Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue12.Dec2000:

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. The Journal of Essential Oil Research 7, 49±52.
Alexopoulos, J.C., Mims, C.W., Blackwell, M., 1996. Introductory Mycology, Wiley and Sons, New York.
Almendros, G., GonzaÂlez-Vila, F.J., Martin, F., Sanz, J.,
AÂlvarez-Ramis, C., 1998. Appraisal of pyrolytic techniques
n di€erent forms of organic matter from a Cretaceous
basement in Central Spain. Organic Geochemistry 28, 613±
623.
Arpino, P., Ourisson, G., 1971. Esteri®cation and transesteri®cation induced in sediments by methanol and ethanol.
Analytical Chemistry 43, 1656±1657.
Baker, E.A., 1980. Chemistry and morphology of plant epicuticular waxes. In: Cutler, D.F., Alvin, K.L., Price, C.E.

(Eds.), The Plant Cuticle. Academic Press, London, pp. 39±
165.
Barbosa, A.M., Dekker, R.F.H., St Hardy, G.E., 1996.
Veratryl alcohol as an inducer of laccase by an Ascomycete,
Botrosphaeria sp., when screened on the polymeric dye Poly
R-478. Letters in Applied Microbiology 23, 93±96.
Baset, Z.H., Pancirov, R.J., Ashe, T.R., 1980. Organic compounds in coal: Structure and origins. In: Douglas, A.G.,
Maxwell, J.R. (Eds.), Advances in Organic Geochemistry
1979. Pergamon, Oxford, pp. 619±630.
Bending, G.D., Read, D.J., 1997. Lignin and soluble phenolic
degradation by ectomycorrhizal fungi. Mycological Research
10, 1348±1354.
Bendoraitis, J.G., 1974. Hydrocarbon and biogenic origin in
petroleum-aromatic triterpenes and bicyclic sesquiterpenes.
In: Tissot, B., Bienner, F. (Eds.), Advances in Organic
Geochemistry 1973. Technip, Paris, pp. 209±224.
Bianchi, G., 1995. Plant waxes. In: Hamilton, R.J. (Ed.),
Waxes: Chemistry, Molecular Biology and Functions. The
Oily Press, Dundee, pp. 175±222.

T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754
Biellmann, J.F., Wennig, R., 1971. Degradation microbienne
de l'acide deÂhydroabietique. Bulletin de la SocieÂte Chimique
de France 5, 1676±1686.
Bray, E.E., Evans, E.D., 1961. Distribution of n-parans as a
clue to recognition of source beds. Geochimica et Cosmochimica Acta 22, 2±15.
Breccia, J.D., Bettucci, J.D., Piaggio, M., SinÄeriz, F., 1997.
Degradation of sugar cane bagasse by several white-rot
fungi. Acta Biotechnologica 17, 177±184.
Chibnall, A.C., Piper, S.H., Pollard, A., Williams, E.F., Sahai,
P.N., 1934. The constitution of the primary alcohols, fatty
acids and parans present in plant and insect waxes. The
Biochemical Journal 28, 2189±2208.
CleÂment-Demange, A., Nicolas, D., LegnateÂ, H., Rivano, F., Le
Guen, V., Gnagne, M., Chapuset, T., 1995. HeÂveÂa, strateÂgies
de seÂlection. Plantations, Recherche, DeÂveloppement 2, 5±19.
Connolly, J.D., Hill, R.A., 1991. Dictionary of Terpenoids,
Chapman and Hall, London.
Cranwell, P.A., 1981. Diagenesis of free and bound lipids in
terrestrial detritus deposited in a lacustrine sediment.
Organic Geochemistry 3, 79±89.
Cranwell, P.A., 1984. Lipid geochemistry of sediments from
Upton Broad, a small productive lake. Organic Geochemistry 7, 25±37.
Dilcher, D.L., 1965. Epiphyllous fungi from Eocene deposits in
western Tenessee, USA. Palaeontographica 116, 1±54.
Eglinton, G., Hamilton, R.J., 1963. The distribution of alkanes.
In: Swain, T. (Ed.), Chemical Plant Taxonomy. Academic
Press, London, pp. 187±217.
Elghazali, B., Gas, G., Fallot, J., 1992. BiodeÂgradation des lignocelluloses de vigne (Vitis vinifera cv. Cabernet Sauvignon)
par Eutypa lata (Pers. Fr.). Tul. Vitis 31, 95±103.
Eriksson, K.-E., Blanchette, R.A., Ander, P., 1990. Microbial
and Enzymatic Degradation of Wood and Wood Components, Springer-Verlag, Berlin.
Evans, C.S., 1987. Lignin degradation. Process Biochemistry
22, 102±105.
Fisher, D.J., Brown, G.A., Holloway, P.J., 1978. In¯uence of
growth medium on surface and wall lipids of fungal spores.
Phytochemistry 17, 85±89.
Giannasi, D.E., Niklas, K.J., 1981. Comparative palaeobiochemistry of some fossil and extant fagaceae. American
Journal of Botany 68, 762±770.
GonÄi, M.A., Nelson, B., Blanchette, R.A., Hedges, J.I., 1993.
Fungal degradation of wood lignins: geochemical perspectives from CuO-derived phenolic dimers and monomers.
Geochimica et Cosmochimica Acta 57, 3985±4002.
Grosclaude, C., Olivier, R., Romiti, C., Pizzuto, J.C., 1990.
Action antagoniste, sur bois in vitro, de quelques basidiomyceÁtes lignicoles vis-aÁ-vis du Ceratocystis ®mbriata f platani
preÂsent dans les tissus ligneux. Agronomie 10, 403±405.
Han, J., Calvin, M., 1969. Hydrocarbon distribution of algae
and bacteria and microbiological activity in sediments. Proceedings of the National Academy of Sciences of the U.S.A
64, 436±443.
Hedges, J.I., Blanchette, R.A., Weliky, K., Devol, A.H., 1988.
E€ect of fungal degradation on the CuO oxidation products
of lignin: a controlled laboratory study. Geochimica et Cosmochimica Acta 52, 2717±2726.
Herbin, G.A., Robins, P.A., 1969. Patterns of variation and
development in leaf wax alkanes. Phytochemistry 8, 1985±1998.

