Organic Geochemistry 31 (2000) 1743±1754 www.elsevier.nl/locate/orggeochem E€ects of fungal infection on lipid extract composition of higher plant remains: comparison of shoots of a Cenomanian conifer, uninfected and infected by extinct fungi Thanh Thuy Nguyen Tu a,b,*, Sylvie Derenne b, Claude Largeau b, Andre Mariotti a, Herve Bocherens a, Denise Pons c a Laboratoire de BiogeÂochimie Isotopique, Universite Paris VI-INRA-CNRS, UMR 7618, Case courrier 120, 4 Place Jussieu, 75 252 Paris Cedex 05, France b Laboratoire de Chimie Bioorganique et Organique Physique, ENSCP-CNRS, UMR 7573, 11 Rue Pierre et Marie Curie, 75 231 Paris Cedex 05, France c Laboratoire de PaleÂobotanique et PaleÂoeÂcologie, Universite Paris VI, 12 Rue Cuvier, 75 005 Paris, France Abstract The lipid fraction extracted from uninfected shoots of a fossil conifer, Frenelopsis alata, was analysed by gas-chromatography±mass-spectrometry, and compared with shoots of the same conifer infected by extinct epiphyllous fungi, so as to study the e€ects of fungal infection on the chemical composition of extracts from higher plant remains. The extracts from the uninfected shoots appeared to be composed of (i) common constituents of higher plant lipids such as n-alkanes and fatty acids, (ii) elemental sulphur, and (iii) substantial amounts of terpenoids characteristic of conifers, such as cadalene, beyerane, dehydroabietane and related compounds. Comparison of this extract with that of fungalinfected shoots revealed, in addition to the aforementioned compounds, the presence of substantial amounts of hydroxysuccinic acid and functionalised benzoic compounds that were interpreted as degradation products of lignin by fungi. This study a€orded preliminary indications of the composition of extracts from higher plant remains infected by fungi. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Frenelopsis alata; Lipids; Cenomanian; Fungi; Lignin degradation 1. Introduction The investigation of fossil ¯ora by organic geochemical methods has been widely applied during the last decade and numerous studies of extracts have established the precise chemical composition of lipids from fossil plants (e.g. Logan and Eglinton, 1994; Otto et al., 1994; Huang et al., 1996). By identifying speci®c biomarkers of higher plants, such studies have provided a better understanding of the origin of organic matter in various sediments (Cranwell, 1984; Rieley et al., 1991; * Corresponding author at present address: Department of Geochemistry, Organic Geochemistry Group, Utrecht University, Faculty of Earth Sciences, Budapestlaan 4, Postbus 80021, 3508 TA Utrecht, The Netherlands. Tel.: +31-30-2535068; fax: +31-30-253-5030. E-mail address: nguyentu@geo.uu.nl (T.T. Nguyen Tu). Logan and Eglinton, 1994; Huang et al., 1995). The chemical composition of lipids from fossil plants have also been compared with that of their modern counterparts in order to test, on a chemical basis, the phylogenetic link between the species studied (Giannasi and Niklas, 1981; Huang et al., 1995). Assessing the nature and extent of the changes in lipid composition associated with diagenesis is crucial for such studies. Diagenesis of leaf lipids has been carefully studied in a few cases (Cranwell, 1981; Wannigama et al., 1981; de Leeuw et al., 1995). In addition, Logan et al. (1995) have shown, with fossil plants from the Miocene Clarkia Formation, that leaf waxes do not move into the surrounding sediment. To date, little is known about the e€ects, on lipid composition, of the biodegradation of higher plant remains by saprophytic organisms, especially fungi, although the latter widely invade plants as saprobes or parasites (Alexopoulos et al., 1996). In 0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00077-2 1744 T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 contrast, the ability of fungi to degrade lignin is well documented (Evans, 1987; PelaÂez et al., 1995; Breccia et al., 1997; Bending and Read, 1997) and the consequences of such alteration processes for the fate of lignin have been established (e.g. Hedges et al., 1988; GonÄi et al., 1993). In the present work, we have compared remains of a conifer infected by extinct fungi with uninfected remains of the same conifer, as a ®rst approach to determining the e€ects of fungal infection on extracts of fossil plant remains. The fossil ¯oras from the Middle Cenomanian of France and the Czech Republic provide exceptional