Chemotypes and Biomarkers of Seven Species of New Caledonian Liverworts from the Bazzanioideae Subfamily

Volatile components of seven species of the Bazzanioideae sub-family (Lepidoziaceae) native to New Caledonia, including three endemic species (Bazzania marginata, Acromastigum caledonicum and A. tenax), were analyzed by GC-FID-MS in order to index these plants to known or new chemotypes. Detected volatile constituents in studied species were constituted mainly by sesquiterpene, as well as diterpene compounds. All so-established compositions cannot successfully index some of them to known chemotypes but afforded the discovery of new chemotypes such as cuparane/fusicoccane. The major component of B. francana was isolated and characterized as a new zierane-type sesquiterpene called ziera-12(13),10(14)-dien-5-ol (23). In addition, qualitative intraspecies variations of chemical composition were very important particularly for B. francana which possessed three clearly defined different compositions. We report here also the first phytochemical investigation of Acromastigum species. Moreover, crude diethyl ether extract of B. vitatta afforded a new bis(bibenzyl) called vittatin (51), for which a putative biosynthesis was suggested.


Introduction
Liverworts are part of Bryoflora (mosses: 14,000 species, liverworts: 6000 species and hornworts: 300 species), considered as the first terrestrial plants and taxonomically indexed between algae and pteridophytes. Bryophytes possess archaic characteristics such as the absence of seeds and vascularized leaves [1]. Morphological traits such as small size of organs or relatively simple structure or high intraspecies variability and fugacity of some microscopic details (which may disappear within the plant dryness such as oil bodies), add difficulties for liverwort's taxonomic identification. Nevertheless, many liverworts have unique organelles called oil bodies in their cells which are linked to the biosynthesis of original secondary metabolites such as mono-, sesqui-and diterpenes or phenolic compounds that could be cladistic biomarker. Most of liverwort's sesquiterpenes are enantiomers of those found in higher plants [2].
New Caledonia is an archipelago of 18,600 km 2 located in South Pacific region and considered as a hotspot of biodiversity [3]. In New Caledonia, 482 species and infraspecific taxa of liverworts have been described in a recent checklist [4]. The rate of endemism is comprised between 13% and 39% that makes New Caledonia as one of the richest liverwort areas in the world, together with Japan, New Zealand, and Costa Rica [5].
Volatile compounds of 27 specimens belonging to seven different species, including three endemics (marked with an asterisk) were investigated in order to check intra-and inter-variability of molecular contents. Studied Bazzanioideae species belong to the following genera: Acromastigum (A. tenax*, A. caledonicum*) and Bazzania (B. parisii, B. marginata*, B. vittata, B. francana, B. bernieri and B. serrifolia). B. serrifolia is a synonym of B. bernieri according to Kitagawa (1973) [8] but we studied here its putative specific status by separating the samples in two lots of specimens on the basis of their morphological traits.
Volatile components of diethyl ether extracts were analyzed by GC-MS-FID in order to index these plants into known Bazzania chemotypes. In the present work, two new molecules: an oxygenated dimer of lunularic acid (51) and an alcohol zierane-type sesquiterpene (23) are characterized for the first time (Tables 1 and 2). They were respectively isolated from diethyl ether extract of B. vittata and B. francana. To our best knowledge, this is the first phytochemical investigation of all these species and the first chemical analysis of those belonging to the Acromastigum genus.
Zierane-type sesquiterpenes are very rare in nature: zierene had been found in four different Plagiochila species [25] and three different zierane-type sesquiterpenes had been found in Saccogyna viticulosa [26], zierane-type sesquiterpene lactone had been found in Chandonanthus hirtellus [27]. This is the first report of zierane-type sesquiterpene regarding Bazzania genus.
