In vitro culturing and resynthesis of the mycobiont ... · tact (appressoria) and is followed by...

The Lichenologist 45(1): 65–76 (2013) 6 British Lichen Society, 2013doi:10.1017/S002428291200059X

In vitro culturing and resynthesis of the mycobiont Protoparmeliopsismuralis with algal bionts

Beata GUZOW-KRZEMINSKA and Elfie STOCKER-WORGOTTER

Abstract: The widespread and ubiquitous lichen-forming fungus Protoparmeliopsis muralis is able toform a thallus with Trebouxia species. In this study, several photobiont strains were isolated fromdifferent specimens of P. muralis and cultured in vitro. The compatibility of Trebouxia spp. andAsterochloris algae with P. muralis were investigated in in vitro resynthesis experiments and the re-lichenized bionts were observed with the scanning electron microscope. It was found that, in additionto compatible photobionts, also a presumably incompatible Asterochloris sp. was able to interact withthe mycobiont. The life strategy that enables the mycobiont to form associations with a wider rangeof photobionts could be advantageous for the survival of the lichen and successful colonization ofnew habitats.

Key words: compatibility of bionts, ITS rDNA sequencing, lichen, photobiont, scanning electronmicroscopy

Accepted for publication 31 July 2012

Introduction

Lichens are composed of a fungal (mycobiont)and a photosynthetic partner (photobiont). Itis estimated that less than 150 species ofphotobiont are described so far from lichens(Honegger 2009). About 85% of lichen-forming fungi associate with green algae, andunicellular Trebouxia spp. are among the mostcommon photobionts being found in c. 20%of all lichen species (Friedl & Budel 1996).Terms ‘selectivity’ and ‘specificity’ refer tosymbiotic associations and are differently de-fined in the literature, leading to confusion assummarized by Honegger (2008). Accordingto Galun & Bubrick (1984) ‘selectivity’ is thepreferential association between symbioticpartners while ‘specificity’ means interactionwith absolute exclusivity. Smith & Douglas(1987) defined ‘specificity’ as the degree oftaxonomic difference between partners with

which an organism associates, and used ‘selec-tivity’ to refer to their availability in naturalecosystems. Beck et al. (2002) proposed touse the term ‘selectivity’ for the characteriza-tion of the range of possible partners that canbe selected by this biont (viewed from the per-spective of one biont only), while ‘specificity’should be used for the symbiotic associationas a whole. Later, Yahr et al. (2006) defined‘specificity’ as the phylogenetic range of com-patible partners for a given symbiont, and‘selectivity’ as the frequency of associationwith any of the compatible partners. Herewe refer to the term ‘specificity’ as the num-ber of partners that can be selected by onebiont, and ‘selectivity’ as the degree of pref-erential interaction between symbiotic part-ners. Many lichens form vegetative propa-gules that enable the joint propagation offungal and algal partners, such as sorediaand isidia. In others, however, both biontsdisperse as separate units. In lichens discharg-ing sexual fungal spores, the re-lichenizationhas to be established with an appropriate algalstrain in each generation de novo (Beck et al.1998; Sanders & Lucking 2002). Althoughlichen-forming algae in the genus Trebouxia

B. Guzow-Krzeminska: Department of Molecular Biology,University of Gdansk, Wita Stwosza 59, 80-308 Gdansk,Poland. Email: [email protected]. Guzow-Krzeminska and E. Stocker-Worgotter: De-partment of Organismic Biology, University of Salzburg,Hellbrunner Str. 34, 5020 Salzburg, Austria.

