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Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research I. Morphological and Anatomical Characteristics

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Lichens are symbioses of two organisms, a fungal mycobiont and a photoautotrophic photobiont. In nature, many lichens tolerate extreme environmental conditions and thus became valuable models in astrobiological research to fathom biological resistance towards non-terrestrial conditions; including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. All studies demonstrated the high resistance towards non-terrestrial abiotic factors of selected extremotolerant lichens. Besides other adaptations, this study focuses on the morphological and anatomical traits by comparing five lichen species-Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, Buellia frigida, Pleopsidium chlorophanum-used in present-day astrobiological research. Detailed investigation of thallus organization by microscopy methods allows to study the effect of morphology on lichen resistance and forms a basis for interpreting data of recent and future experiments. All investigated lichens reveal a common heteromerous thallus structure but diverging sets of morphological-anatomical traits, as intra-/extra-thalline mucilage matrices, cortices, algal arrangements, and hyphal strands. In B. frigida, R. geographicum, and X. elegans the combination of pigmented cortex, algal arrangement, and mucilage seems to enhance resistance, while subcortex and algal clustering seem to be crucial in C. gyrosa, as well as pigmented cortices and basal thallus protrusions in P. chlorophanum. Thus, generalizations on morphologically conferred resistance have to be avoided. Such differences might reflect the diverging evolutionary histories and are advantageous by adapting lichens to prevalent abiotic stressors. The peculiar lichen morphology demonstrates its remarkable stake in resisting extreme terrestrial conditions and may explain the high resistance of lichens found in astrobiological research.
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Extremotolerance and Resistance of Lichens:
Comparative Studies on Five Species Used
in Astrobiological Research I. Morphological
and Anatomical Characteristics
J. Meeßen & F. J. Sánchez & A. Brandt & E.-M. Balzer &
R. de la Torre & L. G. Sancho & J.-P. de Vera & S. Ott
Received: 1 February 2013 /Accepted: 26 March 2013 /
Published online: 20 July 2013
Springer Science+Business Media Dordrecht 2013
Abstract Lichens are symbioses of two organisms, a fungal mycobiont and a photoauto-
trophic photobiont. In nature, many lichens tolerate extreme environmental conditions and
thus became valuable models in astrobiological research to fathom biological resistance
towards non-terrestrial conditions; including space exposure, hypervelocity impact simula-
tions as well as space and Martian parameter simulations. All studies demonstrated the high
resistance towards non-terrestrial abiotic factors of selected extremotolerant lichens. Besides
other adaptations, this study focuses on the morphological and anatomical traits by com-
paring five lichen speciesCircinaria gyrosa, Rhizocarpon geographicum, Xanthoria
elegans, Buellia frigida, Pleopsidium chlorophanumused in present-day astrobiological
research. Detailed investigation of thallus organization by microscopy methods allows to study
the effect of morphology on lichen resistance and forms a basis for interpreting data of recent
and future experiments. All investigated lichens reveal a common heteromerous thallus struc-
ture but diverging sets of morphological-anatomical traits, as intra-/extra-thalline mucilage
matrices, cortices, algal arrangements, and hyphal strands. In B. frigida, R. geographicum,
and X. elegans the combination of pigmented cortex, algal arrangement, and mucilage seems to
enhance resistance, while subcortex and algal clustering seem to be crucial in C. gyrosa,aswell
as pigmented cortices and basal thallus protrusions in P. c h l o ro p h an u m . Thus, generalizations
Orig Life Evol Biosph (2013) 43:283303
DOI 10.1007/s11084-013-9337-2
J. Meeßen (*)
A. Brandt
E.<M. Balzer
S. Ott
Institut für Botanik, Heinrich-Heine Universität (HHU), Universitätsstr. 1, 40225 Düsseldorf, Germany
F. J. Sánchez
R. de la Torre
Instituto Nacional de Técnica Aeroespacial (INTA), Ctra. de Ajalvir km. 4, Torrejón de Ardoz,
28850 Madrid, Spain
L. G. Sancho
Departamento de Biología Vegetal II, Facultad de Farmacia,
Universidad Complutense de Madrid (UCM), 28040 Madrid, Spain
J.<P. de Vera
Institut für Planetenforschung, Deutsches Zentrum für Luft- und Raumfahrt (DLR),
Rutherfordstraße 2, 12489 Berlin, Germany
on morphologically conferred resistance have to be avoided. Such differences might reflect the
diverging evolutionary histories and are advantageous by adapting lichens to prevalent abiotic
stressors. The peculiar lichen morphology demonstrates its remarkable stake in resisting
extreme terrestrial conditions and may explain the high resistance of lichens found in
astrobiological research.
Keywords Buellia frigida
Circinaria gyrosa
Lichen morphology
Lichen anatomy
CLSM Confocal laser scanning microscopy
EPS Extracellular polymeric substances
LEO Low Earth orbit
LTSEM Low temperature scanning electron microscopy
PAR Photosynthetically active radiation (400700 nm)
PSII Photosystem II
SEM Scanning electron microscopy
SLC Secondary lichen compound
UVR Ultra-violet radiation (100400 nm)
Since the millennium lichens became valuable model organisms in astrobiological research
(Sancho et al. 2008). Remarkable progress to fathom their limits and limitations in resisting
harsh environmental conditions was made in recent experimental approaches. This includes
space exposure experiments as LICHENS II on BIOPAN 5/FOTON M2, LITHOPANSPERMIA
and STONE on BIOPAN6/FOTON M3, and LIFE on EXP OSE-E/EuTEF (Sancho et al. 2007;de
la Torre et al. 2007, 2010a;deVera2012; Raggio et al. 201 1; Onofri et al. 2012;Scalzietal.2012)
and experiments simulating various space conditions such as vacuum and UVR, (de Vera et al.
2003, 2004a, b, 2007, 2008, 2010;delaTorreetal.2002, 2004, 2007, 2010a, b; Sánchez et al.
2012) as well as hypervelocity impacts (Stöffler et al. 2007; Horneck et al. 2008;deVeraandOtt
2010). An overview of previous astrobiological studies on lichens is given in Table 1.All
experiments demonstrated high resistance towards space and Martian environmental parameters
of those lichens that are adapted to harsh abiotic factors in their natural habitat. Furthermore, in
April 2014 two lichen species will be exposed to LEO-space conditions and on-board simulated
Mars conditions for 15 to 18 months during the BIOMEX-experiment on EXPOSE-R2/Zve zda
(ESA call ILSRA-AO 2009). The viability after exposure was assessed by various methods, as
determination of photosynthetic activity by chlorophyll-α-fluorescence of PSII and by light
dependent gas exchange analyses, live/dead-staining of both symbionts utilizing CLSM (apo-
as well as eusymbiotic), LTSEM, cultivation experiments and germination capacity analyses of
the fungal spores (see references above).
Lichens are symbiotic associations formed by two organisms, the heterotrophic fungal
partner (mycobiont) and the photoautotrophic partner (photobiont) which is an eukaryotic
green alga or a prokaryotic cyanobacterium. Many lichens tolerate extreme environmental
conditions, such as scarcity of soil substratum and nutrients, high levels of (toxic) metal ions
(review by Bačkor and Fahselt 2008
), cold and long-term snow coverage (Kappen 1993;
Dyer and Crittenden 2008), heat, intensive insolation with high levels of UVR (Lange 1992;
Nybakken et al. 2004), low water availability, infrequent water supply and extreme drought
284 J. Meeßen et al.
Table 1 Overview of past and recent astrobiological investigations on lichens
Experiment Lichen Facility Duration Tested parameters or
exposure conditions
Viability analysis Reference
Lichens I R. geographicum,
X. elegans
short several samples recovered after
explosion during launch
PSII activity de la Torre et al. 2004
Lichens II R. geographicum,
X. elegans
16 d space exposure, λ >110, >200,
>290, >400 nm, (UVA + UVB
at 2.2×10
kJ m
PSII activity, LTSEM Sancho et al. 2007;
Sancho et al. 2008
Lithopanspermia C. gyrosa, R.
X. elegans
10 d space exposure, λ >110, >200,
>290, >400 nm
PSII activity, LTSEM,
light dependent gas
exchange analysis
de la Torre et al. 2010a;
Raggio et al. 2011
Stone R. geographicum, BIOPAN 6/
10 d space exposure, followed by
atmospheric re-entry
samples totally destroyed
during re-entry
de la Torre et al. 2010a
Life R. geographicum,
X. elegans
559 d LEO temp. + irradiation
(>110 nm, 7×10
kJ m
UV (200400 nm, 9.2×10
kJ m
2), GCR
( 190 mGy), vac. (10
or Mars atm. (100 % CO
, 7 mbar)
Life/Dead staining
(FunI, Bac Light), PSII
activity, fxSEM, TEM,
biont growth assays,
Onofri et al. 2012;
Scalzi et al. 2012,
analysis in progress
Biomex B. frigida,
C. gyrosa
1218 months LEO temp. + irradiation
(>190 nm, up to 8×10
kJ m
vac. (10
Pa) or Mars atm.
