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Water Distribution in Foliose Lichen Species: Interactions between Method of Hydration,
Lichen Substances and Thallus Anatomy
VIRGINIA SOUZA-EGIPSY*, FERNANDO VALLADARES and CARMEN ASCASO
Centro de Ciencias Medioambientales CSIC, Serrano 115 dpdo., 28006 Madrid, Spain
Received: 21 March 2000 Returned for revision: 12 May 2000 Accepted: 6 June 2000 Published electronically: 3 August 2000
Three lichens (Neofuscelia pokornyi,N. pulla and Xanthoria parietina) from a semi-arid habitat were examined using
low-temperature scanning electron microscopy to evaluate the eects of hydration method, lichen substances and
thallus anatomy on the water distribution of hydrated thalli. In the Neofuscelia species, extracellular water within the
thallus was observed in association with cracks in its otherwise impervious upper cortex, while X. parietina showed
abundant extracellular water between medullary hyphae. Spraying the thalli followed by maintenance for 14±20 h in
a water-saturated atmosphere led to the disappearance of the external water ®lm in X. parietina but not in the
Neofuscelia species. Surface water was abundant in specimens of all species immediately after spraying for 15 min. No
extracellular water was observed inside the thallus 14±20 h after spraying, but after rinsing with acetone its presence
was detected in all three species. Hydric strategy correlated with cortex hygroscopicity: X. parietina, an aero-
hygrophytic species, had a more hygroscopic upper cortex than the Neofuscelia species, which are substrate-
hygrophytic. The hygroscopicity of the upper cortex was linked with the amount of extracellular water in the thalline
interior. Dierences between X. parietina and Neofuscelia in the polarity and distribution of their lichen substances
agreed with species dierences in the presence and distribution of free water both as a ®lm over the surface and inside
the thallus. Lichen substances appear to play a role in the maintenance of air-®lled intrathalline spaces in species
whose anatomy, habitat, or both, favour water-logged conditions. #2000 Annals of Botany Company
Key words: Lichen, water relations, semi-arid, lichen substances, LTSEM, thallus anatomy, extracellular water,
Neofuscelia pokornyi (Ko
Èrb.) Essl., Neofuscelia pulla (Ach.) Essl., Xanthoria parietina (L.) Th. Fr.
INTRODUCTION
Water relations are of major ecophysiological interest in
lichenology (Galun, 1988). Numerous studies report how
lichens of dierent morphology respond to a frequently and
rapidly changing availability of water (Kappen and
Valladares, 1999). Only a limited number of thallus types
have been the subject of intrathalline water localization
studies (see Scheidegger et al., 1997). Previous reports
indicate that free water is not usually present in the internal
parts of the lichen thallus and that all the strati®ed foliose
and fruticose lichens examined to date keep their medullary
space air-®lled even under water-saturated conditions
(Brown et al., 1987;Ott and Scheidegger, 1992;Scheidegger,
1994;Scheidegger and Schroeter, 1995;Scheidegger et al.,
1995;Schroeter and Scheidegger, 1995). The possibility that
water is able to accumulate in extracellular spaces inside the
thallus has important implications for lichen physiology, in
particular with regard to the increased resistance to CO2
diusion in water. It has been suggested that free extra-
cellular water inside the thallus could be of less importance
as a CO2diusion barrier than super®cial water ®lms
(Scheidegger, 1995). In some Lasallia species, certain
structural features combine intercellular water storage
with air-®lled spaces where gas exchange is facilitated
(Valladares et al., 1998). In disagreement with the suggest-
ions of other authors, it seems that not all lichen species
have a hydrophobic thalline interior (Honegger and Peter,
1994;Honegger et al., 1996).
The water relations of lichens also seem to be aected by
the accumulation of secondary compounds (lichen sub-
stances). In 1926, Goebel suggested that lichen substances
encrusted in the walls of fungal hyphae could make the
medulla water repellent. Green et al. (1985) suggested that
certain lichen substances maintained water-free zones thus
facilitating gas exchange for photosynthesis. However,
Lange et al. (1997) recorded no change in high net photo-
synthesis rates maintained at high water contents in
Diploschistes muscorum following the removal of lichen
substances.