Puymaly were postulated not to occur infree-living stages (Ahmadjian 1988, 1993),a limited number of reports on free-livingTrebouxia spp. are available (e.g. Tschermak-Woess 1978; Bubrick et al. 1984; Mukhtaret al. 1994; Sanders 2005). However, free-living Trebouxia spp. are neither an abundantnor a dominant element of the community,and in most ecosystems the most commonaerophilic algae are non-symbiotic algae(Honegger 2009). A germinating fungal sporemay survive on a substratum for a longerperiod of time if it is able to form an associa-tion with a non-compatible photobiont. Thistemporary state lasts until the appropriatealgal strain is available (Ott 1987). In thepast decade, our knowledge of fungal-algalassociations has improved with an increasein the number of studies that have focusedon the selectivity and population structureof photobionts (e.g. Beck et al. 1998;Piercey-Normore & DePriest 2001; Guzow-Krzeminska 2006; Piercey-Normore 2006;Hauck et al. 2007; Muggia et al. 2008;Wornik & Grube 2010). These studies pro-vide information on the potential range ofphotobiont partners that associate with lichenfungi.

Two different experimental approachesare used to investigate selectivity of thelichen symbionts. The first one involves invitro resynthesis of the axenically culturedmycobiont with different photobiont species.Another approach is based on the analysisof the specimens collected in different geo-graphical regions and the identification ofthe symbionts present in the lichen thalli(Honegger 1996, 2008). Our present knowl-edge on the range of acceptable photobiontsis mainly based on this type of investigation,especially as molecular approaches facilitateidentification of algal bionts in thalli andresolve relationships among photobionts. Assummarized by Honegger (2008), availabledata suggest that morphologically advancedlichen taxa are specific to moderately specificas most green-algal lichen-forming fungiassociate with different genotypes of onephotobiont species (e.g. Ohmura et al. 2006;Hauck et al. 2007). However, some myco-bionts may accept a wide range of Trebouxia

strains (e.g. Blaha et al. 2006; Guzow-Krzeminska 2006). Low specificity and selec-tivity have been suggested as a strategy thatfacilitates lichen symbionts surviving un-favourable environmental conditions (e.g.Romeike et al. 2002; Piercey-Normore 2006).

Schaper & Ott (2003) proposed a modelof the lichenization process for which theyrecognized different levels of compatibilityof the symbionts. In vitro studies give us theopportunity to observe initial stages of thelichenization process. The first step is pre-contact that takes place prior to physical con-tact. Symbionts are regarded as compatible ifthey enter the second stage of development,characterized by an early, tight contact ofthe mycobiont and photobiont cells andfollowed by the envelopment of the algal cellsby the fungal hyphae (Ahmadjian et al. 1978;Galun 1988; Joneson & Lutzoni 2009). Theprocess begins with simple wall-to-wall con-tact (appressoria) and is followed by theformation of haustoria-like structures thatis a further step in the establishment of asymbiotic contact between compatible sym-bionts. Initial contact may also be character-ized by the formation of mucilage and is fol-lowed by the development of soredia-likestructures embedded in a gelatinous matrix.However, with decreasing compatibility ofthe partners, the recognition process is muchslower and for less compatible bionts only aloose structure of the algal-fungal associationis formed (with less intimate contact betweensymbionts). For non-compatible photobionts,no recognition is observed and the develop-mental stages of initial lichenization do notoccur (Schaper & Ott 2003).

Protoparmeliopsis muralis [syn. Lecanora mu-ralis (Schreb.) Rabenh.] is a green-algal lichencontaining Trebouxia photobionts [note thevalidity of the name Protoparmeliopsis muralisis under discussion (Laundon 2010)]. A re-cent molecular study showed that P. muralisforms a strongly supported monophyleticgroup with other lobate species (Perez-Ortegaet al. 2010). It is a widespread lichen thatcan colonize different types of substrata. Nu-merous apothecia are always present on themature thalli of this lichen which suggestspredominant propagation of the lichen fungus

THE LICHENOLOGIST66 Vol. 45

by meiotic fungal spores. It was previouslyfound that different photobionts are compat-ible algal partners for P. muralis, but these arelimited to the genus Trebouxia. Based on ITSrDNA diversity, the following photobiontshave so far been identified in the thalli of thislichen: Trebouxia asymmetrica, T. gigantea, T.cf. impressa, T. incrustata and an unidentifiedlineage of Trebouxia named T. sp. ‘muralis I’(Guzow-Krzeminska 2006). The low level ofspecificity and selectivity may constitute animportant reason why Protoparmeliopsis mura-lis is one of the most successful urban lichensin the world.

