PALEVO 1631-0683 Elsevier S1631-0683(07)00100-5 10.1016/j.crpv.2007.09.005 Research article Systematic Palaeontology (Palaeobotany) Chemotaxonomical investigations of fossil and extant beeches. I. Leaf lipids from the extant Fagus sylvatica L. Étude chimiotaxonomique de hêtres fossiles et actuels. I. Lipides foliaires du Fagus sylvatica L. actuel Ricqlès Jean Broutin, Armand de Nguyen-Tu Thanh Thuy ttnguyen@snv.jussieu.fr a Egasse Céline a Zeller Bernd b Derenne Sylvie c Paléobiodiversité et paléoenvironnements, UMR 5143 MNHN, bâtiment de géologie, B.P. 48, 57, rue Buffon, 75231 Paris cedex 05, France Laboratoire de biogéochimie des écosystèmes forestiers, UR 1138 INRA, centre INRA de Nancy, 54280 Champenoux, France Laboratoire de chimie bioorganique et organique physique, BioEMCo UMR 7618, ENSCP, 11, rue Pierre-et-Marie-Curie, 75231 Paris cedex 05, France 6 6-7 S1631-0683(07)X0038-1 La paléobotanique et l'évolution du monde végétal : quelques problèmes d'actualité Palaeobotany and evolution of the plant's world: some current problems 451 461 © 2007 Académie des sciences. Published by Elsevier B.V. All rights reserved. 2007 Académie des sciences Full (PDF)

The chemistry of fossil plants can help in precising their taxonomic affinities as well as their taphonomic history. The lipids from mature leaves of Fagus sylvatica L. were thus characterised to identify the components potentially informative that might be preserved in fossil beeches. Gas chromatography–mass spectrometry analyses of the lipids revealed a complex mixture comprising more than 60 identified constituents, belonging to diverse chemical families (e.g., sterols, fatty lipids, acyclic isoprenoids, triterpenes, glycerols, esters). Although most of the identified components are reported here for the first time in the European beech, they predominantly correspond to rather common compounds in Angiosperms. Nevertheless, their co-occurrence may constitute a useful fingerprint in further chemotaxonomic investigations of beeches. The newly reported compounds also include degradation products of plant lipids and fungal markers, showing that these degradation markers are to be considered as molecules related to the leaves in further taphonomic studies.

La chimie des plantes fossiles peut aider à préciser leurs affinités taxinomiques et leur histoire taphonomique. Les lipides de feuilles matures de Fagus sylvatica L. ont donc été caractérisés, afin d’identifier les composés potentiellement informatifs qui pourraient être préservés dans les hêtres fossiles. L’analyse de ces lipides par spectrométrie de masse a révélé une mixture complexe de plus de 60 molécules identifiées, appartenant à des familles chimiques variées. La plupart des composés identifiés sont décrits ici pour la première fois chez F. sylvatica. Ils correspondent majoritairement à des constituants communs chez les Angiospermes, mais leur présence concomitante pourrait constituer une empreinte chimique pertinente pour les futures études chimiotaxonomiques sur le hêtre. Certains des composés nouvellement identifiés correspondent à des produits de dégradation de lipides végétaux et à des marqueurs fongiques, montrant que de tels marqueurs de dégradation peuvent contribuer significativement aux lipides des fossiles.

 Fagus sylvatica L., Fagaceae, Lipids, Leaf, Gas chromatography, Mass spectrometry, France Fagus sylvatica L., Fagaceae, Lipides, Feuille, Chromatographie en phase gazeuse, Spectrométrie de masse, France presented Written on invitation of the Editorial Board Introduction

Chemotaxonomy has been widely used to help in precising the taxonomical affinities of plants (e.g. [6] and [59]). It is mainly based on the comparative characterisation of the chemistry of the studied taxa and the identification of biomarkers, characteristic compounds typical of a given taxonomic rank. Chemotaxonomy can also be successfully applied to fossil plants, provided that organic matter is preserved, which is often the case (e.g., [13], [24] and [40]). Among plant chemicals, lipids are the most commonly studied in palaeobotany. Indeed, due to their hydrophobic nature, lipids are expected to remain in close association with a buried macrofossil rather than migrating into the embedding sediment. Consequently, several studies have established the chemical composition of lipids extracted from fossil plants and demonstrated their chemotaxonomic value, based on comparison with extant counterparts (e.g., [32] and [38]).