Since water relations are aected by thallus anatomy
(Larson, 1979), and given the suggestion that the distribu-
tion of lichen substances may be responsible for medullary
hydrophobic properties (Honegger, 1993,1994), inter-
actions between these two factors may make generalizations
on the in¯uence of each factor alone on water storage
invalid. The present study was designed to explore these
interactions by observing water storage in lichens of similar
morphology that dier in their anatomy and type and
distribution of lichen substances. The eects of lichen
substances on water storage and distribution were assessed
by extraction of the substances with acetone.
In nature, lichens undergo hydration in several ways
(dew, rain, high relative humidity), and similarly in
experimental studies lichens are hydrated according to
dierent protocols. In particular, the presence of external
Annals of Botany 86: 595±601, 2000
doi:10.1006/anbo.2000.1224, available online at http://www.idealibrary.com on
0305-7364/00/090595+07 $35.00/00 #2000 Annals of Botany Company
* For correspondence. Fax 34 91 564 08 00, e-mail ebvs307@ccma.
csic.es
FIGS 1±6. Legend on facing page.
596 Souza-Egipsy et al.Ð Water Distribution in Foliose Lichens
water ®lms and the maintenance of internal extracellular
water can be aected by the time permitted for the dierent
lichen compartments, which show dierent starting levels of
hydration (e.g. cell walls, cell interior, extracellular spaces),
to reach equilibrium. This prompted us to include the speed
of hydration (rapid vs. slow) as an additional factor
possibly aecting water distribution.
Suprasaturated conditions (high hydration levels that
reduce net photosynthesis through increased resistance to
diusion) are not equally relevant for lichens of dierent
microhabitats. Thus, it would be expected that lichens from
dierent types of environment develop dierent mechan-
isms to avoid suprasaturation. One of the species selected
for the present study (Xanthoria parietina) was found grow-
ing on microsites where the main source of water was the air
(direct rain, water vapour) and may thus be described as an
aero-hygrophytic species. The other species (Neofuscelia
sp.) were obtained from less exposed areas where water was
available mostly from the substrate (run-o) and they may
therefore be considered substrate-hygrophytic. Based on
these features, it was hypothesized that thallus anatomy
and lichen substances could play a role in avoiding supra-
saturation conditions.
MATERIALS AND METHODS
Species and habitat
Lichen specimens were collected from the semi-arid habitat
of Almeria (southeastern Spain) on the Mediterranean
coast. This area has a mean annual temperature of 16.58C
and mean annual rainfall in the range 250±330 mm. The
coastal species Neofuscelia pulla (Ach.) Essl. and Xanthoria
parietina (L.) Th. Fr. were collected from the Cerro de
Enmedio, San Jose
Â(UTM 30SWF80696) and the Barranco
del Monsul (UTM 30SWF748657). These two species grow
on volcanic rocks (andesite) on northern hill slopes;
specimens of X. parietina were found growing on exposed
rock surfaces (e.g. sharp edges of boulders), while N. pulla
grows on relatively protected rock surfaces close to the soil.
The inland, terricolous species Neofuscelia pokornyi
(Ko
Èrb.) Essl., was collected from marls of the Barranco
del Cautivo, Tabernas (UTM 30SWF493962) and from
gypsum crusts of the Venta de Yesos, Tabernas (UTM
30SWG629049).
Hydration method
Before observation of lichen thalli using low-temperature
scanning electron microscopy (LTSEM), specimens were
hydrated with distilled water via two methods. In the ®rst,
referred to as rapid hydration, dry specimens were abun-
dantly sprayed for 15 min and kept on wet ®lter paper at
room temperature and under low light (approx. 50 mmol
mÿ2sÿ1PPFD) until LTSEM examination. In the second,
slow hydration, method, dry samples were sprayed with
distilled water for 15 min as for the rapid hydration method
but were subsequently incubated at 58C in the dark in a
saturated atmosphere for 14±20 h prior to LTSEM
observation. The original substrate of the specimen samples
was retained until the moment that a small fragment was
cut for LTSEM.