opportunities to undertake studies on the e€ects of fungal infection on the lipid composition from higher plant remains. Indeed, one of the most abundant species in both ¯oras is Frenelopsis alata. This conifer belongs to an extinct family, the Cheirolepidiaceae, and exhibits a number of xerophytic characteristics such as a thick cuticle and sunken stomata (HlusÏ tõÂk and KonzalovaÂ, 1976; Pons, 1979). While no fungi have been reported on shoots of F. alata from the Czech Republic, several species of extinct epiphyllous fungi have been observed on the shoots of F. alata from France (Pons and Boureau, 1977). These extinct fungi belong to the phylum Ascomycota. Their excellent morphological preservation and the presence of reproductive stages allowed a precise identi®cation of the two most abundant species: Mariusia andegavensis, a parasitic Microthyriaceae species, and Stomiopeltites cretacea, an epiphytic Micropeltidaceae species (Fig. 1; Pons and Boureau, 1977). Such an excellent preservation of fungi is quite rare in the fossil record; however, several authors have reported similar observations previously on samples of Devonian or Eocene age (e.g. Kidston and Lang, 1921; Dilcher, 1965). Moreover, these species are extinct now so post-excavation contamination by living fungi can be rejected. Indeed, M. andegavensis and S. cretacea have only been described in from the Cretaceous of Europe (Pons and Boureau, 1977). Both French and Czech palaeo¯oras are exceptionally well preserved, thus making them especially suitable for chemical analyses. The remarkable degree of preservation of the fossil shoots is illustrated by: (1) their overall appearance (they are entire or slightly fragmented and look like modern autumnal shoots), (2) their colouration (initially the fossil leaves are brown but rapidly turn black upon exposure to air) and (3) the presence of remnants of mesophylle, which is generally degraded in fossils, between the cuticles. The exceptional preservation of these fossil plant remains provides evidence that sedimentation occurred rapidly near the area where the plant grew (Louail, 1984; UlicÏny et al., 1997a). Finally, both palaeo¯oras and geological settings are similar in France and the Czech Republic, which allows comparison of the same species from the two regions. The surrounding sediments in both France and the Czech Republic are made up of clays deposited under anoxic conditions in a salt marsh setting during the beginning of the Cenomanian transgression in Europe (Pons et al., 1980; UlicÏny et al., 1997a). Both palaeo¯ora exhibit similar compositions and they include leaves, wood and reproductive organs of Pteridophytes, Angiosperms and a number of Gymnosperms that are dominated by the same genera: Frenelopsis and Eretmophyllum (Pons et al., 1980; UlicÏny et al., 1997a; KvacÏek, 1999). Furthermore, the palaeoecology of both ¯oras inferred from stable carbon isotope compositions are almost identical, i.e. a salt marsh vegetation with F. alata growing in the most saline part of the marsh (Nguyen Tu et al., 1999a,b). Lipid extracts from uninfected shoots of F. alata (samples from the Czech Republic) were analysed using gas chromatography±mass spectrometry (GC±MS) and compared to extracts from shoots of F. alata infected by extinct fungi (samples from France) as a ®rst approach to a study of the e€ects of fungal infection on extracts of fossil plant remains. 2. Material and methods 2.1. Sampling sites (Fig. 2) Uninfected fossil shoots of F. alata were collected from the ``Peruc'' member in the PecõÂnov quarry located west of Praha (Czech Republic). The geological setting for the site has been previously described in detail (UlicÏny et al., 1997a). The ``Peruc'' member lies on Carboniferous sandstones and is overlain by littoral sediments of the ``Korycany'' member and marine sediments from the ``PecõÂnov'' member of Late Cenomanian age (UlicÏny et al., 1997a). Fossil shoots of F. alata infected by extinct fungi were collected from the ``Argiles du Baugeois'' member at the locality called ``Le Brouillard'', located north of Angers (France). The geological setting of the site has been previously reported in detail (Louail, 1984). The ``Argiles du Baugeois'' unit lies directly on Brioverian schists and is overlain by a marine formation called ``Marnes aÁ OstreÂaceÂes'' of Late Cenomanian age (Louail, 1984). These two deposits consist of similar sediments, i.e. grey and ®nely laminated