The genus Fagus L. (Fagaceae) is well represented in the fossil record since the Early Cenozoic (e.g., [10]). It includes 10 species in two sub-genera distributed in North America and Eurasia. Fagus exhibits a high intraspecific morphological and genetic variability that may lead to an overestimation of its past and present specific variability (e.g., [9] and [10]). Moreover, as stressed by Denk and Meller [11], the preservation of a number of the most-diagnostic characteristics of beeches is not favoured during the taphonomy and fossilisation processes. Therefore, chemotaxonomic studies of fossil and extant specimens may provide further insights into the taxonomy of the genus Fagus.

This initiating study focuses on the chemical characterisation of leaf lipids of the European beech, Fagus sylvatica L. While the chemistry of F. sylvatica leaves was abundantly studied in the past, no authors detailed the chemical composition of its total lipids, in spite of the quantitative importance of this lipid pool in leaves (e.g., [37] and [66]). Indeed, previous studies focused on the analysis of selected classes of leaf constituents: volatiles [27], [56] and [61], cutin [48], tannins [2], epicuticular waxes [19], [20], [21], [25] and [35], vitamins [28], and the main components of a Bligh–Dyer-type extract [49] and [50]. Moreover, previous studies mainly dealt with the chemistry of summer leaves. However, total leaf lipids probably reach their full development at the beginning of autumn, as do epicuticular waxes [45] and [47]. Furthermore, for deciduous trees, autumn leaves have a much higher probability to be fossilised than summer ones. Therefore, this study aims at characterising the chemistry of total lipids extracted from autumn leaves of F. sylvatica, in order to further help in chemotaxonomic and taphonomic investigations of fossil and extant beeches.

 Materials and methods Plant materials

Samples of F. sylvatica were collected in a rural area (experimental forest of Breuil-Chenue, ‘Parc naturel régional du Morvan’, France; [46]) where urban pollution is minimal. Several leaves were collected on several branches at the same height (approximately 1.5 m) of several trees, in order to minimize biases linked to intraspecific variability and obtain an average chemical composition representative of the leaves. The leaves were collected in October 2004, examined under microscope the day after collection and then oven dried for 48 h at 45 °C. The dried leaves were subsequently stored in the dark at 5 °C until analysis (less than two weeks). A voucher specimen has been deposited at the herbarium of the ‘Laboratoire de paléobotanique, université Paris-6’, France.

 Lipid extraction and fractionation

The extraction was performed combining 20 leaves that corresponded approximately to 1 g of dry biomass. Leaves were crushed in a mortar and powdered to <500 μm. The leaf powder was ultrasonically extracted for 20 min with 30 ml dichloromethane/methanol (2:1; v/v). The mixture was centrifuged (10 min at 4000 rpm) and the extracted lipids were recovered with the supernatant. The procedure was repeated six times on the obtained residue. The combined extracts were concentrated by rotary evaporation and then dried under nitrogen gas. The extract yield was determined by weighting the dried combined extracts.

The total extract was then fractionated using column chromatography on alumina (13 g, Sigma-Aldrich 507C ∼ 150 mesh) deactivated to Brockmann grade IV by adding 0.1 weight % of distilled water. The non-polar fraction (1) was recovered eluting with 52 ml of heptane. The slightly polar fraction (2) was recovered eluting with 39 ml of toluene. The medium polar fraction (3) was recovered eluting successively with 13 ml of dichloromethane and 52 ml of dichloromethane/methanol (9:1; v/v). The obtained fractions were concentrated by rotary evaporation and further dissolved in approximately 3 ml of heptane, toluene, and dichloromethane, respectively.

