While the diversity of foliicolous lichen-forming fungi has been explored in substantial depth, relatively little attention has been paid to their algal symbionts. We studied the unicellular green phycobionts of the lecanoralean lichens Bacidina (Ramalinaceae), Byssoloma, Fellhanera and Tapellaria (Pilocarpaceae) and graphidalean Gyalectidium (Gomphillaceae) from two extratropical foliicolous communities in continental Spain and the Canary Islands. We examined the pyrenoids of algal symbionts within thalli using TEM, and obtained several algal nrSSU and rbcL sequences from whole thalli, and also from cultures isolated from some of these lichens. Pyrenoid structure and molecular sequence data provided support for recognizing Chloroidium (Watanabeales, Trebouxiophyceae) as phycobiont in thalli of Byssoloma subdiscordans and Fellhanera bouteillei (Pilocarpaceae) in both communities. Bacidina apiahica (Ramalinaceae) and Tapellaria epiphylla (Pilocarpaceae) likewise appeared to partner with Chloroidium based on the presence of the same pyrenoid type, although we were able to obtain a phycobiont sequence only from a culture isolate of the latter. These results contrast with those obtained previously from a foliicolous lichen community in southern Florida, which revealed only strains of Heveochlorella (Jaagichlorella) as phycobiont of foliicolous Pilocarpaceae and Gomphillaceae. On the other hand, the pyrenoid we observed in the phycobionts associated with Gyalectidium setiferum and G. minus corresponded to that of Heveochlorella (Jaagichlorella). However, the poor quality of the phycobiont sequence data obtained from G. minus, probably due to the presence of epibiontic algae, could not provide additional perspective on the pyrenoid structure observations. Nonetheless, clear differences in pyrenoid ultrastructure can allow Chloroidium and Heveochlorella phycobionts to be distinguished from each other in TEM. Our results indicate a greater diversity of unicellular green-algal symbionts in foliicolous communities from Spain than previously observed in other geographical areas, and suggest that further studies focused on symbiont pairing in these communities might reveal distinctive and varied patterns of phycobiont preference.

A review of algal (including cyanobacterial) symbionts associated with lichen-forming fungi is presented. General aspects of their biology relevant to lichen symbioses are summarized. The genera of algae currently believed to include lichen symbionts are outlined; approximately 50 can be recognized at present. References reporting algal taxa in lichen symbiosis are tabulated, with emphasis on those published since the 1988 review by Tschermak-Woess, and particularly those providing molecular evidence for their identifications. This review is dedicated in honour of Austrian phycologist Elisabeth Tschermak-Woess (1917–2001), for her numerous and significant contributions to our knowledge of lichen algae (some published under the names Elisabeth Tschermak and Liesl Tschermak).

The Trebouxia photobiont freshly isolated from Xanthoria parietina (L.) Th. Fr. was used to develop a live cell chondriome (mitochondrial DNA) labelling method. In the initial phase six candidate dyes were tested and compared for mitochondrial labelling utility as assessed by the signal to noise ratio (SNR) of the mitochondrial signal to the adjacent cellular background in standardized confocal images of 30 labelled cells. DIOC7, JC-1 and MitoTracker orange (MTO) dyes showed some labelling ability. MTO had significantly higher utility than the other dyes. In a second phase, MTO concentration was optimized. The final labelling protocol was a 30 minute incubation with 1 μM of MTO. The resultant labelling was suitable for both widefield and confocal microscopy. Both 2D thresholding and 3D volume construction are demonstrated using the resultant data. The protocol can therefore be utilized for both qualitative research and for quantitative measurement of the chondriome in Trebouxia photobionts. This will facilitate a wide range of mitochondrial investigations in lichenology.