clays and sand layers. In both deposits, the occurrence of gypsum, marcassite and pyrite crystals in the clays provides evidence of anoxic environments and geochemical studies showed that the sediments are immature (UlicÏny et al., 1997b; Nguyen Tu et al., 1999a). Moreover, both deposits were located at close palaeolatitudes, 355 N and 375 N for France and the Czech Republic, respectively (Fig. 2; Philip et al., 1993) and in the same semi-arid climatic zone (Parish et al., 1982). T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 2.2. Samples The French and the Czech samples were collected in May 1996 and in July 1999, respectively. Approximately seventy shoots of F. alata were collected from several 1745 representative levels of each deposit. Blocks of sediments containing shoots were taken from the ®eld. Shoots were removed in the laboratory, cleaned with pre-extracted cotton wool and dried overnight at 50 C. Fossil shoots were analysed as soon as removed from Fig. 1. (a) Scanning electron microscopy and (b)±(h) light microscopy observations of fungal-invaded shoots of F. alata (samples from the French deposit): (a) mycelian hyphae invading F. alata epidermis; (b) fungal stroma invading F. alata epidermis; (c) stroma of M. andegavensis; (d) mycelian hyphae and intercalated stygmocytes of M. andegavensis; (e) perforation of the cuticle by a canaliculus (arrow) of M. andegavensis; (f) stroma of S. cretacea; (g) globulous stroma of an unidenti®ed parasitic Ascomycete; (h) internal mycelium of an unidenti®ed parasitic Ascomycete. 1746 T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 Fig. 2. Palaeoenvironments during the Cenomanian in Europe and location of the sampling sites (Modi®ed after Philip et al., 1993): pl: palaeolatitudes, EL: exposed lands, TS: terrigenous shelf and shallow terrigenous basin, SP: shallow platform, CP:chalky platform, SM: slope and deep marine basin; ? sampling sites. the sediment blocks. Twenty shoots of F. alata from the Czech Republic were prepared for light microscopy examination (i.e. cuticles were mounted on a slide after moderate KOH attack) and no trace of fungal infection could be detected. Also no trace of fungal infection could be detected under scanning electron microscopy (SEM) on ten additional shoots. The absence of any fungi on F. alata from the Czech deposits is in agreement with numerous microscopic observations previously made by palaeobotanists (KvacÏek, pers. comm.). The Czech samples were thus used as reference samples representing uninfected shoots. Among the 20 shoots of F. alata from France which were also examined by light microscopy, 12 appeared to be invaded by the epiphyllous fungi (Fig. 1b±h). Moreover, SEM observations on ten additional shoots revealed the presence of a concentrated network of hyphae on most of the examined shoots (Fig. 1a). Thus, the French samples were compared with the Czech ones to study the e€ect of fungal infection on higher plant remains. corresponding methyl esters due to the catalytic activity of clays (Arpino and Ourisson, 1971). 2.3.2. Gas chromatography GC analyses were carried out using an Intersmat IGC 121 FL ®tted with a fused silica capillary column, coated with CP-SIL-5CB (25 m  0.32 mm i.d., 0.23 mm ®lm thickness; Chrompack). The temperature program used was 100 to 300 C at 4 C/min and then 300 C isothermal for 20 min, using helium as carrier gas, split injector temperature and FID detector temperature being 300 C. 2.3.3. GC±MS The chromatographic conditions were the same as above using a Hewlett-Packard 5890A chromatograph coupled to a Hewlett-Packard 5980 SeÂrie II mass spectrometer, scanning from 40 to 800 Da, electron energy 70 eV. Compounds were identi®ed by comparison of their retention times and mass spectra with those of reference compounds or with literature data. 2.3. Lipid analyses 3. Results and discussion 2.3.1. Extraction Approximately 50 shoots, corresponding to approximately 600 mg, of each batch of F. alata were crushed in a mortar. Lipid extraction was performed by stirring in 30 ml of CH2Cl2/CH3OH (2/1, v/v) overnight at room temperature. Extracts were recovered after centrifugation (10 min at 4000 rpm). During the overnight extraction free acids were transformed into their 3.1. Uninfected shoots The soluble fraction, recovered after dichloromethane/methanol extraction of the F. alata shoots from the Czech deposit, accounts for 3.8 wt.