Morphological, chemical and genetic differentiation of two subspecies of Cistus creticus L. (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus)

Phytochemistry 70 (2009) 1146–1160

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Morphological, chemical and genetic differentiation of two subspecies of Cistus creticus L. (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus) Julien Paolini a,*, Alessandra Falchi b, Yann Quilichini c, Jean-Marie Desjobert a, Marie-Cecile De Cian d, Laurent Varesi d, Jean Costa a a

UMR-CNRS 6134 SPE, Université de Corse, Laboratoire Chimie des Produits Naturels, 20250 Corti, France INSERM, U707, 75012 Paris, France c UMR-CNRS 6134 SPE, Université de Corse, Service d’Etude et de Recherche en Microscopie Electronique, 20250 Corti, France d UMR-CNRS 6134 SPE, Université de Corse, Laboratoire de Génétique Moléculaire, 20250 Corti, France b

a r t i c l e

i n f o

Article history: Received 15 January 2009 Received in revised form 15 June 2009 Available online 5 August 2009 Keywords: Cistus creticus SEM Labdane diterpene GC/MS HS-SPME ISSR Statistical analysis

a b s t r a c t Cistus creticus L., an aromatic species from the Mediterranean area, contains various diterpenes bearing the labdane skeleton. The production of essential oil from this species has potential economic value, but so far, it has not been optimized. In order to contribute to a better knowledge of this species and to its differentiation, the morphological characters, volatile chemical composition and genetic data of two subspecies (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus) were investigated. The leaf trichomes were studied using scanning electron microscopy. The chemical composition of Corsican essential oil (C. creticus subsp. corsicus) has been reported using GC, GC/MS and 13C NMR; the main constituents were oxygenated labdane diterpenes (33.9%) such as 13-epi-manoyl oxide (18.5%). Using plant material (54 samples) collected from 18 geographically distinct areas of the islands of Corsica and Sardinia, the basis of variation in the headspace solid-phase microextraction volatile fraction and an inter-simple sequence repeat genetic analysis were also examined. It was shown that the two subspecies of C. creticus differed in morphology, essential oil production, volatile fraction composition and genetic data. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Cistus L. is a relatively small genus (19 Mediterranean and Macaronesic species are currently recognized), but shows a noteworthy morphological diversification (Greuter et al., 1984). Cistus is a complex genus, because of the polymorphism of a number of species and the hybridization between related species. Indeed, hybridization has been reported to be an active process in Cistus (Ellul et al., 2002), and many hybrid combinations within and among pink- or white-flowered species have been recorded in the field, based on intermediate morphological characters (Grosser, 1903). There are only a few investigations that address the genetic structure of the genus Cistus (Batista et al., 2001; Farley and McNeilly, 2000; Guzmán and Vargas, 2005; Quintela-Sabarís et al., 2005). Cistus creticus L. (syn. Cistus incanus subsp. creticus or syn. Cistus villosus) is a subshrub (1 m high) abundant in the oriental Mediterranean basin with simple opposed leaves and pink flowers (blossoming time, May–June) (Coste, 1937). C. creticus is a highly variable species; three subspecies from various Mediterranean areas of distribution (northern Africa, western Asia, Caucasus, East Europe and southwestern and southeastern Europe) have been re* Corresponding author. Tel.: +33 04 95 45 01 93; fax: +33 04 95 45 01 62. E-mail address: [email protected] (J. Paolini). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.06.013

ported (Greuter et al., 1984): C. creticus subsp. creticus, C. creticus subsp. eriocephalus and C. creticus subsp. corsicus. In the geographical area chosen for this study (Corsica and Sardinia islands), C. creticus is only represented by two subspecies: C. creticus subsp. eriocephalus and C. creticus subsp. corsicus (Greuter et al., 1984; Jeanmonod and Gamisans, 2007). The leaf trichomes of Cistus species have been previously investigated for taxonomic purposes (Tattini et al., 2007). Jeanmonod and Gamisans (2007) have reported the morphological characters (density of stellate hairs, shape of leaf edge and petiole morphology) used to distinguish the two subspecies. Moreover, the morphological studies of C. creticus subspecies (Gulz et al., 1996) showed the presence of variable characters on the leaf surface such as the size and density of stellate trichomes. Gulz et al. (1996) also reported the presence of glandular trichomes (sites of secondary metabolites biosynthesis, secretion and storage). However, Demetzos et al. (2002) suggested that the presence of several glandular trichomes on the leaf surface of C. creticus subsp. creticus could explain the production of essential oil and resin labdanum. Indeed, for this subspecies, gene and proteins expression showed that the enzyme geranylgeranyl diphosphate synthase is developmentally- and tissue-regulated, showing maximum expression in trichomes and in the smallest leaves (0.5–1.0 cm) (Pateraki and Kanellis, 2008).

J. Paolini et al. / Phytochemistry 70 (2009) 1146–1160

The essential oil and resin secreted by the glandular structures of C. creticus may have economic value. Indeed, the natural products of C. creticus showed cytotoxic activity against a number of human leukemia cell lines (Dimas et al., 1998, 1999) and various biological activities (Chinou et al., 1994; Demetzos et al., 1999, 2001; Sassi et al., 2008). Moreover, several authors (Angelopoulou et al., 2001; Chinou et al., 1994; Dimas et al., 1999, 2001; Matsingou et al., 2006) have reported a possible medical application of semisynthetic components derived from metabolites of C. creticus. Several chemical studies have been published concerning the solvent extracts and essential oils from aerial parts of C. creticus, and the major components were diterpenes with labdane skeletons (Anastasaki et al., 1999; Demetzos et al., 1990, 1994a,b,c, 1997, 1999; Demetzos and Loukis, 1995; Hatzellis et al., 2004 ). The chemical composition of essential oil from two C. creticus subspecies from Greece has been reported by Demetzos et al. (2002); C. creticus subsp. eriocephalus oil was characterized by a high amount of sesquiterpenes (dcadinene, a-cadinene, a-copaene, bulnesol, viridiflorol and ledol), whereas C. creticus subsp. creticus oil was dominated by diterpenes (manoyl oxide and 13-epi-manoyl oxide). Trichomes were shown to be useful tools for recent trends in chemosystematics considering information from biosynthetic pathways in plants. Indeed, enzymes extracted from living glandular cells of plants were successfully employed to study the biosynthesis of terpenoids (Spring, 2000). Several groups have suggested that plant terpene synthases (Tps) share a common evolutionary origin based upon their similar reaction mechanism and conserved structural and sequence characteristics (Trapp and Croteau, 2001). Moreover, published studies (Skoula et al., 2000; Tetenyi, 2002) have shown that variation in essential oil composition from wild populations is accompanied by population-related differences in genetic profile. For instance, inter-simple sequence repeat (ISSR) markers have been successfully applied in a wide range of species for determining intra- and interspecific diversity (Godwin et al., 1997). A few chemotaxonomic studies have suggested that the chemical variability of C. creticus subspecies is affected by various ecological parameters (Demetzos and Perdetzoglou, 1999, Demetzos et al., 2002) such as light (solar radiation) or insect defense/attraction. These studies have indicated a high degree of variability in the quantity and composition of essential oil and resin among populations growing wild in Crete (Greece) (Demetzos et al., 2002). However, the chemical composition of many subspecies remains unrecorded and wild populations from the northwest Mediterranean have been poorly explored. This is especially true for the essential oils of C. creticus from the islands of Corsica and Sardinia. This work is the first attempt to study the detailed analysis of essential oil from C. creticus subsp. corsicus harvested in Corsica. This study is also the first to combine morphological characteristics of the leaf structure, volatile profile and genetic data in order to propose a method for better differentiation of the C. creticus subspecies. To investigate the biosynthesis of diterpenes and their regulation in C. creticus subspecies, 54 leaf samples selected from 18 geographically distinct areas of Corsica and Sardinia were studied by scanning electron microscopy, headspace solid-phase microextraction (HS-SPME) and ISSR markers. The hypotheses of this study were that: (i) the biosynthesis of secondary metabolites of C. creticus is related to morphological and genetic aspects and (ii) the selection, conservation and exploitation of genetic resources of C. creticus should present an economical interest for some Mediterranean regions. The study aims to address the following questions: (i) Is C. creticus subspecies differentiation based on morphological characteristics related to volatile composition and (ii) do ISSR markers provide similar conclusions? What is the implication of the detected genetic variation for conservation strategies?

