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Endolithic growth of two

Lecidea

lichens in granite from continental Antarctica detected by molecular and

microscopy techniques

A. De los Ríos

1

, L. G. Sancho

2

, M. Grube

3

, J. Wierzchos

4

and C. Ascaso

1

1

Centro de Ciencias Medioambientales (CSIC), Serrano 115 dpdo., Madrid-28006, Spain;

2

Dpto Biología Vegetal II, Universidad Complutense de Madrid,

Madrid-28040, Spain;

3

Institut für Pflanzenwissenschaften, Karl Franzens-Universität Graz, Holteigasse 6, Graz-8010, Austria;

4

Servei de Microscopia

Electrónica, Universitat de Lleida, Rovira Roure 44, Lleida-25198, Spain

Summary

• Through the combined use of molecular and microscopy techniques, the endolithiclichens

Lecidea cancriformis

and

Lecidea

sp. were identified, even in the absence offruiting bodies, and positioned under epilithic lichens. Cells of both algal and fungalsymbionts were observed in fissures and cracks of the lithic substrate with no clearheteromerous structure. At the ultrastructural level, the two lichens differed in termsof their algal–fungal relationships.• Only one genotype of

Trebouxia

ITS sequence was identified from specimensof

Lecidea

sp.,

Umbilicaria aprina

and

Buellia frigida

from the same zone,which could be mainly determined by low availability of alga in these extremeenvironments.• These lichens showed features typical of both chasmoendolithic and euendolithicmicroorganisms. Signs of biogeophysical and biogeochemical action on thesubstrate were detected close to fungal cells. This action seemed to be mainlyconditioned by the local physico-chemical features of the substrate. Evidencefor the biomobilization of elements by these endolithic lichens was found.

L. cancriformis

was observed to accumulate substantial amounts of calcium-richbiominerals.• The combined approach proposed is useful for mapping the distribution of endolithiclichens and analysing the processes that occur in their microscopic environment.

Key words:

Antarctica, endolithic lichens, granitic rock, ITS rDNA,

Lecidea

, ultrastructure.

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doi

: 10.1111/j.1469-8137.2004.01199.x

Author for correspondence:A. De los Ríos Tel:

34-917452500

Fax:

34-915640800

Email: arios@ccma.csic.es

Received:

13 May 2004

Accepted:

2 July 2004

Introduction

The lithic substrate is being increasingly viewed as animportant and largely unexplored part of the biosphere.Life underneath the rock surface is particularly common inthe most extreme terrestrial habitats on Earth (Friedmann,1980; Gross

et al

., 1998; Kidron, 2000). However, apart fromits ubiquitous presence in this environment and the factthat its colonizers set off the soil stabilization process (Wynn-Williams, 1993) and participate in the elemental cycles ofdiverse ecosystems (Ferris & Lowson, 1997; Blum

et al

.,

2002; Hoffland

et al

., 2002), our knowledge of the endolithichabitat is still at a rudimentary stage.

Fungi and algae living in symbiotic association as lichensare common internal colonizers of the lithic substrate(Friedmann, 1982; Friedmann

et al

., 1988; Ascaso

et al

., 1995,1998; De los Ríos

et al

., 2002). The endolithic habitat sheltersthe lichen cells from both excessive drought and temperatureextremes (Fry, 1922; Pinna

et al

., 1998; Kidron, 2000).Organisms that live within hard rocky substrates acquirespecialized adaptive features, which in turn determine differentendolithic ecological niches. These organisms are able to