Lichen substances
Lichen substances were removed according to a modi®ca-
tion of the method of Solhaug and Gauslaa (1996)
employing dry lichen fragments carefully detached from
the substrate. Each fragment was rinsed in pure acetone
(maximum 0.2 % water content) four times, for 5 min each
time at room temperature. Fragments were then left for at
least 12 h at room temperature in an open container to
ensure vaporization of any residual acetone. The four
acetone extracts were kept at 48C for subsequent analysis by
thin layer chromatography (TLC). The TLC protocol
followed was that of Culberson (1972) with modi®cations
by Manrique and Crespo (1983). Lichen substances were
identi®ed by TLC on the basis of their performance in three
solvent systems (A, toluene±dioxane ±acetic acid 180 : 60 : 8;
B, hexane ±diethyl ether ±formic acid 130: 100 : 20; and C,
toluene±acetic acid 200: 30) with respect to the performance
of two standards (atranorine, Sigma Chem. Co.) and
norstictic acid (extracted from Parmelia acetabulum
(Neck.) Duby.) on 60 F254 silica gel plates (Merck 5554).
To explore the possible eects of detaching the thalli from
their substrate, a second set of specimens of N. pulla were
rinsed in acetone attached to the original substrate ®ve
times, for 5 min each time.
Low temperature scanning electron microscopy
Small fragments of hydrated and dehydrated thallus from
each specimen were cut with a razor blade, mounted using
O.C.T. compound (Gur BDH, UK) and mechanically ®xed
onto a specimen holder at room temperature. The
fragments were immediately plunge-frozen in slushed
nitrogen and directly transferred onto the cryo-chamber,
pre-cooled to ÿ1808C, via an air-lock transfer device. The
frozen fragment was then fractured with a blade pre-cooled
in the cryo-chamber to observe a transverse section of the
thallus interior. The specimen was then sputter coated with
gold for 2 min 15 s using a 10 mA current in the cryo-
chamber, and transferred to the SEM-chamber, pre-cooled
FIGS 1±6. LTSEM micrographs of freeze-fractured saturated thalli of Neofuscelia pulla (Figs 1, 3 and 5) and Xanthoria parietina (Figs 2, 4 and 6)
following rapid (Figs 1 and 2) and slow hydration (Figs 3 and 4). Figures 5 and 6 correspond to slowly hydrated thalli subsequently rinsed in
acetone. Fig. 1. Asterisks indicate the surface water ®lm; the arrow indicates extracellular water in the upper cortex; note lichen substances on algal
surfaces. Figure 2 shows surface water ®lms, indicated by arrows; internal extracellular water is indicated by asterisks. Fig. 3. The arrow indicates
surface water ®lm; note lichen substances and calcium oxalate crystals in the algal layer and upper medulla. Figure 4 illustrates the absence of
a surface water ®lm and internal extracellular water in X. parietina hydrated slowly. Fig. 5. Asterisks indicate internal extracellular water; the
arrow points to calcium oxalate crystals on medullary hyphae. Fig. 6. Asterisks indicate internal extracellular water in the medulla. Bars 10 mm
(Figs 1 and 5) and 20 mm (Figs 2±4 and 6).
Souza-Egipsy et al.Ð Water Distribution in Foliose Lichens 597
to ÿ150±1608C, where it was observed at an accelerating
voltage of 10±15 kV. Duplicate specimens were slowly
heated to ÿ908C in the SEM-chamber after fracturing, and
kept at this temperature for 2 min to sublimate the ®rst
super®cial micrometers of water (etching). This step aids
observation of the lichen ultrastructural elements and
permits identi®cation of the water fraction and con®r-
mation of its location. The sublimation process was
monitored on the SEM screen to con®rm that the empty
spaces in the etched specimens were originally water-free.
After this step, the specimen was returned to the cryo-
chamber, sputter coated with gold as before, and returned
to the SEM-chamber. All the micrographs shown in this
study were obtained from etched samples except for the
dehydrated specimens. The instrumental set up consisted of
a CT 1500 Cryotrans system (Oxford Instruments, UK)
mounted on a Zeiss 960 scanning electron microscope.
