Seasonal variations in the fine structure ofHypogymnia physodes (lichenizedAscomycetes) and itsTrebouxia photobiont

The lichenized ascomyceteHypogymnia physodes was collected every second month during a one year period at the same site in a wood near the city of Zürich and investigated with light and electron microscopy techniques. Temperature and relative humidity were measured at the collecting site. Seasonal variations in the germination rate of soredia, in photobiont cell size and cell number, in type and amounts of storage products, and in the density of intramembranous particles of the plasma membrane of theTrebouxia photobiont were observed. Highest germination rates and intense aplanospore formation were observed in January, highest particle densities in both external and internal fracture faces of the plasma membrane in March. Lipids and starch were most abundant in spring to early summer. In summer and autumn numerous photobiont cells died off.This study is dedicated to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday.     

HEAVILY LICHENIZED PHYSOLINUM (CHLOROPHYTA) FROM A DIMLY LIT CAVE IN MISSOURI1

Heavily lichenized Physolinum monile (De Wildem.) Printz from damp limestone walls in a dimly lit cave located in Missouri was studied from fresh collections and specimens fixed in situ, and from cultures. The narrow (7-13 μm wide thallus), profusely branched plant consisted of filaments of the alga P. monile ensheathed by clear fungal cells (5-8 in a single layer) that adhered tightly to each other and completely covered the algal cells. Cells of P. monile filaments were uninucleate, each containing a single massive chloroplast with numerous tightly packed thylakoids and lipid droplets and surrounded by a thin layer of cytoplasm. No plasmodesmata occurred in the cellulosic crosswalls between adjacent cells. The ensheathing fungal cells contained concentric bodies, produced haustoria that penetrated the algal cells, and developed hyphae (the tips of which formed clusters of conidia). Ensheathing fungal cells were well situated and constructed to concentrate light on the algal cells. Colonies of blue-green algae were firmly attached to the surface of the fungal cells. The association was slow growing but frequently produced and released aplanospores from the algal cells. Aplanospores were single (not attached to each other) with smooth walls or united in groups of two or more. Structures resembling lichen soredia, composed of aplanospore-like cells attached to one or more comdia-like cells, commonly occurred among the lichenized Physolinum filaments. The single chloroplast that occupies most of the cell's volume, the numerous, tightly packed thylakoids, and light focusing by ensheathing fungus cells may enable the organism to survive in a dimly lit environment. Because the filamentous alga reproduces only by aplanospores, we propose resurrection of the genus Physolinum. The lichenized Physolinum somewhat resembles the lichens Coenogonium moniliforme Tuck. and Cystocoleus Thwaites.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus <Emphasis Type="Italic">Tephromela atra</Emphasis> from the Mediterranean region

Recent DNA sequence analyses have revealed the diversity of algal partners in lichen symbioses. Although morphologically similar, different genetic lineages of photobionts are detected in wide geographic ranges of the same lichen fungal species. We studied the photobiont of the genus Trebouxia, which are known as partners of diverse lichen-forming fungal species in the Mediterranean region. We studied the phylogeny of these algae with a multilocus dataset including three loci: ITS, rbcL, and actin type I gene. The two lineages found, informally named Trebouxia sp. 1 and Trebouxia sp. 2, are related to Trebouxia arboricola/decolorans. The cultivation under axenic conditions succeeded only for one of them so far. We used light microscopy, confocal laser scanning microscopy and transmission electron microscopy for phenotypic characterisation. The ultrastructural characters currently used to describe species in the genus do not support the segregation of Trebouxia sp.1 from Trebouxia arboricola. The preferential presence in Mediterranean climates of these strains suggests eco-physiological adaptation. Despite their asexuality in long living lichen symbioses, coccoid algal lichen partners have apparently diversified genetically and physiologically.     