1147

2. Results and discussion 2.1. Leaf trichomes The aerial parts of C. creticus subsp. eriocephalus and C. creticus subsp. corsicus were harvested from 16 localities in Corsica and two in North Sardinia (Fig. 1). The observation of two subspecies of C. creticus, realized by scanning electron microscopy, has highlighted the presence of trichomes on their leaf surface (Fig. 2). The leaf surface of C. creticus subsp. corsicus is characterized by the presence of two trichome shapes: stellate hairs (Fig. 2A) and long ball-headed tubes (Fig. 2B). The distribution of these two trichomes is homogeneous on the upper (Fig. 2C) and the lower side (Fig. 2D). The leaf surface (upper and lower side) of C. creticus subsp. eriocephalus presents only one trichome shape: stellate hairs (nonsecretorial trichome). Their distribution is homogeneous on the upper side (Fig. 2E), while on the lower side, they are mostly concentrated on the veins and their branches are longer (Fig. 2F). The arrangement of the stellate hairs is denser on the lower side. We noticed that the number and the length of stellate hair branches are greater in C. creticus subsp. eriocephalus. These observations confirm the interest of the use of leaf trichomes for a taxonomic classification of Cistus species (Gulz et al., 1996). 2.2. Essential oil composition No essential oil was obtained for the samples of C. creticus subsp. eriocephalus from the seven corresponding localities (Bc, Fv, Fa, No, Pc, Re and St), whereas the essential oil yields of C. creticus subsp. corsicus from the 11 stations (Al, Ba, Bo, Bt, Ca, Cf, Er, Fm, Ol, Pa and Pv) were 0.03%. The presence of long ball-headed tubes on the leaf surface of C. creticus subsp. corsicus, which have a secretorial function, could explain the variability of the production of essential oils between these two subspecies (Spring, 2000). The essential oils of C. creticus subsp. corsicus were qualitatively rather similar, but differed in the amounts of major components. The major volatile constituents were 13-epi-manoyl oxide (4.4– 51.0%) 152, manool 156 (0.8–21.0%) and labdan-7,14-dien-13-ol 157 (0.2–9.9%). It was obvious from the chromatographic profile that C. creticus subsp. corsicus produced a diterpene rich oil (33.9%) containing a large number of components of various families in low proportions. Consequently, the essential oil was first submitted to repeated chromatography and all the fractions were investigated by GC and GC/MS. Some fractions selected based on their chromatographic profile were also analyzed by 13C NMR. Combined analysis of the essential oil from aerial parts of C. creticus subsp. corsicus led to the identification of 164 compounds (Table 1): – 132 components by comparison of their mass spectra and their retention indices with those of our own library. Some oxygenated diterpenes were also identified by 13C NMR (ambrox 137, manoyl oxide 149, 13-epi-manoyl oxide 152 and sclareol 159); – 30 components by comparison of their mass spectra and their retention indices with those of commercial libraries. Among them, two diterpenes bearing the labdane skeletons were confirmed by comparison of their chemical shifts measured in the spectra of fractions with those reported in the literature: norambrenolide 154 (F2.19: 4.5%) (Dolmazon et al., 1995) and manool 156 (F3.5: 62.7%) (Lu et al., 1994). Identification of labdan7,14-dien-13-ol 157 was confirmed using 13C NMR by comparison of the unassigned signals in the spectrum of fraction F3.8 (157: 41.9%) with the chemical shifts of manool (Lu et al., 1994) and compound 147 (Gray et al., 2002);

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J. Paolini et al. / Phytochemistry 70 (2009) 1146–1160

Fig. 1. Sample locations of C. creticus subsp. corsicus (d) and C. creticus subsp. eriocephalus (N).

– the occurrence of two labdane ketones, 13-oxo-15,16-bis-norent-labd-8(17)-ene 142 and 13-oxo-15,16-bis-nor-ent-labd7(8)-ene 147, which were not present in available libraries, were identified by 13C NMR by comparison of chemical shifts in the spectra of fractions F2.11 (142: 70.2%) and F.2.14 (147: 40.4%), respectively, with those reported in the literature. 13C NMR spectral data of compounds 142 and 147 were previously respectively reported in solvent extracts from Ruppia maritima (Dellagreca et al., 2000) and in the synthesis of rhinocerotinoic acid (Gray et al., 2002). To our knowledge, these compounds were reported for the first time in the genus Cistus. In all, 164 components were identified in C. creticus subsp. corsicus, accounting for 83% of the total amount of the oil (Table 2). This oil was characterized by high contents of oxygenated diterpenes (33.9%). The main components were 13-epi-manoyl oxide (18.5%), manool (7.2%), labda-7,14-dien-13-ol (3.8%), manoyl oxide (4.2%) and slareol (2.7%). It should be noted that there is no com-

mercial interest for C. creticus subsp. eriocephalus, compared with C. creticus subsp. corsicus, due to its absence of essential oil production. These results were in accordance with those previously reported on the populations of C. creticus from Greece. Indeed, Demetzos et al. (1997) have observed that C. creticus subsp. creticus produces more resin labdanum than C. creticus subsp. eriocephalus. 2.3. HS-SPME and chemical variability HS-SPME is a simpler and more rapid procedure for extraction of the volatile fraction from aromatic plants (Belliardo et al., 2006; Demirci et al., 2005; Flamini et al., 2003; Pellati et al., 2005) in comparison to hydrodistillation, which is time consuming and needs a large amount of sample. HS-SPME analysis allowed a qualitative estimate of volatile compounds using a small quantity of material (Paolini et al., 2008). The HS-SPME has also been used for the characterization of chemical variability of aromatic plants and for the study of volatile fractions