1753

HlusÏ tõÂk, A., KonsalovaÂ, M., 1976. Frenelopsis alata (K.
Feistm.) Knobloch (Cupressaceae) from the Cenomanian of
Bohemia, a new plant producing Classopolis pollen.
Evolutionary Biology 5, 125±131.
Huang, Y., Lockheart, M.J., Collister, J.W., Eglinton, G.,
1995. Molecular and isotopic biogeochemistry of the Miocene Clarkia Formation: hydrocarbons and alcohols.
Organic Geochemistry 23, 785±801.
Huang, Y., Lockheart, M.J., Logan, G.A., Eglinton, G., 1996.
Isotope and molecular evidence for the diverse origins of
carboxylic acids in leaf fossils and sediments from the
Miocene Lake Clarkia deposit, Idaho, U.S.A. Organic Geochemistry 24, 289±299.
Jakucs, E., Vetter, J., 1992. Comparative studies on the lignocellulose degrading ability of various fungus species.
International Journal for Mycology and Lichenology 5, 217±
235.
Kidston, R., Lang, W.H., 1921. On Old Red Sanstone plant
showing structure, from the Rhynie Chert Bed, Aberdeenshire. Part V. The Thallophyta occurring in the peat-bed, the
succession of the plants through a vertical section of the bed,
and the conditions of accumulation and preservation of the
deposit. Transactions of the Royal Society of Edinburgh 52,
885±902.
Kirk, T.K., Farrell, R.L., 1987. Enzymatic ``combustion'': the
microbial degradation of lignin. Annual Review of Microbiology 41, 465±505.
KoÈgel-Knaber, I., de Leeuw, J.W., Tegelaar, E.W., Hatcher, P.,
Kerp, H., 1994. A lignin-like polymer in the cuticle of spruce
needles: implications for the humi®cation of spruce litter.
Organic Geochemistry 21, 1219±1228.
Kohlmeyer, J., Bebout, B., Volkmann-Kohlmeyer, B., 1995.
Decomposition of mangrove wood by marine fungi and teredinids in Belize. Marine Ecology 16, 27±39.
Kolattukudy, P.E., Croteau, R., Buckner, J.S., 1976. Biochemistry of plant waxes. In: Kolattukudy, P.E. (Ed.), Chemistry
and Biochemistry of Natural Waxes. Elsevier, Amsterdam,
pp. 289±347.
KvacÏek, J., 1999. New data and revision of three gymnosperms
from the Cenomanian of Bohemia Ð Segenopteris variabilis
(VelenovskyÂ) VelenovskyÂ, Mesena bohemica (Corda) comb.
n. and Eretmophyllum obtusum (VelenovskyÂ) comb. n.. Acta
Musei Nationalis Pragae, Series B, Historia Naturalis 55, 15±
24.
de Leeuw, J.W., Rijpstra, W.I.C., Nienhuis, P.H., 1995. Free
and bound fatty acids in the living and decomposing eelgrass
Zostera marina L. Organic Geochemistry 23, 721±728.
Logan, G.A., Eglinton, G., 1994. Biochemistry of the Miocene
lacustrine deposit, at Clarkia Northern Idao, U.S.A. Organic
Geochemistry 21, 857±870.
Logan, G.A., Smiley, C.J., Eglinton, G., 1995. Preservation of
fossil leaf waxes in association with their source tissues,
Clarkia, northern Idaho, U.S.A. Geochimica et Cosmochimica Acta 59, 751±763.
Louail, J., 1984. La transgression creÂtaceÂe au sud du Massif
Armoricain, CeÂnomanien de l'Anjou et du Poitou, CreÂtaceÂ
supeÂrieur de VendeÂe. MeÂmoires de la socieÂte geÂologique et
mineÂralogique de Bretagne 29, 1±333.
Medel, R., ChacoÂn, S., 1992. Ascomycetes lignicolas de Mexico
III. Algunos sphaeriales. International Journal for Mycology
and Lichenology 5, 253±260.