 Quantification

The main components of the three studied fractions were quantified using (1) an internal standard (i.e. tetratriacontane for fraction 1 and 1-tricosanol for fractions 2 and 3) introduced in the fractions in known amount, (2) the GC peak areas of the component and the standard, and (3) the response factor in gas chromatography (GC) of the measured component. The latter was obtained from calibration curves of the standard and the measured component, or compound with similar chemical structure, polarity, and molecular weight if no reference compound was available.

 Derivatization

The hydrocarbon fraction was analysed as such, while the other two fractions were converted into trimethylsilyl derivatives prior to analyses. Aliquots (30 μl) of the fractions were dried in a stream of nitrogen and then trimethylsilylated by reaction with 30 μl of N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) for 1 h at 80 °C. After cooling, the aliquot was dried under a gentle stream of nitrogen and redissolved in 30 μl of appropriate solvent (toluene for fraction 2 and dichloromethane for fraction 3).

 Gas chromatography–mass spectrometry analysis

Gas chromatography (GC) analyses were performed on a Varian GC 3900 gas chromatograph fitted with a fused silica capillary column coated with VF5-MS (50 m × 0.32 mm i.d., 0.25 μm film thickness). The GC operating conditions were as follows: increase from 80 °C to 100 °C at 10 °C min−1, then increase to 325 °C at 4 °C min−1, followed by an isothermal period of 30 min at 325 °C. Helium was used as carrier gas at a constant flow of 2 ml min−1. The splitless injector and FID detector temperatures were held at 350 °C.

Gas chromatography–mass spectrometry (GC–MS) analyses were carried out using an Agilent 6890N gas chromatograph coupled with an Agilent 5973N mass spectrometer, scanning the range 35–800 Da, electron impact voltage 70 eV, interface temperature 250 °C, source temperature 220 °C. The chromatographic conditions were the same as above. The compounds were identified by comparison of their retention time and mass spectrum with those of reference compounds or literature data (see Table 1 for references), and interpretation of mass spectrometry fragmentation patterns.

 Results and discussion

The total lipids recovered after dichloromethane/methanol extraction of F. sylvatica leaves accounted for 11.3 wt % of the dried leaves. These lipids comprise 0.5% (0.6 mg) of non-polar fraction, 7.8% (9.4 mg) of slightly polar fraction and 17,1% of (20.7 mg) of medium polar fraction (i.e. eluted with heptane, toluene and a mixture of dichloromethane and methanol, respectively, see experimental section). The GC–MS analyses of these fractions revealed that beech lipids correspond to a complex mixture of more than 60 identified constituents (Table 1), and the chromatogram of the most abundant fraction is shown in Fig. 1. When all the fractions are considered, sterols constitute the main compound class of the extract, with β-sitosterol being by far the dominant molecule, with 1276 μg g−1 of dry biomass (Table 2, Fig. 1). Several sterols identified here in the beech lipids correspond to widespread phytosterols (i.e. brassicasterol, stigmasterol, campesterol, cycloartenol). Except brassicasterol, all have been previously described in beech leaves or, at least, in other parts of F. sylvatica, such as wood or seeds [20], [33], [43] and [50]. In addition, classical oxidation and biodegradation products of these sterols (i.e. hydroxy-, oxo- and unsaturated derivatives; [26] and [41]) are detected in significant amounts (Table 1, Fig. 1). Fungal sterols, such as ergosterol and obtusifoliol [14] and [23], and derivatives, are also identified in beech leaves (Table 1). Their occurrence can be explained by the presence of epiphytic fungi on several leaves, as revealed by microscopic examination (Fig. 2). Indeed, as most deciduous leaves, beech leaves host a number of endophytic and epiphytic fungi (e.g., [39]). The presence of degradation products of phytosterols and of fungal sterols were not previously reported in the common beech, which is likely due to the lower maturity of previously investigated leaves.