Several investigations on lichen photobionts (PBs) after exposure to simulated or real-space parameters consistently reported high viability and recovery of photosynthetic activity. These studies focused on PBs within lichen thalli, mostly exposed in a metabolically inactive state. In contrast, a recent study exposed isolated and metabolically active PBs to the non-terrestrial stressor UVC254 nm and found strong impairment of photosynthetic activity and photo-protective mechanisms (Meeßen et al. in 2014b). Under space and Mars conditions, UVC is accompanied by other stressors as extreme desiccation and low temperatures. The present study exposed the PBs of Buellia frigida and Circinaria gyrosa, to UVC in combination with desiccation and subzero temperatures to gain better insight into the combined stressors' effect and the PBs' inherent potential of resistance. These effects were examined by chlorophyll a fluorescence which is a good indicator of photosynthetic activity (Lüttge & Büdel in 2010) and widely used to test the viability of PBs after (simulated) space exposure. The present results reveal fast recovery of photosynthetic activity after desiccation and subzero temperatures. Moreover, they demonstrate that desiccation and cold confer an additional protective effect on the investigated PBs and attenuate the PBs' reaction to another stressor – even if it is a non-terrestrial one such as UVC. Besides other protective mechanisms (anhydrobiosis, morphological–anatomical traits and secondary lichen compounds), these findings may help to explain the high resistance of lichens observed in astrobiological studies.

In this study we examined photobiont identity, diversity and selectivity in the genus Bryoria. We focused on B. fremontii and section Implexae in order to determine whether secondary chemistry is correlated with photobiont identity. DNA from two loci for photobionts and three loci for mycobionts was sequenced for both parsimony and Bayesian phylogenetic analyses. A comparison of photobiont and mycobiont phylogenies reveals that most Bryoria species associate exclusively with lineages of the Trebouxia simplex group; only B. smithii was associated with a different photobiont. We conclude that most Bryoria species included in our study are highly selective in their choice of algal partners and that the presence/concentration of different secondary compounds does not correlate with photobiont identity either in section Implexae or in B. fremontii.

The lichen Xanthoria elegans has been exposed to space conditions and simulated Mars-analogue conditions in the lichen and fungi experiment (LIFE) on the International Space Station (ISS). After several simulations and short space exposure experiments such as BIOPAN, this was the first long-term exposure of eukaryotic organisms to the hostile space conditions of the low Earth orbit (LEO). The biological samples were integrated in the EXPOSE-E facility and exposed for 1.5 years outside the ISS to the combined impact of insolation, ultraviolet (UV)-irradiation, cosmic radiation, temperatures and vacuum conditions of LEO space. Additionally, a subset of X. elegans samples was exposed to simulated Martian environmental conditions by applying Mars-analogue atmosphere and suitable solar radiation filters. After their return to Earth the viability of the lichen samples was ascertained by viability analysis of LIVE/DEAD staining and confocal laser-scanning microscopy, but also by analyses of chlorophyll a fluorescence. According to the LIVE/DEAD staining results, the lichen photobiont showed an average viability rate of 71%, whereas the even more resistant lichen mycobiont showed a rate of 84%. Post-exposure viability rates did not significantly vary among the applied exposure conditions. This remarkable viability is discussed in the context of particular protective mechanisms of lichens such as anhydrobiosis and UV-screening pigments.

Spilonema was originally described to accommodate an unusual group of cyanolichens with thread-like, cushion-forming thalli, and has long been placed in Coccocarpiaceae based on ascomatal development. However, Spilonema is the only genus of Peltigerales to include species lichenized with the cyanobacterial genus Stigonema, and the evolutionary relationships of Spilonema to other genera in the family have yet to be tested using molecular data. We present evidence from combined nuclear 28S, 18S and mitochondrial 12S rDNA to confirm the placement of the core species of Spilonema (S. paradoxum and S. revertens) in Coccocarpiaceae. Our data further show that despite possessing a different genus of photobiont (Scytonema), the north Pacific endemic genus Spilonemella must be included within Spilonema, suggesting that closely related species of the genus have changed photobionts in the course of evolution. However, we recovered Spilonema dendroides, one of the only lichens known to associate with the cyanobacterial genus Hyphomorpha, as only distantly related to the Coccocarpiaceae. The evolutionary relationships of this species are as yet unclear but it may occupy a basal position in the Peltigerales. We create for this species the new genus Erinacellus T. Sprib., Muggia & Tønsberg.