(100 % CO
, 7 mbar, 100 % rH)
Life/Dead staining (FunI,
Bac Light), PSII activity,
biont growths assays,
scheduled 20142015,
EVT, SVT in progress
Simulation F. bracteata,
X. ele- gans,
X. parietina
Space Sim. Test
Beds, DLR
4, 8, 16 h 1) vac. (10
Pa), 2) UV (160400 nm,
2.8+8.8+0.7 W m
), 3) vac. + UV (10
Pa, 160400 nm, 2.8+8.8+0.7 W m
Life/Dead staining
(FunI, Sytox Green),
ascospore germination rate
de Vera et al. 2003;
de Vera et al. 2004a
Simulation F. bracteata,
X. elegans
Space Sim.
Test Beds,
DLR Cologne
1, 5, 15,
30 min, 1,
2, 4, 8, 16 h
1) vac. (10
Pa), 2) UV (200400 nm;
40, 466, 3,347 kJ m
) 3) vac. +
UV (10
Pa), 200400 nm; 40, 466,
3,347 kJ m
) 4) vac. + UV (10
160400 nm, 8.1 W m
Life/Dead staining
(FunI, Sytox Green),
ascospore germination rate
de Vera et al. 2004b
Simulation B. frigida,
P. aphthosa
Space Sim.
Test Beds,
DLR Cologne
5, 15, 30, 60,
120, 240,
480 min
UVC (254 nm, 2.1201.8 J m
) Life/Dead staining
(FunI, Sytox Green)
de Vera and Ott 2010
Extremotolerance and Resistance of Lichens 285
Table 1 (continued)
Experiment Lichen Facility Duration Tested parameters or
exposure conditions
Viability analysis Reference
Simulation X. elegans Space Sim.
Test Beds,
DLR Cologne
5 min, 2, 4,
8, 16 h
vac. (2.8×10
Pa) and
UV (200400 nm, 2.8 W m
Life/Dead staining (FunI,
Sytox Green), biont
growths assays
de Vera et al. 2008
Simulation X. elegans HUMIDITY LAB,
DLR Berlin
22 d (1),
1) Mars atm. (95 % CO
, 6 mbar,
100 % rH, 55+20 °C) 2) Mars atm.
(95 % CO
, 6 mbar, 100 % rH, +15 °C) 3)
Mars atm. (95 % CO
, 6 mbar, 100 % rH,
+5 °C) 4) Earth atm. (100 % rH, +15 °C)
PSII activity, Life/Dead
staining (FunI)
de Vera et al. 2007;
de Vera et al. 2010
Simulation X. elegans Shock Recovery
Exp., EMI
< 1 min hypervelocity impact simulations
5, 10, 15, 20, 30, 41.5, and 50 GPa)
Life/Dead staining
(FunI, Sytox Green)
Stöffler et al. 2007;
Horneck et al. 2008
Simulation P. chlorophanum Mars Sim.
DLR Berlin
35 d Mars radiation (2002,500 nm,
kJ m
Mars atm. (95 % CO
, 6 mbar,
49+25 °C, 080 % rH)
PSII activity de Vera et al. 2012
Simulation R. geographicum,
X. elegans
Test, INTA
10, 20 h 1) UV + VIS + NIR (200900 nm) 2)
vacuum + UV + VIS + NIR (3.0×10
bar, 200900 nm)
PSII activity de la Torre et al. 2004
Simulation C. gyrosa PASC at CAB,
120 h 1) Mars UV (200400 nm,
30 mW cm
), 93 °C 2) Mars atm.
(95 % CO
, 0.6 % H
O, 7 mbar),
93 °C 3) Mars UV (200400 nm,
30 mW cm
), vac. (10
bar), 93 °C
PSII activity, light
dependent gas
exchange analysis
Sánchez et al. 2012
286 J. Meeßen et al.
as observed in cold and hot deserts like the dry valleys in continental Antarctica (Marchant
and Head 2007; Hara ńczyk et al. 2008; Sun et al. 2010) and the Andean Atacama Desert
(McKay et al. 2003).
Several adaptive strategies are discussed to contribute to the resistance of lichens towards
extreme environmental conditions and therefore they are of interest for astrobiological
research. Among other factors, such as the photobionts capacity to protect its photosynthetic
apparatus during and regenerate it after stressful conditions (e. g. drought and cold,
Sadowsky et al. 2012), three aspects constituting the extremophile character of many lichen
species should be highlighted: the first one is the poikilohydric nature of lichens.
Poikilohydry allows lichens to tolerate extreme desiccationbut also high UVR/PAR-levels
(Nybakken et al. 2004) and high or low temperatures that may accompany droughtby
passing into an ametabolic state which is referred to as anabiosis or, more specifically,
anhydrobiosis (Kranner et al. 2005). Re-hydration activates the lichen metabolism, what
usually occurs under more moderate and therefore physiologically favourable conditions. It
is known that morphological properties affect thallus water-uptake and water-retention,
which influence the duration of physiological activity (refer to Lange et al. 1999). A second
aspect is the vast variety of secondary lichen compounds (SLCs) formed in high amounts
almost exclusively in the symbiotic state (Henssen and Jahns 1974). They cause PAR- and
UVR-shielding by absorption (Solhaug and Gauslaa 1996; Huneck and Yoshimura 1996;
Solhaug and Gauslaa 2004; Mc Evoy et al. 2006) and are proposed to protect particularly the
The present study focuses on a third aspect: the morphological and anatomical traits that
shape a lichen thallus and adapt it to the habitats dominant (micro)climatic and orographic
factors. As all fungi, mycobionts are modular organisms (Carlile 1995) characterized by
poor differentiation of cell types and plectenchymatic tissues; the photobiont cells show no
differentiation. The low degree of functional cell differentiation is compensated on the next
higher level of organismic organization by an intriguing complexity of lichen morphology. This
is displayed by various growth types (e. g. crustose, placoid, foliose, fruticose, umbilicate) as
well as functional morpho-anatomical structures (e.g. heteromerous thallus stratification,
pseudocyphellae, rhizines, cephalodia, and reproductive structures as apothecia, perithecia,
pycnidia, soredia, and isidia). Valuable introduction into lichen morphology is provided by
Jahns (1988) or Büdel and Scheidegger (1996) while the link between morphological traits and
their adaptative or eco-physiological significance is still an issue of vivid debate in lichenology.
Variation of morphological and anatomical traits unquestionably represents lichen adaptation to
the environmental conditions of the respective ecological niche (Jahns 1988; Büdel and
Scheidegger 1996). Such traits directly influence major physiological aspects of the lichens
biology as effectiveness of photosynthesis, nutrient uptake, nutrient distribution, gas exchange,
but also water uptake and water retention which are fundamental aspects of the poikilohydric
life strategy (Lange et al. 2001). Consequently, morphology and anatomy are crucial factors in
understanding the extremotolerance of many lichen species.
To date, astrobiological studies were performed with eight lichen species. The mycobiont
of all investigated species is an ascomycetes (class Lecanoromycetes). Seven lichens harbour
a coccal green alga of the genus Trebouxia sp. as a photobiont (phylum Chlorophyta, class
Trebouxiophyceae) while Peltigera aphthosa harbours Coccomyxa sp. (Trebouxiophyceae).
All lichens are composed of two eukaryotic symbionts, making the selected species valuable
models to investigate the capacity of eukaryotes and of symbioses to resist non-terrestrial
environmental factors. The species preferentially used in astrobiological research are
Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and currently Buellia
frigida. Recently, promising Martian environment simulations were performed with
Extremotolerance and Resistance of Lichens 287
Pleopsidium chlorophanum (de Vera et al. 2012). Simulation studies were also performed
with Fulgensia bracteata and Xanthoria parietina (de Vera et al. 2004a, b) but due to their
anatomical analogies with X. elegans, both are not addressed by the present study. P.
aphthosa as a mere control to B. frigida in UVC-exposure experiments (de Vera and Ott
2010) was also excluded.
The results of astrobiological research depicted above justify continuing research on the
resistance of lichens and to focus on the questions, if and to what extend the morphological
organization of the symbiotic association contributes to its potential of resistance. Therefore,
detailed investigations of thallus structure and anatomical organization were made by light-
microscopic analysis and fixed scanning electron microscopic analysis. The presented re-
sults give decisive insight into the morphology and anatomy of five out of eight lichens used
to date in astrobiological studies. They allow determining the portion of thallus organization
in the lichens potential of resistance and will give a valuable basis for interpreting the data
of recent and future experiments. The results demonstrate that the peculiar morphology and
anatomy of lichens have a remarkable stake in their resistance towards extreme environ-
mental conditions on Earth as well as towards space and Mars conditions.