Molecules 2018, 23, x viticulosa [26], zierane-type sesquiterpene lactone had been found in Chandonanthus hirtellus [27]. This is the first report of zierane-type sesquiterpene regarding Bazzania genus. Compound 23 was obtained as a light-orange oil. Its molecular formula was determined to be C15H26O based on the molecular ion peak at m/z 220.1830 [M•] + (calcd. for C15H26O, 220.1827) as observed in the GC/HR-EI-MS, which corresponds to four degrees of unsaturation. The IR spectrum of 23 showed absorption at 3397.5 cm −1 (hydroxyl), 2985.3, 2923.9, 2854.1 cm −1 (alkane), 1377.4 cm −1 (methyl), 1437.5 cm −1 (methylene), 3086.3, 1636.7 cm −1 (alkene), 1149.8 cm −1 (ter-alcohol). The 13 C-NMR (Table 1) and HSQC spectra revealed the presence of 15 carbon resonances including three quaternary carbons, three methine, seven methylene and two methyl groups. Among the three quaternary carbons, one was an oxygenated carbon according to its chemical shift at δC 87.2 and two were exo-methylene carbons according to their chemical shifts at δC 151.1, 150.1. All methine groups are alkane carbons according to their chemical shifts at δC 57.6, 49.9, 46.1. Among the seven  (Table 1) and HSQC spectra revealed the presence of 15 carbon resonances including three quaternary carbons, three methine, seven methylene and two methyl groups. Among the three quaternary carbons, one was an oxygenated carbon according to its chemical shift at δ C 87.2 and two were exo-methylene carbons according to their chemical shifts at δ C 151.1, 150.1. All methine groups are alkane carbons according to their chemical shifts at δ C 57.6, 49.9, 46.1. Among the seven methylenes, two of them were assigned as exo-methylene carbons according to their chemical shifts respectively at δ C 112.5 and 110.2, then so indicative of the presence of two rings. Then, linkages to a cyclopentane ring was deduced with HMBC correlations between H-3, H-2 and H-1 with C-5 at δ C 87.2 ( Figure 4a).

Bazzania serrifolia, Fusicoccane-and Cuparane-Type
Six specimens of B. serrifolia were investigated. Volatile compounds content of the specimens MET092 and MET099 were quite different from the others, so these specimens were pooled apart under the appellation BS1 and the other four ones (MET041, 051-053) were pooled under the appellation BS2 as presented in Tables 4-10.
Volatile compounds contents of the two specimens were similar even if 10 constituents were identified for MET060, and 18 ones for MET049. B. vittata was characterized by the presence of aromadendrane-type sesquiterpenes mostly by the co-occurrence in both specimens of viridiflorol (21) (10.8-13.5%) and guaiol (22)  Observed neophytadiene (10.2-13.0%) could be an artifact from phytol degradation (moiety of esterified side chain of chlorophyll-a) during GC-FID-MS analysis [29].
Large amount of a new natural compound called "vittatin", a dimeric form of lunularic acid (51) was isolated from MET049 (47% of the crude ether extract, structural identification is described below). Lunularic acid (49a) was detected in numerous liverworts and algae but rarely in vascular plants and was known to play a similar biological role than abscisic acid found in vascular plants such as growth inhibitory [30]. Lunularic acid (49a) is known to possess fungicide, algaecide and anti-hyaluronidase activities [31]. Presence of this compound in MET060 was confirmed.
Biaryl meta-meta junction observed for vittatin (51) is a criterion for bis(bibenzyl)s structural classification. The methylenedioxy bond observed between the two bibenzyl units of vittatin (51) is very rare in bis(bibenzyl)s structures [32]. Putative pathway of vittatin (51) is proposed below as well as the role of its hypothetic precursor in the structural biosynthesis scheme of natural bis(bibenzyl)s.  