Identification of photobionts from the lichen family Physciaceae using algal-specific ITS rDNA sequencing

Abstract:The identity of photobionts from 20 species of the Physciaceae from different habitats and geographical regions has been determined by ITS rDNA sequence comparisons in order to estimate the diversity of photobionts within that lichen group, to detect patterns of specificity of mycobionts towards their photobionts and as a part of an ongoing study to investigate possible parallel cladogenesis of both symbionts. Algal-specific PCR primers have been used to determine the ITS rDNA sequences from DNA extractions of dried lichens that were up to 5 years old. Direct comparisons and phylogenetic analyses allowed the assignment of Physciaceae photobionts to four distinct clades in the photobiont ITS rDNA phylogeny. The results indicate a diversity within the genus Trebouxia Puymaly and Physciaceae photobionts that is higher than expected on the basis of morphology alone. Physciaceae photobionts belonged to 12 different ITS lineages of which nine could unambiguously be assigned to six morphospecies of Trebouxia. The identity of the remaining three sequences was not clarified; they may represent new species. Specificity at the generic level was low as a whole range of photobiont species were found within a genus of Physciaceae and different ranges were detected. The photobionts ofPhyscia (Schreb.) Michaux were closely related and represented one morphospecies of Trebouxia, whereas the algal partners of Buellia De Not and Rinodina (Ach.) S. Gray were in distant lineages of the ITS phylogeny and from several Trebouxia morphospecies. Photobiont variation within a genus of Physciaceae may be due to phylogeny, geographical distance or because photobionts from neighbouring lichens were taken (‘algal sharing’). At the species level Physciaceae mycobionts seem to be rather selective and contained photobionts that were very closely related within one morphospecies of Trebouxia.     

THE EFFECT OF AGRONOMIC PHOTOSYSTEM-II HERBICIDES ON LICHENS

The sensitivity ofHypogymnia physodes,Lobaria pulmonariaandPeltigera aphthosaH. physodesto six photosystem II herbicides and to DBMIB was tested in the laboratory by chlorophyll flouresence and oxygen-exchange measurements. in addition, experiments with freshly isolated photobiont cells fromH. physodesandL. pulmonariawere performed. Generally, the lichens were most sensitive to the urea herbicides diuron and isoproturon, whereas the triazines atrazine, terbuthylazine, and simazine and the triazinone metamitron wre less inhibitory. Among the three lichen species invesigated,H. physodeswas the most sensitive to the urea herbicides. For the other agents, no signifiant differences between lichen species could be found. The highest pI₅₀values obtained from dose response curves were around 6.5 for isolated photobionts, but most values for lichen thalli were in the range 5-6. Thus, there is no particular sensitivity of green algal lichen photobionts to photosytem II herbicides as compared to other algae, higher plant chloroplasts or protoplasts. In nature, we observed recovery from (damaging) treatment with 10⁻⁵mol diuron 1⁻¹forH. physodeswithin weeks. Therefore, damage to lichens fromt he use of photosystem-II herbicides in agriculture is probably only of very local occurence.     

Early development of Hypogymnia physodes (L.) Nyl. in response to emissions from a copper smelter

Abstract:The early development of Hypogymnia physodes from soredia to the formation of stratified lobes has been studied experimentally in the vicinity of a copper-smelting plant in the Middle Urals. SEM investigations combined with life table analyses of early developmental stages revealed decreases in soredial survival and developmental rate in polluted localities. Non-stratified pre-thallus stages without an epicortex were tolerant to toxic impact and were able to survive even in the zone with the highest pollution (lichen desert zone). The sensitivity of developmental stages increased after stratified lobes had developed.     

Photobiont selectivity leads to ecological tolerance and evolutionary divergence in a polymorphic complex of lichenized fungi

Background and Aims

The integrity and evolution of lichen symbioses depend on a fine-tuned combination of algal and fungal genotypes. Geographically widespread species complexes of lichenized fungi can occur in habitats with slightly varying ecological conditions, and it remains unclear how this variation correlates with symbiont selectivity patterns in lichens. In an attempt to address this question, >300 samples were taken of the globally distributed and ecologically variable lichen-forming species complex Tephromela atra, together with closely allied species, in order to study genetic diversity and the selectivity patterns of their photobionts.

Methods

Lichen thalli of T. atra and of closely related species T. grumosa, T. nashii and T. atrocaesia were collected from six continents, across 24 countries and 62 localities representing a wide range of habitats. Analyses of genetic diversity and phylogenetic relationships were carried out both for photobionts amplified directly from the lichen thalli and from those isolated in axenic cultures. Morphological and anatomical traits were studied with light and transmission electron microscopy in the isolated algal strains.

Key Results

Tephromela fungal species were found to associate with 12 lineages of Trebouxia. Five new clades demonstrate the still-unrecognized genetic diversity of lichen algae. Culturable, undescribed lineages were also characterized by phenotypic traits. Strong selectivity of the mycobionts for the photobionts was observed in six monophyletic Tephromela clades. Seven Trebouxia lineages were detected in the poorly resolved lineage T. atra sensu lato, where co-occurrence of multiple photobiont lineages in single thalli was repeatedly observed.