Material and Methods
Buellia frigida Darb. (1910) is a frequent, endemic, crustose lichen of maritime and
especially continental Antarctica, colonizing habitats down to 84°S. The thalli grow on
rocks being fully exposed to wind, low temperatures and high irradiation levels during
Antarctic summer and in altitudes up to 2015 m a.s.l. and intact thalli of up 1520 cm are
frequent found on Inexpressible Island (Øvstedal and Lewis Smith 2001). For the present
study, B. frigida was collected on two sites in continental Antarctica: In 1996/1997 at
Inexpressible Island (74°54S, 163°43E) in Terra-Nova-Bay, Ross Sea (by R. L. Smith),
and in 2009/2010 in the vicinity of the German Gondwana Station at Gerlache Inlet (74°38
S, 164°13E), in North Victoria Land (by S. Ott). Air-dried samples were stored at 25 °C
until use. For BIOMEX on EXPOSE-R2/Zvezda samples from Gondwana Station will be
Circinaria gyrosa Sohrabi (2012) was recently revised from Aspicilia fruticulosa
(Sohrabi 2012) and used under its previous name for several space and simulation experi-
ments (de la Torre et al. 2010a; Raggio et al. 2011; Sánchez et al. 2012). C. gyrosa originates
from continental deserts and arid areas of Middle Asia, Eurasia, North America and
Northern Africa. It is adapted to harsh environmental conditions, including heat, drought,
and high levels of solar UVR (Sancho et al. 2000). Samples were collected from clay soils in
high basins of central Spain: Guadalajara, Zaorejas, 1260 m a.s.l. (40°4540N, 02°1208E).
The samples were collected in June 2010 and kept under dark and dry conditions until
Rhizocarpon geographicum (L.) DC (1805) (R. geo.) is a widespread, bipolar lichen,
mostly distributed on northern hemispherical arctic and alpine habitats but also found in
maritime and continental Antarctica. It is found on exposed, often dry, siliceous or granite,
non-calciferous rock in montane, subalpine and alpine or polar zones. The specimen
investigated in this study are collected on two distinct montaneous, respectively alpine sites:
In Spain at the Sierra de Guadarrama mountains near Navacerrada (about 40°4720N,
04°0012W, at about 1400 m a.s.l.), and in Valais, Switzerland at Col du Sanetsch,
N, 07°1711E, at 2140 m a.s.l.) as well as in the vicinity of Zmutt (46°00N,
07°71E, at 1950 m a.s.l.).
288 J. Meeßen et al.
Xanthoria elegans (Link) Th. Fr. (1860) is a cosmopolitic lichen colonizing open sites on
diverse substrata in various habitats, including harsh environments as alpine (up to
7,000 m.s.l. in Himalaya), peninsular Antarctic (Berry Hills, Cape Lachman, James Ross
Island, 64°S) and continental Antarctic inland sites (near Wood Bay, Victoria Land, 74°S,
Øvstedal and Lewis Smith 2001). It is frequent on volcanic, silicate and limestone rock, at
nitrophilic sites, and occasionally found on concrete and other anthropogenic substrata.
Habitats of X. elegans are usually exposed to high levels of UVR (de Vera 2005). For the
present study, thalli of X. elegans were collected in June 2005 at Col du Sanetsch, Valais,
Switzerland (46°2148N, 07°1751E, at 2140 m a.s.l.) and stored at 25 °C until investi-
gation. Thalli of X. elegans from this and from adjacent collection sites (Zermatt, 46°00N,
07°71E, at about 1950 m a.s.l.) were used before in astrobiological studies (de Vera et al.
2003, 2004a, b, 2007, 2008, 2010; Stöffler et al. 2007; Horneck et al. 2008) and in the LIFE
experiments on EXPOSE-E/EuTEF (see de Vera et al. 2012; Onofri et al. 2012).
Pleopsidium chlorophanum (Wahlenb.) Zopf (1855) is a bipolar distributed species found
in North Europe and North America, but also throughout maritime and continental
Antarctica (Øvstedal and Lewis Smith 2001). It is most frequent in continental Antarctica,
where it is widespread down to far inland nunataks at altitudes of up to 2500 m a.s.l. P.
chlorophanum colonizes dry, shaded rocks and cracks. It forms hyphal strands which
penetrate 1015 mm into the rock and facilitate bio-weathering. The samples used for the
present investigation were collected in 2009/2010 next to Gondwana Station at Gerlache
Inlet, North Victoria Land (74°38S, 164°13E). The air-dried samples were stored at 25 °C
until further use.
Explanation of lichenological terminology: heteromerous: internally stratified; pseudo-
cyphellae: cortical pores; hypo-/prothallus: basal/marginal layer of crustose thalli; plecten-
chyma: general term for interwoven hyphal tissues; paraplectenchyma: tissue of isodiametrical
hyphae; prosoplectenchyma: tissue of elongated periclinal hyphae; apothecium: fungal
fruitingbody; hymenium: asci-bearing layer in apothecia; epi-/hypothecium: layer above/below
the hymenium; ascus: ascospore-bearing cell; paraphyse:
sterile hymenial hypha; pycnidium:
conidiospore-forming fungal structure.
Light-Optical Microscopic Studies and Stratification Measurements
Five to twelve specimen of each lichen species were screened on their morphological
properties and representative ones were prepared for further investigations under a stereo-
scopic microscope (M8, Wild Heerbrugg AG). Photo-documentation of the respective lichen
morphology were performed with a Nikon D80 (AF Micro-NIKKOR macro lense, 60 mm
focal length). For each lichen species thin layer sections (1220 μm) were obtained from at
least 4 representative specimen at marginal (distal) and central (proximal) lobes or areolae,
respectively (Table 2), by using a freezing microtome (ca. 30 °C, Frigomobil 1206,
Reichert-Jung) and subsequently stained with 5 % lactoglycerol cotton blue. Analysis of thallus
anatomy and further photo documentation were performed under a digital lightmicroscope
(Axio imager A1). and stratification measurements were done by proprietary software tools
(AxioVision Rel. 4.8.2).
Scanning Electron Microscopy of Fixed Thallus Samples
For each lichen species, thin layer sections (5080 μm) of 23 different lobes/areolae of 24
representative thalli were prepared (as described above), transferred into 2.5 % glutaralde-
hyde in 200 mM cacodylate-buffer, fixed under vacuum and under ambient pressure (30 min
Extremotolerance and Resistance of Lichens 289
each), and washed for 10 min in pure cacodylate-buffer. Afterwards the samples were
mounted in microporous capsules (plano GmbH) and dehydrated by subsequent dilution
series of graduated ethanol (in water) and acetone (in ethanol). Submerged in acetone, the
thallus sections were brought into screw cab containers (Dr. W. Hert Mikrotechnik), closed
with 3 mm copper grids, transferred to a critical point drying device (CPD 020, Balzers
Union), washed thrice, and dried at the critical point temperature of 34.5 °C. The dehydrated
lichen sections were transferred to specimen holders, air dried for 8 h, and gold-sputter
coated for 180 s at 35 mA (Sputter Coater, Agar Scientific Ltd.), followed by detailed
examination of thalline morphology and anatomy by scanning electron microscopy (SEM;
LEO 1430(VP), LEO Electron Microscopy Ltd.).
Buellia frigida
Morphology B. frigida is an epilithic crustose to placoid lichen (Physiciaceae) which forms
greyish to black circular thalli up to 50 cm (Fig. 1a). The thallus margin is composed of
rectangular areolae (max. 0.6×3.0 mm) of black colour and represents the growth zone. The
older central region is formed by angular grey to black coloured areolae and numerous,
black, convex and lecideine apothecia (max. 1 mm). The apothecia bear apically thickened
and pigmented paraphyses and club-shaped asci containing eight highly melanized, two-
celled ascospores each. Asexual reproductive structuresas soredia and isidiaare not
formed while immersed pycnidia are frequently formed in central areolae.
In the hydrated state, the thallus is significantly swollen by water uptake, reducing the
density of its blackish melanin pigmentation in the cortex (Fig. 4a). Consequently, the
photobiont in the algal layer becomes effectively exposed to light in the wet state. During
dehydration, the thallus shrinks, leading to densification of the melanin pigmented cortex
(Fig. 4b). This effect is most obviously observed in the algal rich marginal zones of B.
frigida thalli where the number of algae is highest (Fig. 2a). The thallus surface is mostly
covered by a mucilageous epicortex (Fig. 5) which may appear white in the dehydrated state
(Fig. 1a), usually more prominent in the depigmented central parts of the thallus (Fig. 2a,
Anatomy The stratification is measured at thalli from two collection sites (Inexpressible
Island and Gondwana Station) and for the specimen of the latter habitat in marginal (MS)
and central (CS) thallus sections (Table 2). Despite a decrease of algal layer thickness and
pigmentation in central thallus sections, the differences between sites and sections are low.
Below the epicortex, which is occasionally interrupted in more central areolae, a
paraplectenchymatous cortex of swollen and melanin-pigmented apical cells is located.
From the marginal (e.g. younger) towards the central (e.g. older) thallus sections the cortex
structure remains stable while its pigmentation ceases and the conglutination of the cortical
cells increases, leading to a patchy pattern of pigmented and unpigmented areas. Below, the
algal layer is composed of homogeneously dispersed algal clusters and interwoven hyphae,
both strongly gelatinized (Fig. 3a). The algal layer is more pronounced at the margin but
reduced to singled, patchy clusters in the centre. The occurrence of algal clusters in the
central parts is clearly correlated to the residual melanin-pigmented areas above (Fig. 2a).