The widespread and ubiquitous lichen-forming fungus Protoparmeliopsis muralis is able to form a thallus with Trebouxia species. In this study, several photobiont strains were isolated from different specimens of P. muralis and cultured in vitro. The compatibility of Trebouxia spp. and Asterochloris 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 addition to compatible photobionts, also a presumably incompatible Asterochloris sp. was able to interact with the mycobiont. The life strategy that enables the mycobiont to form associations with a wider range of photobionts could be advantageous for the survival of the lichen and successful colonization of new habitats.

Lichens are a fascinating example of a symbiotic mutualism. It is still uncertain which processes guide fungal-photobiont interactions, and whether they are random or of a more complex nature. Here, the fungal-algal interactions in Ramalina menziesii and co-occurring taxa are analyzed by using DNA sequences of the algal Internal Transcribed Spacer region (ITS), to investigate fungal-algal associations in juvenile R. menziesii and allied species. Algal species were identified by a combination of BLAST searches, median-joining network analysis, and Bayesian phylogenetics. Fungal-algal networks were analyzed for nestedness, both at the species and haplotype level (fungal species vs. algal haplotypes), and the networks were inspected for evidence of compartmentalization. Bayesian phylogenetic trees indicated that the widespread green alga Trebouxia decolorans associated with R. menziesii, as well as six other fungal species. Four additional fungal species interacted with four different species of Trebouxia. Only in one out of ten samples were algal haplotypes shared with the nearest neighbours of juvenile R. menziesii. Fungal-algal species interactions were compartmentalized, while at the level of algal haplotypes, nestedness was found. This pattern is similar to the compartmentalization found in other intimately interacting mutualists.

Algal genotypes should freely associate with different lichen fungi that grow in the same confined habitat giving the appearance of low levels of selectivity and specificity. If genetic compatibility between algal and fungal partners limits the combination of partners, then some degree of taxonomic specificity should be evident. This study examined the photobiont composition in a community of epiphytic lichens on Jack Pine to investigate selectivity and specificity. The objectives of the study were to infer algal identity, to infer photobiont dispersal, and to investigate the distribution of algal genotypes relative to the fungal partner. Photobiont variability was determined by Restriction Fragment Length Polymorphism (RFLP) and nucleotide sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA (rDNA). Seven species of lichen-forming fungi are reported to associate with five divergent algal genotypes, with only one species, Evernia mesomorpha, showing some degree of selectivity and specificity. The algae represent at least two species (Trebouxia jamesii and T. impressa) for the area confined to 200cm2 on the north side of 20 Jack Pine trees. Gene flow was inferred in this tightly defined community of lichenized algae. The algal sharing and inferred gene flow may suggest that soredia provide a means of algal transport and distribution among lichen-forming fungi in the habitat.

The genetic diversity of photobionts in the lichen Protoparmeliopsis muralis was examined to determine the identity of the photobiont and the level of selectivity for the photobiont by the mycobiont. Forty ITS rDNA sequences of photobionts from thirty nine specimens of Protoparmeliopsis muralis have been determined. The photobiont species were identified both by direct comparison with the available sequences and by phylogenetic analysis. The sequences obtained from P. muralis specimens belonged to different Trebouxia clades. The most common photobionts were T. incrustata and an unidentified lineage, here named as “muralis I”. Less common photobionts were T. asymmetrica and T. gigantea. Two unidentified Trebouxia spp. and a photobiont that belonged to the clade that includes T. impressa, T. flava and T. potteri were also found to be associated with P. muralis. Such flexibility has not been reported to date. Moreover, two different photobionts were found in a single specimen. They belonged to the T. gigantea clade and “muralis I” lineage. The low level of selectivity with respect to the photobiont may constitute an important aspect in that Protoparmeliopsis muralis is one of the most successful urban lichens in the world.

The assertion that the blue-greens (‘cyanobacteria’) are bacteria and not algae has led to alternative concepts for both of the latter terms. Bacteria is now often used to designate the prokaryote kingdom, that includes the blue-greens, while in other contexts it means specifically those prokaryotes that are not blue-greens. The term algae, on the other hand, has no biosystematic implications; it encompasses many phylogenetically independent lineages characterized by oxygen-evolving photosynthesis. The algal status of the blue-greens is therefore not compromised by their classification among the bacteria. Current lichenological terminology, however, reflects the view that the blue-greens are not algae, thereby diverging from the concept of algae employed in phycology. The purpose and utility of terms that group organisms according to structural, functional and ecological criteria are insufficiently appreciated. These concepts do not compete with phylogenetic/biosystematic classifications, but rather complement them.