The major objective of this study was toisolate the photobionts and mycobiont fromProtoparmeliopsis muralis thalli and test theoptimal conditions for in vitro growth of themycobiont. Moreover, it was our aim to ob-serve re-lichenization events with compatible,and presumably incompatible, algae. Forthis reason, an artificial resynthesis of the P.muralis mycobiont with different photobiontswas performed.

Materials and Methods

Selected specimens

For the mycobiont isolation we used a specimen ofProtoparmeliopsis muralis (BGK 232) collected in Osowiec,which is located in the Biebrza National Park (Poland).This particular specimen contained Trebouxia sp. ‘muralisI’ as the photobiont. Other specimens of P. muralis wereused for photobiont isolation (as listed in Table 1). Thefollowing algae were used in resynthesis experiments:Trebouxia asymmetrica Friedl & Gartner, T. gigantea(Hildreth & Ahmadjian) Gartner, T. incrustata Ahmad-

jian & Gartner, Trebouxia sp. ‘muralis I’, and Asterochlorissp. (isolated from Cladonia confusa R. Sant. and kindlyprovided by S. Wornik, Graz). The strains used in theexperiments are deposited at the Department of Molec-ular Biology of the University of Gdansk, and detailedinformation on these strains is given in Table 1.

Isolation and culture of photobionts

Prior to isolation, the lichens were examined under astereomicroscope and pieces of thalli were washed insterile water with Tween 80. Photobionts were isolatedfollowing the thallus fragmentation procedure of Yama-moto (1990), modified according to Stocker-Worgotter(2002). The fragments were inoculated on agar slantsin tubes containing solid medium. After 4–6 weeks thealgae were transferred to a sterile Petri dish containingsolid medium with a sterile inoculation loop. The photo-bionts were sub-cultured on Bold’s Basal Medium(BBM) (Deason & Bold 1960; Bischoff & Bold 1963)and Modified Bold’s Basal Medium (MBB) (Friedl1989) in order to provide optimal conditions for thegrowth of algae (from 2 to 5 replicates on each medium).The cultures were grown in a culture chamber undera light-dark cycle at 20�C for 14 h and 15�C for 10 h,and a light intensity of 50–100 mmol m–2 s–1 (standardconditions).

Isolation and culture of mycobiont

The mycobiont was isolated from the spores usingthe Ahmadjian method (Ahmadjian 1993), with an addi-tional pre-washing step of the apothecia in water (a smalldrop of Tween 80 detergent was added to bi-distilledwater in a 50 ml beaker). In order to remove contamina-tions and dirt particles by rotations the beaker wasplaced on a magnetic stirrer.

The cultures obtained were multi-spore cultures. Themycobiont was sub-cultured on the following media:the modified Bold’s Basal Medium with 1% glucose(Deason & Bold 1960; Bischoff & Bold 1963), Murashige-Skoog (Stocker-Worgotter 2001a), G-LBM (Brunaueret al. 2007) and Saboraud 2% glucose medium (Fluka

Table 1. List of mycobiont and algal species used in resynthesis experiments with details of the lichens specimens that were usedfor the isolation of the biont. GenBank accession numbers of the ITS rDNA sequences obtained in this study are provided.