Three morphological aspects, (i) depigmentation, (ii) increase of mucilage in the epicortex,
and (iii) ceasing of the algal layer, point to thallus degeneration in the central (e.g. older
290 J. Meeßen et al.
Table 2 Stratification of five lichen species relevant in astrobiological research
Thickness of thalline strata in [μm] ± standard deviation
Lichen (from) Mucilage layer Cortex Sub-cortex Algal layer Medulla Lower cortex Hypo-thallus
pigm. parapl.
B. frigida (from Gondwana Station, North Victoria Land and Inexpressible Island, both Antarctica)
Gondwana 9.4±2.9 13.3±2.9 –– 104.6±51.5 206.1±35.5 ––
Inexpr. Isl. 12.4±2.9 15.8±5.5 –– 68.0±15.3 204.8±43.8 ––
Marginal 8.2±1.8 14.5±1.8 –– 116.9±52.4 205.5±37.2 ––
Central 12.6±2.8 14.8±5.6 –– 67.1±14.3 205.3±41.9 ––
C. gyrosa (from Zaorejas, Guadalajara, Central Spain)
Proximal 20.8±0.9 18.3±1.3 125.7±13.0 69.2±12.0 385.9±12.2 ––
Distal 24.3±9.5 21.4±2.4 147.1±29.0 78.3±39.7 346.3±111.0 ––
R. geographicum (from Navacerrada, central Spain and Col du Sanetsch, Valais, Switzerland)
Valais 28.1±3.5 –– 94.7±15.1 159.1±61.3 66.0±37.7
Navacerr. 5.0±1.5 16.1±11.9 –– 123.6±4.4 71.7±12.2 61.9±15.5
X. elegans (from Col du Sanetsch, Valais, Switzerland)
Marginal 22.1±4.5 18.8±8.2 65.4±10.6 146.6±46.8 24.2±3.7
Central 35.7±20.2 20.5±6.5 96.6±72.6 230.0±150.3 29.5±14.2
P. chlorophanum (from Gondwana Station, North Victoria Land, Antarctica)
Gondwana 4.1±1.8 15.0±5.0 11.7±2.3 228.2±102.7 n.d. ––
Numbers indicate the mean of n=44 to 110 independent measurements of thallus stratification with standard deviation. For B. frigida, marginal and central stratification properties
were examined with samples from Inexpressible Island, Antarctica. marginal = lobe sections at the margin of a thallus, central = lobe sections from centrally located lobes,
proximal = sections from the inward parts of a thallus, distal = sections near the branch tips of C. gyrosa
Extremotolerance and Resistance of Lichens 291
parts) of the thallus. Thalline lobes, pycnidia, and apothecia are frequently formed in the
inner parts. The medulla is the largest stratum of B. frigida consisting of strongly interwoven
hyphae, stabilizing the thallus, and acting as a layer of water retention and gas exchange. The
thallus is tightly fixed to the substratum by medullary hyphae, often incorporating small rock
particles. A lower cortex or rhizine strands were not observed.
Fig. 1 Habitus macrographs of the five investigated lichens. 1a thallus detail of B. frigida from marginal
zones (left) to the center (right ) with black rectangular areolae at the margin and angular areolae and black
apothecia (arrows) in the center. 1b vagrant, spherical, fruticose, and compact thallus of C. gyrosa with
pseudocyphellae as white tips (arrow) at the end of sympodial branchings. 1c habitus of a R.geographicum
thallus with yellowish areolae, black interspersed apothecia and black prothallus (left margin, arrow). 1d
placoid thallus of X. elegans with protruding, branched, and narrow lobes at the margin (upper part) and disc-
like apothecia in the center (lower part, arrows). 1e: thallus detail of P. chlorophanum, showing yellow,
irregular, distinct, convex to pulvinate areolae with verrucose openings of the pycnidia (arrows)
292 J. Meeßen et al.
Fig. 2 Thin sections of five investigated lichens. 2a B. frigida, high amounts of algal cells below a pigmented
cortex at younger marginal areolae (upper row) and deceased algal numbers below the depigmented cortex in
older, central areolae (lower row). 2b C. gyrosa, distal cross section, stratification from outside to the center:
pigmented and paraplectenchymatous cortex, pronounced subcortex, evenly arranged algal clusters and loose
central medulla. 2c section through an areola of R. geographicum, showing the algae arranged in vertical lines
below the highly pigmented cortex. 2d heteromerous lobes of X. elegans 2e lobe section of P. chlorophanum,
two types of photobionts, in the algal layer of the lobe (a) and at the base of the rhizine-like strand (left ,b)
Extremotolerance and Resistance of Lichens 293
Circinaria gyrosa
Morphology C. gyrosa is a vagrant, spherical, fruticose, and compact lichen of brownish to
ochre colour with a diameter of max. 2.5 cm (Fig. 1b). It has been recently assorted to the
family of the Megasporaceae (Sohrabi 2012). The outer branches end in nearly circular
pseudocyphellae that appear as white tips, exposing the medulla directly to the atmosphere
(Fig. 1b, as described in Sancho et al. 2000). The surface is formed by a brownish epinecral
layer containing no detectable amounts of SLCs (Raggio et al. 2011). The detailed study of
morphological-anatomical traits reveals insight that might explain its high potential of
Fig. 3 Fixed SEM micrographs of the five investigated lichens. Algal cells highlighted with transparent green
spots. 3a B. frigida 3b C. gyrosa 3c R.geographicum 3d X.elegans 3e P. chlorophanum. Legend as follows:
AC: algal cluster (in C. gyrosa), AL: algal layer, cM: medullary hyphae, sheathed with whewellite crystals (in
C. gyrosa), loC: lower cortex (in X. elegans), M: medulla, paC: paraplectenchymatous Cortex, piC:
pigmented cortex, S: subcortex (in C. gyrosa). Mucilageous epicortex (B. frigida, R. geographicum, P.
chlorophanum) not visible in the choosen micrographs
Fig. 4 Macrographic top view on the thallus margin of B. frigida under wet and dry conditions. 4a In the wet
and swollen state (left) the green colour of the algal layer is dominant, while in the dry and shriked state (4b,
anhydrobiosis) the black melanin pigmentation of the upper cortex shields lower strata of the thallus from
excess irradiation
294 J. Meeßen et al.
resistanceas demonstrated in previous astrobiological studies (de la Torre et al. 2010a;
Raggio et al. 201 1; Sánchez et al. 2012). The porous pseudocyphellae at the tips facilitate gas
exchange between the atmosphere and the interior gas space (Sánchez et al. 2012). Apothecia
are rare and no subject of astrobiological studies; asexual reproduction was not observed.
Anatomy Thin sections (15 μm) of proximal and distal parts of sympodial branches revealed
particular anatomical structures and internal stratification of C. gyrosa (Table 2, Figs. 2b and
3b). Minor differences between distal and proximal parts were detected. The outer stratum is
formed by a brown epinecral layer, followed by a vivid paraplectenchymatous layer of
roughly isodiametric cells. Below this layer, C. gyrosa forms a particular, extended, and
compact periclinal prosoplectenchymatous subcortex. This pronounced thallus structure
consists of tightly arranged fungal hyphae that are conglutinated by high amounts of
extracellular mucilage (Fig. 3b). It is supposed to significantly contribute to the lichens
mechanical stability, to act as a diffusion barrier for gas exchange, and to contribute to the
high resistance of C. gyrosa. Below, singled, dense, and evenly distributed clusters of
photobiont cells are located which are lowly abundant and do not form a continuous algal
layer as it is observed in other lichens (Figs. 2b and 3b;e.g.Xanthoria- and Peltigera-
species). The algal clusters are more frequent in distal parts next to the pseudocyphellae. The
spaces between the clusters consist of fungal tissue similar to the subcortex while inward,
loose medullary hyphae connect the medulla to the clusters. The central branch tubes are
formed by medullary fungal tissue that is rich in inner aerial spaces and connected to the
atmosphere by apical pseudocyphellae. To prevent complete soaking with water under wet
conditions and to enable efficient gas exchange when wet, the medullary hyphae were
covered with extracellulary deposited whewellite-crystals (Fig. 6, Böttger et al. unpubl.).
Rhizocarpon geographicum
Morphology R. geographicum (Rhizocarpaceae) forms epilithic, crustose thalli of lime-
green, angular to rounded, flat to convex areolae, situated upon a well-developed black
hypothallus surrounded by a marginal prothallus (Fig. 1c). Usually several thalli fuse into
large colonies. The frequently formed apothecia between the areoles are black and disc-
shaped, with a black epithecium, a thin margin and up to 1 mm. The asci form eight large
(2535 μm), melanized and septate spores. Asexual reproduction was not observed.