% of the dried material. Analysis of the extract by GC±MS led to the identi®cation of a number of constituents (Table 1). 1747 T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 Table 1 Composition of the lipid extracts of F. alata fossil shoots Uninfected shoots a Fungi-infected shoots Peak label Constituents Distribution Relative abundanceb Distributiona Relative abundanceb & !! * h b 1 2 3 4 5 ^ 13 6 11 7 8 12 9 10 n-Alkanes n-Acids Sulphur Hydroxybenzaldehyde Benzoic acid Cadalene Beyerane Dehydroabietane Ferruginol Oxoferruginol a, o Diacids Hydroxysuccinic acid p-Anisic acid Dimethylbenzoic acid p-Hydroxybenzoic acid m-Hydroxybenzoic acid Dimethoxypropyl benzene Vanillic acid Dihydroxymethoxybenzoic acid C14±C33 (C29, C17) C10±C32 (C16, C26) S6±S8 (S6) 1.0 (0.4) 0.6 (0.1) 0.4 0.5 0.3 0.7 0.3 0.3 0.3 0.5 ± ± ± ± ± ± ± ± ± C16±C35 (C31, C20) C10±C32 (C16, C30) S6±S8(S6) 1.0 (0.5) 8.4 (0.8) 1.3 0.4 1.0 0.1 ±c ± 0.2 0.1 2.3 0.9 0.3 0.7 0.5 1.3 0.5 1.8 1.3 C4±C10 (C10) a (Cmax., Csubmax.). Relative abundance of the maximum (sub-maximum) of the series, or of the considered compound, calculated with respect to the dominant n-alkane. c Not detected. b The total ion current (TIC) trace (Fig. 3) revealed the presence of low amounts of elemental sulphur, from S6 to S8. Since elemental sulphur was also identi®ed in extracts of Eretmophyllum obtusum, a fossil Ginkgo from the same Cenomanian deposit (Nguyen Tu et al., 1999b), we conclude that the sulfur originated from the surrounding sediment and was likely adsorbed on the shoots. The main series of compounds in the TIC trace (Fig. 3) corresponds to C14 to C33 n-alkanes. It shows a bimodal distribution with a maximum at C29 and a strong odd-over-even predominance in the C23±C33 range [CPI of 2.4 calculated according to Bray and Evans (1961)] and a sub-maximum at C17 and a CPI of 1.2 in the C14±C22 range (Fig. 4). Odd long chain nalkanes are typical lipids of the cuticular waxes of higher plants (e.g. Chibnall et al., 1934; Eglinton and Hamilton, 1963; Bianchi, 1995) and are believed to originate from F. alata shoots. The origin of the short chain n-alkanes without any marked odd/even predominance is less clear. Several origins can be considered. Firstly, they could constitute original constituents of F. alata since short chain n-alkanes without any predominance have been described in leaf lipids from some higher plants such as Pinus and Eucalyptus (Herbin and Robins, 1969). Secondly, they may correspond to degradation products of the fatty acids described below. Indeed, reduction and decarboxylation of fatty acids are known to lead to the formation of hydrocarbons during diagenesis (e.g. Tissot and Welte, 1978). Finally, at least a part of these short chain n-alkanes could correspond to bacterial hydrocarbons since even short chain n-alkanes are characteristic constituents of these organisms (e.g. Oro et al., 1967, Han and Calvin, 1969; Saliot, 1981) which are ubiquitous in soils and sediments. Accordingly, the occurrence of such compounds suggests that even if these shoots of F. alata have not been subjected to fungal activity in the Czech deposit, they probably underwent some microbial degradation. The second most abundant series of compounds after the n-alkanes corresponds to fatty acids ranging from C10 to C18 and maximising at C16 with a strong even/ odd predominance (CPI=0.2). A series of methyl esters of C13 to C32 fatty acids was also detected. It shows a maximum at C16, a sub-maximum at C26 and a strong even/odd predominance similar to that observed for the fatty acids (CPI=0.2). Such a distribution of methyl esters is similar to that usually observed for the fatty acids in higher plants. Esteri®cation of carboxylic acids upon extraction of sediments with solvent mixtures containing methanol was previously reported and is 1748 T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 Fig. 3. TIC trace of the lipid extract of uninfected F. alata shoots. Peak labels refer to Table 1. Symbols in bracket represent minor compounds in a coelution. likely due to the catalytic activity of clays (Arpino and Ourisson, 1971). To test this possibility, a standard acid (C14) was added to a piece of fossil shoot and submitted to the extraction procedure. The corresponding methyl ester was formed in substantial amounts. As a result, it can be considered that (i) the free fatty acids of the extract from F. alata were partially methylated upon extraction and (ii) some of the detected esters