Strain SpeciesSource

(specimens collected by BGK, unless otherwise stated)GenBank

Acc. Number

BGK232 Protoparmeliopsis muralis P. muralis, Poland, Biebrza National Park, Osowiec,2005, Wegrzyn BGK232

HM209239

BGK3956 Trebouxia asymmetrica P. muralis, Poland, Zarzecze, Kukwa UGDA-L 3956 JQ359769

BGK29 T. gigantea P. muralis, Poland, Malbork, BGK29 JQ359768

BGK190 T. incrustata P. muralis, Poland, Przywidz, BGK190 JQ359766

BGK243 T. sp. ‘muralis I’ P. muralis, Austria, Graz, 2006, Kukwa BGK243 GU339206

BGKT5 Asterochloris sp. Cladonia confusa (algal strain provided by S. Wornik, Graz) JQ359767

2013 Resynthesis of Protoparmeliopsis muralis—Guzow-Krzeminska & Stocker-Worgotter 67

N. 84086). The cultures were kept in the dark in theculture chamber at 20�C for 14 h and 10�C for 10 h.Well-developed mycelia were used for further resynthesisexperiments.

Resynthesis of Protoparmeliopsis muralis withdifferent photobionts

Axenically grown mycelia were used for the resynthesisexperiments and different algal species were combinedwith the mycobiont. Fungal colonies were homogenizedin sterile double-distilled water with a pestle in a mortar.Then a small amount of algal cells (about 1/3 of thevolume of the mycobiont suspension) was taken froman axenic culture and added to the homogenized myco-biont. The suspension containing mixed bionts wastransferred with a Pasteur pipette to a new Petri dishcontaining nutrient media. The following media wereused for resynthesis experiments: Murashige-Skoog(Stocker-Worgotter 2001a), G-LBM (Brunauer et al.2007), Saboraud 2% glucose medium (Fluka N. 84086)and BBM (Deason & Bold 1960; Bischoff & Bold 1963)enriched with 0�5% mannitol. The cultures were kept inthe culture chamber under a day/night cycle at 20�C for14 h and 10�C for 10 h and a light intensity of 50–100mmol m–2 s–1 (standard conditions). The effect of thegrowth of the bionts in the resynthesis culture wasobserved with the stereomicroscope every week andassessed visually.

Microscopic investigations

In order to examine the identity of isolated photo-bionts, light microscopy studies were performed using aNikon Eclipse E800 microscope. The identification ofalgae was carried out following a determination key byEttl & Gartner (1995).

Scanning electron microscopy (SEM)

The sample, a small piece of the resynthesis culture,was attached to the holder, frozen in liquid nitrogen andsputtered with platinum. Afterwards, the selected speci-mens from the resynthesis experiments were investigatedwith a JEOL JSM-7401F Field Emission Scanning Elec-tron Microscope (Biology Centre - Institute of Parasitol-ogy, Ceske Budejovice, Czech Republic) at an accelerat-ing voltage of 3�0 kV.

Control samples, aposymbiotically grown Trebouxiasp. ‘muralis I’ and P. muralis, were cut from the agarmedium and chemically fixed according to the followingprotocol: samples were fixed for 2 h at room temperatureand then overnight at 4�C in the solution consisting of5% glutaraldehyde and 0�5 M cacodylate buffer, pH7�0. The samples were then washed twice with 0�5 Mcacodylate buffer, pH 7�0, followed by washing in dis-tilled water (twice for 15 min). In the next step thesamples were gradually dehydrated in 10%, 20%, 30%ethanol (15 min each), then 50%, 75% ethanol (30 mineach), followed by 96% and finally twice in anhydrousethanol (1 h each). Dehydrated samples were subjectedto critical point drying using EMITECH K850 at 35–

40�C and a pressure of 73 Atm. The samples were thensputtered with gold. The selected control specimenswere investigated using a Philips XL30 Scanning Elec-tron Microscope (Laboratory of Electron Microscopy,University of Gdansk, Gdansk, Poland) at an accelerat-ing voltage of 15�0 kV.