Two shade-adapted (Lobaria pulmonaria and Cetraria islandica) and two sun-adapted lichen populations (Xanthoria parietina and Cetraria nivalis) were exposed to three irradiance regimes (1: photosynthetic radiation—PAR, 2: PAR+UV-A, 3: PAR+UV-A+UV-B) and two hydration regimes (1: no hydration, 2: daily hydration) in a growth cabinet for three weeks. Shade-adapted thalli had transparent upper cortices without coloured pigments, whereas sun-adapted thalli had coloured UV-B absorbing cortical pigments masking the photobiont. Manipulation of pigment concentration was the third factor used in the factorial design (1: pigments intact, 2: pigments non-destructively extracted from air-dry living thalli by acetone). Inhibition of the photobiont due to PAR alone was severe in the two shade-adapted populations, but no applied UV wavelength bands caused additional aggravation of photoinhibition. Shade-adapted thalli of the ubiquitous C. islandica were more PAR-susceptible than of the rare old forest lichen L. pulmonaria, suggesting that screening by the mycobiont rather than photobiont characteristics, account for their different success in sun-exposed localities. Hydration of shade-adapted species during exposure reduced their photoinhibition substantially, probably because of moisture-activated repair mechanisms. On the contrary, the sun-adapted X. parietina was most phototolerant in the desiccated state, whereas hydration caused increased photoinhibition. When removing the orange cortical pigment parietin, the photoinhibition in moist thalli was aggravated, confirming a PAR-protective function of parietin. No effects of irradiance treatment, pigment extraction (usnic acid), or hydration level were observed in C. nivalis.

Molecular sequence data of the nuclear ITS region was used to investigate the diversity of photobionts in Polish samples of Flavocetraria nivalis. The samples came both from alpine habitats, as well as from lowland localities near the coast. All green algal symbionts were identified as members of the Trebouxia simplex aggregate. These were compared with those of additional samples from Flavocetraria nivalis collected in different parts of Europe and also with photobionts assigned to T. simplex from other lichens. Within the T. simplex aggregate, the Trebouxia ITS sequences from F. nivalis formed four clades. In the Polish lowland populations only a single clade of T. simplex was detected which also occurs in Polish mountains, south Sweden and Austria. A further clade of T. simplex is present in F. nivalis from Polish mountains and is also known from F. nivalis further north in Scandinavia and Greenland, as well as from other lichens in Sweden, the Austrian Alps, and Antarctica.

The lichen symbiotic organisms Fulgensia bracteata and Xanthoria elegans as well as their isolated photobionts and mycobionts were exposed to conditions simulating the extreme parameters of outer space in order to assess their limits of survival, e.g. during a potential interplanetary transfer or on the surface of an extraterrestrial body. Using the space simulation facilities at DLR, the test parameters under investigation were vacuum (10−3 Pa) and ultraviolet (UV) radiation at wavelength ranges from vacuum-UV to UV-A, applied separately or in combination. An analysis of vitality was made using confocal laser scanning microscopy and LIVE/DEAD staining using FUN I or SYTOX green. Intact lichens were extremely resistant to vacuum exposure as well as to UV radiation up to doses of about 160 kJ m−2 (200<λ<400 nm). Removal of the upper-cortex structure significantly increased the sensitivity of the lichens to the space parameters: already a relatively short treatment of about 5 h and a UV dose of about 50 kJ m−2 reduced the vitality rate to nearly 50%; however, the dose effect curves levelled off and continued treatment did not reduce the vitality further. Similar survival curves levelling off at higher UV doses were observed for the isolated photobionts; however, in the latter cases, the saturation occurred at five times lower doses (full UV spectrum). Also spores of the mycobionts showed a remarkable UV (254 nm) resistance up to doses of about 3 kJ m−2. The data suggest that the symbiotic features peculiar to lichens allow them to cope with the extreme conditions of outer space or even with Martian surface conditions provided suitable niche habitats are available to serve as refuges and complementary endogenous or exogenous protection mechanisms are established.