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colonize existing cracks and fissures (chasmoendolithic), internalpores (cryptoendolithic) or penetrate actively into the rock(euendolithic) (Golubic

et al

., 1981). Water, light and nutrientscan be rather sparse in the endolithic habitat, but in extremeenvironments its microclimate can be less severe than at theexposed rock surfaces where harsher environmental conditionsexist. At this rock–atmosphere interface, lichenized alga and fungi,along with free-living microorganisms, are able to form different‘biofilms’ (Warscheid & Braams, 2000; de los Ríos

et al

., 2002;Ascaso & Wierzchos, 2003). The biofilm organization provideprotection to the resident microorganisms against the environ-mental conditions, since they can maintain conditions insidethat are radically different to those of the external environment(Little

et al

., 1997; De los Ríos

et al

., 2003). In continentalAntarctica, lithobiontic communities are the predominant formsof terrestrial life (Friedmann, 1982; Ascaso & Wierzchos, 2003;De la Torre

et al

., 2003; De los Ríos

et al

., 2003) and are thus anessential target for any evaluation of Antarctica’s biodiversity.However, the study of lithobiontic microorganisms is met withnumerous hurdles since these microorganisms are embeddedin a hard substrate and are extremely difficult to culture. Someendolithic lichens can be observed with the naked eye whentheir mature fruiting organs appear at the rock surface, but intheir absence endolithic lichens are not so easily determined(Fry, 1922). Besides, the factors determining colonization andsuccession in the lithic substrate are not completely knownand it is difficult to foresee the presence of particular endo-lithic forms when there are no associated epilithic forms. Theuse of molecular methods to identify Antarctic endolithicmicroorganisms has recently been reported after their labora-tory culture (Smith

et al

., 2000; Hughes & Lawley, 2003) orwithout the need for previous culture (De la Torre

et al

.,2003). These molecular studies are starting to provide insightinto the existing biodiversity, yet the lack of microscopy studiescarried out in parallel to these approaches means there is littleinformation on the specific microsites inhabited by the micro-organisms identified, or their interactions or organization.The aim of the present study was to apply both microscopyand molecular techniques in an effort to simultaneously iden-tify and characterize endolithic lichen symbionts in rock sam-ples from Granite Harbour (Antarctica).

Materials and Methods

Materials

Pieces of granite rock sparsely colonized by lichens and mosseswere collected from the Ross Sea coast, Granite Harbour(77

°

00

′

-S, 162

°

34

′

-E) across a range of altitudes from the coastto the summit of Discovery Bluff (5–500 m a.s.l). In this zone,epilithic forms of life were only present in places regularly exposedto melt water (Seppelt

et al

., 1995). Samples were collectedunder natural conditions and stored at

−

20

°

C until processingfor microscopy, molecular, or microanalytical procedures.

Microscopy studies

The rock samples were prepared according to a proceduredeveloped for observing the rock–microorganism interfaceby scanning electron microscopy with backscattered electronimaging (SEM–BSE, Wierzchos & Ascaso, 1994). In brief,the pieces of rock were fixed in glutaraldehyde and then inosmium tetroxide, dehydrated in a series of ethanol solutions,and embedded in LR-White resin. Blocks of resin-embeddedrock samples were finely polished, carbon coated and observedusing DMS 960 and DMS 940 A Zeiss SEM microscopes.Microprobe analyses were performed using an energy dispersiveX-ray spectroscopy (EDS) instrument fitted with a LinkISIS microanalytical system during SEM observation. Themicroscopy and/or microanalytical operating conditions wereas follows: 0

°

tilt angle, 35

°

take-off angle, 15 kV accelerationpotential, 6 or 25 mm working distance and 1–5 nA specimencurrent. The EDS method allows qualitative and quantitativemicroanalysis by providing element spatial distribution maps.These maps indicate the relative concentrations of elementsaccording to a colour scale: dark blue represents a concentrationof absolute zero and white denotes the maximum absoluteconcentration of the corresponding pure-component spectrum.

In addition, endolithic lichen masses were removed fromthe same rock under the stereomicroscope using a sterileneedle and blocked in 2% (w/v) agar. Small pieces of agarcontaining the microbial cells were fixed in glutaraldehyde andosmium tetroxide solutions, dehydrated in a graded ethanolseries, and embedded in Spurr’s resin following the protocoldescribed by De los Ríos and Ascaso (2001). Several ultrathinsections (15–20) from the different samples were post-stainedwith lead citrate (Reynolds, 1963) and observed using a ZeissEM910 transmission electron microscope.