could be derived from transesteri®cation of acid moieties linked via ester functions to non GC-amenable constituents. Fig. 4. Distribution of n-alkanes and n-acids in the extracts of uninfected and fungi-infected shoots of F. alata. T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 The total distribution of the fatty acids of the fossil conifer was obtained by adding the distribution of methyl esters to the one of fatty acids: C10-C32 with a maximum at C16 and a sub-maximum at C26 (Fig. 4, Table 1). While long chain even fatty acids are typical of higher plant lipids (e.g. Kolattukudy et al., 1976; Bianchi, 1995), short chain even fatty acids maximising at C16 constitute ubiquitous compounds in the geosphere and their origin can be as diverse as that of short chain n-alkanes, including lipids from higher plants, microalgae and bacteria (e.g. Shaw and Johns, 1986; Meyers and Eadie, 1993). In addition to the fatty acid and n-alkane series, a number of cyclic compounds were identi®ed in the lipid extract from F. alata shoots: (i) benzoic acid and hydroxybenzaldehyde which are rather ubiquitous compounds in higher plant remains and (ii) several terpenoids: cadalene 1 (appendix), beyerane 2, dehydroabietane 3, ferruginol 4 (12-hydroxydehydroabietane) and 7-oxoferruginol 5 (7-oxo-12hydroxydehydroabietane). Cadalene 1 is known to occur as a natural product in higher plant lipids (Adams, 1995a). It is usually considered to be derived from sesquiterpenoids such as farnesol or cadinene present in a number of conifers and has been identi®ed in various sediment samples such as Deep Sea Drilling Project cores and Eocene sediments (Bendoraitis, 1974; Simoneit, 1986). Although beyerane 2 has never been reported in living plants, beyerene occurs commonly in higher plant lipids (Adams, 1995a). Beyerane 2 has been reported from Miocene to Permian crude oils where it was used as an indicator of conifer contribution (Noble et al., 1985). It is considered to be derived from the C20 tetracyclic diterpenoids which occur widely in the leaf resins of conifers (Noble et al., 1985). Dehydroabietane 3 occurs as a natural product in resins but may also be diagenetically derived from abietic acid (Simoneit, 1986). Ferruginol 4 occurs as a natural product in higher plants (e.g. Adams, 1995a) and was previously identi®ed in coal extracts (Baset et al., 1980). Oxoferruginol was previously identi®ed in the lipids of several conifers (e.g. Connolly and Hill, 1991) but may also correspond to a degradation product since microbial oxidation at position 7 of the abietane skeleton has been previously reported (Biellmann and Wennig, 1971). Sedimentary compounds with an abietane skeleton are widely used as conifer indicators (Philp, 1985; Simoneit, 1986). F. alata belongs to the Cheirolepidiaceae, an extinct family. However, some authors include it in the Cupressaceae (e.g. Taylor, 1981) and the presence of a number of terpenoids such as farnesol, cadinene and several C20 tetracyclic terpenoids with abietic skeleton has been reported in this family (e.g. Connolly and Hill, 1991; Adams, 1995b). Thus, the presence of these terpenoids in lipid extracts from F. alata provides additional evidence that these compounds are biomarkers of 1749 conifers. The presence of functional groups of poor stability in the structure of some of these terpenoids is consistent with the excellent morphological preservation of the F. alata shoots. 3.2. Fungal-infected shoots The soluble fraction, recovered after dichloromethane/methanol extraction of F. alata shoots infected by fossil fungi, is slightly more abundant than in the uninfected material; it accounts for 5.1 wt.% of the dried material. Analysis by GC±MS shows that most of the compounds identi®ed in the uninfected samples are also present in the infected shoots (Table 1, Fig. 4). However, these compounds exhibit di€erences in their distributions and relative abundances which can be attributed to (i) di€erences in the environment in which the plants grew since it is well documented that the chemical composition of leaf lipids can vary in order to adapt to environmental variations (e.g. Baker, 1980; Bianchi, 1995) or (ii) the fungal infection. Indeed, the abundance of the acids, recognised as methyl esters in the infected sample, is markedly higher with respect to the n-alkanes when compared to the uninfected shoots (Table 1). Moreover, while a similar range (C10±C32) is observed for the acids in