The second most abundant compound class after the sterols corresponds to acyclic isoprenoid molecules; among them, phytadienes are predominant (Table 1, Table 2, Fig. 1). Phytadienes were shown to be artificially produced from phytol by numerous analytical procedures such as acid-hydrolysis, saponification, and high-temperature GC injection [17]. However, phytadienes probably constitute original components of F. sylvatica since (1) they are detected in the trimethylsilylated extracts, and (2) Grossi et al. [17] suggested that analysis of trimethylsilylated extracts does not produce any phytadiene as artefacts. Along with phytadienes, phytol, phytone, phytenals, and other oxygenated phytyl compounds are detected in substantial amounts in the leaf lipids extracted from F. sylvatica (Table 1). These compounds mainly correspond to various intermediates in the degradation of the phytyl chain of chlorophylls. To a lesser extent, they also may be derived from other compounds comprising a phytyl chain (e.g., tocopherols, phylloquinone, phytylalkanoates; [3], [8], [51], [52], [54], [55] and [63]). These phytyl degradation compounds are reported here for the first time in beech lipids. Phytol is also present as a series of wax esters of phytol with acid moieties comprising 6 to 20 carbons and maximizing at C16 (Table 1); such a series has been described previously in leaf lipids of F. sylvatica, with a similar distribution pattern [50].

Other acyclic isoprenoid compounds are identified, the most abundant being the ubiquitous squalene, which has been described previously in beech seeds and wood (Table 1 and Table 2; [20] and [43]). Polyunsaturated acyclic isoprenoid ketones and aldehydes are also identified here for the first time in F. sylvatica (Table 1). These compounds are rather common in plant oils and can correspond either to degradation products or to biosynthetic intermediates of squalene or other terpenes (e.g., [12], [60] and [62]). Vitamins are also detected in the lipids extracted from beech leaves (Table 1 and Table 2): α-tocopherol (i.e. vitamin E), which has been previously reported in beech leaves [28], and phylloquinone (i.e. vitamin K1), which is described here for the first time. Tocopherols are also present as wax esters (C14, C16) of α- and β-tocopherols (Table 1). These compounds that are described here for the first time in F. sylvatica have not been frequently reported in the literature, probably because their high molecular weight made them difficult to detect at the temperatures generally used for GC analyses. Nevertheless, they may correspond to rather common plant compounds since (1) their moieties are widespread among plants and (2) they were detected in various Angiosperms (e.g., [42]).

Several series of aliphatic homologues are also detected in substantial amounts in the leaf lipids extracted from F. sylvatica; most of them have been previously described in its leaf lipids, with similar distribution patterns [20], [31], [35], [49] and [50]. These series exhibit a marked carbon number predominance. The most abundant series show an even over odd predominance; they correspond to n-alkan-1-ols, n-aldehydes, n-acids, and, to a lesser extent to methyl esters, isopropyl esters and n-alkenes (Table 1 and Table 2, Fig. 3). The absolute abundance determined for octacosanal should be considered cautiously, since Naß et al.[35] showed that quantification of aldehydes is often non-reproducible. However, these authors pointed out that the quantification biases were remarkably low in the case of F. sylvatica extracts. Otherwise, the natural occurrence of methyl esters in lipids is uncommon. Esterification of carboxylic acids upon extraction with solvent mixture containing methanol was previously attributed to the presence of clays in the experimental mixture [1] and [36]. To test the methylation of acids without clays, a standard acid (C17) was added to a piece of leaf and submitted to the extraction procedure. The corresponding ester was formed in substantial amounts and it was concluded that the methyl esters detected corresponded to a part of the original fatty acids that had been methylated upon extraction. Hence, the distribution showed in Fig. 3 is a combination of fatty acid and methyl ester distribution. Series with an odd over even predominance are also present: n-alkanes, n-alkan-2-ols and n-alkan-2-ones (Table 1 and 2, Fig. 3). n-Alkan-2-ones are generally considered as oxidation products of n-alkanes, n-alkan-2-ols corresponding to intermediates in this conversion [7], [49], [50] and [65]. The similarity in the distribution of these three series of compounds supports this hypothesis (Fig. 3). The occurrence of such degradation products in beech lipids may be due to the maturity of the studied leaves. As previously noticed by Lockheart [30], the n-alkane distribution of F. sylvatica leaf lipids is markedly dominated by the C27 homologue, which seems to be typical of the Fagus species living in the European cool temperate forest (e.g., F. sylvatica, F. crenata) when compared with species living in the warmer areas of Asia and Eurasia (e.g., F. grandifolia, F. japonica, F. orientalis), which exhibit significant amounts of the C25 and C29 homologues along with the C27. Nonacosan-10-ol is also identified for the first time in the European beech (Table 1), although commonly observed in several Embryophytes [4] and [22]. Additionally, three series of long-chain esters are detected: wax esters, benzyl esters and p-coumaric esters (Table 1); their distribution is in agreement with previous characterisation of leaf lipids from F. sylvatica [20], [35] and [50].