Conclusions

Low selectivity apparently allows widespread lichen-forming fungi to establish successful symbioses with locally adapted photobionts in a broader range of habitats. This flexibility might correlate with both lower phylogenetic resolution and evolutionary divergence in species complexes of crustose lichen-forming fungi.     

Lichen substance concentrations in the lichen Hypogymnia physodes are correlated with heavy metal concentrations in the substratum

Lichen substances (i.e. lichen-specific carbon-based secondary compounds) are known to be involved in the uptake and immobilization of metal ions, though the biochemical mechanisms of this interaction are largely unexplained. Previous research on potential effects of lichen substances on heavy metal uptake and tolerance mostly focused on lichens in heavily polluted areas with exceptionally high metal concentrations. In the present study, we aimed at gathering information as to whether lichen substances might be involved in the fine-tuning of metal uptake even at not or low-polluted sites. Therefore, we studied lichen substance concentrations in the epiphytic lichen Hypogymnia physodes and metal concentrations in its substratum in a montane spruce forest of Germany. H. physodes produces two depsides and five depsidones, which had been shown to be involved in metal homeostasis, namely in Cu and Mn uptake, in previous laboratory experiments. The amount of lichen substances increased with increasing heavy metal concentration in the substratum, though the latter varied only in the range of a few μmol g⁻¹ between the sample trees. Variability of lichen substance concentrations in H. physodes within the individual trees was low. Among the different lichen substances of H. physodes, the amount of the depsidone physodalic acid relative to the total of lichen substances was most closely correlated to the concentrations of Cu and Mn in the substratum, whereas the amount of the depsidone 3-hydroxyphysodic acid decreased both with increasing concentrations of these two metals and physodalic acid. Thus, our data suggest that lichen substances contribute to metal homeostasis not only in heavy metal-rich habitats, but also at not or low-polluted sites where the lichen substances apparently help to maintain constant intracellular metal concentrations despite of spatially varying availabilities of metal ions.     

Drought-induced structural alterations at the mycobiont-photobiont interface in a range of foliose macrolichens

Summary Cryotechniques, such as low temperature scanning electron microscopy (LTSEM) and freeze-substitution for transmission electron microscopy (TEM), were applied to two cyanobacterial and three green algal macrolichens in order to locate free water and to visualize drought-induced structural alterations at the mycobiont—photobiont interface. The following species were examined:Peltigera canina/Nostoc punctiforme, Sticta sylvatica/Nostoc sp. (both Peltigerales),Parmelia sulcata/Trebouxia impressa, Hypogymnia physodes/Trebouxia sp. (both Lecanorales), andXanthoria parietina/Trebouxia arboricola (Teloschistales). In all species free water was confined to the symplast and the apoplast. No intercellular water reservoirs were found in the gas-filled thallus interior. Thalline fluctuations in water content reflect fluctuations in apoplastic and symplastic water. All the taxonomically diverse lichen photobionts have access to water and dissolved nutrients via the fungal apoplast only. Drought stress (i.e., water content 20%/dw and below) caused dramatic shrinkage and deformation in all cell types. At any level of hydration the fungal and algal protoplast maintained close contact with the cell wall. This applied to the cyanobacterial photobionts and their murein sacculus and gelatinous sheath too. Although the cytoplasm of both partners was strongly condensed in desiccated lichens the cellular membrane systems, usually negatively contrasted, were very well preserved. The significance of these data is discussed with regard to the functioning of the symbiotic relationship.     