Anatomy Thalli samples from the Spanish location Navacerrada occasionally show an
interrupted gelatinous epicortex that is missing in samples from the Swiss Alps (Table 2,
Fig. 5 SEM-micrograph (top view)
on the surface of a marginal B.
frigida areola. The smooth areas are
covered with mucilage, the rugged
areas in between show the
unsheathed hyphae of the cortex
Extremotolerance and Resistance of Lichens 295
Fig. 2c). The samples collected in Spain were covered by a gelatinous layer upon the cortex
which is thinner compared to samples from the Alps while the algal layer is thicker and the
medulla is thinner. Samples from both locations display densely arranged cortical cells
(Fig. 7) that are intensely coloured and incrusted with SLCs. The algal layer below is
characterized by rows of algal cells and interjacent hyphae both arranged antiklin to the
surface and highly gelatinized (Figs. 2c and 3c). Upon the blackish prothallus, the medulla is
formed by densely aggregated and highly gelatinized hyphae of antiklin orientation with few
interior gas spaces (Fig. 3c).
Xanthoria elegans
Morphology X. elegans (Teloschistaceae) is a placoid to crustose lichen (Fig. 1d) that may
cover large areas of the substrate. At the margin, the thallus protrudes narrow, convex,
densely arranged, and overlapping lobes that are lifted above the substrate or attached to it
by rhizine-like strands. Thalli are often fusing to form large colonies. The bright yellow-
orange to red colour is produced by SLCs in the upper cortex. The intensity of the colour
depends on the degree of insolation in the respective habitat (Nybakken et al. 2004) and is
effectuated by superficial formation of parietin-crystalsa SLC also found in abundant,
orange, lecanorine apothecia ( 13 mm) in central thallus parts. The asci are formed among
straight to branched paraphyses and bear eight elliptic ascospores. Asexual reproductive
structures are missing.
Fig. 6 SEM-micrograph , cross
section of the medulla of C. gyrosa.
The medullary hyphae are dense
ensheathed with whewellit e crystals
(arr ows)
Fig. 7 SEM-micrograph (side view)
on the surface of a R. geographicum
areola. The cortex is partially
removed, exposing the algal layer
and illustrating the clear vertical
orientation of the hyphae in the
cortex and the algal layer
296 J. Meeßen et al.
Anatomy The anatomy of X. elegans is investigated with marginal (i. e. younger) and central
(i.e. older) lobes of representable thallus samples (Table 2). The inner structure is
heteromerous (Figs. 2d and 3d). The upper surface is coated by a well-developed upper
cortex, which is formed by iso-diametric anticlinal paraplectenchymatous cells and
pigmented by parietin in the outer parts. The algal layer is composed of distinct but evenly
arraged clusters of photobiont cells with gelatinized interjacent hyphae (Fig. 3d). The
medulla consists of a spongy, loose network of long periclinal prosoplentenchymatous
hyphae forming a large, gas filled interior space (Fig. 3d). In contrast to the other investi-
gated lichens X. elegans forms a lower cortex. The comparison of marginal and central
sections indicates an extension of cortical, algal and medullary layers with age.
Pleopsidium chlorophanum
Morphology P. chlorophanum (Acarosporaceae) is a crustose, effigurate, morphologically
variable lichen with irregular, distinct, convex to pulvinate areolae ( 1020 mm), and a
smooth to verrucose surface (Fig. 1e). The colour is sulphuric yellow on mature or exposed
areolae to lime green on young or shaded areolae. Apothecia of up to 2.5 mm are reported
to be frequent (Øvstedal and Lewis Smith 2001), but not found in samples collected at
Gondwana Station. This might be correlated to extreme environmental conditions preventing
sexual reproduction by extremely short periods of favourable growth conditions.
Nonetheless, large numbers of pycnidia are formed in the thalli releasing the pycnospores
through bottle-neck apertures in verrucose elevations. Penetration of rock fissures by
outgrowing hyphal strands is a common observation; it fixes the thallus to the substratum
and promotes rock colonization as well as bio-weathering.
Anatomy The cortex is divided into a pigmented upper layer (Table 2, Fig. 3e). The
pigmentationwhich is missing in premature areolae but develops by timeis due to
extracellular deposits of needle-shaped yellow crystals while the unpigmented layer is
constituted of paraplectenchymatous and intensively gelatinized hyphae. The cortex is
covered by an epicortex which is more pronounced above the pycnidia. Below this layer,
the large numbers of photobiont cells are situated (Fig 2e). They are not consistently
arranged in a distinct and uniform algal layer, but fill large areas of the globose areolae
more or less densely or clustered. Depending on that, the extremely loose medulla is
irregularly shaped. In the vicinity of and within the basal thallus strands the aggregation
of hyphae becomes denser and more gelatinized. In these basal parts of the areolae, a second
type of photobiont is found which is smaller, blueish-green, and shows a different prolifer-
ation pattern (Fig. 2e). For clarification of photobiont identity molecular phylogenetics are in
As represented by the different growth types (crustose, placoid, and fruticose) and the
diverse morphological and anatomical traits (i. a. the prevalence of different strata,
Table 2), it is not a peculiar growth type or trait but an individual set of features that enables
lichens to brave harsh environmental conditions and explains the high potential in resisting
extreme environmental factors. Protection against excess PAR and UVR is often considered
one of the most crucial factors in research on lichen extremotolerance (Solhaug and Gauslaa
2004), therefore the following paragraphs pre-dominantly focus on photoprotective effects.
Extremotolerance and Resistance of Lichens 297
By the comparative approach, it is possible to identify some features that contribute to the
resistance of astrobiologically relevant lichens. While the discussion focuses on thalline
structures, the fruiting bodies of the investigated lichens reveal additional features to protect
the fungal spores inside: melanized paraphyses, a gelatinous matrix in and on the hymenium,
deposits of SLCs in the epithecium, the hypothecium and the apothecial margins (parietin in
X. elegans, melaninic substances in B. frigida and R. geographicum). In the case of B.
frigida and R. geographicum the spores themselves are highly melanized implying that they
are not only protected within the apothecium but also beyond, being an advantage for
successful establishment at highly insolated habitats.
Mucilage Matrices The formation of extracellular polymeric substances (EPS, i. e.
mucilageous or gelatinous matrices) is a basic property of the investigated lichens.
Besides being the basic biont contact interface (Honegger 1992), mucilage covers the
surface, conglutinates cortical and subcortical cells, ensheaths algal clusters and covers
medullary hyphae. Two predominant appearances of gelatinous substances are observed:
the formation of a gelatinous epicortex (partially in P. chlorophanum and X. elegans, site-
dependent in R. geographicum, frequently in B. frigida) and the formation of gelatinous
substances in peculiar strata (in the subcortex of C. gyrosa or the algal layer of B. frigida).
Besides aspects of water-uptake and -retention, the mucilage in the epicortex and the
(sub)cortex might promote resistance: It was discussed that gelatinous substances have
UVR-screening properties (Lütz et al. 1997; Belnap et al. 2001; de Vera et al. 2003, 2010 ;
Flemming et al. 2007; Ortega-Retuerta et al. 2009) and that mucilage might act as a
radiation-protective layer. Studies with bacterial exopolymer biofilms show that they are
only transmitted by minor proportions of UVR (13 % of UVC, 31 % of UVB, 33 % of
UVA), protecting the cells from exposure and suggesting that EPS is a natural defense
against UVR (Elasri and Miller 1999). With up to 12.6 μminB. frigida, 5.0 μminR.
geographicum, and 4.1 μminP. chlorophanum the mucilageous epicortices of these
extremotolerant lichens are more extended than in the more temperate distributed
Parmeliaceae (0.61.0 μm, Büdel and Scheidegger 1996). The epicortex may also change
the reflection properties of the surfacedue to refractive and dispersive effectsand reduce
the intensity of PAR and UVR in the thallus. The remarkable amounts of gelatinous
substances in the subcortex of C. gyrosa as well as in the algal layer of B. frigida and R.
geographicum may cause additional shielding against PAR and/or UVR.
Cortices As in most lichens, a pigmented and conglutinated cortex is found in all five
investigated species followed by an unpigmented paraplectenchymatous cortex in C. gyrosa,
X. elegans, and P. chlorophanum. In these cortices, the vivid fungal cells are found in the
lower part while the upper pigmented part occasionally lacks vivid cell lumina, forming an
epinecral layer of pigment incrusted dead cell remnants (as observed in X. elegans and P.
chlorophanum). In all cases the pigmentation is confined to fungal cell walls of the apical
hyphae, ceasing with increasing depth. The SLCs of astrobiologically relevant lichens are
addressed elsewhere (Meeßen et al., unpubl.), but also the cortical morphology contributes
to resistance. Besides physiological limitation, herbivore defence, and mechanical stabiliza-
tion, protection of the photobiont is considered a main function of the cortex (Ertl 1951;
Jahns 1988; Kappen 1988). In general, lichen cortices are able to absorb 2643 % of the
incident light while shade- and light-adapted thalli of the same species may vary in cortical
organization due to the different light regimes (Ertl 1951; Büdel and Scheidegger 1996).