J. Paolini et al. / Phytochemistry 70 (2009) 1146–1160

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Fig. 2. SEM of untreated leaves of C. creticus subsp. corsicus and C. creticus subsp. eriocephalus. (A) Stellate hairs of C. creticus subsp. corsicus (lower side). Bar = 100 lm. (B) Long ball-headed tubes of C. creticus subsp. corsicus (upper side). Bar = 100 lm. (C) Upper side of subsp. C. creticus corsicus. Bar = 500 lm. (D) Lower side of subsp. C. creticus corsicus. Bar = 500 lm. (E) Upper side of subsp. C. creticus eriocephalus. Bar = 500 lm. (F) Lower side of subsp. C. creticus eriocephalus. Bar = 500 lm. LTH long ball-headed tubes; S stoma; SH satellite hairs; V vein.

emitted by species without essential oil (Petrakis et al., 2005). Thus, the chemical variability of C. creticus subsp. eriocephalus and C. creticus subsp. corsicus has been studied using the volatile composition sampled by HS-SPME. The volatile fraction of 54 samples (three taxa from 18 localities) of C. creticus was analyzed by HS-SPME. The GC and GC–MS analysis allowed the identification of 28 components, including eight acyclic nonterpenic components (2, 3, 4, 21, 45, 46, 81 and 134), three monoterpene hydrocarbons (7, 11 and 14), 10 sesquiterpene hydrocarbons (56, 58, 66, 75, 80, 84, 87, 89, 93 and 95), three oxygenated sesquiterpenes (109, 125 and 130) and four labdane diterpenes (142, 147, 149 and 152). The volatile fractions of the two subspecies showed important differences from a qualitative and quantitative viewpoint (Fig. 3). C. creticus subsp. eriocephalus was characterized by a significant amount of monoterpene hydrocarbons (myrcene 11 and limonene 14) and linear nonterpenic components ((Z)-hex-3-en-1-ol 2, (Z)-hex-3-en-1-ol 3, hexanol 4 and nonanal 21) (Fig. 3A). C.

creticus subsp. corsicus was characterized by high contents of labdane diterpenes (13-epi-manoyl oxide 149, manoyl oxide 152, 13-oxo-15,16-bis-nor-ent-labd-8(17)-ene 142 and 13-oxo15,16-bis-nor-ent-labd-7(8)-ene 147) and sesquiterpene hydrocarbons ((E)-b-caryophyllene 66, c-cadinene 93 and d-cadinene 95) (Fig. 3B). Several studies have previously made extensive use of statistical methods to interpret different aspects of the metabolism of aromatic plants, demonstrating the usefulness of principal component analysis (PCA) and canonical analysis (CA). For instance, PCA and CA have been recently used for determining the chemical variability of essential oil compositions from Citrus reticulata (Fanciullino et al., 2006), Lavandula latifolia (Muñoz-Bertomeu et al., 2007) and Hypericum species (Nogueira et al., 2008). To synthesize the chemical composition data, PCA was applied to examine the relative distribution of samples according to their production of different volatile compounds. Figs. 4 and 5 were obtained from the correlation matrix calculated with the standard-

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J. Paolini et al. / Phytochemistry 70 (2009) 1146–1160

Table 1 Chemical composition of the essential oil of Cistus creticus subsp. corsicus. Noa

Components

I lb

I ac

I pd

%e

Identificationf

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Hexanal (Z)-hex-3-en-1-ol (Z)-hex-2-en-1-ol Hexanol Heptanal Tricyclene a-Pinene Camphene b-Pinene 2-Pentyl furan Myrcene Decane para-Cymene Limonene c-Terpinene Octanol (E)-THF-linalool oxide para-Cymenene Terpinolene Linalool Nonanal Undecane a-Campholenal (E)-non-3-en-2-one Camphre trans-Pinocarveol b-Terpineol Pinocarvone Isoborneol Borneol Lavandulol Cryptone para-Cymen-8-ol Terpinen-4-ol Safranal a-Terpineol Decanal myrtenol b-Cyclocitral Dodecane trans-Carveol Geranial Nonanoic acid Bornyl acetate Undecan-2-one Undecanal (E,E)-2,4-decadienal Undecan-2-ol Tridecane (E)-undec-3-en-2-one Eugenol a-Cubebene Decanoic acid Methyl eugenol a-Ylangene a-Copaene Dodecanal b-Bourbonene 1,5-di-epi-bourbonène (a and/or b) b-Elemene Tetradecane 7,8-Dihydro-a-ionone Nopyl acetate a-Gurjunene Neryl acetone (E)-b-caryophyllene b-Ylangene b-Copaene trans-a-Bergamotene a-Guaiene Aromadendrene Isogermacrene-D a-Himachalene a-Humulene allo-Aromadendrene

780 851 861 837 882 927 936 950 978 981 987 993 1015 1025 1051 1063 1058 1075 1082 1086 1076 1100 1105 1112 1123 1126 1137 1137 1142 1150 1150 1160 1169 1164 1182 1176 1180 1178 1195 1200 1200 1244 1263 1270 1273 1290 1291 1284 1300 1315 1331 1355 1347 1369 1376 1379 1389 1386 1390 1389 1392 1396 1413 1412 1421 1420 1430 1434 1440 1443 1445 1450 1455 1462

759 822 835 839 876 924 933 947 974 980 984 1000 1015 1024 1052 1055 1060 1077 1083 1085 1086 1100 1110 1118 1127 1129 1134 1138 1144 1155 1159 1163 1162 1166 1171 1177 1180 1182 1185 1201 1202 1241 1254 1266 1274 1282 1288 1290 1299 1322 1334 1352 1355 1373 1375 1379 1383 1387 1391 1391 1400 1401 1402 1414 1415 1422 1425 1431 1436 1439 1442 1445 1450 1455 1463

