Taxonomy of prickly juniper (Juniperus oxycedrus group): A phytochemical–morphometric combined approach at the contact zone of two cryptospecies

Phytochemistry 141 (2017) 48e60

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Taxonomy of prickly juniper (Juniperus oxycedrus group): A phytochemicalemorphometric combined approach at the contact zone of two cryptospecies Francesco Roma-Marzio a, *, Basma Najar b, John Alessandri a, Luisa Pistelli b, Lorenzo Peruzzi a a b

Department of Biology, University of Pisa, Via Derna 1, 56126, Pisa, Italy Department of Pharmacy, University of Pisa, Via Bonanno 33, 56126, Pisa, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 February 2017 Received in revised form 10 May 2017 Accepted 18 May 2017

Based on different essential oil composition paralleling different genotypes, Juniperus deltoides was recently segregated from Juniperus oxycedrus. Despite a clear phytochemical and molecular differentiation, J. deltoides resulted not clearly morphologically discernible from J. oxycedrus, so that it was defined as a cryptospecies. Italy represents the contact zone of their distribution, but the ranges of the two species are not sufficiently known, due to unsatisfactory morphological characterisation. To further complicate the picture, a third closely related species (ecotype), J. macrocarpa, occurs all across the Mediterranean coasts. After a preliminary phytochemical analysis to ascertain the (chemo-)identities of the studied populations, we performed a morphometric investigation to test the degree of morphological distinctiveness among the taxa. According to our analysis, some character (e.g. leaf mucro length, leaf width, seed-cone size and seed size) resulted useful to discriminate these cryptic taxa. Finally, based on these characters, an extensive revision of herbarium specimens allowed us to redefine the distribution pattern of the investigated species in the Central Mediterranean area. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Juniperus deltoides Juniperus oxycedrus Juniperus macrocarpa Cupressaceae Essential oil composition Herbarium specimens Identification key Morphometrics Italy

1. Introduction It is difficult to provide a general and unambiguous definition of cryptospecies, due to the absence of a general definition of species itself. Cryptospecies are morphologically indistinguishable, but genetically differentiated, reproductively isolated (Paris et al., 1989; Whittall et al., 2004; Bickford et al., 2007), and allopatric (Arrigoni, 1988). On the contrary, cryptic species living in sympatry are more appropriately defined as ‘sibling species’ (Grant, 1971). Cryptospecies are frequently recognized and described based on DNA sequence data (Bickford et al., 2007), but phytochemical investigations can also provide valuable information, helping to disentangle taxonomically critical groups (Harborne, 2000; Hadacek, 2002; Stuessy, 2009; Carta et al., 2015; Tundis et al.,

* Corresponding author. E-mail address: [email protected] (F. Roma-Marzio). http://dx.doi.org/10.1016/j.phytochem.2017.05.008 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

2016; Passalacqua et al., 2017). A paradigmatic case where both DNA and chemosystematics have been jointly used to recognise a cryptospecies is the so-called Juniperus oxycedrus group (prickly juniper), i.e. J. deltoides R.P.Adams, J. macrocarpa Sm., and J. oxycedrus L. s.str. (Adams et al., 2005). The genus Juniperus L. (Cupressaceae, gymnosperms) consists of about 60 dioecious woody species, and it is divided into three sections: J. sect. Caryocedrus Endl., J. sect. Juniperus, and J. sect. Sabina Spach (Adams, 2012). The Juniperus oxycedrus group is included within J. sect. Juniperus (Adams, 2014a, 2014b). Juniperus oxycedrus was described by Linnaeus (1753) based on plants from the western part of the Mediterranean basin (“Habitat in Hispania, G. Narbonensi”), while Juniperus deltoides was segregated from the former species based on plants from the eastern Mediterranean basin (Greece) (Adams, 2004). The description of J. deltoides as a distinct species relied mostly on the different composition of essential oil (EO): the leaf oil of east Mediterranean

F. Roma-Marzio et al. / Phytochemistry 141 (2017) 48e60

populations was poor in a-pinene and rich in limonene, compared to west Mediterranean populations (Adams et al., 2003, 2005; Raj
cevi c et al., 2013, 2015; Adams, 2014b). Interestingly, this phytochemical differentiation was supported and paralleled by DNA molecular markers: nrITS (Adams et al., 2003, 2005, 2015; Mao et al., 2010; Adams and Schwarzbach, 2012); ISSR (Adams et al., 2003); RAPD (Adams et al., 2003, 2005); cpDNA (Mao et al., 2010; Rumeu et al., 2011; Adams and Schwarzbach, 2012; Adams  ski et al., 2014). Despite this, Juniperus et al., 2015); SSR (Boratyn deltoides is hardly morphologically discernible from J. oxycedrus  ski et al., 2014), (Adams et al., 2005; Adams, 2014b; Boratyn although the lineages including the two taxa have been estimated to diverge since the late Miocene (8e10 Ma) (Mao et al., 2010;  ski et al., 2014). Boratyn The occurrence of both J. oxycedrus and J. deltoides was reported in Italy: the first one in the west portion of Liguria (NW Italy) and Sardinia, the second one supposed to occur in the whole peninsular Italy, from Tuscany to Calabria (Adams, 2014a, 2014b). The recent record of J. oxycedrus s.str. for peninsular Italy (Lazzeri et al., 2015; Bartolucci et al., 2017), based on some putatively discriminant morphological characters (Adams, 2014b), was the occasion to deeper investigate the morphological distinction and distribution of this species pair. Tuscany, in particular, seems to represent the main contact zone between the ranges of J. deltoides and J. oxycedrus (Roma-Marzio et al., 2016). All across the Mediterranean coasts, also J. macrocarpa Sm. occurs. The latter species represents an ecotype, often treated as an ecologically vicariant subspecies of J. oxycedrus (Farjon and Filer, 2013), chemotaxonomically and phylogenetically much closer to J. oxycedrus s.str. than to J. deltoides (Adams et al., 2003, 2005, 2015; Mao et al.,  ski et al., 2014). 2010; Adams and Schwarzbach, 2012; Boratyn The present study aims to clarify the taxonomy and distribution of the Juniperus oxycedrus group in a critical geographical area,

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where the ranges of the three cited above taxa are coming in contact, i.e. the Central Mediterranean area, and peninsular Italy in particular. A phytochemical approach was followed to detect the (chemo-)identity of the sampled populations and, based on the obtained results, a morphometric analysis was performed on selected Juniperus populations, to test the degree of morphological distinctiveness among the taxa, and to highlight the most useful and significant diagnostic morphological characters. Finally, based on these characters, a critical review of herbarium specimens was performed, in order to clarify the distribution of the involved taxa.

