(14), 15- diene t t 0.1 t t 0.2 1978 manoyl oxide 28.0 25.4 22.0 14.0 13.3(18.2) 32.9(39.2) 2009 epi-13-manoyl oxide t t 0.1 t t 0.2 2055 abietatriene 0.3 0.3 0.1 0.2 0.2 0.4 2087 abietadiene 0.1 0.2 0.1 0.1 t t 2298 4-epi-abietal 0.2 0.4 0.2 0.4 0.2 0.2 2314 trans-totarol 0.9 1.2 0.2 0.4 0.5 1.9 2331 trans-ferruginol 0.1 0.1 t t t 0.3 total % cedarwood cpds. 0.2 0.1 0.0 0.7 36.7 19.2 KI = linear Kovats Index on DB-5 column. Compositional values less than 0.1% are denoted as traces (t). Unidentified components less than 0.5% are not reported.