Molecular studies

Total DNA was extracted from the epilithic lichens, fruitingbodies of endolithic lichens and microorganisms colonizinginternal fissures of the rock, by the method of Cubero

et al

.(1999). In this last case, small fragments of the endolithicbiofilm were selected under the stereomicroscope after fracturingthe rock along the lines of fissure. Two methods were used toamplify endolithic fungal or algal rDNA. In the first method,small fragments of lithic substrate containing endolithicfungal masses were placed in a 0.5 ml-Eppendorf tube fordirect PCR-amplification of the ITS regions of fungal rDNA,modified after Wolinski

et al

. (1999). In the second method,PCR was performed on total DNA isolated from identifiedapothecia and unidentified endolithic fungal masses. In total,12 endolithic fungal ITS sequences corresponding to threedifferent altitudes were determined. The primers used foramplification of the DNA from the fungal partner were ITS1Fand ITS4 (White

et al

., 1990) and from the algal partner wereITS1T and ITS4T (Kroken & Taylor, 2000). In each case, the

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50 µl PCR volume (10 m

Tris pH 8.3/50 m

KCl/1.5 m

MgCl

2

/50 µg gelatine) contained 1.25 units of Dynazyme Taqpolymerase (Finnzymes, Espoo, Finland), 0.2 m

of each of thefour dNTPs, 0.5 µ

of each primer and

c

. 10–50 ng genomicDNA. Products were cleaned using the QiaQuick Spin kit(Qiagen, Vienna, Austria). Both complementary strands weresequenced using the BigDye Terminator Cycle SequencingReady Reaction Kit (Applied Biosystems, Vienna, Austria)according to the manufacturer’s instructions. Sequences wererun on an ABI310 capillary sequencer (Applied Biosystems).The sequences are submitted to GenBank (nos AY667580,AY667581, AY667582 and AY667583).

Results

Epilithic thalli were scarce in the study area. Thalli of

Buelliafrigida

were mainly observed in zones of lower altitude. Afew thalli of

Umbilicaria aprina

and

Caloplaca

sp. were alsogrowing on rocks. Epilithic black apothecia correspondingto endolithic lichens could be detected in samples collectedacross the entire range of altitudes. Detailed light microscopyanalysis of these apothecia showed that these can be assignedto the species

Lecidea cancriformis

C.W. Dodge & G.E. Bakerand

Lecidea

sp. (Øvstedal & Lewis Smith, 2001). While

L.cancriformis

showed a more or less brown hypothecium andsmall and thin ascospores (7–9

×

2–3 µm), the hypotheciumwas colourless and ascospores were broadly elliptic (14–17

×

6–9 µm) in

Lecidea

sp. The second species cannot beassigned to any of the species cited for Antarctica. Neither arethe broadly elliptic ascospores of this

Lecidea

consistentwith

Lecidea

sp. A (Øvstedal & Lewis Smith, 2001), which isdescribed to have subglobose ascospores. No fruiting bodieswere observed in further samples but endolithic growths visibleto the naked eye as white masses were seen when the rockswere fractured along their fissures. The ITS regions of genomicDNA extracted from the two apothecia described above wereamplified to yield two PCR products of approximate size900 bp and 650 bp (lines 3 and 6 in Fig. 1). Products ofsimilar size were also obtained by PCR amplification of singleITS regions of genomic DNA from different endolithic masses(with and without fruiting bodies) found in rock fissures acrossthe altitude range (lines 2 and 5 in Fig. 1). We also tried directPCR on very small rock fragments colonized by endolithicforms with comparable results to those obtained after DNAisolation (line 4 in Fig. 1). The analysis of the obtained sequencesdetected only two distinct fungal sequences corresponding tothe two different product sizes. These sequences of similar lengthwere almost identical, but differed from those obtained forthe epilithic lichens found in the zone. Nevertheless, they wereidentical to those obtained through amplification of genomicDNA isolated from the two types of apothecium, allowing theidentification of endolithic growths.