Table 2) and HSQC spectra revealed the presence of only 16 carbon resonances including seven quaternary carbons, six methine and three methylene groups, suggesting a dimer form. Among the seven quaternary sp 2 hybridized carbons, one was attributed to a carbonyl carbon according to its chemical shift at δ C 170.7 and six were attributed to aromatic carbons according to their chemical shifts at δ C 156.6, 153.0, 141.0, 137.8, 128.3 and 119.9. All methine groups corresponded to aromatic carbons according to their chemical shifts at δ C 130.9, 128.8, 128.3, 120.6, 120.4 and 114.0. Among the three methylene groups, one was methylenedioxy carbon according to its chemical shift at δ C 98.9. The 1 H and COSY correlations of (51) (Figure 5b) exhibited the presence of two aromatic systems, an ABX spin system as observed in the aromatic protons at δ H 7.07 (2H, d, 8.2 H-6), 7.17 (2H, dd, 8.2, 2.0, H-5) and 7.51 (2H, d, 2.0, H-3) and a second aromatic system like an AX2 spin system as observed at δ H 7.20 (2H, t, 7.9, H-13), 6.75 (2H, d, 7.9, H-12) and 6.76 (2H, d, 7.9, H-14), which indicated the presence of two tri-substituted aromatic rings 1,2,4 and 1,2,3 respectively. The 1 H and COSY spectra showed also an ethylene group at δ H 2.97 (4H, m, H-8), 2.89 (4H, m, H-7) attached to C-4 and C-9 by the presence of 3 J HMBC correlation between H-7 and C-3 at δ C 128.3, C-5 at δ C 128.8 and between H-8 and C-10 at δ C 119.9, C-14 at δ C 120.4 ( Figure 5a). NOE correlations were observed between H-7 and H-3 and between H-8 and H-14 ( Figure 5b). The HMBC spectrum showed two small 4 J correlations between H-12 at δ H 6.75, H-14 at δ H 6.76 and C-15 at δ C 170.7 (Figure 5a) which put in evidence the attachment of carboxylic group at C-10. A hydroxyl group was fixed on C-11 within a characteristic 13 C chemical shift at δ C 156.6 and a 1 H chemical shift at δ H 10.43 due to an hydrogen bond established with the the carboxylic acid group at C-15. The signal on 1 H-NMR spectrum at δ H 5.55 (2H, s, H-16) was attributed to a methylenedioxy attached on C-1 determined by the presence of 3 J HMBC correlation between H-16 and C-1 at δ C 153.3 (Figure 5a). The 13 C-and 1 H-NMR data of this monomer structure were similar of lunularic acid NMR data [33], the dimer form corresponded to two monomers of lunularic acid attached at C-2 by meta-meta junction within a methylenedioxy bridge at C-1. The structure of this new compound (51) was named vittatin. Spectroscopic data are available in Supplementary Materials.   (51) Bis(bibenzyl)s are biosynthesized from lunularin (49b) or its precursor lunularic acid (49a). This assumption was supported by feeding experiments using radioactive and 13 C-labelled precursors [34]. Marchantin C synthase (isolated from a cell culture of Marchantia polymorpha) was supposed to be involved in the coupling mechanism of two molecules of lunularic acid (49a) leading to marchantin C (48) (type II) [35].

Putative Biosynthesis of Vittatin
Due to the functionalization of vittatin (51), hypothesis can be proposed suggesting that this bis(bibenzyl) should be formed by biaryl meta-meta coupling at the aromatic ring A (or C) of lunularic acid (49a) leading to a putative intermediate (50a). It is interesting to note that Momordica charantia peroxidase catalyzes biaryl meta-meta coupling with dihydroresveratrol as substrate [36]. The last step could be the formation of a methylenedioxy junction between the two phenol functions in para position of the aromatic cycle (A and C) leading to vittatin (51) as shown in Figure 6.  (51) Bis(bibenzyl)s are biosynthesized from lunularin (49b) or its precursor lunularic acid (49a). This assumption was supported by feeding experiments using radioactive and 13 C-labelled precursors [34]. Marchantin C synthase (isolated from a cell culture of Marchantia polymorpha) was supposed to be involved in the coupling mechanism of two molecules of lunularic acid (49a) leading to marchantin C (48) (type II) [35].

Putative Biosynthesis of Vittatin
Due to the functionalization of vittatin (51), hypothesis can be proposed suggesting that this bis(bibenzyl) should be formed by biaryl meta-meta coupling at the aromatic ring A (or C) of lunularic acid (49a) leading to a putative intermediate (50a). It is interesting to note that Momordica charantia peroxidase catalyzes biaryl meta-meta coupling with dihydroresveratrol as substrate [36]. The last step could be the formation of a methylenedioxy junction between the two phenol functions in para position of the aromatic cycle (A and C) leading to vittatin (51) as shown in Figure 6.