Cellular injuries in epiphytic lichens transplanted to air polluted areas

The epiphytic lichens Hypogymnia physodes (L.) W. Wats, and Bryoria capillaris (Ach.) Brodo & D. Hawksw. growing on spruce branches were transplanted from a clean rural area to the environment of a fertilizer plant and a pulp mill in central Finland. The common major pollutants in these environments are SO₂, NO_x and ammonia but the fertilizer plant also emits fluorides. In the transmission electron microscope two main types of cellular injuries were observed both in algal and fungal cells in both species and in both environments. The first type, characterized by rapid degeneration of cell organelles, was an apparently acute injury leading to plasmolysis and rapid death of the cells. The second type was considered chronic injury and involved changes in chloroplast shape, swelling of mitochondria and increased density of cytoplasm in algal cells, and increased vacuolization and appearance of dark vacuolar accumulations in fungal cells. The cytoplasmic storage droplets decreased gradually in size both in algae and fungi. The acute injury was mainly seen in the lichens transplanted to sites with higher pollution levels near the sources, and was more usual in algal than in fungal cells. B. capillaris was more susceptible to acute injury than H. physodes. An additional injury type was detected in algal cells of both lichen species in the vicinity of the fertilizer plant. This type was characterized by severe swelling of thylakoids and their interspaces and granulation of thylakoid membranes, and was suspected to be related to the effects of fluorides. The injuries as seen in light microscope, and expressed as increased proportion of dead algae and visible bleaching of thallus, were usually observable simultaneously. The injuries as seen in the electron microscope always preceded other injuries; they were clearly observable already after one week of transplantation in the more polluted sites but developed more slowly in the less polluted ones. The time lack between these injuries and the visible ones was 2–3 weeks in the more polluted sites, but several months or even years in the less polluted ones.     

Association mapping of magnesium and manganese concentrations in the seeds of C. arietinum and C. reticulatum

Chickpea (Cicer arietinum L.) is one of the oldest and most important pulse crops grown and consumed all over the world, especially in developing countries. Magnesium (Mg) and manganese (Mn) are essential plant nutrients in terms of human health and many health problems arise in their deficiencies. The objectives of this study were to characterize genetic variability in the seed Mg and Mn concentrations and identify single nucleotide polymorphism (SNP) markers associated with these traits in 107 Cicer reticulatum and 73C. arietinum genotypes, using a genome wide association study. The genotypes were grown in four environments, characterized for Mg and Mn concentrations, and genotyped with 121,841 SNP markers. The population showed three-fold and two-fold variation for the Mg and Mn concentrations, respectively. The population structure was identified using STRUCTURE software, which divided 180 genotypes into two (K = 2) groups. Principal component analysis and neighbor joining tree analysis confirmed the results of STRUCTURE. A total of 4 and 16 consistent SNPs were detected for the Mg and Mn concentrations, respectively. The identified markers can be utilized in breeding of chickpea to increase Mg and Mn levels in order to improve human and livestock nutrition.     

Dissociation and horizontal transmission of codispersing lichen symbionts in the genus Lepraria (Lecanorales: Stereocaulaceae)

Lichenized fungi of the genus Lepraria lack ascomata and conidiomata, and symbionts codisperse by soredia. Here, it is determined whether algal symbionts associated with Lepraria are monophyletic, and whether fungal and algal phylogenies are congruent, both of which are indicative of a long-term, continuous association between symbionts. The internal transcribed spacer (ITS) and part of the actin type I locus were sequenced from algae associated with Lepraria, and the fungal ITS and mitochondrial small subunit (mtSSU) were sequenced from fungal symbionts. Phylogenetic analyses tested for monophyly of algal symbionts and congruence between algal and fungal phylogenies. Algae associated with Lepraria were not monophyletic, and identical algae associated with different Lepraria individuals and species. Algal and fungal phylogenies were not congruent, suggesting a lack of strict codiversification. This study suggests that associations between symbionts are not strictly maintained over evolutionary time. The ability to switch partners may provide benefits similar to genetic recombination, which may have helped this lineage persist.     

Fungal specificity and selectivity for algae play a major role in determining lichen partnerships across diverse ecogeographic regions in the lichen‐forming family Parmeliaceae (Ascomycota)