both sets of samples, a much higher relative abundance is noted for the shortest (C10±C18) compounds in the infected samples (Fig. 4). Although environmental variations, especially hydric stress, are known to stimulate fatty acid synthesis (Weete et al., 1978), the above di€erences in saturated fatty acid distribution are more likely to re¯ect a fungal contribution. Indeed, fungal acids have been shown to mostly comprise C16 and C18 fatty acids (Turner, 1971; Weete, 1976; Ratledge and Wilkinson, 1988). When the distribution of the n-alkanes is compared between both sets of samples, there can be noticed, as for the acids, a relative increase in the abundance of the shortest chain compounds (C16±C22) in the case of the infected shoots (Figs. 4 and 5). Moreover, these short nalkanes now exhibit an even-over-odd carbon number predominance (CPI=0.7 in the C16±C24 range). Little is known about fungal hydrocarbons but short chain nalkanes have been reported in fungi (Fisher et al., 1978; Ratledge and Wilkinson, 1988). Moreover, all the fossil fungi described on F. alata shoots belong to the Ascomycota phylum and predominantly even hydrocarbons have been found previously in extracts from the mycelia of fungi from this phylum (Weete, 1976). A number of compounds, not detected in the uninfected samples, occur in the extract from the fungalinfected shoots, including a, o-dicarboxylic acid methyl esters, ranging from C4 to C10 and maximising at C10 (Fig. 5). As discussed before, these diesters in fact correspond to diacids methylated upon extraction due 1750 T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 Fig. 5. TIC trace of the lipid extract of F. alata shoots infected by extinct fungi. Peak labels refer to Table 1. Symbols in bracket represent presence of a minor coeluting compounds. to the catalytic activity of clays. Such diacids are generally considered as bacterial degradation products (Otto et al., 1994) and, as con®rmed by their absence in the uninfected shoots, are unlikely to correspond to primary constituents of F. alata lipids. Moreover, such diacids were also detected in lipid extracts of a fossil Ginkgo collected from the same French deposit (Nguyen Tu et al., 1999b). Since this fossil Ginkgo was not invaded by fungi, these diacids are unlikely to be linked to the fungal infection and likely re¯ect bacterial activity. A mono-unsaturated C18 fatty acid occurs in relatively high amount in the infected sample whereas it was absent from the uninfected one. Based on the relatively high contribution of this acid to fungal acids (Turner, 1971; Weete, 1976; Ratledge and Wilkinson, 1988), we suggest that its presence re¯ects a fungal contribution. The main di€erence between the two extracts is found in the ®rst part of the chromatogram. Indeed, it is dominated by functionalised benzoic compounds which were not detected in the uninfected samples; most contain a methyl ester function corresponding to an acid function present before extraction: p-anisic acid 6, phydroxybenzoic acid 7, m-hydroxybenzoic acid 8, vanillic acid 9, tentatively identi®ed dihydroxymethoxybenzoic acid 10, dimethylbenzoic acid 11 and dimethoxypropyl benzene 12 (Fig. 5). To the best of our knowledge such compounds have not been described previously among the free lipids of higher plants, or in lipid extracts from fossil plants. The substitution pattern of the aromatic ring of some of these acids is similar to that of lignin basic units. Moreover, some of these compounds such as vanillic acid 9 or hydroxybenzoic acids 7, 8 correspond to lignin basic units. Lignin is an ubiquitous constituent of conifers shoots and needles (Sarkanen and Ludwig, 1971) and lignin-like polymers have been reported in the cuticle of spruce needles (KoÈgel-Knaber et al., 1994). Moreover, pyrolytic studies of Cretaceous remains of Frenelopsis oligostomata showed that they consisted mainly of heavily altered lignin (Almendros et al., 1998). Many fungi are known to metabolise lignin more or less intensively, owing to the action of enzymes such as laccase or peroxydases (Kirk and Farrell, 1987; Eriksson et al., 1990). These enzymes can lead to the oxidation of alcohol groups, cleavage of Ca-Cb bonds, cleavage of aryl±Ca bonds, demethoxylation and aromatic ring cleavage (Evans, 1987). The aromatic compounds identi®ed in F. alata could therefore be derived from lignin via degradation by fungi. Indeed, p-anisic acid 6, hydroxybenzoic acids 7, 8, vanillic