2. Materials and methods 2.1. Phytochemical investigation Populations of Juniperus deltoides R.P.Adams, J. macrocarpa Sm., and J. oxycedrus L. (Cupressaceae) were collected during 2016 from thirteen localities: ten from Italy, two from Tunisia and one from Croatia (Table 1). For each locality, a herbarium voucher was prepared and deposited at PI (herbarium acronym follows Thiers, 2017). All the EOs were obtained by hydrodistillation from dry aerial parts, using a Clevenger-type apparatus according to the Italian Pharmacopoeia (AOAC, 1990). The GC/MS analyses were performed with a Varian CP-3800 apparatus, equipped with a DB-5 capillary column (30 m
0.25 mm i.d., film thickness 0.25 mm) and a Varian Saturn 2000 ion-trap mass detector. The oven temperature was programmed rising from 60  C to 240  C at 3  C/min; injector temperature, 220  C; transfer-line temperature, 240  C; carrier gas, He (1 ml/min). The identification of the EO compounds was based on the comparison of their retention times (Rt) with those of pure reference samples, and their linear retention indices (LRIs) determined

Table 1 Sampled localities and source of data used for the phytochemical investigation. For the populations sampled in the present study, decimal degrees coordinates (WGS84) and altitude (m a.s.l.) are provided. ID Locality

Locality

Country

Source of data

Coordinates

Altitude

EL SI PE VE FL MV MP LI OR SB SA BH KR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Elba Island (Livorno, Tuscany) Castiglione d’Orcia (Siena, Tuscany) Monte Pelato (Livorno, Tuscany) Marina di Vecchiano (Pisa, Tuscany) Finale Ligure (Savona, Liguria) Monte Vaso (Pisa, Tuscany) Monte Pisano (Pisa, Tuscany) Calignaia (Livorno, Tuscany) Oriolo (Cosenza, Calabria) Rosignano Solvay (Livorno, Tuscany) Sidi Ameur Bir Hannun
Miljeva
cki Bogati ci (Sibenik)

Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Tunisia Tunisia Croatia Greece Greece Spain Spain Italy Morocco Greece Greece Portugal Spain Turkey France Bulgaria Italy Greece Croatia Turkey

Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Adams 1999 Adams 1999 Adams 1999 Adams 1999 Adams et al., 2005 Adams et al., 2005 Adams et al., 2005 Adams et al., 2005 Adams et al., 2005 Adams et al., 2005 Adams et al., 2005 Adams et al., 2005 Adams and Tashev 2012 Adams and Tashev 2012 Adams and Tashev 2012 Raj
cevi c et al., 2013 Hayta and Bagci 2014

43.431740, 42.998589, 43.435018, 43.797410, 44.177760, 43.435018, 43.736920, 43.426810, 40.010620, 43.376270, 35.879110, 36.083330, 43.904543,

80 600 295 2 187 560 314 10 315 0 800 800 230

Lemo Crysoritsi El Penon Tarifa Raiano (Abruzzo) Marrakech Lemos Archova Vila Nova de Foz Coa El Penon Eskisehir Peyruis Devin region Raiano (Abruzzo) Archova Benkovac Bursa

10.430017 11.605017 10.610169 10.266640 08.343000 10.610169 10.582490 10.398950 16.499150 10.438326 9.479410 9.250000 15.989145

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Table 2 Measured morphological characters. N

ID

Character

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

L W Wb W10 W25 W50 W80 W90 dW Mu LBs P A BS Hs H D T S Sl Sw St

Leaf length (mm) Leaf maximum width (mm) Leaf basal width (mm) Width of leaf on 10% of leaf's length from base up (mm) Width of leaf on 25% of leaf's length from base up (mm) Width of leaf on 50% of leaf's length from base up (mm) Width of leaf on 80% of leaf's length from base up (mm) Width of leaf on 90% of leaf's length from base up (mm) Distance from the leaf's base to the point of maximum width (mm) Mucro length (mm) Stomatal band maximal width (mm) Leaf perimeter (mm) Leaf area (mm2) Maximum height of stomatal band concavity (mm) Leaf thickness (mm) Seed cone height (mm) Seed cone diameter (mm) Presence or absence of seed cone scale tips Number of seeds for seed cone Seed length (mm) Seed width (mm) Seed thickness (mm)

relatively to a series of n-alkanes. The mass spectra were compared with those listed in the commercial libraries NIST 2011 and ADAMS, together with a homemade mass-spectral library built up from pure substances and compounds of known oils, and with MS data from the literature. Data from the EO composition of all collected plants, integrated with data available in literature from other accessions of prickly juniper (Table 1), were used to build a matrix with mean percent values of all compounds. A default percentage of 0.01 was assigned

to those compounds detected in traces (Supplemental material S1). To evaluate the relationships among taxa, this matrix was subjected to Principal Component Analysis (PCA). 2.2. Morphometric investigation Based on the results of the phytochemical investigation, we selected five populations showing an EO profile typical (Adams et al., 2005) of Juniperus oxycedrus (MV), J. deltoides (SI and KR), and J. macrocarpa (VE, growing on sandy dunes and LI, growing on rocky coast). Populations of J. macrocarpa were also checked by qualitative morphological and ecological characters provided by Adams (2014a). Ten female individuals for each population have been sampled, with the exception of KR, where the sampling of only four individuals was possible. For each individual, five reproductive branches were collected, and morphological characters were measured on a randomly sampled leaf and cone for each branch. Totally, 220 leaves and 220 seed cones were sampled (50 for each population, 20 for KR). We measured 22 characters (15 for the leaves, 4 for the seed cones, and 3 for the seeds), selected based on previous works (Klimko et al., 2004, 2007; Brus et al., 2011, 2016; Morando et al., 2012; Adams, 2014a) (Table 2 and Fig. 1). To measure vegetative characters, leaves were scanned (characters #1e13), or photographed under a stereomicroscope (characters #14e15), and then measured by means of ImageJ 1.47 software (Rasband, 1997). Seeds and seed cones were measured by means of a digital calliper (accuracy ± 0.1 mm). Quantitative data have been used to build a data matrix (Supplemental material S2), then subjected to multivariate Discriminant Analysis (DA), an identification optimization procedure based on the probability of identification using a priori classification (Tundis et al., 2014; Peruzzi et al., 2015). Each character was

Fig. 1. Graphical representation of the measured leaf and seed cone characters (for description see Table 2).

F. Roma-Marzio et al. / Phytochemistry 141 (2017) 48e60

also subjected to univariate analysis (non parametric KruskalWallis test with Bonferroni correction for multiple comparisons). For the binomial character T, not included in the multivariate analysis, a c2 test was carried out. Only P values  0.01 have been considered significant. All the above cited analyses (including those for phytochemical data) have been carried out by means of the software PAST version 3.15 (Hammer et al., 2001; Hammer, 2017) and R version 3.3.1 (R Core Team, 2016). 2.3. Morphological investigation (updating the geographic distribution) Based on the morphometric results, we selected those morphological characters showing less overlapping values among taxa, for identification purposes. Then, using these characters, we performed a morphological analysis on 512 herbarium specimens (Supplemental material S3). Specimens from CLU, FI, MSNM, PI, SIENA were studied directly, whereas specimens in APP, BOLO, CAME, PAD, ROV, UTV were studied through high resolution digital images (acronyms follow Thiers, 2017). Finally, using QGIS 2.18 software, we georeferenced all the specimens in order to draw an updated distribution map of the Juniperus oxycedrus group. 3. Results 3.1. Phytochemical investigation One hundred sixty-six compounds have been identified (Table 3), representing from 85.5% to 97.5% of the total oil composition. Monoterpenes, with a percentage ranging between 34.94% and 81.25%, were the main class of constituents of terpene profiles of all populations studied, except those collected in Tunisia (BH and SA). The oxygenated monoterpenes did not exceed 12.14%, while the largest fraction was attributed to monoterpene hydrocarbons, ranging from 22.8% to 76.16%. The EOs from leaves collected in Tunisia showed sesquiterpenes as main class of constituents (ranging from 34.78 to 44.16%), while in all the other accessions sesquiterpenes occurred with percentages between 8.42% and 38.1%. According to the PCA results (Fig. 2; 78% of variance explained by the first two axes), Juniperus deltoides accessions were clearly separated from both J. oxycedrus and J. macrocarpa (Fig. 2). On the contrary, no clear phytochemical separation was evident between J. oxycedrus and J. macrocarpa. The three compounds which mainly contributed to the observed variation pattern were: a-pinene (90% of explained variance on axis 1), limonene (87.3%, axis 2), and manoyl oxide (16%, axis 2). The populations from Siena (SI) and Croatia (KR) showed a chemical composition typical of J. deltoides, although the population from Croatia displayed a percentage of limonene lower than the population from Siena. Low levels of apinene (<25%) have been found in all the populations attributed to J. deltoides, in contrast to the populations attributed to J. oxycedrus, showing high levels of a-pinene (>30%). All the sampled populations of J. oxycedrus and J. macrocarpa showed limonene levels below 6%, with the exception of the J. macrocarpa population from Marina di Vecchiano (VE). Tunisian populations, although showing low levels of a-pinene, were characterized by low amount of limonene and by a percentage of manoyl oxide comparable with other populations of J. oxycedrus. The Italian populations of J. oxycedrus and J. macrocarpa showed levels of germacrene D > 2%, whereas in J. deltoides the amount was generally lower than 1%, except for the Croatian population ‘16’ from the literature (4.26%, see Supplemental material S1).