Hydrated, physiologically active thalli of B. frigida are coloured intensively green by the
algal layer below the cortex (Fig. 2a). The swelling of cortical cells by water-uptake reduces
298 J. Meeßen et al.
the density of the cortical pigmentation, and exposes the algae to higher light intensities. If
B. frigida passes into anhydrobiosis, the thallus becomes intensively coloured black by
shrinking cortical cells and densifying melanin incrustations in the upper cortex (Fig. 4b).
This effect of increasing cortical absorbance substantially reduces excess light levels
reaching the photobiont and might be an adaptation comparable to the pruina, a superficial
layer of crystalline deposits or dead cells that increases reflection when dry (Jahns 1988;
Büdel and Scheidegger 1996) and resembles a protective adaptation (Kappen 1973). Both
effects protect lichens most effectively in anhydrobiosis, in which it experiences consider-
ably long periods of insolation (Lange et al. 1999), its repair mechanisms are dormant, and
harmful effects of excess PAR/UVR are accumulative (Solhaug and Gauslaa 2004).
Astrobiological investigations stress the role of the lichen cortex in protecting fungal and
algal bionts: for R.geographicum it was found that the removal of the cortex before
exposition reduces the relative PSII activity of the photobiont depending on the type of
UVR-transmission filter used (de la Torre et al. 2010a). In other experiments the thallus
tissue viability decreased at about 15 % in F. bracteata and 25 % in X. elegans if the cortex is
removed before UVR-exposure (de Vera et al. 2003; de Vera and Ott 2010). However, in
such experiments the effect of the cortical structure itself was not separated from the effect of
the deposited SLCs.
Subcortex In the present study, a subcortex is found in C. gyrosa only. It measures up to
150 μm and is characterized by dense fungal hyphae that are highly conglutinated with
mucilage but lack any pigmentation. This structure is exclusively formed by the very lichen
species that showed no sign of major SLC production (Raggio et al. 2011). The thick and
dense fungal cortex was found to protect the algal populations within the cluster while the
contribution of the single layers (cortex and subcortex) is not yet quantified. We conclude
that the highly conglutinated subcortex does not only deal with the mechanical stress of the
lichens vagrant life style, but also compensates the lack of photoprotective SLCs.
Especially, if its location above the algal layer, the lack of SLCs in the cortex (Raggio et
al. 2011), and its high resistance towards UVR-exposure (de la Torre et al. 2010a; Sánchez et
al. 2012) are taken into account. Several factors may contribute: the sheer thickness, the high
amount of mucilage (with the shielding properties discussed above), and the protective effect
of the densely packed hyphae themselves.
Algal Layer Investigations on the viability of both symbionts of X.elegans and P. aphthosa
after exposure to UVC
at 2.1201.8 J×m
(de Vera and Ott 2010) showed a higher
decrease in viability of the photobiont compared to the mycobiont, supporting the hypothesis
that the photobiont is the more sensitive partner of the symbiosis. Recent observations show
that the resistance of lichens to high UVR and vacuum can be attributed to the mycobiont (de
Vera et al. 2008; de Vera 2012), while additional results indicate that also the arrangement of
the photobiont contributes to resistance: In B. frigida, C. gyrosa, P. chlorophanum and X.
elegans the algae are clustered in more or less dense aggregates which are enveloped by a
layer of gelatinous substances. In R. geographicum the algae are surrounded by highly
gelatinized hyphae and arranged in rows vertical to the surface of the thallus and thus, in line
with the direction of most intensive insolation. Both arrangementsclustering and
alignmentcan be interpreted as protective strategies to avoid excess insolation.
Live/Dead analysis after several simulation experiments supports this hypothesis, clearly
showing that inner cells of algal clusters are more vital than outer ones after exposure to
UVR or UVR + vacuum (de Vera et al. 2003, 2004a, b), even if isolated photobionts were
tested (de Vera et al. 2008).
Extremotolerance and Resistance of Lichens 299
Basal Thallus Strands Out of the five investigated species, only P. chlorophanum forms
basal thallus strands penetrating the upper layer of the rock substrate, integrating an
endolithic characteristic to an usually epilithic lichen. This feature may not only substantially
contribute to the lichens potential of substrate colonization and bioweathering but may also
reflect an adaptation towards extremotolerance. Growing inside the substrate and using its
structure as a protection is a strategy of many organismsincluding lichensto colonize the
most extreme terrestrial habitats (Sun et al. 2010). For P. chlorophanum, the endolithic
strands resemble a reservoir of hyphal biomass which might allow regeneration if the
epilithic thallus is damaged by stressors as UVR and abrasion. This is stressed by the fact
that a second, morphologically distinct type of algal partner is found to be located in the
basal zone, suggesting not only a regenerative capacity of the mycobiont but also of an
alternative photoautotrophic partner.
General Aspects All investigated lichens reveal the same anatomical blueprint of a
heteromerous thallus (Jahns 1988; Büdel and Scheidegger 1996) but show diverging sets
of morphological-anatomical traits represented by the presence and properties of different
strata and anatomical structures. Besides other factors (poikilohydry, SLCs), the results
indicate that these traits help to explain lichen extremotolerance towards abiotic factors as
well as their resistance towards space and Mars parameters (de Vera et al. 2003, 2004a, b,
2007, 2008, 2010; de la Torre et al. 2004; 2007; Sánchez et al. 2012). From a morphological
point of view, in B. frigida, R. geographicum, and X. elegans the combination of cortex (with
varying SLCs), algal arrangement, and mucilage seems to be fundamental to constitute
resistance, while in C. gyrosa the subcortex seems to play a crucial role, as well as the
rhizine-like strands in P. chlorophanum.
All lichens tested to date showed high viability in astrobiological experiments. However,
experimental attempts to test the protective effects of the distinct morphological and
anatomical thallus structures are scarce. In UV-exposure experiments (λ>160 nm) with X.
elegans and F. bracteata, lichen thalli with intact and with removed cortex were compared
(de Vera et al. 2003). Samples with removed cortex showed a loss in viability of 1535 % in
X. elegans and 1540 % in F. bracteata indicating a protective effect of the cortex. In the
LITHOPANSPERMIA experiment, R. geographicum and X. elegans were exposed to space
with intact and with removed or depigmented cortices, respectively (de la Torre et al. 2010a),
revealing a post-flight reduction of PSII activity of 6.981 % in R. geographicum and of
0.143 % in X. elegans. Comparing the effect of removed (R. geographicum) and
depigmented (X. elegans
) cortices, a more severe effect is found if the cortex is removed.
However, such results do not help much to separate the protective effect of the cortex itself
and the adjacent SLCs, as both lichens also reveal anatomical differences in terms of cortical
structure (Table 2) and algal arrangement. Nonetheless, the high viability in both studies
indicates additional protective features, e. g. as discussed above. In C. gyrosa, the post-flight
reduction of PSII activity was low (04.5 %, de la Torre et al. 2010a) what might be
correlated to the protective effect of its extended subcortex, as its cortex is supposed to lack
sufficient amounts of SLCs (Raggio et al. 2011). The differences in the reduction of PSII
activity in X. elegans and R. geographicum after the LIFE experiment are evident (Onofri et
al. 2012). Post-flight dark control samples of X. elegans showed a reduction of PSII activity
of 2 % and irradiated post-flight samples showed a reduction of about 55 %, in R.
geographicum the reduction is 97.5 % and 99.5 %, respectively. What morphological-
anatomical features of X. elegans might help to explain such difference? Especially as both
lichens are crustose, form pigmented cortices of about 20 μm thickness, and bear a
photobiont of the same genus (Trebouxia). Besides the different predominant SLCs in both
300 J. Meeßen et al.
lichens (parietin in X. elegans compared to rhizocarpic acid in R. geographicum), two
features may give an explanation: the additional 20 μm-wide paraplectenchymatous cortex
and the densely clustered photobiont cells, presumably shielding each other more effectively
than the aligned photobiont cells in R. geographicum. Nonetheless, a different level of
desiccation resistance among the exposed lichen species is also supposed to contribute to the
diverging survival rates.
The present study shows that generalizations concerning the resistance of lichens towards
extreme conditions have to be avoided. The differences reflect the diverging evolutionary
histories of their lineages which led to different adaptations to the respective ecological
niches (Jahns 1988). Such adaptations enable the symbiosis to successfully cope with
prevalent abiotic stressors and support its persistence in extreme habitat. Alpine and polar
regions are characterized by high levels of insolation and the dominance of lichens in these
regions can be explained by their ability to endure UVR (Solhaug and Gauslaa 2004).
Several studies highlight the mycobiont to be more resistant towards UVR-exposure than the
photobiont (de la Torre et al. 2002; de Vera et al. 2008; de Vera and Ott 2010; de Vera 2012),
and thus protects the photosynthesizing partner. Nonetheless, studies with isolated
mycobionts stress that undifferentiated axenic fungal tissue is more susceptible to the
damaging effects of UVR than complete lichen thalli (de Vera and Ott 2010). These results
give a clear hint on the importance of distinct differentiated thallus structuresas demon-
strated in the present studyrather than mere fungal biomass.