acid 9 and dihydroxymethoxybenzoic acid 10, detected here in the lipid extract of F. alata shoots of T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 the French deposit, have been described as depolymerisation products of lignin or of lignin model compounds by fungi (Odier and Rouau, 1985; Kirk and Farrell, 1987; Ribbons, 1987; Young and Frazer, 1987; Youn et al., 1995). It should also be noticed that the benzoic acid previously detected in uninfected samples, exhibits here a higher abundance relative to hydroxybenzaldehyde than in the uninfected samples. It could therefore also partially correspond to a lignin degradation product. Moreover, hydroxysuccinic acid 13, present at the beginning of the chromatogram (Fig. 5), could originate from the oxidative cleavage of the ring in aromatic acids, such as those mentioned above, formed through lignin depolymerisation (Odier and Rouau, 1985; Ribbons, 1987). Fungi from the Basidiomycota phylum are considered as the most widely implicated in lignin degradation (Ribbons, 1987; Jakucs and Vetter, 1992; PelaÂez et al., 1995; Breccia et al., 1997); however, a number of Ascomycetes are also able to degrade lignin (Grosclaude et al., 1990; Elghazali et al., 1992; Jakucs and Vetter, 1992; Medel and ChacoÂn, 1992; Whalley, 1996). M. andegavensis and S. cretacea, the fungi identi®ed in F. alata, belong to extinct species. Nonetheless, they belong to the Ascomycete families Microthyriaceae and Micropeltidaceae which belong to the orders of Dothideales and Pleosporales, respectively (Pons and Boureau, 1977). Members of these two orders have been described as lignin-degrading fungi (Zare-Maivan and Shearer, 1988; CleÂment-Demange et al., 1995; Kohlmeyer et al., 1995; Barbosa et al., 1996). Microcyclus ulei, a modern parasitic Dothideale, is even known to invade hevea leaves (Hevea brasiliensis) where it provokes large lesions, leading to leaf loss and tree death (Rivano, 1992). As a result, it can be considered that the epiphyllous fungi associated with F. alata shoots were able to degrade lignin and that the aromatic compounds identi®ed in the lipid extract may constitute a signature of such a lignolytic activity. Moreover, lignin degradation probably occurred prior to leaf fall because (1) the fungi associated with F. alata were parasitic or epiphytic (Pons and Boureau, 1977) and (2) 1751 sedimentation occurred rapidly after leaf fall (Louail, 1984; UlicÏny et al., 1997a). 4. Conclusions GC±MS analysis of extracts of uninfected shoots of F. alata has led to the identi®cation of (i) typical components of higher plant waxes, i.e. long chain nalkanes and fatty acids, (ii) sulphur from the surrounding sediment, and (iii) cyclic components including substantial amounts of terpenoids characteristic of conifers, such as cadalene, beyerane and dehydroabietane and related compounds. Comparison of these extracts with extracts from fungal-invaded shoots revealed, in addition to the above compounds, the presence of substantial amounts of hydroxysuccinic acid and functionalised benzoic compounds which were interpreted as lignin degradation products released by the fungi. These results give, for the ®rst time, preliminary indications of the in¯uence on the composition of extracts from fossil higher plant remains that can be associated with fungal infection. Acknowledgements We thank J. KvacÆek for providing the Czech samples and for his hearty reception in Praha. We are grateful to M. Grably, C. Girardin, G. Bardoux and especially Y. Pouet for technical assistance and GC±MS facilities. Thanks to M.F. Ollivier-Pierre, J. Sakala and R. Grasset for their assistance in ®eldwork. We are also indebted to B. Allard, N Augris, S. Bourdon, J. Broutin, F. Mariotti and F. Baudin for helpful discussions. Thanks should also go to the two anonymous referees for constructive review of the manuscript and to P.F. van Bergen for helpful comments. The study was supported by a grant from C.N.R.S. (FeÂdeÂration de Recherche en Ecologie Fondamentale et AppliqueÂe) and N.E.B. Appendix on next page 1752 T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754 Appendix References Adams, R.P., 1995a. Identi®cation of Essential Oil Components by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, Carol Stream. Adams, R.P., 1995b. Comparisons of the volatile leaf oils of Juniperus rigidita Mig. from Northern China, Korea and Japan. 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