51

Plants from Tunisia, despite coming from two neighbouring localities, presented a different EO composition: in Sidi Ameur (SA) a-pinene was the major compound, followed by manoyl oxide. On the contrary, abietadiene was the major compound in Bir Hannoun (BH) sample, followed by germacrene D. Finally, b-atlantone, eudesma-4(15),7dien-1-b-ol and dolabradiene have been found only in these Tunisian accessions. Concerning the Croatian population (KR), EO was mainly represented by manoyl oxide, followed by limonene, a-pinene and caryophyllene oxide. The occurrence of cis-dihydrocarvone, acedrene, 1,7-di-epi-b-cedrene, and isobornyl N-butyrate were unique to this accession. 3.2. Morphometric investigation Discriminant Analysis resulted in 96.82% (jackknifed) correct classification of individuals, a priori attributed to the three species based on phytochemical profiles (Fig. 3). The nine characters most contributing to the discriminant function (loading values higher than j0.09j) resulted A, dW, L, Mu, P, H, Sl, St, Sw. In particular, high values of Mu and low values of dW, St, and Sw contribute to separate J. deltoides from J. oxycedrus, while high values of A, L, P, H, and Sl contribute to neatly separate J. macrocarpa from the other two taxa. Just a small overlapping among individuals of J. deltoides (from Croatia) and individuals of J. oxycedrus was found (3 out of 120 not correctly classified), as well as a limited possible confusion between J. oxycedrus and J. macrocarpa (3 out of 150), and J. macrocarpa over J. deltoides (1 out of 170). Results of univariate analysis of continuous characters are summarized in Table 4. The states of eleven characters (A, W, W25, W50, W80, W90, Hs, Mu, H, D, and Sl) showed significant differences among the three taxa (P < 0.01). In addition, the states of further six characters (L, Wb, W10, LBs, P, and S) were significantly different between J. macrocarpa and the other two taxa (P < 0.01). Finally, J. deltoides showed significant differences from other taxa concerning the character-states of dW, Sw, and St. No significant difference in BS and T was found. Among the statistically significant characters, those with less overlapping among the three taxa were Mu, W90, W, LBs, H, and Sl (Fig. 4). 3.3. Morphological investigation (updating the geographic distribution) By measuring Mu, W90, W, LBs, H and Sl on herbarium specimens, we were able to revise their identification. Consequently, the distribution of Juniperus oxycedrus and J. deltoides in the Mediterranean basin (Fig. 5), and particularly in Italy (Fig. 6), was updated. Outside Italy, the occurrence of J. deltoides was confirmed for Croatia (including Istria), whereas J. oxycedrus was confirmed to occur in Tunisia. In Italy, J. oxycedrus is found in the North-western regions (i.e. Liguria, Piedmont, Emilia-Romagna), Sardinia, but also in Puglia, Basilicata, and Calabria. In extreme North-eastern Italy (Friuli Venezia Giulia) and in some region of central-southern Italy (Marche, Umbria, Lazio, Molise and Campania) only J. deltoides occurs, whereas in Tuscany and Abruzzo both species occur, but they have been never recorded to co-occur at the same site, so far (Fig. 6). 4. Discussion Our phytochemical results confirmed that Juniperus oxycedrus and J. macrocarpa are closer to each other than J. deltoides, as highlighted in all the previous studies based on EO characterisation and DNA molecular markers (Adams et al., 2003, 2005, 2015, Adams 2014b; Mao et al., 2010; Rumeu et al., 2011; Adams and

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Table 3
Comparison of the chemical composition (% of EO) of sampled populations. KR ¼ Miljeva
cki Bogati ci (Sibenik, Croatia); SI ¼ Castiglione d’Orcia (Tuscany, Italy); VE ¼ Marina di Vecchiano (Tuscany, Italy); LI ¼ Calignaia (Tuscany, Italy); SB ¼ Rosignano Solvay (Tuscany, Italy); FL ¼ Finale Ligure (Liguria, Italy); MP ¼ Monte Pisano (Tuscany, Italy); MV ¼ Monte Vaso (Tuscany, Italy); PE ¼ Monte Pelato (Tuscany, Italy); EL ¼ Elba Island (Tuscany, Italy); OR ¼ Oriolo (Calabria, Italy); SA ¼ Sidi Ameur (Tunisia); BH ¼ Bir Hannun (Tunisia). mh: monoterpene hydrocarbons, om: oxygenated monoterpenes, sh: sesquiterpene hydrocarbons, os: oxygenated sesquiterpenes, dh: diterpene hydrocarbons, od: oxygenated diterpenes, nt: non-terpene derivatives. Red columns ¼ J. deltoides, yellow ¼ J. macrocarpa, blue ¼ J. oxycedrus.

Compounda, N (E)-2-hexenal α-thujene tricyclene α-pinene camphene thuja-2,4(10)-diene sabinene β-pinene myrcene δ-2-carene α-phellandrene δ-3-carene p-cymene limonene β-phellandrene (E)-β-ocimene γ-terpinene meta-cymene terpinolene p-cymenene 6-camphenone linalool (N)-nonanal trans-p-mentha-2,8-dien-1-ol cis-p-menth-2-en-1-ol α-campholenal cis-limonene oxideN trans-pinocarveol trans-sabinol cis-sabinol camphor cis-verbenol trans-verbenol (E)-2-nonenalN trans-pinocamphone pinocarvone p-mentha-1,5-dien-8-ol cis-pinocamphone (= iso-pinocamphone) 4-terpineol m-cymen-8-ol p-cymen-8-ol trans-p-mantha-1(7),8-dien-2-ol α-terpineol cis-dihydrocarvone myrtenal + mirtenol verbenone trans-carveol cis-p-mentha-1(7),8-dien-2-ol

Class nt mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh mh nt om om om om om om om om om om nt om om om om om om om om om om om om om om

LRIb 860 932 938 940 955 959 978 981 993 1001 10 06 101 2 1028 1032 1033 1053 1062 1085 1090 1090 1097 1102 1104 1125 1126 1130 1137 1142 11 4 2 1143 114 8 1149 1150 1 164 1165 1166 1170 1177 1180 1180 1183 1189 1192 1193 1195 1214 1221 1230

KR 0.24 ˗ ˗ 6.55 0.4 0.14 ˗ 0.62 0.71 ˗ 0.37 2.26 0.87 9.98 ˗ ˗ ˗ ˗ 0.43 0.35 ˗ 0.12 0.27 0.36 ˗ 0.78 ˗ 0.76 ˗ ˗ ˗ 0.41 0.96 ˗ 0.16 0.52 ˗ 0.32 0.45 ˗ 0.6 0.62 0.52 0.45 0.32 0.47 1.48 0.21