Several pentacyclic triterpenoids can be identified at the end of the chromatogram of fraction 3; they exhibit ursane or oleanane carbon skeleton (Table 1 and Table 2, Fig. 1). Except α-amyrin that has been previously described in beech wood [43], all these triterpenoids are reported here for the first time in F. sylvatica. Most of them correspond to terpenoids rather common in plants, especially in Angiosperms, or to their degradation product. A number of monoacylglycerols is also detected in the leaf lipids of beech (Table 1, Fig. 1). These intermediates in the lipolysis and biosynthesis of triacylglycerols were previously described in the lipids extracted from the seeds of F. sylvatica, although with a narrower range of chain length [44]. Finally, four small terpenoids are detected in small amount (ionene, α-ionone, β-ionone, loliolide), they correspond to common compounds of Angiosperm essential oils, or their degradation products, and are reported here for the first time in F. sylvatica.

 Conclusion and implications

This study thus allowed to precise the chemical composition of free lipids extractable from autumn leaves of the European beech (Fagus sylvatica). In addition to the constituents previously reported in specific lipid pools of F. sylvatica, a number of compounds are described here for the first time in this plant. These compounds are fairly abundant and generally correspond to molecules rather common in Angiosperms; they were not previously reported in beech lipids, probably because previous authors investigated more restricted lipid pools. Nevertheless, the co-occurrence and relative abundances of some of these compounds (e.g., phylloquinone, brassicasterol, nonacosan-10-ol, tocopheryl alkanoates, dodecandral, oleanonic triterpenoids, ursolic acid, hederagenin) may constitute a chemical fingerprint to be used in further chemotaxonomic investigations of fossil and extant beeches. Otherwise, a number of the newly reported compounds correspond to degradation products of the main components of the leaves, emphasizing the quantitative importance of such products in mature leaf lipids. In the same way, the few fungal sterols detected in this study show that the epiphytic microflora might contribute significantly to the lipids extracted from leaves. The occurrence of such maturity markers in lipids extracted from leaves collected on trees in October are to be taken into account in further taphonomic studies; it may help to clarify the degradation/preservation stage of fossils. This investigation of total lipids from autumn leaves of F. sylvatica thus led to a description of beech lipids more complete than previously and should constitute a reference dataset in further chemotaxonomic and taphonomic investigations of fossil and extant beeches.

 Acknowledgments

We gratefully acknowledge the whole team in charge of the experimental site (INRA, Forêt de Breuil-Chenue), especially D. Gelhaye and J.-P. Calmet for their assistance in the fieldwork. Special thanks are also due to C. Anquetil for GC–MS facilities. We are indebted to P. Metzger, J. Broutin, F. Zanetti, S. Lemettre, N. Cao, and V. Malécot for helpful discussions, and to S. Peigné for technical support. This study was supported by a grant from FNS (ACI-JC 10051).

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Chromatogram of the main fraction of the lipids extracted from Fagus sylvatica L. leaves. This fraction was eluted with dichloromethane and methanol and compounds were analysed as trimethylsilyl-derivatives. IS: internal standard.