DNA analyses

Total genomic DNA was extracted directly from algaland fungal cultures using the CTAB method (Armaleo& Clerc 1995). DNA was re-suspended in sterile distilledwater. PCR amplifications were performed using TetradMJ Research thermal cycler or GeneAmp 9700 PCRThermal Cycler (Applied Biosystems). One unit ofRedTaq polymerase (Sigma) was used for each 50 ml ofmaster mix containing 5 ml of 10� Taq polymerase reac-tion buffer, 0�2 mM of each of the four dNTP’s and 0�5mM of each primer. The primers Al1500bf (Helms et al.2001) and LR3 (Friedl & Rokitta 1997) were used asPCR primers for amplification of algal nuclear InternalTranscribed Spacer (ITS) ribosomal DNA (rDNA). Inseveral cases, a semi-nested-PCR was performed usingAl1500bf (Helms et al. 2001) and ITS4M (Guzow-Krzeminska 2006) primers. For the fungal DNA,ITS1F (Gardes & Bruns 1993) and ITS4 (White et al.1990) primers were used. After an initial denaturationstep at 95�C for 10 min, the PCR ran for 35 cycles(95�C for 1 min, 51�C for 40 s, 72�C for 1 min) with afinal extension step at 72�C for 10 min. PCR productswere resolved on agarose gels in order to determineDNA fragment lengths and then purified using HighPure PCR Product Purification Kit (Roche) and se-quenced using Macrogen (Korea) sequencing service(www.macrogen.com). For sequencing of the algal ITSregion, the following primers were employed: ITS1(White et al. 1990) and ITS4M (Guzow-Krzeminska2006). For sequencing of fungal DNA, ITS1F (Gardes& Bruns 1993) and ITS4 (White et al. 1990) primerswere used.

The newly determined ITS rDNA sequences fromphotobionts and mycobionts of P. muralis (Table 1)were compared to the sequences available in GenBankusing BLAST (Altschul et al. 1990).

Results

The symbionts

The following Trebouxia species were iso-lated from Protoparmeliopsis muralis thalli andgrown aposymbiotically: T. asymmetrica, T.gigantea, T. incrustata and unknown Trebouxiasp. ‘muralis I’ (here named after Guzow-Krzeminska 2006). Despite several attempts,we failed to isolate T. impressa from the thalliof P. muralis although it was previously re-ported to be associated with this lichen(Guzow-Krzeminska 2006). However, this


www.macrogen.com

particular photobiont was identified in thethallus of P. muralis only once based on ITSrDNA sequencing. The specimen that wasreported to contain T. impressa as photobiontwas collected in 2003 (Guzow-Krzeminska2006), and during the present work it wastoo old to provide a source of the algal strainand it failed to grow in culture.

The algal cultures were identified under alight microscope by comparing morphologi-cal features and by using a determinationkey (Ettl & Gartner 1995). Moreover, thealgal ITS rDNA region was amplified, se-quenced and the identity of the isolates waschecked using BLAST search (Altschul et al.1990). ITS rDNA sequences of algal strainsthat were used for the resynthesis experimentswere deposited in GenBank and their acces-sion numbers are provided in Table 1.

The mycobiont cultures obtained werepoly-spore cultures as the mycelium was grownfrom multiple spores released from a singleapothecium (Fig. 1A). The mycobiont inves-tigated was associated in nature with Tre-bouxia sp. ‘muralis I’ that was identified inthe thallus of this specimen based on ITSrDNA sequencing. Protoparmeliopsis muraliswas successfully sub-cultured on Murashige-Skoog (Stocker-Worgotter 2001a), G-LBM(Brunauer et al. 2007) and BBM medium(Deason & Bold 1960; Bischoff & Bold1963) enriched with glucose. The myceliawere well developed on these media (Fig.1B–D), but the growth of the mycobiontwas slightly inhibited on Saboraud 2% glu-cose medium. The identity of the sub-cultured mycobiont was confirmed by BLASTanalysis of its nuclear ITS rDNA sequence(GenBank accession number HM209239).