SI VE 0.3 ˗ 0.1 0.19 ˗ ˗ 24.2 42.28 0.5 0.96 0.2 ˗ 0.6 0.64 1 1.71 1.1 3.05 0.2 0.18 0.7 1.28 2.7 9.86 1.2 1.02 32.6 11.89 ˗ ˗ ˗ 0.38 0.1 0.13 ˗ ˗ 0.7 2.36 0.3 ˗ ˗ ˗ ˗ 0.23 0.3 ˗ 0.6 ˗ ˗ 0.12 1.1 0.56 0.5 0.15 ˗ 0.28 ˗ ˗ 0.8 ˗ ˗ 0.24 ˗ ˗ 2.5 0.54 ˗ ˗ ˗ ˗ 0.6 0.21 0.4 ˗ ˗ 0.12 0.5 0.53 ˗ ˗ ˗ ˗ ˗ ˗ 0.2 1.85 ˗ ˗ 0.7 ˗ 0.4 ˗ ˗ ˗ 0.3 ˗

LI 0.11 ˗ 38 0.43 ˗ 0.24 2.45 2.44 0.26 0.37 0.29 0.36 4.1 ˗ 0.1 ˗ ˗ 0.49 ˗ ˗ ˗ ˗ ˗ ˗ 0.72 ˗ 0.29 0.14 ˗ ˗ ˗ 0.96 0.1

0.24 ˗ ˗ 0.12 ˗ ˗ ˗ 0.15 ˗ 0.27 0.12 ˗ ˗

PE EL SB FL MP MV OR SA 0.16 0.16 0.25 0.19 0.1 ˗c 0.3 ˗ 0.19 0.17 0.24 0.19 0.11 0.18 0.3 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 ˗ 37.98 44.72 57.12 49.02 30.13 49.12 59.4 10.85 0.33 0.55 0.59 0.52 0.45 0.53 0.6 0.35 ˗ 0.13 0.4 ˗ 0.04 0.23 0.14 ˗ 0.2 0.36 0.24 0.51 0.31 0.24 0.3 0.76 1.48 2.72 2.15 2.29 1.67 1.44 2.3 ˗ 1.98 3.37 2.88 2.36 2.08 1.34 2.6 0.69 0.1 0.13 0.16 0.12 0.55 ˗ 0.2 ˗ 0.3 1.32 1.08 ˗ ˗ 1.15 0.95 0.9 ˗ ˗ 3.07 0.52 ˗ ˗ 0.1 3.87 0.64 0.53 0.41 0.89 1.21 0.18 0.9 0.42 ˗ 5.18 3.78 2.46 3.4 1.34 4.4 0.88 4.33 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.29 ˗ ˗ ˗ ˗ ˗ 0.2 0.15 ˗ ˗ ˗ ˗ 0.14 0.13 0.1 0.16 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.11 0.4 0.79 0.86 0.44 0.87 1.03 0.55 0.5 ˗ ˗ 0.17 ˗ 0.16 ˗ ˗ ˗ ˗ 0.43 ˗ ˗ ˗ ˗ ˗ ˗ 0.31 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.2 0.1 0.11 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.73 0.38 1.29 0.92 0.65 1.87 1.9 1.61 ˗ 0.07 ˗ ˗ ˗ ˗ 0.1 0.17 0.4 ˗ 0.27 0.33 0.26 0.56 0.9 0.92 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 0.5 0.36 0.24 0.18 0.39 0.29 0.15 ˗ ˗ 0.25 0.2 0.41 ˗ 1 ˗ 0.87 0.32 0.64 0.77 0.23 0.94 0.4 1.14 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.2 ˗ ˗ 0.13 ˗ ˗ ˗ 0.2 0.19 ˗ 0.3 0.28 0.17 0.51 0.4 0.59 ˗ ˗ ˗ 0.32 ˗ ˗ ˗ ˗ ˗ ˗ 0.1 0.14 ˗ 0.24 0.1 0.32 0.3 0.34 0.29 0.2 0.2 0.28 0.18 0.12 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.12 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.11 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.92 0.45 0.21 0.57 0.79 0.31 0.1 0.17 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 0.31 0.28 ˗ 0.37 0.3 0.31 0.18 0.12 0.16 ˗ ˗ ˗ 0.3 0.73 0.3 ˗ ˗ ˗ ˗ ˗ 0.2 0.45 ˗ ˗ ˗ ˗ ˗ 0.17 ˗ ˗

BH ˗ ˗ ˗ 6.6 ˗ ˗ 0.1 0.44 0.48 ˗ ˗ ˗ 0.13 0.66 ˗ ˗ ˗ ˗ 0.19 0.12 ˗ ˗ ˗ ˗ ˗ 0.33 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗

F. Roma-Marzio et al. / Phytochemistry 141 (2017) 48e60

cis-carveol carvacrol methyl etherN cuminaldehyde carvone piperitone (E)-2-decenal p-mentha-1-en-7-al perilla aldehyde bornyl acetate iso-bornyl acetate (Z)-undec-9--en-1-alN trans-carvyl acetateN α-cubebene α-terpenyl acetate cis-carvyl acetate neryl acetateN α-copaene β-bourbonene β-cubebene 7-epi-sesquithujene β-elemene cypereneN β-longipinene longifolene α-cedreneN 1,7-di-epi-cedrene N α-gurjunene β-cedrene β-caryophyllene (= (E)-caryophyllene) β-copaene cis-thujopsene aromadendrene N cis-muurola-3,5-diene α-humulene (E)-β-farnesene cis-muurola-4(14),5-diene trans-cadina-1(6),4-diene iso-bornyl N-butyrate N γ-muurolene germacrene D ar-curcumene valencene viridiflorene epi-cubebol γ-amorphene N 2-tridecanone α-alaskene Β-alaskene α-muurolene cuparene β-bisabolene trans-γ-cadinene cubebol δ-cadinene trans-cadina-1(2),4-diene (E)-γ-bisabolene N α-cadinene α-calacorene elemol germacrene B

om om om om om nt om om om om nt om sh om om om sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh nt sh sh sh sh sh os sh nt sh sh sh sh sh sh os sh sh sh sh sh os sh

1233 1237 1244 1248 1258 1266 1 27 6 1280 1285 1287 1324 1340 1351 1352 1365 1368 1376 1383 1390 1391 1392 1398 1401 1404 1408 1409 1410 1418 1418 1429 1431 1445 1448 1456 1460 1463 1470 1470 1477 1481 1484 149 3 1495 1496 1496 1497 1 49 8 1498 1499 1502 1509 1513 1518 1523 1533 1535 1537 1542 155 3 1556

0.39 ˗ 0.14 1.08 ˗ 0.23 0.16 ˗ ˗ 0.5 ˗ 0.24 ˗ ˗ 0.24 ˗ 0.88 0.41 ˗ 0.13 ˗ ˗ ˗ ˗ 0.18 0.3 ˗ ˗ 1.1 ˗ 0.22 ˗ 0.51 ˗ ˗ ˗ 0.19 ˗ 0.73 4.3 ˗ ˗ ˗ ˗ ˗ 1.24 0.44 0.51 0.61 0.12 1.61 ˗ 2.21 ˗ ˗ ˗ 2.44 ˗ ˗

0.3 ˗ ˗ 1.3 ˗ ˗ ˗ 0.1 ˗ 0.6 ˗ 0.2 ˗ ˗ 0.2 ˗ 0.5 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.8 ˗ ˗ ˗ 0.5 ˗ ˗ ˗ ˗ ˗ 0.5 0.1 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.4 ˗ ˗ 0.6 ˗ 1.3 ˗ ˗ ˗ 0.4 ˗ ˗