Due to the functionalization of vittatin (51), hypothesis can be proposed suggesting that this bis(bibenzyl) should be formed by biaryl meta-meta coupling at the aromatic ring A (or C) of lunularic acid (49a) leading to a putative intermediate (50a). It is interesting to note that Momordica charantia peroxidase catalyzes biaryl meta-meta coupling with dihydroresveratrol as substrate [36]. The last step could be the formation of a methylenedioxy junction between the two phenol functions in para position of the aromatic cycle (A and C) leading to vittatin (51) as shown in Figure 6.  According to the literature, bis(bibenzyl)s are classified into four structural types (I-IV, Figure 7), each structure is composed of two bibenzyl units which differ from linkages between these units [37]. Due to its structure, the putative biosynthetic intermediate of vittatin (50) should be added into a previous global scheme of bis(bibenzyl) biosynthesis pathway [38] (Figure 7). This scheme highlights that the dimer of lunularin (50b) (=isoperrottetin A, isolated from Radula perrottetii [39]) and the dimer of lunularic acid (50a) might play the role of the precursor of the bis(bibenzyl)s of types I and III which encompassed more than 30 compounds [32].  Figure  7), each structure is composed of two bibenzyl units which differ from linkages between these units [37]. Due to its structure, the putative biosynthetic intermediate of vittatin (50) should be added into a previous global scheme of bis(bibenzyl) biosynthesis pathway [38] (Figure 7). This scheme highlights that the dimer of lunularin (50b) (=isoperrottetin A, isolated from Radula perrottetii [39]) and the dimer of lunularic acid (50a) might play the role of the precursor of the bis(bibenzyl)s of types I and III which encompassed more than 30 compounds [32]. A survey of the literature showed that bis(bibenzyl)s from liverworts of types I and III were found only in the Jungermanniopsida class. These structures were detected in liverworts from the genera Herbertus, Lepidozia, Mastigophora, Plagiochila and Bazzania, which belong to the Lophocoleineae sub-order (Jungermanniales). Nevertheless, bis(bibenzyl) compounds with a (m-m)-(C-C) bond linkage had been detected in two species which do not belong to the Lophocoleineae sub-order: Jamesoniella colorata (Jungermanniineae) [40] and Radula perrottetii (Radulineae) [39].

Discussion
Important sesquiterpene-type diversity illustrated by the Figure 3 was observed among the studied samples of New-Caledonian liverworts. The genus Bazzania had been widely investigated in phytochemistry and most of detected sesquiterpene types belong to bazzanane-, cuparane-, barbatane-, aromadendrane-, bicyclogermacrane-, calamenane-, drimane-, chamigrane-, pinguisane-, myltaylane-and cyclomyltaylane-type [41]. Our results are consistent with the literature data except for the bicyclogermacrane-, calamenane-and drimane-type sesquiterpenes which do not seem to be widespread in Bazzania species from New Caledonia.
Drimane-type sesquiterpenes were detected within moderate relative percentages in one chemotype of B. francana. Calamenane-type, which is considered as a valuable chemotype for several Japanese Bazzania species [42], is the only sesquiterpene-type belonging to the (Z,E)-germacradienyl cation that had been detected in the New-Caledonian Bazzania species studied herein: calamenane-type sesquiterpene was detected with a moderate relative percentage in only one specimen of B. serrifolia. Thus, calamenane-type chemotype seemed to be rare in the present studied New-Caledonian Bazzania species: none of the analyzed specimen compositions could be chemically classified into the known chemotype II.  11.9 5.0     Isolepidozene (25) was detected in two specimens of B. serrifolia as a minor compound, but seemed to be a good biomarker for the Acromastigum genus which had been studied for the first time in this work. The new compound ziera-12(13),10(14)-dien-5-ol (23), belonging to the (E-E) germacradienyl cation sounds to be an important biomarker for two specimens of B. francana (MET062 and MET065).
Multivariate PCA analysis of sesquiterpene type distribution (Figure 8) highlights the striatane/monocyclofarnesane-type chemotype for B. vittata and one specimen of B. francana (MET032). This fact is noteworthy since this sesquiterpene-type is very rare in the Jungermanniales order and seemed to be more specific to the Porellales order [15].
several Japanese Bazzania species [42], is the only sesquiterpene-type belonging to the (Z,E)-germacradienyl cation that had been detected in the New-Caledonian Bazzania species studied herein: calamenane-type sesquiterpene was detected with a moderate relative percentage in only one specimen of B. serrifolia. Thus, calamenane-type chemotype seemed to be rare in the present studied New-Caledonian Bazzania species: none of the analyzed specimen compositions could be chemically classified into the known chemotype II.
Isolepidozene (25) was detected in two specimens of B. serrifolia as a minor compound, but seemed to be a good biomarker for the Acromastigum genus which had been studied for the first time in this work. The new compound ziera-12(13),10(14)-dien-5-ol (23), belonging to the (E-E) germacradienyl cation sounds to be an important biomarker for two specimens of B. francana (MET062 and MET065).