Fig. 1. Chromatogramme de la principale fraction des lipides extraits de feuilles de Fagus sylvatica L. Cette fraction a été éluée au dichlorométhane et au méthanol et ses composés identifiés en tant que dérivés triméthylsilylés. IS: standard interne.

Typical zones of Fagus sylvatica L. leaves invaded by fungi. (a) Fructifications of epiphytic fungi breaking through the epidermis (e.g., arrows). (b) Mycelium growing between the main nerve and the margins (e.g., arrows).

Fig. 2. Zones des feuilles de Fagus sylvatica L. typiquement envahies par des champignons. (a) Fructifications de champignons épiphytes perçant l’épiderme (par exemple, flèches). (b) Mycélium se développant entre la nervure principale et les marges (par exemple, flèches).

Distribution of the main series of fatty lipids extracted from Fagus sylvatica L. Numbers in abscissa refer to chain length and arrows indicated when a homologue is detected but below the quantification level.

Fig. 3. Distribution des principales séries de lipides gras, extraites de Fagus sylvatica L. Les numéros en abscisse désignent la longueur de chaîne, et les flèches indiquent un homologue identifié, mais en dessous du seuil de quantification.

Composition of the lipids extracted from Fagus sylvatica L. mature leaves (dichloromethane/methanol extract)

Tableau 1 Composition des lipides extraits de feuilles matures de Fagus sylvatica L. (extrait dichlorométhane/méthanol)