˗ 0.12 ˗ ˗ 0.23 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.14 ˗ ˗ 0.1 ˗ ˗ ˗ ˗ 0.3 ˗ ˗ ˗ ˗ ˗ ˗ 0.24 ˗ ˗ ˗ 0.22 ˗ ˗ ˗ ˗ ˗ 2.27 ˗ ˗ ˗ 0.12 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 0.36 0.46 ˗ ˗ ˗ ˗ ˗ ˗

˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.45 ˗ ˗ 0.3 ˗ ˗ ˗ 0.4 0.24 0.2 ˗ ˗ 1.35 ˗ ˗ ˗ ˗ ˗ ˗ 0.89 0.19 ˗ 0.11 0.76 ˗ 0.2 0.25 ˗ 0.85 7.39 ˗ ˗ ˗ 1.09 ˗ ˗ ˗ ˗ 0.44 ˗ ˗ 0.47 3.05 3 ˗ 0.15 0.11 0.15 ˗ ˗

˗ ˗ ˗ ˗ 0.17 ˗ ˗ ˗ ˗ ˗ 0.16 ˗ ˗ ˗ ˗ ˗ ˗ 0.12 ˗ ˗ ˗ ˗ 0.13 ˗ ˗ ˗ ˗ ˗ 0.47 ˗ ˗ ˗ 0.28 0.46 ˗ ˗ ˗ 0.11 4.04 ˗ ˗ ˗ ˗ ˗ 0.23 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.2 ˗ ˗ ˗ ˗ ˗ 1.22

˗ ˗ ˗ ˗ 0.11 ˗ ˗ ˗ ˗ 0.77 ˗ ˗ ˗ 0.49 ˗ 0.15 ˗ 0.4 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 1.03 0.11 ˗ 0.08 ˗ 1.02 ˗ ˗ ˗ ˗ 0.68 11.17 ˗ ˗ ˗ ˗ 0.15 ˗ ˗ ˗ 0.4 ˗ ˗ 2.11 ˗ 1.5 ˗ 0.16 0.19 ˗ ˗ ˗

53

˗ 0.3 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.42 ˗ ˗ 0.15 ˗ ˗ 0.1 ˗ 0.32 ˗ ˗ ˗ 0.1 ˗ 0.41 ˗ ˗ ˗ ˗ 0.4 ˗ ˗ ˗ 0.29 ˗ ˗ ˗ ˗ 0.12 2.47 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.68 ˗ 0.49 ˗ ˗ ˗ ˗ ˗ ˗

˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.58 ˗ ˗ ˗ 0.41 ˗ ˗ ˗ 0.15 ˗ ˗ ˗ ˗ ˗ ˗ 0.5 ˗ ˗ ˗ 0.53 ˗ ˗ ˗ ˗ 0.24 6.04 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.3 ˗ ˗ 0.56 0.17 0.57 ˗ 0.1 ˗ ˗ ˗ ˗

˗ 0.27 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 1.1 ˗ ˗ 0.35 0.39 ˗ ˗ ˗ 1.12 ˗ ˗ ˗ ˗ ˗ ˗ 0.79 0.18 ˗ ˗ 0.82 ˗ ˗ ˗ ˗ 0.51 8.49 ˗ 0.38 ˗ 0.56 ˗ ˗ ˗ ˗ 0.33 ˗ ˗ 1.42 0.63 2.04 ˗ ˗ 0.19 ˗ ˗ 0.22

˗ 0.15 ˗ ˗ ˗ ˗ ˗ ˗ 1.14 ˗ ˗ ˗ 0.23 ˗ ˗ ˗ 0.13 1.18 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.75 0.18 ˗ ˗ 0.71 ˗ ˗ ˗ ˗ 0.69 6.43 ˗ ˗ 0.12 ˗ ˗ ˗ ˗ ˗ 0.36 ˗ ˗ 0.41 0.26 0.84 ˗ 0.45 ˗ ˗ ˗ ˗

˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.7 ˗ ˗ ˗ 0.1 ˗ ˗ 0.1 0.5 ˗ ˗ ˗ 0.1 ˗ ˗ ˗ ˗ ˗ ˗ 1.2 0.1 ˗ ˗ 0.4 ˗ ˗ ˗ ˗ 0.3 2.9 ˗ 0.1 ˗ ˗ ˗ ˗ ˗ ˗ 0.2 ˗ ˗ 0.8 ˗ 0.4 ˗ ˗ ˗ ˗ 0.1 ˗

˗ 0.1 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.19 ˗ ˗ ˗ 0.61 0.68 0.19 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 2.27 0.4 ˗ 0.21 ˗ ˗ 0.24 ˗ ˗ 0.12 ˗ 0.48 2.24 ˗ ˗ ˗ 1.33 ˗ 1.18 ˗ ˗ ˗ ˗ 0.19 1.21 4.02 1.46 0.14 ˗ 0.12 0.19 0.19 2.23

˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.63 ˗ 0.17 ˗ 0.46 ˗ ˗ 0.12 1.13 0.15 ˗ 0.1 0.22 ˗ ˗ ˗ ˗ ˗ ˗ 0.87 0.16 ˗ ˗ 0.76 ˗ ˗ ˗ ˗ 0.38 7.08 ˗ ˗ ˗ ˗ ˗ 1.6 ˗ ˗ ˗ ˗ ˗ 1.79 ˗ 0.73 ˗ ˗ 0.19 0.11 0.12 2.31

54

F. Roma-Marzio et al. / Phytochemistry 141 (2017) 48e60

β-calacorene (E)-nerolidol (= trans-nerolidol) germacrene D-4-ol spathulenol caryophyllene oxide globulol β-copaen-4-α-ol cis-β-elemenone iso-aromadendrene epoxide N ethyl dodecanoate N cedrol β-oplopenone humulene epoxide II 1,10-di-epi-cubenol humulane-1-6-dien-3-ol N 1-epi-cubenol γ-eudesmol epoxy-alloaromadendrene N T-cadinol (= epi-α-cadinol) 3-iso-thujopsanone N β-eudesmol α-muurolol (torreyol) α-eudesmol α-cadinol valerianol N (E)-7-tetradecenol N β-atlantoneN aromadendrene epoxide 1 N cis-calamenen-10-ol (= calamen-10-β-ol) (Z)-α-santalol cadalene aromadendrene oxide 2 N khusinol N cis-α-santalol N eudesma-4(15),7-dien-1-β-ol ledene oxide II N acorenone 2- pe n t ade c an one 10-norcalamenen-10-one (E,E)-farnesol (E)-nerolidol acetate (E,Z)-farnesol 14-hydroxy-α-muurolene N 1-octadecane nootkatone (E,E)-farnecyl acetate (N)-hexadecanol N cembrene cyclohexadecanolide N sandaracopimara-8(14),15-diene dolabradiene N manoyl oxide epi-13-manoyl oxide abieta-8,12-diene Kaur-16-ene N kaurene abietatriene abietadiene abieta-8(14),13(15)-diene Monoterpene hydrocarbons

sh os os os os os os os os nt os os os os os os os os os os os os os os os nt os os os os sh os os os os os os nt os os os os os nt os os nt dh nt dh dh od od dh dh dh dh dh dh mh

1566 1566 1 57 6 1577 1582 1584 1590 1590 1595 1 59 5 1601 1606 1607 1614 1615 1630 1634 1641 1642 1646 1649 1651 1654 1655 1658 1 6 63 1670 1672 1672 1675 1676 1678 1680 1683 1688 1688 1689 169 9 1702 1719 1721 17 48 1780 1800 1807 1843 1 8 76 1929 1935 1959 1974 1987 2010 2023 2033 2043 2055 2079 2154