Acknowledgments The authors would like to express their sincere gratitude to the German Federal Ministry
of Economics and Technology (BMWi) and the German Aerospace Center (DLR) for funding the work of
Joachim Meeßen (50BW1153) and Annette Brandt (50BW1216), to the Spanish Instituto Nacional de Técnica
Aeroespacial (INTA) for granting a PhD scholarship to Francisco Javier Sánchez Iñigo, and to the German
Aerospace Center (DLR) for supporting the ESA-space experiment BIOMEX (ILSRA ESA-ILSRA 2009
0834, P-I Dr. J.-P. de Vera). Samples of B. frigida and P. chlorophanum were collected by S. Ott during the
GANOVEX 10 expedition which was funded by the German Research Foundation (DFG, OT 96/10-3) in the
framework of the Antarctic Priority Program 1158. We would also like to thank the reviewers for their comments
and suggestions. Results of this study were presented on the 12th European Workshop on Astrobiology (P6.16,
EANA 2012).
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Extremotolerance and Resistance of Lichens 303
... Lichens represent one of the most successful symbiotic associations on Earth and are capable of surviving in extreme environments (Kranner et al. 2008;Grube 2010;Meeßen et al. 2013aMeeßen et al. , 2013b, where life faces its limits (de Vera et al. 2008;de la Torre et al. 2010). Lichens comprise a fungal partner, the "mycobiont", associated with at least one or more photoautotrophic partner(s), the "photobiont", mostly green algae ("chlorobionts"), and/or cyanobacteria ("cyanobionts") (Honegger 1991;Sanders 2001;Henskens et al. 2012). ...
... Lichens are also inhabited by bacteria and other microfungi (Grube and Berg 2009;Spribille et al. 2016;Muggia and Grube 2018;Hawksworth and Grube 2020). The fungal partner, which gives the name to the lichen, is responsible for building the complex structure of the lichen thallus, although it can only achieve this in symbiosis with a compatible photobiont (Honegger 1993;Kranner et al. 2005;Meeßen et al. 2013a). ...
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Lichens and their isolated symbionts are potentially valuable resources for biotechnological approaches. Especially mycobiont cultures that produce secondary lichen products are receiving increasing attention, but lichen mycobionts are notoriously slow-growing organisms. Sufficient biomass production often represents a limiting factor for scientific and biotechnological investigations, requiring improvement of existing culturing techniques as well asmethods for non-invasive assessment of growth. Here, the effects of pH and the supplement of growth media with either D-glucose or three different sugar alcohols that commonly occur in lichens, D-arabitol, D-mannitol and ribitol, were tested on the growth of the axenically culturedmycobiont isolated from the lichen Xanthoria parietina. Either D-glucose or different sugar alcohols were offered to the fungus at different concentrations, and cumulative growth and growth rates were assessed using two-dimensional image analysis over a period of 8 weeks. The mycobiont grew at a pH range from 4.0 to 7.0, whereas no growth was observed at higher pH values. Varying the carbon source in Lilly-Barnett medium (LBM) by replacing 1%D-glucose used in the originally described LBM by either 1%, 2%or 3%of D-mannitol, or 3% of D-glucose increased fungal biomass production by up to 26%, with an exponential growth phase between 2 and 6 weeks after inoculation. In summary, we present protocols for enhanced culture conditions and non-invasive assessment of growth of axenically cultured lichen mycobionts using image analysis, which may be useful for scientific and biotechnological approaches requiring cultured lichen mycobionts.
... Lichens can thrive in harsh environments, often withstanding extreme heat, desiccation, or cold [1,2]. Moreover, some experiments demonstrated that lichens can survive in space, as well as in a simulation chamber that mimics an environmental condition of Mars [3,4]. About 20% of all known fungi are thought to have been lichenized and nearly 98% of them belong to the phylum Ascomycota [5][6][7]. ...
... Only the first twenty nucleotides were shown.2 Length of flanking region of the right border of T-DNA identified by TAIL-PCR analyses.3 Numbers before and after colon refer to scaffolds and the position of nucleotide where T-DNA was inserted in scaffolds, respectively. ...
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Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.
... Some of these play a key role in photoautotrophic metabolism (carotenes and chlorophylls) and structural integrity (cellulose and chitin) of cells, but our chief criterion for selecting these molecules was the advantage they can confer upon organisms when it comes to resisting the harsh martian environment; -carotene, melanin, quercetin, and naringenin, for instance, are potent scavengers of stress-induced radicals. In addition, these molecules were found in a wide range of organisms hosted in the BIOMEX experiment (16): A significant number of bacteria contain carotenoids and some bacteria and cyanobacteria produce cellulose (39); algae, cyanobacteria, lichens, and bryophytes contain carotenoids (11), chlorophyll, and flavonoids (such as quercetin and naringenin) (40,41); and fungi and lichens contain melanin (42) and chitin (43). Chlorophyll was replaced with its derivative, the copper sodium salt of chlorophyllin, which produces very distinguishable Raman spectra when excited at 532 nm (44). ...
Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments , Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable bio-molecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.
... Lichen species have been selected for space experiments because they are able to colonize terrestrial habitats exposed to high intensity solar radiation [4,5], long periods of desiccation, and wide temperature variation [6,7]. Some lichens are remarkably resistant to non-terrestrial conditions, a characteristic that is attributed to a range of morphological adaptations [8], a set of protective secondary compounds [9], and their ability to pass into anhydrobiosis (an ametabolic state) when desiccated [10][11][12][13]. C. gyrosa, previously included in the space exposure experiment LITHOPANSPERMIA exhibited a remarkable post-exposure viability [2,14], demonstrating a conspicuous resistance to space conditions. ...
The extremophile lichen Circinaria gyrosa (C. gyrosa) is one of the selected species within the BIOMEX (Biology and Mars Experiment) experiment. Here we present the Raman study of a biohint found in this lichen, called whewellite (calcium oxalate monohydrate), and other organic compounds and mineral products of the biological activity of the astrobiologically relevant model system C. gyrosa. Samples were exposed to space- and simulated Mars-like conditions during the EXPOSE-R2 mission parallel ground reference experiment MGR performed at the space- and planetary chambers of DLR-Cologne to study Mars’ habitability and resistance to real space conditions. In this work, we complete the information of natural C. gyrosa about the process of diagenesis by the identification of carbonate crystals in the inner medulla together with the biomineral whewellite. The analysis by Raman spectroscopy of simulated Space and Mars exposed samples confirm alterations and damages of the photobiont part of the lichen and changes related to the molecular structure of whewellite. The conclusions of this work will be important to understand what are the effects to consider when biological systems are exposed to space or Mars-like conditions and to expand our knowledge of how life survives in most extreme conditions that is a prerequisite in future planetary exploration projects.
... Our FE-EPMA observation showed few micron-sized particles that were caught in parts of the pores of epicortex on the thallus (Fig 6). The mucilageous epicortex covers the thallus surface with a thickness of 0.6-1 μm in some species of Parmeliaceae [58,59], and may also contribute to particle trapping capacity. Thus, we suggested that particles deposited in the initial fallout were captured and retained on the thallus surface and interspace of the medullary hyphae for a long period owing to adhesion with the mucilage and integration with hyphae. ...
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We investigated the radiocaesium content of nine epiphytic foliose lichens species and the adjacent barks of Zelkova serrata (Ulmaceae, "Japanese elm") and Cerasus sp. (Rosaceae, "Cherry tree") at the boundary of the Fukushima Dai-ichi Nuclear Power Station six years after the accident in 2011. Caesium-137 activities per unit area (the 137 Cs-inventory) were determined to compare radiocaesium retentions of lichens (65 specimens) and barks (44 specimens) under the same growth conditions. The 137 Cs-inventory of lichens collected from Zelkova serrata and Cerasus sp. were respectively 7.9-and 3.8-times greater than the adjacent barks. Furthermore, we examined the radiocaesium distribution within these samples using autoradiography and on the surfaces with an electron probe micro analyzer (EPMA). Autoradiographic results showed strong local spotting and heterogeneous distributions of radioactivity in both the lichen and bark samples, although the intensities were lower in the barks. The electron microscopy analysis demonstrated that particulates with similar sizes and compositions were distributed on the surfaces of the samples. We therefore concluded that the lichens and barks could capture fine particles, including radiocaesium particles. In addition, radioactivity was distributed more towards the inwards of the lichen samples than the peripheries. This suggests that lichen can retain 137 Cs that is chemically immobilised in particulates intracellularly, unlike bark.
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The grit crust is a recently discovered, novel type of biocrust made of prokaryotic cyanobacteria, eukaryotic green algae, fungi, lichens and other microbes that grow around and within granitoid stone pebbles of about 6 mm diameter in the Coastal Range of the Atacama Desert, Chile. The microbial community is very well adapted towards the extreme conditions of the Atacama Desert, such as the highest irradiation of the planet, strong temperature amplitudes and steep wet-dry cycles. It also has several other striking features making this biocrust unique compared to biocrusts known from other arid biomes on Earth. It has already been shown that the grit crust mediates various bio-weathering activities in its natural habitat. These activities prime soil for higher organisms in a way that can be envisioned as a proxy for general processes shaping even extra-terrestrial landscapes. This mini-review highlights the potential of the grit crust as a model for astrobiology in terms of extra-terrestrial microbial colonization and biotechnological applications that support human colonization of planets.