1754

T.T. Nguyen Tu et al. / Organic Geochemistry 31 (2000) 1743±1754

Meyers, P.A., Eadie, B.J., 1993. Sources, degradation and
recycling of organic matter associated with sinking particles
in Lake Michigan. Organic Geochemistry 20, 47±56.
Nguyen Tu, T.T., Bocherens, H., Mariotti, A., Baudin, F.,
Pons, D., Broutin, J. et al., 1999a. Ecological distribution of
Cenomanian terrestrial plants based on 13C/12C ratios. Palaeogeography, Palaeoclimatology, Palaeoecology 45, 79±93.
Nguyen Tu, T.T., Mariotti, A., Bocherens, H., Derenne, S.,
Largeau, C., Pons, D. et al., 1999b. Isotopic analysis of individual compounds: a reliable tool to investigate fossil plant
isotopic signal. Abstracts, 19th Meeting on Organic Geochemistry, 6±10 September 1999, Istanbul, Turkey, pp. 95-96.
Noble, R.A., Alexander, R., Kagi, R.I., Knox, J., 1985. Tetracyclic diterpenoids hydrocarbons in some Australian coals,
sediments and crude oils. Geochimica et Cosmochimica Acta
49, 2141±2147.
Odier, E., Rouau, X., 1985. Les cellulases et les enzymes de
deÂpolymeÂrisation de la lignine. In: Mouranche, J., Costes, C.
(Eds.), Hydrolases et DeÂpolymeÂrases, Enzymes d'IntereÃt
Industriel. Gauthier-Villars, Paris, pp. 199±237.
OroÂ, J., Tornabene, T.G., Nooner, D.W., Gelpi, E., 1967. Aliphatic hydrocarbons and fatty acids of some marine and freshwater microorganisms. Journal of Bacteriology 93, 1811±1818.
Otto, A., Walter, H., PuÈttmann, W., 1994. Molecular composition of a leaf and root-bearing Oligocene Oxbow Lake clay
in the Weisselster Basin Germany. Organic Geochemistry 22,
275±286.
Parish, J.T., Ziegler, A.M., Scoteses, C.R., 1982. Rainfall patterns and the distribution of coals and evaporites in Mesozoic and Cenozoic. Palaeogeography, Palaeoclimatology,
Palaeoecology 40, 67±101.
PelaÂez, F., MartineÂz, M.J., MartineÂz, A.T., 1995. Screening of
68 species of basidiomycetes for enzymes involved in lignin
degradation. Mycological Research 99, 37±42.
Philip, J., Babinot, J.F., Tronchetti, G., Fourcade, E., Guiraud,
R., Bellion, Y. et-al., 1993. Late Cenomanian Palaeoenvironments (94±92 Ma). In: Dercourt, J., Ricou, L.E., Vrielynck, B. (Eds.), Atlas Tethys Palaeoenvironmental Maps.
BEICIP-FRANLAB, Rueil-Malmaison.
Philp, R.P., 1985. Fossil Fuel Biomarkers, Applications and
Spectra, Elsevier, Amsterdam.
Pons, D., 1979. Les organes reproducteurs de Frenelopsis alata
(K. Feistm) Knobloch, Cheirolepidiaceae du CeÂnomanien de
l'Anjou, France. CongreÁs National des SocieÂteÂs Savantes
Bordeaux 1, 209±231.
Pons, D., Boureau, E., 1977. Les champignons eÂpiphylles d'un
Frenelopsis du Cenomanien moyen de l'Anjou (France).
Revue de Mycologie 41, 349±361.
Pons, D., Lauvergeat, J., Broutin, J., 1980. PaleÂoclimatologie
compareÂe de deux gisements du CreÂtace supeÂrieur d'Europe
occidentale. MeÂmoires de la SocieÂte geÂologique de France,
Nouvelle SeÂrie 139, 151±158.
Ratledge, C., Wilkinson, S.G., 1988. Microbial Lipids, Vol. 1,
Academic Press, London.
Ribbons, D.W., 1987. Chemicals from lignins. Philosophical
Transactions of the Royal Society of London, Series A:
Mathematical and Physical Sciences 321, 485±494.