1070 1367 1382 1347 1183 1014 1016 1057 1102 1252 1147 1000 1275 1195 1239 1534 1419 1423 1292 1530 1377 1100 1463 1492 1495 1632 1523 1645 1677 1663 1643 1815 1582 1705 1675 1462 1763 1704 1197 1863 1699 1922 1536 1558 1568 1850 1701 1301 1693 2177 1451 2024 1980 1474 1483 1673 1510 1510 1580 1401 1793 1758 1538 1824 1585 1561 1579 1597 1643 1593 1665 1653 1653 1630

0.1 0.6 0.1 0.1 tr tr 0.9 0.3 0.3 tr 0.1 tr tr 0.1 0.1 tr tr tr 0.1 tr 0.7 tr tr 0.1 0.1 0.1 tr 0.1 0.1 0.2 tr tr tr 0.3 tr 0.6 0.1 tr tr 0.1 tr 0.1 0.2 tr 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.1 tr 0.1 0.4 0.1 0.7 0.1 0.4 tr 0.1 tr 0.1 tr 2.3 tr 0.3 tr 0.2 0.1 0.1 0.2 0.4 0.5

I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I,

MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS

Fra.g

F2.2 F4.12 F4.11 Adams (2001) F4.10 F2.4 F1.1 F1.1 F1.1 F1.1 König et al. (2001) F1.1 F1.1 F1.1 F1.3 F1.3 F1.1 F4.11 König et al. (2001) F4.6 F1.1 F1.1 F3.9 F2.4 F1.1 F2.5 NIST (2005) F3.3 F3.2 F4.3 McLafferty and Stauffer (1994) F4.4 F2.9 F3.10 F4.6 F3.9 F4.5 F4.8 F3.8 F2.8 F4.10 F2.4 F4.7 König et al. (2001) F2.8 F1.1 F4.5 F3.4 König et al. (2001) F4.2 F2.6 F2.10 F2.4 F2.18 F4.6 F1.1 König et al. (2001) F3.2 F3.9 F1.1 König et al. (2001) F4.2 F3.3 F1.1 F1.1 F2.4 F1.1 F1.1 F1.4 F1.1 König et al. (2001) F2.13 F3.2 F1.1 F3.2 F1.4 F1.1 F1.1 F1.1 F1.1 F1.1 F1.2 F1.1 F1.6 F1.1 (continued on next page)

1151

J. Paolini et al. / Phytochemistry 70 (2009) 1146–1160 Table 1 (continued) Noa

Components

I lb

I ac

I pd

%e

Identificationf

Fra.g

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150

Benzyl tiglate b-Ionone 1,11-Oxydocalamenene Tridecan-2-one c-Muurolene Tridecanal Germacrene-D 4-epi-Cubebol b-Selinene Tridecan-2-ol Drim-8(12)-ene a-Selinene Ledene a-Muurolene 6-epi-Shyobunol Cubebol Drimenene c-Cadinene Calamenene (cis and/or trans) d-Cadinene (Z)-nerolidol Cadina-1,4-diene a-Cadinene a-Calacorène b-Caryophyllene oxide b-Calacorene Germacrene B Spathulenol Gleenol b-Copaen-4a-ol Caryophyllene oxide Salvia-4(14)-en-1-one Germacraden-11-ol Globulol Viridiflorol Tetradecanal Humulene epoxide II b-Oplopenone a-Corocalene Guaiol 1,10-Di-epi-cubenol Ledol Agarospirol epi-Cubenol c-Eudesmol Caryophylla-4(14),8(15)-dien-5a-ol Hinesol Cubenol s-Cadinol s-Muurolol d-Cadinol a-Selin-11-en-4-ol b-Eudesmol Eudesm-4(15)-en-6-ol a-Cadinol Intermedeol Cadalene Eudesma-4(15),7-dien-1b-ol Pentadecanal Mintsulphite Benzyl benzoate Ambrox Drim-8-en-7-one Tetradecanoic acid Isopimara-8,15-diene Sclarene 13-Oxo-15,16-bis-nor-ent-labd-8(17)-ene Pimara-8,15-diene Isopimara-8(14),15-diene Hexadecanoic acid Pimara-8(14),15-diene 13-Oxo-15,16-bis-nor-ent-labd-7(8)-ene (Z)-bimorphene Manoyl oxide Isopimara-7,15-diene

1465 1468 1474 1477 1474 1493 1479 1490 1486 1490 1497 1494 1492 1496 1505 1514 1503 1507 1517 1520 1522 1523 1534 1527 1546 1541 1552 1572 1574 1561 1578 1592 1579 1589 1592 1596 1602 1595 1602 1592 1615 1600 1635 1623 1618 1622 1632 1630 1633 1633 1634 1636 1641 1656 1643 1645 1659 1671 1702 1734 1730 1747 1778 1748 1922 1943 1942 1981 1951 1955 1988 1996 2010

1464 1474 1475 1475 1475 1479 1481 1486 1487 1491 1491 1496 1496 1498 1499 1505 1506 1511 1515 1520 1522 1529 1534 1535 1544 1553 1557 1563 1570 1573 1577 1580 1583 1584 1588 1593 1596 1599 1606 1606 1606 1610 1617 1622 1625 1629 1629 1630 1632 1632 1636 1639 1643 1643 1645 1646 1661 1675 1677 1730 1733 1745 1756 1766 1919 1933 1943 1949 1956 1956 1965 1980 1985 1996 2002

1804 1907 1774 1789 1673 1781 1692 1863 1702 1892 1726 1707 1678 1707 2004 1914 1748 1742 1808 1742 1910 1762 1772 1898 1829 1931 1816 2091 2025 2115 1953 1995 2023 2056 2071 1851 2005 2012 2030 2102 2028 1999 2111 2036 2137 2264 2198 2112 2141 2156 2230 2220 2195 2025 2201 2207 2169 2240 1992 2128 2578 2129 2354 2240 2130 2213 2467 2163 2140 2666 2173 2533 2294 2308 -

tr tr 0.3 tr 1 0.1 0.6 tr 1.7 tr 0.3 1.4 tr 1 tr tr 0.7 1.5 0.3 3.2 0.1 0.3 0.3 0.4 0.5 0.1 0.2 0.2 0.5 tr 1.0 0.2 tr 1.8 0.3 0.1 0.5 1 0.4 tr tr 0.4 tr 0.8 0.8 0.5 tr tr 1.4 1.5 1.0 tr 0.7 tr 3 tr 0.6 0.4 0.3 0.1 0.2 0.3 0.3 0.2 0.3 0.8 5.5 0.1 0.1 tr 0.1 1.6 0.3 4.2 0.1