A preliminary checklist of lichen-forming and lichenicolous fungi of Castilla-La Mancha is presented. A total of 5064 records have been compiled from 204 publications. The number of taxa ascends to 832, of which 820 are species and 12 infraspecific taxa. Of these 763 are lichenized and 69 correspond to lichenicolous fungi.
A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM), in conjunction with light microscopy, to document the structural manifestations of hyphal differentiation during the formation of three lichen tissues that localize between the algal layer and the surface of the thallus: the outer cortex of foliose lobes; the outer layer of fruticose stems; and the enwrapping layer of asexual propagules called soredia that protrude from squamulose podetia and foliose lobes. Our observations document features of outer-layer architecture and the role played by extracellular matrices (ECM). They also lead us to propose the medullary stem-cell hypothesis for lichen organization wherein totipotent medullary hyphae produce lateral branches that undergo specific differentiation pathways in specific domains of the thallus.
A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM), in conjunction with light microscopy, to document the structural manifestations of hyphal differentiation during the formation of three lichen tissues that localize between the algal layer and the surface of the thallus: the outer cortex of foliose lobes; the outer layer of fruticose stems; and the enwrapping layer of asexual propagules called soredia that protrude from squamulose podetia and foliose lobes. Our observations document features of outer-layer architecture and the role played by extracellular matrices (ECM). They also lead us to propose the medullary stem-cell hypothesis for lichen organization wherein totipotent medullary hyphae produce lateral branches that undergo specific differentiation pathways in specific domains of the thallus.
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Gaseous phase hydration properties for thalli of Niebla tigrina from Atacama Desert, and for Umbilicaria antarctica from Isla Robert, maritime Antarctica, were analyzed using ¹ H-NMR relaxometry, spectroscopy, and sorption isotherm analysis. The molecular dynamics of residual water was monitored to distinguish the sequential binding very tightly, tightly, and loosely bound water fractions. These two species differ in hydration kinetics faster for Desert N. tigrina [ A 1 = 0.51(4); t 1 = 0.51(5) h, t 2 = 15.0(1.9) h; total 0.7 for p / p 0 = 100%], compared to Antarctic U. antarctica [ A 1 = 0.082(6), t 1 = 2.4(2) h, t 2 = [26.9(2.7)] h, total 0.6 for p / p 0 = 100%] from humid polar area. The ¹ H-NMR measurements distinguish signal from tightly bound water, and two signals from loosely bound water, with different chemical shifts higher for U. antarctica than for N. tigrina . Both lichen species contain different amounts of water-soluble solid fraction. For U. antarctica, the saturation concentration of water soluble solid fraction, c s = 0.55(9), and the dissolution effect is detected at least up to Δ m / m 0 = 0.7, whereas for N. tigrina with the similar saturation concentration, c s = 053(4), this fraction is detected up to the threshold hydration level equal to Δ M/m 0 = 0.3 only.
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This chapter summarises the functional morphology and anatomy of lichen thalli, the focus being on ultrastructural details such as haustorial types at the mycobiont-photobiont interface in asco- and basidiolichens.
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The Holarctic fruticose lichen Stereocaulon paschale is known to be capable of tolerating at least six weeks of constant submersion under natural conditions in a meltwater pool at about 1000 m above sea level in Rondane in eastern Norway. Its adaptations to temporary submergence involve both physiology and anatomy, differing fundamentally from Cladonia stellaris, the dominant lichen species in the adjacent terrestrial habitat. Comparison of two populations of Stereocaulon paschalefrom the melt water pool and from a dry forest habitat revealed significant physiological differences between them in oxygen uptake and the photosynthetic process. We discuss the role of cephalodia and thallus structure, focusing on gas exchange and water relations.
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Extremophilic microorganisms like lichens and cyanobacteria were collected from tropic deserts and from polar and alpine habitats because of their occurrence in intensely irradiated, very dry and/or cold environments which are supposed to be as close as possible to Martian surface conditions. The collected species were exposed during different experiments either to real space conditions on space exposure platforms like BIOPAN and EXPOSE-E on the International Space Station or to Mars simulation conditions in a Mars simulation chamber. Some of these species were also exposed to both of the extreme environmental conditions. It is a technical challenge to perform Mars simulation experiments with long duration exposure times on microorganisms - the so called extremophiles. One of the challenges is, to take care of measuring and monitoring all environmental parameters including measurements of metabolic activity of the investigated microorganisms. But the first performed one month experiment on the Antarctic lichen Pleopsidium chlorophanum in the Mars simulation facility at the Institute of Planetary Research at the DLR Berlin was successful and shows remarkable results on the adaptation capacity of photosynthetic activity within the simulation time of 34 days. This result will be a starting point to revise the previously analyzed simulation experiments on other lichen species and cyanobacteria and to start new "long duration" experiment series with other polar microorganisms. The outcome of this work might be relevant to classify Mars as a habitable planet by a new experimental and biological approach.
This handbook is an indispensable tool for the isolation, identification and structural analysis of the approx. 700 substances currently known to occur in lichens. The first part covers all necessary methods for the analysis of lichen metabolites; the second part gives the analytical and spectroscopical data of all known lichen substances as well as a key to their identification and differentiation. Besides its high value for all chemists working with these substances as a basis for other products, the book serves as a chemotaxonomical key to the identification of lichen species and as a reference for all those who use lichens for the biomonitoring of environmental pollution.
Symbiotic algae or cyanobacterial partners associated with fungi in a lichen are considered the symbionts most sensitive to abiotic stress. Stress caused by high concentrations of metals produce increased levels of highly reactive oxygen species that result in lipid peroxidation as well as damage to proteins and nucleic acids. Excess metals impact primarily on the photobiont as they decrease the concentration and integrity of chlorophyll and the quantum yield of photosynthesis reduce the output of molecular oxygen. Cells experience plasmolysis, electrolyte leakage, swelling of cristae in mitochondria and thylakoids in chloroplasts, and decrease viability. Although the primary fungal symbiont, the mycobiont, provides a degree of defense by binding metal ions in anionic cell wall sites, photobionts may nevertheless be exposed to super-optimal metal concentrations. However, within photobiont cells chelators and chaperones containing sulfhydryl groups bind and detoxify metals and decrease the impact of metals. Increased cysteine concentrations have been documented in photobionts subjected to metal stress. Proline also forms stable complexes with dangerous free radicals that could otherwise produce damage in the cytosol, and photobionts show increased levels of free proline in response to high concentrations of metals. Heat shock proteins chaperone metal ions to cellular destinations where they will be harmless, and increased concentrations of hsps have been found in photobionts exposed to excess cadmium. Defense mechanisms involving primary and secondary metabolites of the mycobiont have so far been little investigated in lichen photobionts but these may also provide a degree of photobiont protection. Recent literature suggests complex interactions of photobionts with metals that depend upon the metal, its concentration, and the strain of photobiont.
This chapter illustrates the physiological and morphological responses of lichens to extreme environmental stresses, which include drought and desiccation, wetness, temperature, humidity, visible or ionizing radiation, gamma irradiation, radioactive materials, and mechanical influences. The drought resistance of lichen is directly correlated to the intensity of desiccation (relative humidity) and to the length of the desiccation period. The stimulation of respiration after desiccation stress causes considerable loss of carbohydrates. Photosynthesis increases spontaneously with water vapor intake at a relative humidity below 100%. Further, the desiccation resistance varies according to species. The comparative analyses of resistance to submersion shown by terrestrial and aquatic lichens clearly demonstrate the ecological importance of the moisture regime for the distribution of lichens. The life of lichen is characterized by rapid changes between active and inactive states. These changes can be optimally profitable if they allow for sufficient time (continuation) and intensity of metabolism, thus, producing biomass. Their resistance allows lichens to persist under environmental stress either in the active state (plasmatic tolerance) or by diminishing the stress in the anabiotic, desiccated state, which is made possible by their poikilohydric nature (constitutional resistance). Lichen can respond to adverse conditions by morphological adjustments, by pigmentation of the thallus, and by changes in the symbiotic state. Erratic lichens in deserts and steppes indicate the ability to be translocated to more favorable environments.
The number of lichen substances with known structure is about 700. It is the aim of this book to help the lichenologist and the natural product chemist in the identification of this large group of plant metabolites. It comprises methods for the isolation and identification of lichen substances by physical and spectroscopic methods, microcrystallization, thin layer chromatography, high performance liquid chromatography, gas liquid chromatography and derivatization. The main part contains the formulae, molecular weights and data about the melting points, colour reactions, UV, IR, 1H-NMR, 13C-NMR and mass spectra, derivatives, TLC RF-values, microcrystallization, HPLC and the standard lichen (which contains the corresponding compound) with references of all lichen substances described in the literature up to 1995. Tables of molecular weights, melting points and reactions of lichen substances are further auxiliaries. Finally, a key for the identification of lichen substances is presented.