Multivariate PCA analysis of sesquiterpene type distribution (Figure 8) highlights the striatane/monocyclofarnesane-type chemotype for B. vittata and one specimen of B. francana (MET032). This fact is noteworthy since this sesquiterpene-type is very rare in the Jungermanniales order and seemed to be more specific to the Porellales order [15]. Several studied species contain high percentages of cuparane-type sesquiterpenes (namely cuparane-, herbertane-and microbiotane-type sesquiterpenes). Concerned species are the following ones: B. marginata, B. francana (MET106), B. serrifolia (MET092 and MET099) and B. bernieri (MET028, 063, 066, 067, 069) from which we detected from 9.3 to 97.7% of cuparane-type sesquiterpenes. This fact suggested that a "special" chemotype I based only on the cuparane-type should be more appropriate to characterize Bazzania species from New Caledonia. Moreover, the detection of Several studied species contain high percentages of cuparane-type sesquiterpenes (namely cuparane-, herbertane-and microbiotane-type sesquiterpenes). Concerned species are the following ones: B. marginata, B. francana (MET106), B. serrifolia (MET092 and MET099) and B. bernieri (MET028, 063, 066, 067, 069) from which we detected from 9.3 to 97.7% of cuparane-type sesquiterpenes. This fact suggested that a "special" chemotype I based only on the cuparane-type should be more appropriate to characterize Bazzania species from New Caledonia. Moreover, the detection of microbiotane-type (derivative of cuparane-type) sesquiterpene for one specimen of B. francana, is noticeable because this compound is also very rare in liverworts [15].
We have noticed that two samples of B. serrifolia (MET099 and MET092) shared many common characteristics with all specimens of B. bernieri (except MET038) such as the high amount of β-caryophyllene (35) and the presence of chamigrane-, cedrane-and bisabolane-type sesquiterpenes (these four structural-type sesquiterpenes were not detected in the other specimens of B. serrifolia or in the specimen MET038 of B. bernieri). In addition, one specimen of B. bernieri (MET038) shared many characteristics with specimens of B. serrifolia (except MET099 and MET092). These data indicated proximity between B. bernieri and B. serrifolia species. Therefore, we didn't find any evidence of chemospecific status of B. serrifolia, and so our phytochemical data would add more assumption of its taxonomic synonymy with B. bernieri [8].
Amongst the Lepidoziaceae family, the literature data reported that fusicoccane-type diterpenes were only found in Bazzania involuta and Lepidozia concinna species [15], so our findings pointed out that fusicoccane-type diterpenes (mainly fusicocca-2,5-diene (42)) seemed to be specific biomarkers to Bazzania species of New Caledonia. As shown in PCA chart of diterpene-type distribution (Figure 9), fusicoccane-type is a characteristic biomarker of B. serrifolia, B. bernieri and B. vittata while labdane-type diterpene is detected mainly in B. bernieri and in one specimen of B. francana (MET106). The labdane-type diterpenes (mainly (Z)-biformene (45)) seemed to be characteristic biomarkers of Bazzania species from New Caledonia because this structural-type compound is very rare in the Lepidoziaceae family. Kaurane-type diterpenes were only detected in two specimens of herein studied Acromastigum species and may be considered as a good biomarker for A. tenax. microbiotane-type (derivative of cuparane-type) sesquiterpene for one specimen of B. francana, is noticeable because this compound is also very rare in liverworts [15].
We have noticed that two samples of B. serrifolia (MET099 and MET092) shared many common characteristics with all specimens of B. bernieri (except MET038) such as the high amount of β-caryophyllene (35) and the presence of chamigrane-, cedrane-and bisabolane-type sesquiterpenes (these four structural-type sesquiterpenes were not detected in the other specimens of B. serrifolia or in the specimen MET038 of B. bernieri). In addition, one specimen of B. bernieri (MET038) shared many characteristics with specimens of B. serrifolia (except MET099 and MET092). These data indicated proximity between B. bernieri and B. serrifolia species. Therefore, we didn't find any evidence of chemospecific status of B. serrifolia, and so our phytochemical data would add more assumption of its taxonomic synonymy with B. bernieri [8].