I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I, I,

F2.6 F2.8 F2.9 F2.10 F1.2 F2.4 F1.1 F4.3 F1.3 F4.5 F1.1 F1.3 F1.1 F1.2 F3.7 F4.4 F1.1 F1.3 F1.5 F1.1 F3.2 F1.1 F1.3 F1.5 F2.5 F1.1 F1.1 F4.2 F3.1 F3.9 F2.17 F2.8 F3.9 F4.3 F3.3 F2.5 F2.7 F2.9 F1.2 F3.9 F3.2 F3.10 F4.4 F3.4 F4.6 F4.7 F4.9 F3.11 F3.10 F3.11 F4.12 F4.3 F4.7 F2.7 F4.12 F4.10 F1.7 F4.9 F2.4 F2.1 F2.5 F3.3 F3.3 F4.2 F1.2 F1.1 F2.11 F1.1 F1.1 F4.2 F1.1 F2.14 F1.1 F2.3 F1.4

MS König et al. (2001) MS MS König et al. (2001) MS MS MS MS MS MS MS König et al. (2001) MS König et al. (2001) MS MS MS MS MS MS König et al. (2001) MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS König et al. (2001) MS MS MS MS NIST (2005) MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS McLafferty and Stauffer (1994) MS MS MS MS König et al. (2001) MS NIST (2005) MS NIST (2005) MS McLafferty and Stauffer (1994) MS MS König et al. (2001) MS NIST (2005) MS MS MS, NMR MS MS MS NIST (2005) MS MS, NMR MS MS MS König et al. (2001)

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Table 1 (continued) Noa

Components

I lb

I ac

I pd

%e

Identificationf

Fra.g

151 152 153 154 155 156 157 158 159 160 161 162 163 164

(E)-bimorphene 13-epi-Manoyl oxide Labda-7,12(E),14-triene Nor-ambreinolide a-Kaurene Manool (labda-8(20),14-dien-13-ol) Labda-7,14-dien-13-ol Isoabienol Sclareol Tricosane Tetracosane Pentacosane Hexacosane Heptacosane

2017 2023 2036 2034 2070 2096 2124 2231 2301 2400 2498 2598 2700

2013 2021 2030 2031 2046 2054 2086 2100 2227 2301 2400 2500 2600 2700

2339 2326 2361 2949 2320 2638 2689 2706 2545 2300 2400 2500 2600 2700

0.5 8.7 0.3 tr 0.2 7.2 3.8 0.2 2.7 0.4 0.1 0.1 0.1 0.1

I, I, I, I, I, I, I, I, I, I, I, I, I, I,

F1.1 F2.2 F1.4 F2.19 F1.1 F3.5 F3.8 F3.10 F3.2 F1.1 F1.1 F1.1 F1.1 F1.1

MS MS MS MS MS MS MS MS MS MS MS MS MS MS

König et al. (2001) NIST (2005) König et al. (2001), NMR König et al. (2001), NMR

Total identified

83.0

Acyclic nonterpenic hydrocarbon components Acyclic nonterpenic oxygenated components Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Diterpene hydrocarbons Oxygenated diterpenes Others

1.0 3.4 1.9 1.7 20.4 17.2 2.8 33.9 0.7

a b c d e f g

Order of elution are given on apolar column (Rtx-1). I l = retention indices on the apolar column of literature. Retention indices of components 104, 118 and 126 were reported for the first time. I a = retention indices on the apolar column (Rtx-1). I p = retention indices on the polar column (Rtx-Wax). % = Percentages of components are given on the apolar column except for components with identical I a (percentages are given on the polar column), tr = trace (<0.05%). MS = mass spectra in electron impact mode; NMR = Carbon-13 Nuclear Magnetic Resonance. Fra. = fraction.

Table 2 C. creticus: primers used, annealing temperatures and polymorphism percentage. Primer code

Primer repeat

Annealing temperature °C

No. of fragments

No. polymorphic bands

Polymorphism %

UBC808 UBC809 UBC812 UBC815 UBC818 UBC834 UBC841 UBC852 UBC854 UBC856 UBC857 UBC880 UBC884 UBC888

AGAGAGAGAGAGAGAGC AGAGAGAGAGAGAGAGG GAGAGAGAGAGAGAGAA CTCTCTCTCTCTCTCTG CACACACACACACACAG AGAGAGAGAGAGAGAGYT GAGAGAGAGAGAGAGAYC TCTCTCTCTCTCTCTCRA TCTCTCTCTCTCTCTCRG ACACACACACACACACYA ACACACACACACACACYG GGAGAGGAGAGGAGA HBHAGAGAGAGAGAGAG BDBCACACACACACACA

50 52 54 52 54 50 50 54 54 50 54 50 54 52

12 9 5 5 4 11 10 8 9 4 5 7 4 10

10 8 2 3 1 4 9 8 3 2 2 3 2 8

83.33 88.89 40.00 60.00 25.00 36.36 90.00 100.00 33.33 50.00 40.00 42.86 50.00 80.00

Y = (C,T); R = (A,G); H = (A,C,T); B = (C,G,T); D = (A,G,T).

ized matrix. The distribution of variables is shown in Fig. 4A; it can be seen that the principal factorial plane (constructed with axes 1 and 2) summarizes 41.20% of the whole variability. The two PCA axes explained 29.75% and 11.45% of the variance, respectively. Furthermore, two opposite groups of variables are very well represented on axis 1 (Fig. 4A): the monoterpene hydrocarbons and linear nonterpenic components on the one hand, and labdane diterpenes and sesquiterpene hydrocarbons on the other hand. The plot established according to the first two axes suggests the existence of two groups of essential oils (Fig. 4B): – Group I: 21 samples from seven localities (Bc, Fa, Fv, No, Pc, Re and St) corresponding to C. creticus subsp. eriocephalus belonged to this group. It was defined by two variables: monoterpene hydrocarbons (limonene 14 as the major component) and linear nonterpenic components.

– Group II: 33 samples from 11 localities (Al, Ba, Bo, Bt, Ca, Cf, Er, Fm, Ol, Pa and Pv) corresponding to C. creticus subsp. corsicus belonged to this group. It was also defined by two variables: labdane diterpenes (13-epi-manoyl oxide 152 as the major component) and sesquiterpene hydrocarbons. The general structure of the dendrogram produced by the Ward’s method of hierarchical clustering, based on the Euclidean distance between population pairs, was similar to that obtained with PCA, grouping the 54 samples into the two main clusters (Fig. 5). CA suggested the existence of two groups based on the amount of components from four families (monoterpene hydrocarbons, linear nonterpenic, labdane diterpenes and sesquiterpene hydrocarbons): group I (21 samples) and group II (33 samples). Few differences were reported between PCA and CA results. As shown in Figs. 4 and 5, the three samples from each location have a similar volatile composition.