Compound Series distribution a Rt b Fraction c formula M w d Characteristic fragments e reference for MS f steroids brassicasterol 43.8 3 C28H46O 398 TMS: 125, 255, 380, 455, 470 [29] stigmasta-3,5-diene 47.3 1 C29H48 396 81, 147, 381, 396 [15] epiergosterol 49.1 3 C28H44O 396 TMS: 253, 337, 363, 468 [14] ergosterol 49.4 3 C28H44O 396 TMS: 201, 337, 363, 468 [14] campesterol 49.8 3 C28H48O 400 TMS: 129, 343, 382, 472 stigmasterol 50.1 3 C29H48O 412 TMS: 83, 129, 255, 394, 484 [57] 7α-OH-sitosterol 50.3 3 C29H50O2 430 TMS: 129, 253, 484, 574 [26] β-sitosterol 51.2 3 C29H50O 414 TMS: 129, 357, 381, 396, 486 stigmastanol (= dehydrositosterol) 51.6 3 C29H52O 416 TMS: 215, 283, 298, 473, 488 obtusifoliol (= 4,14-dimethylergosta-8,24(28)-dien-3-ol) 51.7 3 C30H50O 426 TMS: 227, 393, 483, 498 [14] cycloartenol 52.0 2 C30H50O 426 TMS: 339, 365, 393, 408, 498 7β-OH-sitosterol 52.3 3 C29H50O2 430 TMS: 129, 484 [26] 24 methylenecycloartan-3-one 52.6 2 C31H50O 438 95, 313, 423, 438 4-stigmasten-3-one (= β-sistostenone) 52.7 2 C29H48O2 412 124, 229, 289, 412 24-methylenecycloartanol 52.9 2 C31H52O 440 TMS: 379, 407, 422, 512 [67] 7-ketositosterol 55.3 3 C29H48O2 428 TMS: 129, 161, 390, 500 [26] phytyl derived compounds neophytadiene 22.7 1,2,3 C20H38 278 68, 82, 95, 123, 278 [16] 6,10,14-trimethyl pentadecan-2-one (phytone) 22.8 2 C18H36O 268 43, 58, 71, 250 (Z)-1,3-phytadiene 23.3 1,2,3 C20H38 278 81, 82, 95, 123, 278 [16] (E)-1,3-phytadiene 23.7 1,2,3 C20H38 278 82, 95, 123, 278 [16] 6,10,14-trimethyl-2-methylenepentadecanal 24.7 2 C19H36O 280 84, 97, 125, 280 [51] 2,6,10,14-tetramethylpentadec-2(E)-enal 26.5 2 C19H36O 280 84, 97, 126, 280 [51] Z-phytenal 29.1 2 C20H38O 294 84, 97, 139, 149 [53] E-phytenal 29.9 2 C20H38O 294 84, 97, 140, 149 [53] phytol 30.6 2,3 C20H40O 296 TMS: 75, 123, 143 3-methylidene-7,11,15-trimethylhexadecan-1,2-diol (phytyldiol) 32.8 3 C20H40O2 312 TMS: 73, 143, 147, 353, 456 [54] phytyl esters alkyl chain: C 6 -C 20 (C 16 ) (54.6) 1,2,3 C n H2n–2O2 (534) 95, 123, …, 278, 296, …, M+ [42] other compounds comprising an acyclic isoprenoid chain geranyl acetone 12.4 2 C13H22O 194 43, 69, 136, 151, 194 farnesyl acetone 24.5 2 C18H30O 262 69, 81, 136, 262 4,9,13,17-tetramethyl-4,8,12,16-octadecatetraenal 32.2 2 C22H36O 316 69, 81, 93, 137, 149, 316 4,8,12,16-tetramethylheptadecan-4-olide 34.3 2 C21H40O2 324 99, 114, 126, 324 [55] squalene 43.0 1,2 C30H50 410 69, 81, 95, 410 α-tocopherol 48.1 3 C29H50O2 430 TMS: 73, 237, 502 α-tocopherol ester alkyl chain: C 14 , C 16 (C 16 ) (76.0) 2 C30+n H52+2n O2 (668) 165, 430, M+ [42] β-tocopherol ester alkyl chain: C 14 , C 16 (C 14 ) (66.5) 2 C29+n H50+2n O2 (626) 151, 416, M+ [42] phylloquinone (vitamin K1) 51.4 2 C31H46O2 450 186, 198, 225, 450 lipids with n -alkyl chain isopropyl esters alkyl chain: C 12 , C 14 (C 12 ) (17.2) 2 C2H2n O2 (242) 43, 60, 102, M-42+ methyl esters alkyl chain: C 14 –C 28 , C 18:1 , C 18:2 (C 16 , C 28 ) (24.9) 2 C n H2n O2 (270) 74, 87, …, M+ n-acids C 9 –C 28 , C 18:1 , C 18:2 , C 18:3 (C 16 , C 28 ) (27.7) 2,3 C n H2n O2 (256) TMS: 73, 117, 132, 145, …, M-15 +, M+ n-alkenes C 14 –C 27, (C 26 ) (39.1) 1 C n H2n (378) 55, 69, 83, …, M+ n-alkanes C 12 –C 33 (C 27 ) (41.1) 1 C n H2n+2 (380) 57, 71, 85, …, M+ n-alkan-2-ols C 23 –C 29 (C 27 ) (45.5) 2,3 C n H2n+2O (396) TMS: 73, 75, 117, …, M-15 +, M+ n-alkan-2-ones C 25 , C 27 , C 29 (C 27 ) (45.5) 2 C n H2n O (394) 58, 59, …, M+ n-aldehydes C 20 –C 32 (C 28 ) (47.0) 2 C n H2n O (408) 82, 96, …, M+ nonacosan-10-ol 47.1 3 C29H60O 424 TMS: 73, 229, 369 n-alkan-1-ols C 14 –C 30 (C 28 ) (48.4) 2,3 C n H2n+2O (410) TMS: 75, 103, …, M-15 +, M+ p-coumaric esters alkyl chain: C 18 –C 22 (C 20 ) (56,8) 3 C n H2n–10O3 (516) TMS: 179, 192, 219, 236, …, M+ [58] benzyl esters alkyl chain: C 24 , C 26 , C 28 (C 28 ) (57.6) 2 C n H2n–8O2 (514) 91, 108, …, M-109+, M-91+ [18] wax esters C 36 –C 46 , C 38:1 , C 40:1 (C 44 ) (72.4) 1,2 C n H2n O2 (648) 57, 257, 313, 341, …, M+ glycerol esters 1-palmitoleylglycerol (1-monopalmitolein) 38.7 3 C19H36O4 328 TMS: 147, 237, 369, 457 1-hexadecanoylglycerol (1-monopalmitin) 38.9 3 C19H38O4 330 TMS: 147, 239, 371, 459 1-heptadecanoylglycerol (margarin) 40.7 3 C20H40O4 344 TMS: 147, 253, 385, 473 1-linolenoylgycerol (1-monolinolenin) 42.0 3 C21H36O4 352 TMS: 147, 261, 393, 481 1-monostearoylglycerol (1-monostearin) 42.4 3 C21H42O4 358 TMS: 147, 267, 399, 487 pentacyclic triterpenoids α-amyrenone 51.0 3 C30H48O 424 189, 203, 218, 424 [34] α-amyrin 51.7 3 C30H50O 426 TMS: 189, 203, 218, 498 [34] dodecandral 53.3 3 C30H46O4 470 203, 216, 496, 614 oleanonic aldehyde 54.2 3,2 C30H46O2 438 189, 203, 232, 409, 438 [64] oleanolic acid 54.3 3 C30H48O3 456 TMS: 73, 203, 320, 482, 585 [5] oleanolic aldehyde 54.6 3,2 C30H48O2 440 TMS: 190, 203, 232, 512 ursolic acid 54.9 3 C30H48O3 456 TMS: 73, 203, 320, 482, 585 [5] hederagenin 58.0 3 C30H48O4 472 TMS: 73, 203, 320, 570, 673 [5] miscellaneous ionene 9.9 1 C13H18 174 159, 174 α-ionone 11.8 2 C13H20O 192 93, 121, 136, 192 β-ionone 13.3 2 C13H20O 192 43, 123, 177, 192 loliolide 21.2 3 C11H16O3 196 75, 178, 212, 253, 268