Lecidea

sp. was detected onlyin rock samples collected on the coast and in close proximityto the corresponding apothecia. In contrast, endolithic

L.

cancriformis

were identified by molecular methods at coastalsites and in zones at 100 and 500 m a.s.l.

Algal rDNA ITS regions from specimens of

Lecidea

sp.,and of

U. aprina

and

B. frigida

from the same zone were alsoamplified and sequenced. However, we were only able toidentify one

Trebouxia

sequence among the three species. Thesequence coincides (98% similarity) with the denominated byRomeike

et al

. (2002) as

Trebouxia

sp. D11. The phylogeneticrelationships of this ITS-variant, which has so far been foundonly in Continental Antarctica, are not still clarified. We didnot manage to amplify algal rDNA from

L. cancriformis

,perhaps because of the high amounts of calcium oxalate insome of these samples.

We then applied the SEM–BSE method to examine howthese endolithic forms were distributed in the rock substrate.Epilithic apothecia showed continuity within the lithic sub-strate. The

Lecidea

sp. apothecium is connected via a cord ofdensely packed hyphae to the endolithic part of the lichen(Fig. 2a). Figure 2b is an enlargement of this apotheciumshowing subhymenium and hymenium layers made up ofparaphyses and asci containing ascospores at different devel-opmental stages. Apothecia were also detected inside fissures(marked by arrowheads in Fig. 2c). Despite the fact that theapothecia observed inside fissures were not as well structuredas the external ones, it is possible to distinguish their cup shape

Fig. 1 PCR products using fungal-specific primers of ITS ribosomal DNA regions of DNA isolated from Antarctic endolithic fungal forms. Lines 1 and 7: size marks; line 2: products of DNA isolated from fungal masses within a fissure in rock from a coastal region; line 3: products of DNA isolated from a Lecidea sp. apothecium; line 4: direct PCR products obtained from endolithic fungal cells. Line 5: products of DNA isolated from endolithic forms collected 500 m a.s.l.; Line 6: products of DNA isolated from a Lecidea cancriformis apothecium.

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Fig. 2 (a–d) Scanning electron microscopy with backscattered electron imaging (SEM–BSE) images of Lecidea sp. (a) Lichen apothecium and a nearby network of fissures filled with fungal hyphae. (b) Detail of the apothecium shown in (a) in which the subhymenium (sh) and hymenium (h) layer can be distinguished. P (paraphyses); A (asci). (c) Apothecium within a fissure indicated by arrowheads and a dotted line. (d) Enlargement of the zone indicated by the black arrow in (a) showing algal and fungal symbiont cells. (e) TEM image showing endolithic Lecidea sp. fungal cells in close contact with mineral fragments (black arrows). (f) SEM–BSE image of Lecidea sp. showing photobiont cells penetrated by fungal haustoria (black arrows).

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(outlined by a dotted line in Fig. 2c). Algal and fungal cellswere observed in fissures close to the apothecium (Fig. 2d).Fragmentation of the mineral substrate was clearly visible inzones occupied by this lichen (Fig. 2a). The symbiont cellsappeared to be closely associated with mineral fragments(Fig. 2e). In zones occupied by Lecidea sp., algal cells couldbe observed by SEM–BSE to be penetrated by fungalcells (arrows in Fig. 2f ). On TEM examination, the interfacebetween the algal and fungal cells of this endolithic lichencould be seen in more detail. Fungal haustoria penetrated thealgal cell wall and plasma membrane (Fig. 3a), pushing on thecytoplasm. These intracellular haustoria were thin at theirextreme ends (arrowhead in Fig. 3a,b) and appeared to besurrounded by a sheath (arrows in Fig. 3a). Most of the algalcells were penetrated and lipid globules were observed at thesites of penetration (Fig. 3b). Bacterial cells were detected closeto these penetrated cells (black arrows in Fig. 3b).