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A 17.27

100

14

7

%

12.49

2

11

8.73

15.03

3

9.06

15 18.89

4

21

20.95

33.47

25.06

0 8.72

13.72

18.72

23.72

28.72

47.26

33.72

69.63

58.98

37.08

38.72

43.72

48.72

53.72

58.7263.72

68.72

B

75.00

100

73.72

Time

78.72

149

74.86

74.80

%

7

12.50

66 42.63

84 46.58

80 45.94

13.13 14.47

0 10.95

40.44

20.95

25.95

30.95

35.95

40.95

48.68

93 51.73

17.24

15.95

95

45.95

50.95

128 129

142

55.62

71.07

147

73.49

54.62

70.64

152

90.69

76.34

55.95

60.95

65.95

70.95

75.95

80.95

85.95

Time

90.95

Fig. 3. GC/MS chromatogram obtained from the leaf HS-SPME of C. creticus subsp. eriocephalus (A) and C. creticus subsp. corsicus (B). Peaks: 2 (Z)-hex-3-en-1-ol; 3 (Z)-hex-2en-1-ol; 4 hexanol; 7 a-pinene; 11 myrcene; 14 limonene; 15 c-terpinene; 21 nonanal; 66 (E)-b-caryophyllene; 80 c-muurolene; 84 b-selinene; 93 c-cadinene; 95 dcadinene; 128 b-eudesmol; 129 eudesm-4(15)-en-6-ol; 142 13-oxo-15,16-bis-nor-ent-labd-8(17)-ene; 147 13-oxo-15,16-bis-nor-ent-labd-7(8)-ene; 149 manoyl oxide; 152 13-epi-manoyl oxide.

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A

1

0.75

84 87

0.5

149 147

0.25

F2 (11.45 %)

152 142

45

66 7

0

3

80

4

125

-0.25

93 130

-0.5

2 14 81 46 21

95 56 109

75

58

11

134

89

-0.75

-1 -1

-0.75

-0.5

0

-0.25

0.25

0.5

0.75

1

F1 (29.75 %)

B

Group II C. creticus

subsp. corsicus

Group I C. creticus

subsp. eriocephalus

Fig. 4. PCA of HS-SPME volatile fractions of C. creticus: (A) PCA distribution of variables (component numbers corresponding to those of Table 1); (B) distribution of sample (coding numbers of locations corresponding to those of Fig. 1).

2.4. ISSR analysis The information of ISSRs in assessing genetic variability in C. creticus is summarized in Table 2. Fourteen primers generated good reproducible patterns and revealed 103 bands in a range of 300– 1800 kb. Among them, 65 bands (63.11%) were polymorphic. The dendrogram (Fig. 6) using Dice genetic distances based on 14 ISSR polymorphisms among the 54 individuals of C. creticus

showed that the bootstrap values were very high, indicating the robustness of the partition. The two subspecies of C. creticus appeared separated in the tree (C. creticus subsp. eriocephalus for group I and C. creticus subsp. corsicus for group II), indicating significant divergence between them. In the analysis of molecular variance (AMOVA) (Table 3) based on groups I and II (Fig. 6), 53.55% of the variation was observed among the groups analyzed. The variation among individuals with-

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C. creticus subsp. corsicus

C. creticus subsp. eriocephalus

Pv 3 Pv 1 Cf 2 Pv 2 Fm 2 Cf 1 Pa 3 Ba 3 Ba 2 Ba 1 Fm 1 Bt 2 Pa 1 Pa 2 Fm 3 Cf 3 Er 3 Er 1 Bt 1 Ca 3 Ca 2 Er 2 Bt 3 Ca 1 Po 3 Bo 2 Ol 2 Ol 1 Al 3 Al 1 Bo 3 Ol 1 Al 2 Re 1 St 1 Re 3 Re 2 No 1 Fa 2 Fa 1 Bc 2 Bc 1 Bc 3 No 3 No 2 St 3 St 2 Pc 3 Fa 3 Pc 1 Pc 2 Fv 3 Fv 2 Fv1

Group II

Group I

0.93905075 0.73905075 0.53905075 0.33905075 0.13905075 -0.0609492 -0.2609492 Fig. 5. CA of HS-SPME volatile fraction of C. creticus. Dendrogram of agnes (x = ch, metric = ‘‘euclidean”, stand = TRUE, method = ‘‘ward”). Coding numbers of sample locations corresponding to those of Fig. 1.

in groups and within all populations was 19.67% and 26.78%, respectively. This high genetic differentiation among groups probably results from taxonomic repartition in two C. creticus subspecies (C. creticus subsp. corsicus and C. creticus subsp. eriocephalus). The genetic diversity over the 14 ISSR polymorphisms has low values (group I: 0.197 and group II: 0.203) in the two groups (Table 4). The low genetic diversity within groups could be attributed to the habitat fragmentation and the limited gene flow among populations. As reported by Jeanmonod and Gamisans (2007), the repartition (thermomediterranean and mesomediterranean) of the two subspecies from Corsica and Sardinia does not appear to be linked to geographical distances and ecological conditions (Fig. 1). There are certainly instances where concentrations of secondary metabolites are affected by variation in the environment. However, it cannot be assumed that an effect of environment is independent of the action of genes. There may be a direct effect of the environment on gene expression, suggesting induction or suppression. There may also be a genotype–environment interaction, suggesting that the degree of induction or suppression varies among genotypes. Differentiating among all of these possibilities is difficult and requires simultaneous manipulation of genotype and environmental factors in homogeneous settings. Furthermore, some authors stress that in the hierarchy of factors driving terpene metabolism, a genetically determined ‘‘basal allocation” mitigates the upstream environmental control on plant secondary metabolism (Hamilton et al., 2001). The numerous factors affecting terpene production represent a significant obstacle for developing a model capable of predicting the quantities of plant secondary

metabolites, such as terpenes, under differing environmental and geographical conditions. 2.5. Conclusion Finally, it appears that C. creticus subsp. eriocephalus and C. creticus subsp. creticus from the Corsican–Sardinian continuum were differentiated by botanical characteristics (absence or presence of glandular trichomes) linked with essential oil production and composition of the volatile fraction (limonene or 13-epi-manoyl oxide, respectively, as the major component). Moreover, data analysis of the HS-SPME volatile fraction (Figs. 4 and 5) confirms the clustering observed using polymorphism of the 65 ISSR loci (Fig. 6). The neutrality analysis using the Tajima test and the Fu test showed no selective effect (Table 4). The Mantel test also confirms this result and shows a significant correlation (r = 0.9215, P < 0.020) between the two matrices (volatile composition and ISSR markers). These results indicate that the observed chemical variability was mainly due to genetic diversity. The concordance between the two data sets is in accordance with the findings from other studies using terpenic and genetic markers (Bazina et al., 2002; Echeverrigaray et al., 2001; Skoula et al., 2000; Zaouali and Boussaid, 2008). These data suggest that the basis of variation in the volatile composition of the two C. creticus subspecies depends more on the genetic background and less on geographical factors. In the two examined subspecies of C. creticus, the distribution of leaf trichomes and the difference in essential oil production may be considered distinctive characters at the subspecies level. This is consistent with the taxonomic classification. The HS-SPME volatile

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Fig. 6. Dendrogram based on Dice’s genetic distance (UPGMA method) of 54 individuals of C. creticus using 12 ISSR primers (coding numbers of sample locations corresponding to those of Fig. 1).