Hygroscopic features of the thallus
The hygroscopic features of the dehydrated upper cortex
were evaluated according to the method described by
Valladares (1994b). This method simulates natural thallus
hydration using liquid water. Comparison between dierent
species shows the anity of the upper surface for liquid
water, or a lesser or greater hydrophobic/hydrophilic
nature. A 5 ml drop of Quink Solv-X royal blue washable
ink (Parker, UK) was deposited on the upper cortex of each
lichen specimen. The sizes of the stains were compared to
those produced by a drop of ink of the same volume on two
reference materials: ®lter paper (Whatman, hardened,
ashless #542) as a porous, hydrophilic surface and
Para®lm#(American National Can TM) as a hydrophobic,
impermeable surface. The maximum diameter attained by
the stains was measured using a binocular lens with the aid
of a camera lucida. The data obtained correspond to the
stains produced by ten drops on ®ve±nine thalli per species.
The Student±Newman±Keuls test (n10) was used to
compare data in each group.
RESULTS
Eects o the hydration method
Surface water ®lms. Hydrated thalli of all three species
had a glossy upper surface due to a water ®lm clearly visible
by LTSEM (Figs 1, 2 and 7). In each specimen, the water
®lm was observed on both the upper and the lower thallus
surface (Fig. 2). The method of hydration had an eect on
the presence of a water ®lm in Xanthoria parietina, but not
in Neofuscelia pokornyi or N. pulla. Spraying X. parietina
thalli and keeping them in a water-saturated atmosphere for
14±20 h before LTSEM observation (slow hydration) led to
the disappearance of the water ®lm (Fig. 4) that was clearly
visible when thalli were observed after 15 min of spraying
(rapid hydration, Fig. 2). A water ®lm, although discon-
tinuous (Fig. 8), was always present regardless of the
hydration method in the other two species (Figs 1,2,7
and 8).
Internal extracellular water. The hydration method had an
eect on the presence or absence of free water in the extra-
cellular spaces within the thallus. Free water was found in
the three species after rapid hydration (Figs 1,2and 7) but
not observed in slowly hydrated specimens (Figs 3,4and 8).
The internal distribution of free water diered between
species. Free water was most abundant in X. parietina and
detected both in the medulla and the vicinity of the
photobiont cells (asterisks in Fig. 2). In N. pokornyi and
N. pulla, it was restricted to the upper cortex and photo-
biont layer (arrows in Figs 1 and 7). Free extracellular water
in the upper thallus zones of N. pokornyi and N. pulla was
associated with ®ssures or discontinuities of the upper
cortex that served as conduits for the water ®lm (Figs 1 and
7). The remaining continuous regions of the upper cortex of
N. pulla were impervious to the water ®lm (Figs 3 and 8).
Distribution of lichen substances and interactions with
hydration method
The thalli of X. parietina did not lose colour after acetone
rinsing, despite the intense colour of the acetone extracts.
Parietin was the main substance in the thin layer
chromatographs (TLCs) of X. parietina, while stenosporic
acid was the main phenolic compound in the TLCs of both
N. pokornyi and N. pulla. Parietin is more polar than
stenosporic acid, as indicated by higher Rfvalues in two out
of three solvents (7, 6, 7±8 vs. 5, 6, 6 for parietin and
stenosporic acid in solvents A, B, and C, respectively).
Parietin was mainly concentrated in the upper cortex of
X. parietina. In the two Neofuscelia species, stenosporic acid
and other minor crystallized lichen substances were found
mainly in the medulla and the algal layer on the surface of
both hyphae and algae (Figs 1 and 7). Lichen substances
could be distinguished from calcium oxalates in the LTSEM
micrographs by dierences in shape: crystallized lichen
substances formed thinner bodies than calcium oxalates,
and while the former tended to be like irregular needles, the
latter were polyhedral (arrow in Fig. 5). LTSEM obser-
vation of samples rinsed in acetone showed that the lichen
substances had not been completely eliminated (arrow in
Fig. 9). The extraction of these substances was more
ecient when the specimens were detached from their
substrate.
Free extracellular water was observed within the hydrated
thallus of specimens of each species rinsed in acetone,
irrespective of the hydration method. The main eect of
the partial extraction of lichen substances with acetone
was the maintenance of free extracellular water inside the
thallus after slow hydration (Figs 5,6and 9). Qualitative
dierences in the amount and distribution of free water
were observed between species and hydration method.