In rock samples from areas of higher altitude, L. cancriformiswas frequently identified by ITS rDNA sequencing, evenwhen epilithic apothecia were lacking. Our SEM–BSE studyof these samples revealed internal fissures of the rock fullyoccupied by fungal and algal cells (Fig. 3c). This time, algalcells were not penetrated by the fungal partner (Fig. 3d).Several mineral deposits were observed closely associated withthe cells in the fissures occupied by this lichen (arrow Fig. 3e).In some zones, these deposits covered extensive areas of thefissure (arrows in Fig. 3f ), coinciding with zones lacking algalcells. EDS analysis revealed their basic composition was cal-cium, carbon and oxygen (black arrows in Fig. 4a) permittingtheir identification as calcium oxalate deposits. A furthermineral alteration was observed close to these calcium oxalatedeposits consisting of numerous holes (white arrows in Fig. 4a).This altered zone was shown by EDS to have a lower amountof calcium than nearby areas of unaltered substrate (whitearrows in Fig. 4a), indicating calcium depletion. Potassiumdepletion in biotites was also observed in the proximity ofthis endolithic lichen, as shown in the EDS element distri-bution map in Fig. 4b. Chemical alterations were also notedin feldspar mineral grains found inside fissures (Fig. 4c). TheEDS scan-line shown in this figure indicates a lower amount ofcalcium in peripheral than in the central areas of these grains.

Using molecular methods we were also able to identifyendolithic lichens positioned under epilithic lichens. Fig. 4d–f show an endolithic form of L. cancriformis from the coastunder an U. aprina thallus. The part of the fissure open to thesurface was occupied by mineral fragments and fungal cells(Fig. 4d). Under this, a second zone could be identified inwhich the first algal cells appeared (Fig. 4e). Beneath this, thedeeper cavities were seen to harbour many algal cells (Fig. 4f ).

Discussion

A link between epilithic apothecia and the endolithic growthform seems evident in many cases but is uncertain in others

(Friedmann et al., 1988). This makes their identification usingonly morphological criteria almost impossible. However,the combined use of molecular and microscopy techniquesenabled the identification of endolithic lichens (even in theabsence of fruiting bodies), which can be precisely localized inthe rock substrate and characterized at the ultrastructural level.High resolution microscopy procedures were needed to localizeand characterize the lichen symbionts whereas moleculartechniques served to identify them. Our approach also enabledus to simultaneously evaluate specific interrelations betweenthe symbionts and the lithic substrate and deepen ourunderstanding of the ecology of the biofilms formed at theinterface of the substrate colonized by these lichens. In thisway, the characterization of these endolithic biofilms can becomplete and precise.

Lecidea cancriformis and Lecidea sp. were detected across analtitudinal range in Granite Harbour (Antarctica). The pres-ence of L. cancriformis had been already reported in differentAntarctic regions (Øvstedal & Lewis Smith, 2001). Severalendolithic lichen thalli show a similar structure to ordinaryepilithic forms when embedded in limestone (Fry, 1922;Pinna et al., 1998; Russell et al., 1998). However, in the endo-lithic lichens growing on granite examined here, it was notpossible to discern all these layers. Indeed, the distribution ofcells in the fissures and cracks of the granite substrate mighthinder this kind of organization. However, while fungal cellsoccurred in all the fissures occupied by the lichen, algal cells onlyappeared in some zones. Different mycobiont–photobiontinterfaces such as appressoria, and intraparietal and intracel-lular haustoria have been identified in the few endolithiclichen species studied so far (Plessl, 1963; Galun et al., 1971;Kushnir & Galun, 1977; Friedmann, 1982). These two endo-lithic Antarctic lichens differed in terms of their alga-fungusrelationship. Frequent fungal penetration into the algal part-ner by means of intracellular haustoria was only observed inLecidea sp. For endolithic growths, this ultrastructural traitwas consistent with molecular identification.