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J. Paolini et al. / Phytochemistry 70 (2009) 1146–1160 Table 3 AMOVA analysis in C. creticus from Corsica and Sardinia Islands. Source of variation

d.f

Sum of squares

Percentage of variation

Among groups Among populations within groups Within populations

1 4 48

133.364 52.755 102.752

53.55*** 19.67*** 26.78**

** ***

P < 0.01. P < 0.001.

Table 4 Gene diversity and neutrality tests of ISSR markers.

Sample size Gene diversity Tajima test, D Fu test, Fs

Group I

Group II

21 0.1927 1.101 P = 0.812 1.279 P = 0.223

33 0.2036 1.274 P = 0.845 2.263 P = 0.189

fractions of samples were allotted to two main groups; the first group (C. creticus subsp. corsicus) had a high content of components belonging to the labdane biosynthetic pathway, while the second (C. creticus subsp. eriocephalus) was characterized by limonene and nonterpenic compounds. In addition, the groups deducted from the volatile fraction composition were remarkably congruent with the groups inferred from genetic data obtained from ISSR markers. Such information would be useful for the development of conservation programs and for the selection of populations in order to consider a potential utilization of these oils. 3. Experimental 3.1. Plant material The aerial parts of C. creticus subsp. eriocephalus and C. creticus subsp. corsicus were harvested in May 2007 (full bloom) from 16 localities in Corsica (France) and two in North Sardinia (Fig. 1): Erbalunga (Er), Patrimonio (Pa), Farinole-mer (Fm), Farinole village (Fv), Poggio d’Oletta (Ol), Bastia (Ba), Casamozza (Ca), Barchetta (Bc), Altiani (Al), Noceta (No), Restonica (Re), Favona (Fa), Porto Vecchio (Pv), Bonifacio (Bo), Capo di Feno (Cf), Basteliccacia (Bt), Santa Teresa (St) and Porto Cervo (Pc). Voucher specimens (reference numbers: CC101–CC118) were deposited in the herbarium of the University of Corsica, Corte, France. For each population, the leaves of three individual shrubs were taken at random, and genetic and volatile fraction analyses were performed on each specimen. 3.2. Scanning analysis Leaf surface morphology was analyzed using the Hitachi S3400-N scanning electron microscope of the SERME (Corsica, France). A BSE detector in the VP-SEM mode (column pressure kept at 30 Pa) was used to observe fresh samples without preparation. For each subspecies, 12 leaves were randomly selected on three individual shrubs from natural environment. Micromorphological analysis was performed on of all 24 leaf samples. Portions of 25 mm2 area located in the leaf center were analyzed at 10 and 15 kV. 3.3. Essential oil isolation For each sample location, the fresh vegetal material was water distilled (5 h) using a Clevenger-type apparatus according to the

method recommended in the European Pharmacopoeia (Council of Europe, 1997). The total oil was obtained by mixing oils from various localities in Corsica (Ba, Pa, Ca, Al, Pv and Bo). 3.4. Oil fractionation The essential oil from C. creticus subsp. corsicus (4.6 g) was first chromatographed on a silica gel column (200–500 lm), and four fractions were eluted with pentane (F1) and a mixture of pentane/diethyl oxide of increasing polarity (F2–F4). The apolar fraction F1 (0.88 g) was fractionated on silica gel (63–200 lm) impregnated with AgNO3 (20%), leading to eight fractions (F1.1– F1.8) by elution with pentane. The polar fractions F2 (1.39 g), F3 (0.97 g) and F4 (1.36 g) were fractionated by flash chromatography (silica gel, 63–200 lm) using pentane with an increasing amount of diethyl oxide as eluent into 19 fractions (F2.1–F2.19), 11 fractions (F3.1–F3.11) and 15 fractions (F4.1–F4.15), respectively. The bulk sample and all the fractions of chromatography were submitted to GC and GC/MS analysis. Some fractions selected based on their chromatographic profile were also analyzed by 13C NMR (Table 1). 3.5. HS-SPME The leaves of C. creticus were subjected directly to HS-SPME. The SPME device (Supelco, Bellefonte, PA, USA) coated with divinylbenzene/carboxen/polydimethylsiloxane (30 lm) was used for extraction of the plant volatiles. Optimization of conditions was carried out using leaves (4 g into a 20 ml vial) from various samples taken separately, and was based on the sum of total peak areas. The equilibrium and extraction temperatures were selected after three different experiments at 30, 60 and 90 °C. The equilibration time was selected after three different experiments at 60, 90 and 120 min. The extraction time was selected after three different experiments at 15, 30 and 45 min. The maximum sum of the total peak area was obtained at a temperature of 90 °C, an equilibrium time of 90 min and an extraction time of 30 min. After sampling, SPME fiber was inserted into the GC and GC–MS injection ports for desorption of volatile components (5 min), both using the splitless injection mode. Before sampling, each fiber was reconditioned for 5 min in the GC injection port at 260 °C. HS-SPME and subsequent analyses were performed in triplicate. 3.6. GC analysis GC analyses were carried out using a Perkin–Elmer (Waltham, MA, USA) Autosystem XL GC apparatus equipped with a dual flame ionization detection system and two fused-silica capillary columns (60 m  0.22 mm I.D., film thickness 0.25 lm), Rtx-1 (polydimethylsiloxane) and Rtx-wax (polyethyleneglycol). The oven temperature was programmed from 60 °C to 230 °C at 2 °C/min and then held isothermally at 230 °C for 35 min. Injector and detector temperatures were maintained at 280 °C. Samples were injected in the split mode (1/50), using helium as the carrier gas (1 ml/min); the injection volume was 0.2 ll of pure oil. For the HS-SPME–GC analysis, only the Rtx-1 (polydimethylsiloxane)