Resynthesis experiments

The compatibility of Protoparmeliopsis mura-lis with different algal species under sterile con-ditions was investigated using in vitro resyn-thesis experiments. The mycobiont used inour study was associated in nature with Tre-bouxia sp. ‘muralis I’. Different media wereused for the resynthesis experiments and itwas observed that the growth of some algae,such as T. asymmetrica (Fig. 2A), was inhibited

on Saboraud 2% glucose medium. From thespecies analyzed, only Trebouxia gigantea andT. incrustata exhibited an increased growth onthis medium (Fig. 2B). Other Trebouxia spp.stopped growing on this medium at the begin-ning of the resynthesis experiment, while thegrowth of Asterochloris sp. was comparable tothat of the fungus until the third week of incu-bation, when it was observed that the growthof the mycobiont was faster, and after sevenweeks the photobiont was finally overgrownby the mycobiont. On the other hand, on othermedia tested (i.e. Murashige-Skoog and G-LBM), the cultures were overgrown by thealgae in most cases (Fig. 2C, D & E). We alsoobserved using SEM that on these media, co-cultures of the fungus and the algae werecovered with a gelatinous matrix as presentedfor G-LBM (Fig. 2F).

It was found that the modified BBMmedium (enriched with mannitol) enabledthe balanced growth of both bionts and themycobiont formed numerous filamentoushyphae. We observed that the mycobiontwas able to establish the primordial stages oflichenization with all Trebouxia photobiontsinvestigated on this medium. After 7 weeksof incubation it was observed that fungalhyphae enveloped the algal cells, forming adense network around the photobionts (Fig.3A–C). Scanning electron microscopy (SEM)was used for studying the early contact be-tween lichen bionts. SEM observations wereperformed after 4 and 10 months of incu-bation and showed a very close contact be-tween hyphae and compatible photobiontsin the resynthesis cultures (Fig. 4A & B).The hyphae enveloped even single cells andformed appressoria around compatible pho-tobiont cells. Moreover, the surface of thecultures, at least partly, was covered with agelatinous matrix. Algal cells were incor-porated within this matrix but there was nothallus-like differentiation on agar media.Furthermore, SEM controls of aposymbioti-cally grown bionts (i.e. P. muralis and Tre-bouxia sp. ‘muralis I’) were also prepared(Fig. 4E & F). The presence of crystals ofsecondary metabolites was observed on thefungal hyphae but no such gelatinous matrixwas noticed on either the algal or fungal cells.


The mycobiont was also able to establishprimordial stages of lichenization with anotherphotobiont, Asterochloris sp., regarded as anincompatible algal taxon. In this study, weobserved that Protoparmeliopsis muralis inter-acted with Asterochloris sp., although the algaltaxa of this genus have never been reportedto form a lichen thallus with this mycobiont.However, it seemed that the growth of thealgae was accelerated in comparison to the

slow growth of the mycobiont. The algaeformed cell aggregates enveloped by fungalhyphae (Fig. 3D). However, the fungal hy-phae did not tightly cover all the algal cells,but were concentrated only in some partsof the resynthesis culture; in other parts, thehyphae were only growing on the algal cellswithout characteristic envelopment or closecontact. The contact observed with the scan-ning electron microscope between P. muralis

Fig. 1. Mycobiont isolated from lichen Protoparmeliopsis muralis. A, germinating fungal spores; B, mycelium grownon BBM medium enriched with 1% glucose after 3 months of incubation; C, mycelium grown on MS medium after3 months of incubation; D, mycelium grown on G-LBM medium after 3 months of incubation. Scales: A ¼ 20 mm;

B–D ¼ 1 cm.


and Asterochloris sp. was close and the hyphaeformed appressoria. Moreover, the algae andfungi were covered with a gelatinous matrix.Finally, some of the algal cell aggregates were

embedded within this matrix which may haveformed as a response to the close contact be-tween the symbionts, and probably also as areaction to environmental influences, such