The liverwort B. francana comprised at least three chemotypes in New Caledonia: (1) striatane; (2) microbiotane; and (3) zierane chemotype. Numerous chemotypes for specimens belonging to the same species, collected in a restricted area, is a rarely observed fact but sometimes may occur, for example analysis of Lepidozia fauriana (Lepidoziaceae) samples collected in Taiwan, led to split them into three different chemotypes : (1) amorphane; (2) chiloscyphane; and (3) eudesmane chemotypes [15]. Amongst the Lepidoziaceae family, the literature data reported that fusicoccane-type diterpenes were only found in Bazzania involuta and Lepidozia concinna species [15], so our findings pointed out that fusicoccane-type diterpenes (mainly fusicocca-2,5-diene (42)) seemed to be specific biomarkers to Bazzania species of New Caledonia. As shown in PCA chart of diterpene-type distribution (Figure 9), fusicoccane-type is a characteristic biomarker of B. serrifolia, B. bernieri and B. vittata while labdane-type diterpene is detected mainly in B. bernieri and in one specimen of B. francana (MET106). The labdane-type diterpenes (mainly (Z)-biformene (45)) seemed to be characteristic biomarkers of Bazzania species from New Caledonia because this structural-type compound is very rare in the Lepidoziaceae family. Kaurane-type diterpenes were only detected in Vittatin (51), as a dimer of lunularic acid, could be indexed in type I and III in the bis(bibenzyl)s classification. Bis(bibenzyl)s from type I and III possess various interesting biological activities such as bactericidal towards methicillin-resistant strains like Staphylococcus aureus [43], antimitotic agents [44], vasorelaxant [45]. Vittatin (51) possesses interesting chemical functions such as carboxylic acid, phenol and a methylenedioxy moiety, these features allow numerous chemical transformation through hemisynthesis. Due to its occurrence and functionalization, vittatin (51) should be a valuable raw material for the synthesis of interesting bis(bibenzyl)s (type I and type III), which could have promising pharmaceutical potential.      (41) 1786

General Experimental Procedure
Plant material was air-dried at room temperature and small amount of samples were crushed and extracted with Et 2 O with mortar and pestle. Extract was then purified through a Pasteur pipette packed with silica gel using Et 2 O as eluent to retrieve polar compounds. Crude extracts have been analyzed by GC-FID-MS. GC-FID-MS analysis was performed using a gas chromatograph coupled with a mass detector (Clarus ® 580, Perkin Elmer Inc, Waltham, MA, USA) and a flame ionization detector (Clarus ® 580 , Perkin Elmer Inc, Waltham, MA, USA) using helium at 1 mL/min. Capillary column was a elite-5MS (30 m × 0.25 mm, 0.25 µm) (Perkin Elmer Inc, Akron, OH, USA). Analyses were performed using EI mode. The injection temperature was set at 250 • C. Analyses were carried out using a temperature program starting from 50 • C, with an initial 3 min hold, to 250 • C with a 5 • C/min heating ramp, and keeping the final temperature stable for 15 min. Mass range was set at m/z 40-500. The individual peaks were identified by comparison of mass spectra from libraries as well as the retention indices (RI), which were calculated for all volatile constituents using a homologous series of n-alkanes C8-C32 and were compared with available literature data.
Mass Finder 2.3 library, NIST library (Gaithersburg, MD, USA), Wiley library (Hoboken, NJ, USA) were used for mass spectra comparison and identification. We used mainly NIST MS Search 2.2 software, Pherobase [46] and literature data [47] for retention index comparison to identify constituents of the crude extracts. Relative percentages of constituents were calculated with the area from the FID GC chromatogram corrected with the number of carbon of the corresponding compound (based on the MS identification).
HR-ESI-MS analyses were measured with a SYNAPT G2 HDMS mass spectrometer (Waters, Manchester, United Kingdom). Accurate mass measurements were performed in triplicate with two internal calibrations.  Table 11.

Extraction and Isolation
Plant material of Bazzania vittata (90 g) was extracted by maceration with Et 2 O (three times during one week). The crude extract was obtained as a green pale powder (2.09 g), was then washed through a Büchner funnel with successively: cyclohexane, dichloromethane, methanol and acetonitrile to yield vittatin (51) (980 mg, 47%).

Statistical Analysis
In order to investigate intra-and inter specific variability from 27 specimens of the Bazzanioideae, two data sets were included in the multivariate analysis using the software program past 3. Principal Component Analysis (PCA) was performed for variance-covariance.
First data was set up for analysis of the different sesquiterpene-types (28 sesquiterpene-types corresponding to 82 identified sesquiterpenes). Second data was set up for analysis of the different diterpene-types (four diterpene-types corresponding to 12 identified diterpenes).