Rieley, G., Collier, R.J., Jones, D.M., Eglinton, G., 1991. The
biogeochemistry of Ellesmere Lake, U.K. I: Source correlation of leaf wax inputs to the sedimentary lipid record.
Organic Geochemistry 17, 901±912.
Rivano. F., 1992. La maladie sud-ameÂricaine des feuilles de
l'heÂveÂa. Etude, en conditions naturelles et controleÂes, des
composants de la reÂsistance partielle aÁ Microcyclus ulei (P.
Henn.) V. Arx. PhD thesis, Universite Paris XI.
Saliot, A., 1981. Natural hydrocarbons in sea water. In:
Duursman, E.K., Dawson, R. (Eds.), Marine Organic
Chemistry. Elsevier, Amsterdam, pp. 327±374.
Sarkanen, K.V., Ludwig, C.H., 1971. Lignins, Wiley Interscience, New York.
Shaw, P.M., Johns, R.B., 1986. Organic geochemical studies of
a recent Inner Great Barrier Reef sediment Ð II. Factor
analysis of sedimentary organic materials input source
determinations. Organic Geochemistry 9, 237±244.
Simoneit, B.R.T., 1986. Cyclic terpenoids of the geosphere. In:
Johns, P.B. (Ed.), Biological Markers in the Sedimentary
Record. Elsevier, Amsterdam, pp. 43±99.
Taylor, T.N., 1981. Palaeobotany, an Introduction to Fossil
Plant Biology, McGraw-Hill, New York.
Tissot, B.P., Welte, D.H., 1978. Petroleum Formation and
Occurrence, Springer-Verlag, Berlin.
Turner, W.B., 1971. Fungal Metabolites, Academic Press,
London.
UlicÏnyÂ, D., KvacÏek, J., SvobodovaÂ, M., SÏpicÏaÂkovaÂ, L., 1997a.
High-frequency sea-level ¯uctuations and plant habitats in
Cenomanian ¯uvial to estuarine succession: PecõÂnov quarry,
Bohemia.. Palaeogeography, Palaeoclimatology, Palaeoecology 136, 165±197.
UlicÆnyÂ, D., HladõÂkovaÂ, J., Attrep Jr., M.J., CÏech, S., HradeckaÂ,
L., SvobodovaÂ, M., 1997b. Sea-level changes and geochemical anomalies across the Cenomanian±Turonian boundary:
PecõÂnov quarry, Bohemia. Palaeogeography, Palaeoclimatology, Palaeoecology 132, 265±285.
Wannigama, G.P., Volkman, J.K., Gillan, F.T., Nichols, P.D.,
Johns, R.B., 1981. A comparison of the fresh and dead leaves
and pneumatophores of the mangrove Avicennia marina.
Phytochemistry 20, 658±666.
Weete, J.D., 1976. Algal and fungal waxes. In: Kolattukudy,
P.E. (Ed.), Chemistry and Biochemistry of Natural Waxe.
Elsevier, Amsterdam, pp. 349±418.
Weete, J.D., Leek, G.L., Peterson, C.M., Currie, H.E., Branch,
W.D., 1978. Lipid and surface wax synthesis in water-stressed cotton leaves. Plant Physiology 69, 675±677.
Whalley, A.J.S., 1996. The xylariaceous way of life. Mycological Research 100, 897±922.
Youn, H.-D., Hah, Y., Kang, S.-O., 1995. Role of laccase in
lignin degradation by white-rot fungi. FEMS Microbiology
Letters 132, 183±188.
Young, L.Y., Frazer, A.C., 1987. The fate of lignin and ligninderived compounds in anaerobic environments. Geomicrobiology Journal 5, 261±293.
Zare-Maivan, H., Shearer, C.A., 1988. Wood decay activity
and cellulase production by freshwater lignicolous fungi.
International Biodeterioration 24, 459±474.