Fig. 2. Cultures from resynthesis experiments of Protoparmeliopsis muralis with Trebouxia asymmetrica and T. giganteagrown on different media after 7 weeks of incubation. A, T. asymmetrica on Saboraud 2% medium; B, T. giganteaon Saboraud 2% medium; C, T. asymmetrica on Murashige-Skoog medium; D, T. gigantea on Murashige-Skoogmedium; E, T. asymmetrica on G-LBM medium; F, scanning electron micrograph of the resynthesis of Proto-

parmeliopsis muralis with T. gigantea on G-LBM medium. Scales: A–E ¼ 1 cm; F ¼ 10 mm.


as protecting the algal cells against drynesson exposed surfaces (Fig. 4C & D).

Discussion

Different levels of specificity and selectivityhave been reported from a considerable num-ber of lichens, representing diverse growth-form types (e.g. Beck & Koop 2001; Romeikeet al 2002; Blaha et al. 2006; Guzow-Krzeminska 2006; Wornik & Grube 2010).A previous study based on ITS rDNA se-quencing of selected samples from the naturalenvironment showed that several Trebouxiaspp. were associated with Protoparmeliopsismuralis (Guzow-Krzeminska 2006).

Although many papers have been publishedon in vitro resynthesis of lichens from their

aposymbiotic bionts (e.g. Stocker-Worgotter& Turk 1991; Stocker-Worgotter 2001a, b,2002, 2010; Brunauer et al. 2007), ourknowledge of the conditions needed for re-lichenization of many species still not in-vestigated has to be complemented by novelapproaches using new methodologies (e.g.Joneson et al. 2011). The issue of ‘resynthesis’of lichens from their partners is, in general,quite difficult to explore experimentally, be-cause each time the experimental system hasto be optimized de novo. However, we foundhere that the modified BBM medium (en-riched with mannitol) enabled the balancedgrowth of both bionts. Joneson et al. (2011)also used BBM medium enriched with MYfor Cladonia grayi resynthesis experiments.Therefore, it might be inferred that a slightly

Fig. 3. Cultures from resynthesis experiments grown on BBM medium enriched with mannitol after 7 weeks ofincubation. A, Trebouxia incrustata; B, Trebouxia sp. ‘muralis I’; C, Trebouxia asymmetrica; D, Asterochloris sp.

Scale ¼ 1 mm.


Fig. 4. Scanning electron microscope (SEM) micrographs. A, co-culture of Protoparmeliopsis muralis with Trebouxiagigantea; B, co-culture of P. muralis with Trebouxia sp. ‘muralis I’. ; C, co-culture of P. muralis with Asterochloris sp.;D, co-culture of P. muralis with Asterochloris sp. Scale bar; E, aposymbiotically grown P. muralis; F, aposymbiotically

grown Trebouxia sp. ‘muralis I’. Scales: A & C ¼ 2 mm; B & D–F ¼ 10 mm.


enriched BBM medium could be used as ageneral medium for resynthesis experimentsfor a variety of lichens in future studies.

Joneson & Lutzoni (2009) observed inter-actions of Cladonia grayi with its Asterochlorisphotobiont and other phototrophs, andshowed that the morphological response ofC. grayi was distinctive only in symbioticgrowth with the compatible biont. Compati-bility of lichen bionts has been discussed pre-viously by many authors (e.g. Beck et al.2002; Joneson & Lutzoni 2009). The non-compatible bionts do not interact to formany of the initial stages of lichen develop-ment. On the other hand, compatible biontsenter into stage two of the development, thatis the envelopment of the alga by the myco-biont hyphae. At the same time, gene expres-sion was also studied and several proteinswere found to play an important role in anearly contact of the bionts (Meessen & Ott2010; Joneson et al. 2011). Moreover, thedifferential gene expression observed in theC. grayi model system suggested that myco-bionts and photobionts communicate beforeas well as during cellular contact ( Joneson etal. 2011). Such findings could explain theprocess of recognition of the compatible biontsduring re-lichenization.