˗ ˗ ˗ 0.88 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 6.38 0.95 ˗ ˗ ˗ 0.9 ˗ ˗ ˗ 0.22 0.55 0.77 0.19 0.21 0.5 ˗ ˗ ˗ ˗ ˗ 0.28 0.27 ˗ ˗ 0.33 ˗ ˗ ˗ ˗ ˗ 0.37 ˗ ˗ 1.4 ˗ 0.4 6.32 3.3 0.31 1.07 0.38 0.56 0.18 0.61 0.82 1.75 0.5 1.51 ˗ ˗ 0.3 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 1.63 ˗ ˗ ˗ ˗ ˗ 0.35 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.51 1.98 1.33 0.19 0.36 1.58 2.65 0.2 ˗ ˗ ˗ ˗ ˗ 0.12 ˗ 0.11 ˗ 0.47 0.45 ˗ ˗ ˗ ˗ ˗ ˗ 1.41 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.12 1.62 1.2 0.24 0.92 0.29 0.45 0.25 0.58 0.64 1.62 0.3 1.36 ˗ ˗ ˗ ˗ 0.17 ˗ ˗ ˗ ˗ 0.43 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.25 ˗ 0.88 1.23 ˗ ˗ ˗ ˗ ˗ 0.82 0.56 0.82 2.47 2.19 0.2 2.31 0.51 3.04 ˗ 0.55 0.32 0.81 ˗ ˗ ˗ ˗ ˗ ˗ 0.1 0.35 ˗ ˗ ˗ ˗ ˗ ˗ 0.65 ˗ ˗ ˗ 0.29 ˗ 1.3 0.21 1.87 0.39 0.98 1.01 0.2 0.26 1.1 0.99 0.4 1.46 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.14 0.39 ˗ ˗ ˗ ˗ 0.16 ˗ ˗ ˗ 1.63 ˗ ˗ ˗ ˗ 0.39 ˗ 0.24 ˗ ˗ ˗ 0.11 0.19 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.63 ˗ ˗ 0.15 0.4 ˗ ˗ ˗ 1.08 0.17 0.68 ˗ ˗ 0.6 ˗ 0.44 0.44 3.01 ˗ ˗ ˗ 0.74 1.57 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 2.69 ˗ ˗ 5.96 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.93 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.91 3.01 ˗ ˗ 0.52 0.33 ˗ ˗ ˗ 0.1 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.18 ˗ ˗ ˗ 0.45 0.45 ˗ ˗ ˗ 0.23 1.6 0.1 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.33 ˗ 0.72 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.63 ˗ 0.39 1.39 3.05 ˗ 0.43 ˗ ˗ ˗ 0.45 ˗ ˗ ˗ ˗ ˗ 0.23 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 2.45 ˗ ˗ ˗ ˗ ˗ ˗ 1.94 0.16 1.28 ˗ ˗ ˗ ˗ ˗ 0.4 ˗ ˗ ˗ ˗ 1.91 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.21 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 1.9 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.48 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.17 0.12 0.33 0.78 1.51 ˗ ˗ 0.27 ˗ 5.95 ˗ ˗ ˗ ˗ 0.86 ˗ ˗ ˗ ˗ ˗ ˗ 1.1 ˗ ˗ 0.21 0.13 0.14 0.27 0.84 1.5 ˗ 0.36 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.12 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.2 ˗ ˗ ˗ ˗ ˗ 0.25 ˗ ˗ ˗ 0.13 0.1 0.19 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.13 ˗ ˗ 0.12 0.15 0.18 ˗ ˗ ˗ ˗ ˗ 0.25 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.19 15.59 6.9 3.08 1.99 5.4 0.61 0.67 2.5 5.06 2.08 1.9 6.65 0.4 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.18 ˗ ˗ ˗ ˗ ˗ 0.3 ˗ 1.68 0.13 0.2 0.51 0.31 ˗ ˗ 0.17 ˗ ˗ ˗ ˗ ˗ ˗ ˗ 0.1 ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ 1.6 ˗ ˗ ˗ ˗ ˗ 0.1 ˗ 3.35 0.6 0.61 0.36 3.25 0.51 1.65 1.41 1.25 0.42 0.6 4.77 1.29 1.9 2.33 0.37 9.59 1.41 3.33 4.79 2.49 0.88 1.1 3.96 ˗ ˗ 0.15 ˗ 0.73 ˗ ˗ 0.21 ˗ ˗ ˗ 0.21 22.8 66.2 76.16 49.64 50.51 59.45 69.06 60.81 44.96 55.05 73.3 19.07

˗ ˗ 0.2 ˗ 1.58 ˗ 0.66 0.94 1.58 ˗ ˗ ˗ 1.47 ˗ 4.18 0.69 ˗ ˗ 1.52 0.19 ˗ ˗ ˗ ˗ ˗ 6.51 0.79 ˗ ˗ ˗ ˗ ˗ 3.62 ˗ 1.99 ˗ ˗ 2.19 ˗ 1.59 3.86 2.9 ˗ ˗ ˗ 0.18 ˗ ˗ ˗ ˗ 0.12 6.28 ˗ 0.82 ˗ ˗ 5.09 8.3 0.12 8.72

F. Roma-Marzio et al. / Phytochemistry 141 (2017) 48e60

Oxygenated monoterpens Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Diterpene hydrocarbons Oxygenated diterpenes Non-terpene derivatives Total a

om sh os dh od nt

55

12.14 11.3 5.09 3.46 4.26 3.2 5.01 4.66 4.49 6.97 7.4 7.5 1.59 9.4 17.23 12.48 7.1 13.17 16.1 20.42 5.2 3.69 17.45 7.03 18.92 5.43 18 11.28 4.8 4.73 20.56 10.16 11.9 10.14 10.67 18.65 3.2 21.61 28.06 5.3 1.3 2 9.3 14.45 7.07 4.23 4.64 2.5 3.39 0.73 17.1 2.05 0.61 0.67 15.99 6.9 3.08 1.99 5.4 2.5 5.24 2.08 1.9 6.65 6.28 0.63 3.59 0.2 0.5 9.53 10.3 0 0.22 0.79 0.21 0.93 0.6 0 88.2 97.5 96.14 94.05 95.25 96.34 95.77 95.74 97.74 96.73 95.4 86.83 85.5

Compounds present with percentage < 1.0 % have been excluded from the table. bLinear retention index on the DB5 column. NCompounds reported

for the first time in the EO from leaves of the Juniperus oxycedrus group.