It is not completely clear how these lichens colonizetheir endolithic habitat. Diversity and distribution patterns oflichen communities in Antarctica could be a combination ofrelict flora, long-distance dispersal and recent colonization(as suggested by Vincent, 2000; Romeike et al., 2002). Theinternal distribution of lichens in the lithic substrate can bethe end result of several factors, differing on a small spatial scale.Local aspects such as rock composition, the presence and con-figuration of cracks, fissures or rock discolorations can alter theamount of light penetrating deeply, and among other factors,condition the presence of particular microorganisms in thesubstrate (Matthes et al., 2001). In addition, the endolithicdevelopment of certain organisms is also influenced by thepresence of epilithic lichens or other endolithic microorgan-isms and their actions on the substrate, inducing the forma-tion of microhabitats and chemical environments in rocksubstrates (De los Ríos et al., 2002). The observation of

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Fig. 3 (a–b) TEM images showing the fungal–algal interface of Lecidea sp. symbionts. Arrowheads point to the thinnest part of the haustoria. (a) Zone of algal cell penetrated by fungal haustoria. Black arrows indicate the haustoria sheath. (b) Trebouxia cell with numerous lipid bodies (L) in the zone of haustoria penetration. Black arrow indicates bacteria cells. (c–f) Scanning electron microscopy with backscattered electron imaging (SEM–BSE) images of Lecidea cancriformis. (c) General view of a network of fissures colonized by these species. (d) Detailed image of photobiont and mycobiont cells. (e) Fissure occupied by algal and fungal cells in which calcium oxalate deposits can be observed (white arrow). (f) Fissure colonized by mycobiont cells showing large deposits of presumptive biominerals in the form of calcium oxalate deposits.

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Fig. 4 (a) SEM–BSE and energy dispersive X-ray spectroscopy (EDS) elemental distribution maps of a zone colonized by Lecidea cancriformis. Black arrow indicates calcium oxalate deposits and white arrows point to an altered zone showing calcium depletion. (b) EDS potassium and iron distribution map showing biotites in areas of potassium depletion starting from the zone indicated by arrows. (c) Feldspar fragment found inside a granite fissure and EDS scan-line showing relative concentration changes in silicon (above) and calcium (below) along the line, indicating the depletion of calcium at the margins of the rock fragment. (d–f) SEM–BSE images of L. cancriformis detected under Umbilicaria aprina thalli in a coastal locality. (d) Zone close to the surface in which only fungal cells intermixed with several mineral fragments appeared. (e) Zone of the fissure showing the first algal cells. (f) Deeper zone showing numerous algal cells.

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epilithic lichen thalli upon substrates colonized by endolithicLecidea with the same photobiont species suggests thatendolithic lichens may provide algal species that facilitatecolonization by other lichens.