Microbial symbionts are instrumental to the ecological and long‐term evolutionary success of their hosts, and the central role of symbiotic interactions is increasingly recognized across the vast majority of life. Lichens provide an iconic group for investigating patterns in species interactions; however, relationships among lichen symbionts are often masked by uncertain species boundaries or an inability to reliably identify symbionts. The species‐rich lichen‐forming fungal family Parmeliaceae provides a diverse group for assessing patterns of interactions of algal symbionts, and our study addresses patterns of lichen symbiont interactions at the largest geographic and taxonomic scales attempted to date. We analysed a total of 2356 algal internal transcribed spacer (ITS) region sequences collected from lichens representing ten mycobiont genera in Parmeliaceae, two genera in Lecanoraceae and 26 cultured Trebouxia strains. Algal ITS sequences were grouped into operational taxonomic units (OTUs); we attempted to validate the evolutionary independence of a subset of the inferred OTUs using chloroplast and mitochondrial loci. We explored the patterns of symbiont interactions in these lichens based on ecogeographic distributions and mycobiont taxonomy. We found high levels of undescribed diversity in Trebouxia, broad distributions across distinct ecoregions for many photobiont OTUs and varying levels of mycobiont selectivity and specificity towards the photobiont. Based on these results, we conclude that fungal specificity and selectivity for algal partners play a major role in determining lichen partnerships, potentially superseding ecology, at least at the ecogeographic scale investigated here. To facilitate effective communication and consistency across future studies, we propose a provisional naming system for Trebouxia photobionts and provide representative sequences for each OTU circumscribed in this study.     

The alanine ester content and magnesium binding capacity of walls of Staphylococcus aureus H grown at different pH values

Recent studies have shown that teichoic acids are functionally involved in the uptake of Mg²⁺ and that their ability to bind Mg is reduced by the presence of alanine ester substituents. Walls of Staphylococcus aureaus H grown at pH 5 contain more ester alanine and bind less magnesium than do walls of S. aureaus grown under similar conditions at pH 6 and 7. The differences in alanine content are largely due to its removal by base catalysed hydrolysis of the labile ester linkages during growth in media of pH 6 and 7 and the incorporation of alanine esters into the walls does not appear to be influenced by the pH of the medium in which the bacteria are grown under the conditions described. The decreased magnesium binding capacity of walls of S. aureus grown at pH 5 correlates with their increased alanine ester content but there is no proportional relationship between these two quantities.     

Photobiont selectivity for lichens and evidence for a possible glacial refugium in the Ross Sea Region, Antarctica

Lichens are a symbiosis consisting of heterotrophic, fungal (mycobiont) and photosynthetic algal or cyanobacterial (photobiont) components. We examined photobiont sequences from lichens in the Ross Sea Region of Antarctica using the internal transcribed spacer region of ribosomal DNA and tested the hypothesis that lichens from this extreme environment would demonstrate low selectivity in their choice of photobionts. Sequence data from three targeted lichen species (Buellia frigida, Umbilicaria aprina and Umbilicaria decussata) showed that all three were associated with a common algal haplotype (an unnamed Trebouxia species) which was present in all taxa and at all sites, suggesting lower selectivity. However, there was also association with unique, local photobionts as well as evidence for species-specific selection. For example, the cosmopolitan U. decussata was associated with two photobiont species, Trebouxia jamesii and an unnamed species. The most commonly collected lichen (B. frigida) had its highest photobiont haplotype diversity in the Dry Valley region, which may have served as a refugium during glacial periods. We conclude that even in these extreme environments, photobiont selectivity still has an influence on the successful colonisation of lichens. However, the level of selectivity is variable among species and may be related to the ability of some (e.g. B. frigida) to colonise a wider range of habitats.     

Characterization of two novel non-reducing polyketide synthase genes from the lichen-forming fungus Hypogymnia physodes

Lichens are known to produce a variety of secondary metabolites including polyketides, which have valuable biological activities. Some polyketides are produced solely by lichens. The biosynthesis of these compounds is primarily governed by iterative type I polyketide synthases. Hypogymnia physodes synthesize polyketides such as physodic, physodalic and hydroxyphysodic acid and atranorin, which are non-reducing polyketides. Two novel non-reducing polyketide synthase (PKS) genes were isolated from a fosmid genomic library of a mycobiont of H. physodes using a 409bp fragment corresponding to part of the reductase (R) domain as a probe. H. physodes PKS1 (Hyopks1) and PKS2 (Hypopks2) contain keto synthase (KS), acyl transferase (AT), acyl carrier protein (ACP), methyl transferase (ME) and R domains. Classification based on phylogeny analysis using the translated KS and AT domains demonstrated that Hypopks1 and Hypopks2 are members of the fungal non-reducing PKSs clade III. This is the first report of non-reducing PKSs containing the R domain-mediated release mechanisms in lichens, which are also rare fungal type I PKS in non-lichenized filamentous fungi.     