Fig. 2. PCA based on phytochemical data (Component 1: 58.4%, Component 2: 21.4% of the observed variance). Full circles represent the populations sampled in the present study (for acronyms see Table 1), while empty squares represent the populations whose EO profile was derived from literature. Acronyms marked with an asterisk represent the populations also used for morphometric analysis. In green the relative contribution of each variables is reported, but only the three compounds showing a contribution greater than 15% are named (i.e. a-pinene, limonene, manoyl oxide). Red ¼ J. deltoides, yellow ¼ J. macrocarpa, blue ¼ J. oxycedrus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 ski et al., 2014). Indeed, monoterpene Schwarzbach, 2012; Boratyn variation in conifers is largely genetically determined, and scarcely influenced by environmental factors (Squillace, 1976). For instance, a single gene controls the biosynthesis of limonene, but the biosynthesis of a-pinene is under the control of several genes (Squillace et al., 1980). Volatile terpenoids are involved in many biological processes, and play a major role in ecosystem dynamics. Some of them act as signals for animals or as defence against pathogens/herbivores, others may interfere with the soil biology, e.g. litter decomposition and allelopathy, influencing intra- and inter-specific competition ~ uelas and Llusia , 2001; Thompson, 2005; among plants (Pen Chomel et al., 2016). At cellular level, these compounds are also involved in the protection of membranes against extreme temperatures or water stress (Loreto and Schnitzler, 2010). Accordingly, many potential environmental drivers may induce a modulation of essential oil production, having a relevant role in plant adaptation and evolution (Thompson, 2005). In some conifer, pathogen coleopterans are strongly attracted by

a-pinene, but limonene is repelling them, resulting in plant protection (Nordlander, 1990; Sanchez-Husillos et al., 2013). Hence, the high percentage of limonene against a-pinene (paralleled by genetic differentiation) in J. deltoides, could have some impact in the ecological interactions of this species. Accordingly, a detailed study of plant-pathogen chemical interactions in the species pair J. deltoides/J. oxycedrus may represent an interesting case study for further investigations on this topic. While the general variation pattern of limonene/a-pinene among prickly junipers (Adams et al., 2005) was fully supported by our data, we failed to confirm the putative exclusiveness of many compounds reported in previous studies. For example, Adams et al. (2005) reported cis-p-mentha-2,8-dien-1-ol as exclusive of eastern populations (J. deltoides), but we found traces of this compound also in some J. oxycedrus accession (EL, PE), while it resulted absent in J. deltoides from Croatia (KR). Other compounds were considered as exclusive of J. deltoides by Adams et al. (2005), but they have been indifferently found in both J. oxycedrus and J. macrocarpa (such as acalacorene and (E)-2-decenal), or were completely absent in the

56

F. Roma-Marzio et al. / Phytochemistry 141 (2017) 48e60

Fig. 3. Discriminant Analysis based on 21 quantitative continuous morphological characters. In blue, the relative contribution of each variable is reported, but only those showing loading values higher than j0.09j are named. Red ¼ J. deltoides, yellow ¼ J. macrocarpa, blue ¼ J. oxycedrus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 4 Comparison of morphological features among Juniperus deltoides (n ¼ 70), J. oxycedrus (n ¼ 50) and J. macrocarpa (n ¼ 100). Quantitative numerical values are expressed as mean ± SD. Qualitative character T (cone tips) is expressed as number of cones showing tips to the total of measured cones. Character states marked by different superscript letters are significantly different (P < 0.01). Characters in bold are also shown in Fig. 4. ID character

J. deltoides

J. oxycedrus

J. macrocarpa

L (mm) W (mm) Wb (mm) W10 (mm) W25 (mm) W50 (mm) W80 (mm) W90 (mm) dW (mm) Mu (mm) LBs (mm) P (mm) A (mm2) BS (mm) Hs (mm) H (mm) D (mm) T S Sl (mm) Sw (mm) St (mm)

13.85 ± 2.08a 1.42 ± 0.18a 1.04 ± 0.17a 1.34 ± 0.18a 1.37 ± 0.18a 1.28 ± 0.18a 0.88 ± 0.18a 0.47 ± 0.13a 3.11 ± 1.31a 1.14 ± 0.33a 0.31 ± 0.04a 35.56 ± 6.4a 16.81 ± 3.62a 0.05 ± 0.02a 0.52 ± 0.09a 8.48 ± 0.95a 9.65 ± 0.97a 63/70a 2.52 ± 0.71a 5.58 ± 0.69a 3.06 ± 0.41a 2.73 ± 0.44a

13.28 ± 2.58a 1.54 ± 0.24b 1.01 ± 0.17a 1.31 ± 0.19a 1.50 ± 0.24b 1.40 ± 0.23b 1.07 ± 0.22b 0.77 ± 0.17b 3.63 ± 0.99b 0.67 ± 0.2b 0.33 ± 0.06a 37.82 ± 7.7a 19.3 ± 5.28b 0.05 ± 0.02a 0.45 ± 0.08b 9.99 ± 1.03b 10.59 ± 1b 30/50a 2.66 ± 0.72a 6.25 ± 0.67b 4.05 ± 0.68b 3.46 ± 0.6b

15.02 ± 2.23b 1.97 ± 0.27c 1.19 ± 0.19b 1.70 ± 0.25b 1.92 ± 0.32c 1.80 ± 0.24c 1.26 ± 0.17c 0.82 ± 0.116b 3.94 ± 0.97b 0.42 ± 0.31c 0.59 ± 0.09b 43.74 ± 6.86b 25.57 ± 4.84c 0.05 ± 0.02a 0.58 ± 0.1c 13.1 ± 1.74c 13.5 ± 1.6c 83/100a 3.05 ± 0.66b 7.47 ± 0.7c 4.23 ± 0.63b 3.82 ± 0.78b

populations studied (e.g. a-copaen-11-ol). Nevertheless, arcurcumene and cadalene have been confirmed to be exclusive compounds of J. deltoides. In addition, a plethora of minor population-specific compounds have been found in the Tunisian populations of J. oxycedrus and in the Croatian population of J. deltoides.

Based on the morphological characters reported by Adams (2014a), the populations from Tunisia are clearly referable to J. oxycedrus s.str. However, their peculiar EO composition (low values for both limonene and a-pinene, but relatively high values of manoyl-oxide) resulted similar to the phytochemical profile typical of J. oxycedrus subsp. badia (H.Gay) Debeaux (Adams, 1999). This subspecies is endemic to western Mediterranean, occurring across the Iberian Peninsula and Algeria (EuroþMed, 2006 onwards;  ski et al., 2014), but never recorded for Tunisia. Despite this, Boratyn some introgression/gene flow among the two taxa cannot be excluded, since these phenomena have been previously documented in Juniperus (Adams, 1983; Adams et al., 2016). Moving on to morphometrics, this kind of quantitative morphological comparison was carried out for the first time in the taxonomy of prickly juniper. According to Adams (2014a,b), the main qualitative characters typical of J. deltoides are: delta-shaped leaves, flat stomatal bands, and the occurrence of cone scale tips. The present study confirmed ‘delta-shaped’ leaves as a significant character, as demonstrated by the comparison between the leaf maximum width (W) and the width on 10% of leaf's length from base up (W10). While W10 per se did not show differences between the two species, W resulted narrower in Juniperus deltoides, resulting in more or less ‘delta-shaped’ leaves. On the contrary, the concavity of the stomatal band, quantified in our study by the character BS (Fig. 1), was not significantly different among the three species. Furthermore, the scale tips on the seed cones resulted only slightly, and not significantly, more frequent in J. deltoides. The traditional morphological distinction between J. macrocarpa and J. oxycedrus relied on wider leaves and larger size of cones and seeds (e.g. Franco, 1964; Pignatti, 1982; Lebreton et al., 1998; Klimko et al., 2004; Schulz et al., 2005; Adams, 2014a). All these characters were quantitatively confirmed by our results. However, we noticed the possible occurrence of gradients of morphological variation from the sea level to higher altitudes along rocky coasts, suggesting that Juniperus macrocarpa might fall within the

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Fig. 4. Boxplots showing those morphological characters less overlapping among the three taxa. A ¼ Mucro length, B ¼ Width of leaf on 90% of leaf's length from base up; C ¼ leaf maximum width; D ¼ stomatal band maximum width; E ¼ seed cone height; F ¼ seed length.

infraspecific variability of J. oxycedrus. More detailed, fine-scale investigations could further clarify the relationships among these two taxa. Other characters were also investigated in previous literature, often concerning single species in different geographic areas, and their general morphological variation is congruent with the data evidenced by our study. Indeed, seed and seed cones dimension in the eastern inland populations (J. deltoides) have been found to be smaller than in western inland populations (J. oxycedrus) (Klimko  ski et al., 2014). In addition, et al., 2007; Brus et al., 2011; Boratyn the variation pattern of some leaf characters such as W and W90  ski et al., 2014) is congruent with (Brus et al., 2011, 2016; Boratyn our results. By means of our morphometric approach, we have been able to reveal new morphological features useful to distinguish the three species. The leaf mucro length (Mu) resulted significantly different among the three taxa: J. macrocarpa is characterized by a very short or absent mucro (average value ¼ 0.42 mm), J. deltoides is the species showing the largest values (average value ¼ 1.14 mm), whereas J. oxycedrus shows intermediate features (average value ¼ 0.67 mm). Leaf thickness (Hs) has been never previously investigated, and it resulted significantly different among the three taxa. According to this feature, Juniperus oxycedrus shows the thinnest leaves, followed by J. deltoides and by J. macrocarpa.