X. parietina contained more free water than the other
species. The water was observed in several spaces in the
medulla (asterisks in Figs 2 and 6). In the other species,
most of the water was restricted to small spaces in the upper
cortex and single points within the photobiont layer (Figs 1,
5,7and 9). In Neofuscelia the quantity of water in the
extracellular spaces inside the thallus depended on the
distribution of the ®ssures in the upper cortex, while in
598 Souza-Egipsy et al.Ð Water Distribution in Foliose Lichens
X. parietina it depended on the connection of spaces
between bundles of medullary hyphae. Acetone rinsing did
not modify the formation of surface ®lms of water in any of
the species. Surface water ®lms were present whatever the
hydration method in Neofuscelia species but only after
rapid hydration in X. parietina, as in the samples without
any previous treatment.
Anatomy and hygroscopic features of the upper cortex
The upper cortex of all three species examined was para-
plectenchymatous and made up of leptodermous fungal
cells. Comparison of dry and wet images of the upper cortex
revealed species dierences in the gelatinous matrix
surrounding the fungal hyphae. The matrix was dense and
homogeneous in the Neofuscelia species, both in the
hydrated and dry state (Figs 1 and 10), while in X. parietina
the matrix was homogeneous when hydrated (Fig. 2) but
reticulate and shrunken when dry (Fig. 11). Occasional
®ssures (e.g. Figs 1 and 7) interrupted the sharply de®ned
upper cortex of N. pulla and N. pokornyi. The upper cortex of
X. parietina was more irregular and wrinkled with areas of
accumulated dead cells and necrotic material giving rise to a
porous texture. The lumina of cortical hyphae were larger
than those of the hyphae of other regions in X. parietina but
were of similar size in the Neofuscelia species.
The hygroscopic features of the upper surfaces of the
three species were dierent, as shown by the application of
drops of water-soluble ink. The drops applied to the surface
of X. parietina thalli produced stains that were of a signi®-
cantly greater diameter than those on the Neofuscelia thalli
surfaces (Fig. 12). The stains observed on the X. parietina
thalli were not signi®cantly dierent from those produced
on ®lter paper, while those on Neofuscelia were not
signi®cantly dierent from those on a impermeable surface
(Para®lm#) (see Fig. 12 all con®dence limits 50.01).
DISCUSSION
The species examined in the present study share a foliose
thallus morphology, but their hydric strategy diers, as
shown by their water distribution pattern and microhabitat
preferences. Free water was found within the extracellular
spaces of the thallus interior after rapid hydration of the
lichens. Species dierences in the presence and amount of
free extracellular water inside the thallus (X. parietina
consistently showed more free water) can be explained by
dierences in the texture and hydrophilic features of the
upper cortex. The reticulate upper cortex of X. parietina was
very hygroscopic, despite the accumulation of lichen sub-
stances (mostly parietin) in this layer (Fig. 12). In contrast,
FIGS 7±9. LTSEM micrographs of freeze-fractured saturated thalli of
Neofuscelia pokornyi. Fig. 7. Rapid hydration. Asterisk indicates the
surface water ®lm; arrows indicate internal extracellular water; note the
irregular crystals of lichen substances. Fig. 8. Slow hydration. Arrow
points to the discontinuous water ®lm. Fig. 9. A slowly hydrated
thallus rinsed in acetone. Asterisk indicates internal extracellular water
and the arrow points to remnants of lichen substances on hyphal cell
walls. Bars 10 mm.
Souza-Egipsy et al.Ð Water Distribution in Foliose Lichens 599
the upper cortex of N. pokornyi and N. pulla was
impervious and only permitted the entry of liquid water
through cracks in the surface (Figs 1 and 7). Since lichen
substances accumulate in the medulla and not in the upper
cortex in these two species, hydrophobic lichen substances
cannot be responsible for the hydrophobic nature of this
surface. The waterproof cortex of Neofuscelia minimizes the
risk of internal water-logged spaces in terricolous and saxi-
colour species exposed to run-o. This impermeability
might be related to ®lms of hydrophobinsÐhydrophobic
proteins secreted by many fungi (DeVries et al., 1993;
Wessels, 1996).
In X. parietina, the external water ®lm disappears on slow
hydration since this surface shows more permeable and
hydrophilic features that favour water absorption. Liquid
water cannot accumulate on the upper cortex as occurs
when this layer is more impervious and hydrophobic.