Pinna et al. (1998) attributed typical features of euendo-lithic organisms to several endolithic calcicolous lichens.The endolithic lichens of our study shared chasmoendolithicand euendolithic features. These lichens colonize fissures andcracks in the rock, but the alterations observed in the proxim-ity of the fungal partner also indicates a perforating capacity.Adopting one particular endolithic ecological niche overanother could depend of the features of the biofilm and theenvironmental conditions. The long-term survival of Antarc-tic endolithic communities is closely linked to weathering(Johnston & Vestal, 1993). The development of apothecia atthe surface and penetration of fungal cells in fissures observedin the material examined can explain the biogeophysicalaction on the substrate related to both lichen species. Physicalweathering by epilithic lichens is frequently associated with abiogeochemical action (Bjelland & Thorseth, 2002; De losRíos et al., 2002). Several signs of biogeochemical substratealterations were also detected in the vicinity of these endo-lithic lichens. Evidence of calcium and potassium biomobili-zation processes, as well as calcium oxalate accumulation, wasobserved around the mycobiont cells. Biomobilization hasbeen frequently associated with the biogeochemical action ofdifferent lithobionts (Wierzchos & Ascaso, 1996, 1998; de losRíos et al., 2003). Extensive calcium oxalate deposits weredetected close to L. cancriformis thalli. However, it was notpossible to attribute the presence of calcium oxalate to thisspecies, since we found no such deposits in the vicinity ofL. cancriformis detected on the coast. The presence of calciumoxalate deposits seemed here to be mainly related to the chem-ical composition of the substrate. Fungi colonizing granitewith high amounts of feldspar plagioclase containing calciumwere found to accumulate this biomineral. Bjelland et al.(2002) have also recently associated the presence of calciumoxalate with specific local geochemical features of the lichen-colonized substrate. The functions of calcium oxalate inlichens remain unclear. Calcium oxalate synthesis was inter-preted by Wadsten & Moberg (1985) as a calcium detoxifica-tion mechanism, but this seems unlikely in these granitecolonizers, since the amount of calcium in the substrate is low.Cells of endolithic biofilms densely accumulate in fissures andcavities where light is scarce. The presence of calcium oxalatein these biofilms could facilitate endolithic life if they actas radiation reflectors (Modenesi et al., 2000), especially in thepresence of extracellular polymeric substances (EPS). It hasbeen recently reported that biofilm organization and, morespecifically, the ‘biofilm gel effect’ of EPS lead to a moreefficient acquisition of solar energy (Decho et al., 2003).

Romeike et al. (2002) established ITS sequences in fourAntarctic Umbilicaria collected from different sites. TheirITS rDNA sequence for Trebouxia, consistent with the

sequence obtained in our study, corresponds to the onlyTrebouxia sequence that to our knowledge has been obtainedfrom lichens collected around the Granite Harbour area. Theseresults could indicate a low incidence of Trebouxia species inthese harsh conditions and/or poor specificity of the mycobi-ont. Mycobionts less specific in their choice of photobiont areable to survive in conditions in which only some photobiontspecies exist. A low degree of selectivity towards the photobi-ont partner has been attributed to some Antarctic lichensfor different reasons. High genetic diversity of Trebouxia wasfound in Umbilicaria species along a transect of the Antarcticpeninsula (Romeike et al., 2002), while low diversity of Nostocwas reported in different cyanolichen species from maritimeAntarctica (Wirtz et al., 2003). The authors related thelow photobiont selectivity found in these mycobionts to theextreme environmental conditions. However, if the occurrenceof algal species depends on environmental conditions, theavailability of algae in the severe environment of this conti-nental Antarctic region would be a more limiting factor forthe mycobiont than its preferences for certain photobionts. Itis likely that only certain resistant strains of Trebouxia are ableto live in these peculiar environmental conditions. Only onedominant green alga rDNA sequence was also found by De laTorre et al. (2003) in cryptoendolithic communities ofBeacon sandstone from the McMurdo Dry Valleys (Antarctica).Fully analysing these microbial communities and approachingthe complex processes that take place within them involvesidentifying their members and understanding their func-tional relationships. We describe the use of molecular andmicroscopy tools to map the distribution of endolithic lichensand evaluate different aspects of their ecology. Across therange of altitudes examined, we identified two Lecidea lichensshowing a different distribution pattern and established theexistence of a close relationship with their immediateenvironment.

Acknowledgements

The authors would like to thank Fernando Pinto and SaraLapole for technical assistance, Dr M. Castello (Trieste, Italy)for helping with taxonomic identification and Ana Burtonfor reviewing the English. We are especially grateful to Prof.A.T.G. Green and Antarctica New Zealand for their logisticsupport and excellent field facilities. This study was funded bygrants REN2003-07366-C02-02, BOS2003-02418 of thePlan Nacional I+D and Acciones Integradas funds (ÖAD/MCyT). MG acknowledges support by FWF P11998.

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