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column was used and volatile components were desorbed in a GC injector with an SPME intel liner (0.75 mm I.D., Supelco, Inc.). Retention indices (I) of the compounds were determined relative to the retention times of the series of n-alkanes (C5–C30) with linear interpolation, using the Van den Dool and Kratz equation (Van den Dool and Kratz, 1963) and software from Perkin–Elmer. Component relative concentrations were calculated based on GC peak areas without using correction factors. 3.7. GC/MS analysis GC/MS conditions: samples were analyzed with a Perkin–Elmer Turbo mass detector (quadrupole), coupled to a Perkin–Elmer Autosystem XL, equipped with the fused-silica capillary columns Rtx-1 and Rtx-Wax (ion source temperature 150 °C; energy ionization 70 eV). EI mass spectra were acquired over the mass range 35– 350 Da (scan time: 1 s). Other GC conditions were the same as described under GC except split 1/80. For HS-SPME–GC–MS analysis, only the Rtx-1 column was used and volatile components were desorbed in a GC injector with an SPME intel liner (0.75 mm I.D., Supelco Inc.). 3.8. NMR analysis Some fractions selected based on of their chromatographic profile were analyzed by 13C NMR on a Bruker (Wissembourg, France) Avance 400 Fourier Transform Spectrometer operating at 100.13 MHz, equipped with a 5 mm probe, in deuterated chloroform, with all shifts referred to internal tetramethylsilane. 13C NMR spectra were recorded with the following parameters: pulse width = 4 ls (flip angle 45°); acquisition time = 2.7 s for 128,000 data table with a spectral width of 25,000 Hz (250 ppm); CPD mode decoupling; digital resolution = 0.183 Hz/pt. The number of accumulated scans was 5000 for each sample (around 40 mg of the oil in 0.5 ml of CDCl3). Exponential line broadening multiplication (1 Hz) of the free induction decay was applied before Fourier transformation. 3.9. Components identification As previously reported (Paolini et al., 2005, 2007), the methodology used for identification of individual components was based on: (a) a comparison of calculated retention indices (I p, I a) on polar and apolar columns with those of authentic compounds or literature data (I l) (Jennings and Shibamoto, 1980; Joulain and König, 1998; NIST, 2005); (b) computer matching with commercial mass spectral libraries (Adams, 2001; König et al., 2001; McLafferty and Stauffer, 1988, 1994; NIST, 1999) and a comparison of mass spectra with those of our own library of authentic compounds or literature data (Jennings and Shibamoto, 1980; Joulain and König, 1998); and (c) by 13C NMR, following the methodology first reported by Formácek and Kubeczka (1982) and based on the comparison of 13C NMR spectral data of components in the mixture with those reported in the literature. 3.10. ISSR–PCR amplifications Total DNA was extracted from approximately 0.1 g of fresh leaves using a DNeasy plant kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. DNA was visualized by 1.7% agarose gel electrophoresis stained with ethidium bromide and quantified with ladder Gel Pilot 200 (Qiagen). Of all the ISSR primers available in set 9 (Biotechnology Laboratory, University of British Columbia, Canada), 14 showed reproducible polymorphism (Table 2). Optimal conditions for DNA amplifications were empirically determined by testing different concentra-

tions of genomic DNA, MgCl2 and primers. The optimal annealing temperature was found to vary according to the base composition (Table 2). Amplification of 10 ng DNA was performed in a 25 ll volume of PCR mixture containing 2 mM of MgCl2, 1 lM of primer and 12 ll of Hot Start DNA Polymerase Master Mix (Qiagen). Amplifications were carried out in a Gene Amp PCR System 9700 from Applied Biosystem (Perkin Elmer, France) with an initial denaturation/activation at 94 °C for 15 min, followed by 35 cycles comprising 30 s at 94 °C, 1 min at annealing temperature (Table 2) and 2 min at 72 °C. A final extension for 10 min at 72 °C was included. Amplified products were analyzed in a 1.2% agarose gel stained with ethidium bromide and visualized and photographed under UV light (Kodak logic gel, France). The size of the fragments was estimated using a ladder Gel Pilot 200 (Qiagen). Reproducibility of the amplification was confirmed by repeating each experiment. 3.11. Data analysis PCA and CA are data mining tools that are useful for providing unsupervised visual classification of multivariate data such as GC data (Brereton, 2003; Massart, 1998). PCA and CA were applied on a matrix linking the volatile fraction composition and sample locations in order to identify possible relationships between subspecies of C. creticus and volatile components. Statistical analyses were performed with the XLSTAT software (Addinsoft, Paris, France), where variables were the relative percentages of volatile compounds. Each ISSR band was considered as a character and the presence or absence of the band was scored in binary code (1 = present, 0 = absent). These data were used to analyze genetic diversity among the populations (Nei, 1973). The software package NTSYSPC (Rohlf, 1989) was used to perform cluster analysis using the Dice coefficient. The Dice coefficient is the most suitable measure of dissimilarity between banding patterns of closely related diploid forms (Kosman and Leonard, 2005). Confidence limits of the tree were estimated with the WINBOOT program with 1000 replications. The genetic distances were computed using the WINDIST program (Yap and Nelson, 1996). Genetic and chemical matrices were analyzed for correlation with the Mantel test implemented on Arlequin software (Schneider et al., 2000). Arlequin software was also used for the AMOVA to partition variance among groups (Weir and Cockerham, 1984), within groups and among individuals within groups, and for the neutrality analysis performed by the Tajima D test and the Fu Fs test. Acknowledgements The authors are grateful to Pr. J. Casanova (Université de Corse, Equipe ‘‘Chimie et Biomasse”, UMR-CNRS 6134) for NMR data acquisition. They are also indebted to the Collectivité Territoriale de Corse for partial financial support. References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing, Carol Stream. Anastasaki, T., Demetzos, C., Perdetzoglou, D., Gazouli, M., Loukis, A., Harvala, C., 1999. Analysis of labdane-type diterpenes from Cistus creticus (subsp. Creticus and subsp. eriocephalus), by GC and GC–MS. Planta Med. 65, 735–739. Angelopoulou, D., Demetzos, C., Dimas, C., Perdetzoglou, D., Loukis, A., 2001. Essential oils and hexane extracts from leaves and fruits of Cistus monspeliensis. Cytotoxic activity of ent-13-epi-manoyl oxide and its isomers. Planta Med. 67, 168–171. Batista, F., Bañares, A., Caujapé-Castells, J., Carque, E., Marrero-Gómez, M., Sosa, P.A., 2001. Allozyme diversity in three endemic species of Cistus (Cistaceae) from the Canary Islands: intraspecific comparisons and implications for genetic conservation. Am. J. Bot. 88, 1582–1592.

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