Although several papers on in vitro re-lichenization have been published to date, inmost cases the morphology of the resynthesisculture differs from the original lichen thallusas far as they are grown on agar substrata.However, the limited number of reportsshowed the ability of forming thallus lobesor even fruiting bodies in in vitro resynthesisexperiments, but in most cases the use ofsoil substratum seems to be crucial for thedevelopment of more differentiated struc-tures (e.g. Bubrick & Galun 1986; Stocker-Worgotter & Turk 1991, 1993; Stocker-Worgotter 2001a, b; Stenroos et al. 2003;Stocker-Worgotter & Elix 2006). In ourshort-term study (regarding the slow growthof the P. muralis lichen fungus), we did notobtain any of the more differentiated struc-tures. However, the formation of the primor-dial stages of lichenization was observed forall Trebouxia spp. investigated here, includ-ing Trebouxia asymmetrica, T. gigantea, T.

incrustata and an unidentified lineage of Tre-bouxia sp. ‘muralis I’ that were previouslyfound to be compatible photobionts for thislichen-forming fungus. Moreover, P. muraliswas able to relichenize with Asterochloris sp.,regarded as an incompatible photobiont,although the contact between fungal andalgal cells was not as tight as with Trebouxiaphotobionts. In the case of Asterochloris, thealgal aggregates were enveloped by fungalhyphae, but in some parts of the culture thehyphae were only growing next to algal cellswithout characteristic close contact of bothbionts. Moreover, no thallus-like structureswere formed. Related findings were presentedby Schaper & Ott (2003) for Fulgensia brac-teata, which was able to establish a symbioticassociation with less compatible algal part-ners. Asterochloris spp. are known to be mainlyassociated with Cladoniaceae and Stereocaula-ceae, but they have also been reported fromother lichen families, such as Parmeliaceae(summarized in Skaloud & Peksa 2010).However, in some lineages of Asterochloris thespecificity to the mycobiont is very low as thealgae were found to associate with about 20fungal species from different genera (Skaloud& Peksa 2010). Our findings suggest thatAsterochloris could also be accepted by theProtoparmeliopsis muralis fungus as a tempo-rary photobiont, until a more suitable algalsymbiont (Trebouxia) is available, and a finalswitch to its ‘preferred’ algal partner may oc-cur. This could be advantageous for survivalof the mycobiont, especially on substratawhere the lichen fungus acts as a pioneer, suc-cessfully colonizing new habitats.

A part of this study was financially supported by theMarie Curie Fellowship within the 6th European Com-munity Framework Programme, project no. 24206, toBGK (fellow) and EST-W (leader of the project) andperformed at the University of Salzburg. BGK alsoacknowledges the financial support of the Marie CurieEuropean Reintegration Grant within the 7th EuropeanCommunity Framework Programme, project no. 239343.EST-W acknowledges the support of FWF projects 18210and 20887. We are grateful to the Austrian Science Foun-dation FWF for supporting Project P23570 (ESt-W).The teachers and instructors from EMBO ElectronMicroscopy and Stereology course held in Ceske Bude-jovice (Czech Republic in 2008) are acknowledged foradvice and help with sample preparations and assistancewith electron microscopy investigations, especially


Martina Tesarova and Jana Nebesarova. Dorota Łuszczek(University of Gdansk) is acknowledged for help withcontrol sample preparation for SEM investigations. BGKalso thanks Grzegorz Wegrzyn, Andreas Beck, GeorgBrunauer, Armin Hager and Sabine Wornik for helpfuldiscussion, and is grateful to the collectors named inTable 1 for providing fresh material. Hans Peter Comesis acknowledged for making his laboratory facilitiesavailable. We are grateful to anonymous reviewers fortheir valuable comments and suggestions to improvethe manuscript.

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