Effects of cadmium and copper on the ultrastructure ofAnkistrodesmus braunii andAnabaena 7120

The effects of brief exposure to, or growth in the presence of, lethal and sublethal concentrations of Cu(NO)₂ and Cd(NO₃) on the ultrastructure of the blue-green algaAnabaena 7120 and the green algaAnkistrodesmus braunii were studied. Exposure to increasing amount of both metal ions led to the appearance of larger proportions of electron-dense cells whose organelles were less well defined than those of untreated cells. Metal-treated cells ofAnabaena 7120 became distorted. Some had a corrugated appearance. Others lysed, leaving a much larger proportion of heterocysts. Such heterocysts were often empty or had a curious collapsed appearance. Growth ofA. braunii in the presence of 10^–4 M Cu(NO₂)₂ produced substantial numbers of multinucleate giant cells with thick walls; such cells result from repeated mitotic division without subsequent cytokinesis. The giant cells contained centrioles, structures not as yet found in normal cells of the genusAnkistrodesmus. Some nuclei of giant, but not of normal, cells contained deep indentations that appeared as holes in cross section. Some giant cells also contained triple parallel strands of endoplasmic reticulum which extended across much of the cell, connecting to the nuclear envelope. Some ultrastructural changes were also noted in algal cells grown over sediment containing Cu or Cd, but these were generally less severe than those occurring when metal ions were added directly to the algal cultures.     

Possible functional roles of cortical depsides and medullary depsidones in the foliose lichen Hypogymnia physodes

Secondary compounds were quantified in 75 thalli of the foliose lichen Hypogymnia physodes collected in habitats along a natural shade-sun gradient from dark spruce forests to sun-exposed sea cliffs. The irradiance in all the 75 lichen sites was estimated from hemispherical photographs. The content of lichen compounds per thallus area correlated positively with irradiance level (r²=0.73), and the mean concentration increased from 6.7% in the spruce forest to 14.4% on sea cliffs. The medullary depsidones, physodic, physodalic and protocetraric acids, constituted >95% of the total pool of extractable secondary compounds, the cortical depsides, atranorin and chloratranorin, represented <5%. Both cortical compounds correlated well with direct and with diffuse radiation, whereas the three medullary compounds correlated better with diffuse than with direct radiation. Mentioned trends are consistent with a solar radiation screening hypothesis of both groups of these colourless compounds occuring as tiny crystals outside fungal hyphae. However, the UV-B protective hypothesis of the compounds was further tested in a lab experiment. Unnaturally high UV-B doses were required to significantly reduce the PSII efficiency (F_V/F_M) of symbiotic algae. Removal of the major pool of secondary compounds with acetone did not increase photobiont susceptibility to UV-B. Therefore, the main functional role of the UV-B absorbing secondary compounds in H. physodes is hardly UV-B screening. Other roles such as PAR-screening and defence against herbivores and pathogenic organisms are discussed.     

High photobiont diversity in the common European soil crust lichen Psora decipiens

The genetic diversity of green algal photobionts (chlorobionts) in soil crust forming lichens was studied as part of the SCIN-project (Soil Crust InterNational). A total of 64 lichen samples were collected from four different sites along latitudinal and altitudinal gradients in Europe (Tabernas/Spain; Hochtor-Großglockner/Austria; Gynge Alvar/Sweden; Ruine Homburg/Germany). The dominant lichen species at all four sites was Psora decipiens, often occurring with Buellia elegans, Fulgensia bracteata, F. fulgens and Peltigera rufescens. Genetic identification of chlorobionts was carried out using the nuclear marker (nrITS) and a chloroplast marker (psbL-J). We found P. decipiens to be associated with several different species of Trebouxia and Asterochloris, although previously described to only have Asterochloris sp. The phylogenetic analyses revealed a high chlorobiont diversity with 12 well supported clades, including Trebouxia asymmetrica, T. jamesii, T. impressa and other, as yet taxonomically unidentified clades (Trebouxia sp. URa1-4, T. sp. URa6, T. sp. URa7-13). Additionally, five clades of Asterochloris were identified (A. magna, A. sp. URa14 -17). Most of the chlorobiont species appeared to be cosmopolitan, but five clades were unevenly distributed between the sampling sites with only Trebouxia being found in the warm and dry Spanish habitats and combinations of Trebouxia and Asterochloris in the cooler and more humid habitats. The wide range of chlorobiont species might contribute to the observed domination of P. decipiens at all four research sites of the SCIN project which range from a desert in Spain to an alpine site in the Alps of Austria.