A focus on the distribution of prickly juniper, after our extensive herbarium revision, confirms that both J. deltoides and J. oxycedrus occur in peninsular Italy, as stated by Adams (2014b). However, a different distribution pattern has been highlighted (Figs. 5 and 6). Besides Juniperus macrocarpa occurring along the coasts, plants from south-eastern Italy turned out to be J. oxycedrus, as well as plants from Sardinia and NW Italy. On the other hand, J. deltoides occurs in the extreme NE Italy and in central Italy, where it is mostly restricted to hilly and mountain inland areas. In central Italy, its range is intermingled with that of J. oxycedrus at the north-eastern and south-eastern edges. The range separation among the two species resulted much less pronounced than previously thought (Adams, 2014b). However, despite a macroparapatric (sensu Smith, 1965) distribution, microallopatry (sensu Smith, 1965) is supported by currently available distributional data, still confirming for J. deltoides and J. oxycedrus the status of cryptospecies. It is interesting to note that east-west Mediterranean distribution pattern can be found in other woody taxa, whose ranges are coming in contact in Italy, as for instance Emerus major Mill. subsp. major and E. major subsp. emeroides (Boiss. & Spruner) Soldano & F.Conti (Fabaceae), or the species pair Oreoherzogia alpina (L.) W.Vent and O. fallax (Boiss.) W.Vent (Rhamnaceae) (EuroþMed, 2006 onwards; Peruzzi and Bartolucci, 2016; Roma-Marzio et al., 2016).

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Fig. 5. Distribution range of Juniperus deltoides (in red) and J. oxycedrus (in blue) according to Adams (2014b, redrawn) (a) and as resulted by the present study concerning central Mediterranean (b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

An ancient east-west migration from the Balkan peninsula might be invoked to explain the presence of Juniperus deltoides in central Apennines, via historical land-connections (Thompson, 2005; Pezzetta, 2010). On the other hand, the current disjunct distribution pattern of Juniperus oxycedrus in Italy could be the result of a double migration route, i.e. from north-west (SE France) and from south-west (North Africa), or the result of a fragmentation from a formerly continuous distribution, possibly induced by competition with J. deltoides in C Italy. Anyway, a detailed phylogeographic study will be necessary to clarify the historical processes and the migratory pathways that led to current macroparapatric (but seemingly microallopatric) distribution of J. deltoides and J. oxycedrus in the Central Mediterranean area. In conclusion, our study represents the first attempt to quantitatively evaluate the morphological differences among prickly junipers, complemented and supported by phytochemical investigation. Incidentally, phytochemical analysis allowed us to highlight 32 compounds (see Table 3) that, at the best of our knowledge, have been reported for the first time as EO components of the leaves in prickly junipers. Our results allowed us to redefine the ranges of the cryptospecies J. deltoides and J. oxycedrus, and to provide new tools for their identification, also taking the similar J. macrocarpa into account. Accordingly, it will be possible to push forward the knowledge about the biology of these species, with potential implications in their conservation (Rupprecht et al., 2011; Farjon, 2013), ecology

and phytosociology (Chiarucci et al., 2000; Baldoni et al., 2004), and reproductive biology (Arista et al., 2001).

4.1. Identification key for Juniperus oxycedrus group Due to well-known gender dimorphism (Brus et al., 2011), the present key applies only to female plants. We recommend the usage of averaged values, by measuring at least 10 different leaves, mature seed-cones and/or seeds. All values reported in the key are expressed as mean (±SD). 1. Leaf maximum width 1.97 (±0.27) mm; stomatal band 0.59 (±0.09) mm wide; leaf mucro generally absent or very short (0.42 ± 0.31 mm); well developed seed-cones with diameter 13.5 (±1.6) mm and height 13.1 (±1.74) mm J. macrocarpa 1. Leaf maximum width 1.42 (±0.18) mm; stomatal band 0.31 (±0.04) mm; leaf mucro always present; well developed seedcones with diameter 9.64 (±0.97) mm and height 8.47 (±0.95) mm 2 2. Mucro 0.67 (±0.2) mm long; leaf on 90% of leaf's length from base up 0.77 (±0.2) mm wide; width of leaf on 10% of leaf's length from base up narrower than maximum width of leaf; seed cone height 10 (±1.03) mm J. oxycedrus 2. Mucro 1.14 (±0.33) mm long; leaf on 90% of leaf's length from base up 0.47 (±0.13) mm wide; width of leaf on 10% of leaf's length from base up more or less as large as maximum width of leaf; seed cone height 8.48 (±0.95) mm J. deltoides

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Fig. 6. Distribution map of J. deltoides (red dots), J. oxycedrus (blue dots), and J. macrocarpa (yellow dots) in central Mediterranean, based on the studied herbarium specimens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Acknowledgements We thank the following personnel of herbaria: APP (Fabrizio Bartolucci, Fabio Conti), BOLO (Annalisa Managlia, Rosa Ranalli), CAME (Domenico Lucarini), CLU (Liliana Bernardo), FI (Chiara Nepi, Egildo Luccioli, Lorenzo Cecchi), MSNM (Gabriele Galasso), PAD (Rossella Marcucci), PI (Lucia Amadei, Simonetta Maccioni), ROV (Filippo Prosser), SIENA (Ilaria Bonini, Gianmaria Bonari), UTV (Anna Scoppola, Sara Magrini). Giovanni Astuti, Marco D'Antraccoli, €nswetBo
zo Frajman, Paola Liguori, Brunello Pierini, and Petr Scho ter are gratefully acknowledged for their kind help in the collection of plant material on the field. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.05.008. References Adams, R.P., 1983. Infraspecific terpenoid variation in Juniperus scopulorum: evidence for Pleistocene refugia and recolonization in western north America. Taxon 32, 30e46. Adams, R.P., 1999. The leaf essential oils and taxonomy of Juniperus oxycedrus L. subsp. oxycedrus, subsp. badia (H.Gay) Debeaux, subsp. macrocarpa (Sibth. & Sm.) Ball. J. Essent. Oil. Res. 11, 167e172. Adams, R.P., 2004. Juniperus deltoides, a new species, and nomenclatural notes on Juniperus polycarpos and J. turcomanica (Cupressaceae). Phytologia 86, 49e53. Adams, R.P., 2012. Taxonomy of Juniperus, section Juniperus: sequence analysis of nrDNA and five cpDNA regions. Phytologia 94, 280e297. Adams, R.P., 2014a. Junipers of the World: the Genus Juniperus, fourth ed. Trafford Publishing, Vancouver, B.C. 422 pp.

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