A porous upper cortex, like that of X. parietina, improves
water uptake from the air but is not an ecient barrier
against water loss by evaporation as discussed in Rundel
(1982) and Valladares et al. (1998). Periods of metabolic
activity (hydration under natural conditions) might be
extended in X. parietina due to water storage in the medulla
observed in the present study, which could counteract the
fast dehydration that is associated with a permeable and
porous upper surface and exposed microhabitat conditions.
This distinction has led to the de®ning of aero-hygro-
phytic and substrate-hygrophytic species (Sancho and
Kappen, 1989). In agreement with the present ®ndings,
aero-hygrophytic species of Umbilicaria have porous upper
cortices, while the reverse is true of substrate-hygrophytic
species (Larson, 1979,1981,1987;Scott and Larson, 1984;
Sancho and Kappen, 1989;Valladares, 1994a,b;Valladares
et al., 1998). Moreover, aero-hygrophytic strategy can also
be linked to the structural and functional features of the
thallus. For instance, the two aero-hygrophytic lichens with
lichen substances in the upper cortex, X. parietina examined
here and Pseudevernia furfuracea explored by Scheidegger
et al. (1995), show upper cortices of dierent hygroscopi-
cities (X. parietina very hygroscopic, P. furfuracea imper-
vious) possibly due to dierences in the nature of the
substances involved. The structural-functional dierences
between these two aero-hygrophytic species can be
explained by water availability in their respective habitats.
Water is very limited in the arid Mediterranean habitats
of X. parietina, while it is much more abundant in the
temperate and mountain forest habitats of P. furfuracea.
FIGS 10 and 11. LTSEM micrographs of free-fractured dehydrated thalli. Fig. 10. Neofuscelia pulla. Bar 20 mm. Fig. 11. X. parietina.
Bar 5mm.
abb
ba
14
12
10
8
6
4
2
0
Xp Npo Npu Pf FP
Maximum diameter (mm)
FIG. 12. Hygroscopic features of the upper cortex of Xanthoria
parietina (Xp), Neofuscelia pokornyi (Npo) and N. pulla (Npu) in
comparison with impermeable Para®lm#(Pf) and porous Whatman
®lter paper (FP) estimated as the maximum diameter (mm) of the stain
produced by a 5 ml drop of water-soluble ink. Species and materials
sharing the same letter showed no signi®cant dierence (ANOVA on
ranks P50.01).
600 Souza-Egipsy et al.Ð Water Distribution in Foliose Lichens
Thus, the risk of suprasaturation is minimized in
P. furfuracea with an impervious upper cortex while, in
the case of X. parietina, suprasaturation conditions are rare
and brief, due to the exposed nature of its microhabitat.
Free extracellular water was observed within the thallus
of slowly hydrated specimens of the three species rinsed in
acetone. This ®nding suggests that even though lichen
substances are not capable of conferring hydrophobic
properties to the upper cortex and medulla of X. parietina
or the photobiont layer of the Neofuscelia species, in the
long run, the presence of lichen substances reduces water-
logging of the thallus interior and provides for a relatively
water-free photobiont layer.
In conclusion, the eects of lichen substances on water
distribution within the thallus dier according to species
since: (1) not all lichen substances are equally hydrophobic
due to dierences in their polar and chemical nature; and
(2) each species has a particular type, amount and thalline
distribution of lichen substances. These ®ndings suggest
that lichen substances may play a role in the maintenance of
air-®lled intrathalline spaces in species whose anatomy,
habitat, or both, favour suprasaturation conditions. Com-
plex interactions between the method of hydration, the type
and distribution of lichen and other substances present in
the cell walls, together with the anatomy of the thallus, give
rise to a highly dierent thalline distribution of free water in
each species. The complexity of the interactions between the
three factors examined here may explain the discrepancies
between previous ®ndings with regard to the existence of
free water inside fully-hydrated lichens and the in¯uence of
this free water on thallus gas exchange.
ACKNOWLEDGEMENTS
The authors thank Fernando Pinto for technical assistance
with the LTSEM procedures, and Estela Serin
Äa
Âand
Rosario Arroyo for help with the TLC analyses. Financial
support was provided by the Spanish Ministry for Educa-
tion and Culture (DIGCYT PB95-0067 and PB98-0679).
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