Carbon number range (maximum, submaximum).

a Gamme de variation de la longueur de chaîne (maximum, sous-maximum).

Retention time (minutes), values in parenthesis correspond to the series maximum.

b Temps de rétention (minutes), les valeurs entre parenthèses correspondent au maximum de la série.

Elution fraction: heptane (1), toluene (2) and dichloromethane/methanol (3).

c Fraction d’élution: heptane (1), toluène (2) et dichlorométhane/méthanol (3).

Molecular weight; weights in parenthesis correspond to the series maximum.

d Masse moléculaire, les masses entre parenthèses correspondent au maximum de la série.

Fragments in bold correspond to the base peak of the mass spectrum, TMS indicate that the compound was identified as trimethylsilyl derivative.

e Les fragments en gras correspondent au pic de base du spectre de masse, TMS indique qu’un composé a été identifié après triméthylsilylation.

When no reference is indicated, the compound was identified by interpretation of mass spectrometry fragmentation pattern and/or comparison with NIST database.

f Quand aucune référence n’est indiquée, le composé a été par interprétation de ses caractéristiques de fragmentation en spectrométrie de masse et/ou par comparaison avec la base de données NIST.

Quantification of the main constituents of Fagus sylvatica leaf lipids

Tableau 2 Quantification des principaux constituants des lipides foliaires de Fagus sylvatica

Constituent Abundance * β-sitosterol 1276.0 neophytadiene 1096.0 oleanolic acid 631.2 (E)-1,3-phytadiene 491.1 phytyl hexadecanoate 456.0 phytol 370.0 n-octacosan-1-ol 314.8 n-octacosanal 290.0 n-hexadecanoic acid 275.2 n-heptadecane 268.3 squalene 109.6 eicosanyl-p-coumarate 97.0 4-stigmasten-3-one 38.9 α-tocopherol 7,0

mg g−1 of dry biomass.

* mg g−1 de biomasse sèche.