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The genetic combinations between mycobionts and photobionts in Parmotrema tinctorum collected from ca. 60 km super(2) of the Shimizu district, of Shizuoka City in Japan was investigated based on ITS rDNA sequences. This lichen produces apothecia quite rarely, and in principle propagates vegetatively by isidia. The genetic diversity of the mycobiont comprised four types, while that of the photobiont comprised 21 types. There were 28 different combinations between mycobiont and photobiont. All the photobionts were identified as Trebouxia corticola (s. lat.), based on both molecular phylogenetic results and morphological observation of culture strains obtained in this study. Therefore, P. tinctorum is considered to be highly selective toward the photobiont. The 28 combinations from the small area represent an unexpectedly high diversity, because P. tinctorum is thought to propagate vegetatively. Four possible mechanisms to account the high genetic combinations are suggested: i.e., photobiont exchange, fusion of thalli, and long-distance dispersal of isidia or ascospores. The genetic diversity of photobionts was poor in the urban area, but rich in suburbs and mountainsides. This might be caused by a bottleneck or founder effect in the population recovering from former damage by heavy air pollution, or variable selectivity of P. tinctorum depending on the environments.

ABSTRACT Lichens are classic models of symbiosis, and one of the most frequent nutritional modes among fungi. The ecologically and geographically widespread lichen-forming algal (LFA) genus Trebouxia is one of the best-studied groups of LFA and associates with over 7000 fungal species. Despite its importance, little is known about its diversification. We synthesized twenty years of publicly available data by characterizing the ecological preferences of this group and testing for time-variant shifts in climatic regimes over a distribution of trees. We found evidence for limited shifts among regimes, but that disparate lineages convergently evolved similar ecological tolerances. Early Trebouxia lineages were largely forest specialists or habitat generalists that occupied a regime whose extant members occur in moderate climates. Trebouxia then convergently diversified in non-forested habitats and expanded into regimes whose modern representatives occupy wet-warm and cool-dry climates. We rejected models in which climatic diversification slowed through time, suggesting climatic diversification is inconsistent with that expected under an adaptive radiation. In addition, we found that climatic and vegetative regime shifts broadly coincided with the evolution of biomes and associated or similar taxa. Together, our work illustrates how this keystone symbiont from an iconic symbiosis evolved to occupy diverse habitats across the globe. Lichen algae convergently evolved to occupy shared climates and habitats at a gradual rate, and broadly coincident with the evolution of these biomes and associated taxa.

The popular dual definition of lichen symbiosis is under question with recent findings of additional microbial partners living within the lichen body. Here we compare the distribution and co-occurrence patterns of lichen photobiont and recently described secondary fungus (Cyphobasidiales yeast) to evaluate their dependency on lichen host fungus (mycobiont). We sequenced the nuclear internal transcribed spacer (ITS) strands for mycobiont, photobiont, and yeast from six widespread northern hemisphere epiphytic lichen species collected from 25 sites in Switzerland and Estonia. Interaction network analyses and multivariate analyses were conducted on operational taxonomic units based on ITS sequence data. Our study demonstrates the frequent presence of cystobasidiomycete yeasts in studied lichens and shows that they are much less mycobiont-specific than the photobionts. Individuals of different lichen species growing on the same tree trunk consistently hosted the same or closely related mycobiont-specific Trebouxia lineage over geographic distances while the cystobasidiomycete yeasts were unevenly distributed over the study area – contrasting communities were found between Estonia and Switzerland. These results contradict previous findings of high mycobiont species specificity of Cyphobasidiales yeast at large geographic scales. Our results suggest that the yeast might not be as intimately associated with the symbiosis as is the photobiont.

Near-infrared spectroscopy (NIRS) is an accurate, fast and safe technique whose full potential remains to be exploited. Lichens are a paradigm of symbiotic association, with extraordinary properties, such as abiotic stress tolerance and adaptation to anhydrobiosis, but subjacent mechanisms await elucidation. Our aim is characterizing the metabolomic NIRS fingerprints of Ramalina farinacea and Lobarina scrobiculata thalli, and of the cultured phycobionts Trebouxia lynnae and Trebouxia jamesii. Thalli collected in an air-dry state and fresh cultivated phycobionts were directly used for spectra acquisition in reflectance mode. Thalli water peaks were associated to the solvation shell (1354 nm) and sugar–water interactions (1438 nm). While northern–southern orientation related with two hydrogen bonded (S2) water, the site was related to one hydrogen bonded (S1). Water, lipids (saturated and unsaturated), and polyols/glucides contributed to the profiles of lichen thalli and microalgae. R. farinacea, with higher desiccation tolerance, shows higher S2 water than L. scrobiculata. In contrast, fresh phycobionts are dominated by free water. Whereas T. jamesii shows higher solvation water content, T. lynnae possesses more unsaturated lipids. Aquaphotomics demonstrates the involvement of strongly hydrogen bonded water conformations, polyols/glucides, and unsaturated/saturated fatty acids in the dehydration process, and supports a “rubbery” state allowing enzymatic activity during anhydrobiosis.

• Premise of the study: Many lichens exhibit extensive ranges spanning several ecoregions. It has been hypothesized that this wide ecological amplitude is facilitated by fungal association with locally adapted photobiont strains.• Methods: We studied the identity and geographic distribution of photobionts of the widely distributed North American lichen Ramalina menziesii based on rbcL (chloroplast DNA) and nuclear ribosomal ITS DNA sequences. To test for ecological specialization, we associate photobiont genotypes with local climate and phorophyte.• Key results: Of the photobiont lineages of R. menziesii, 94% belong to a clade including Trebouxia decolorans. The remaining are related to T. jamesii. The photobionts showed (1) significant structure according to ecoregion and phorophyte species and (2) genetic associations with phorophyte species and climate.• Conclusions: Geography, climate, and ecological specialization shape genetic differentiation of lichen photobionts. One great advantage of independent dispersal of the fungus is symbiotic association with locally adapted photobiont strains.

Lichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches. In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.

Trebouxia sp. (TR9) and Coccomyxa simplex (Csol) are desiccation-tolerant lichen microalgae with different adaptive strategies in accordance with the prevailing conditions of their habitats. The remodelling of cell wall and extracellular polysaccharides depending on water availability are key elements in the tolerance to desiccation of both microalgae. Currently, there is no information about the extracellular proteins of these algae and other aero-terrestrial microalgae in response to limited water availability. To our knowledge, this is the first report on the proteins associated with the extracellular polymeric substances (EPS) of aero-terrestrial microalgae subjected to cyclic desiccation/rehydration. LC-MS/MS and bioinformatic analyses of the EPS-associated proteins in the two lichen microalgae submitted to four desiccation/rehydration cycles allowed the compilation of 111 and 121 identified proteins for TR9 and Csol, respectively. Both sets of EPS-associated proteins shared a variety of predicted biological functions but showed a constitutive expression in Csol and partially inducible in TR9. In both algae, the EPS-associated proteins included a number of proteins of unknown functions, some of which could be considered as small intrinsically disordered proteins related with desiccation-tolerant organisms. Differences in the composition and the expression pattern between the studied EPS-associated proteins would be oriented to preserve the biochemical and biophysical properties of the extracellular structures under the different conditions of water availability in which each alga thrives.

Lichens are poikilohydric symbiotic organisms that can survive in the absence of water. Photosynthesis must be highly regulated in these organisms, which live under continuous desiccation-rehydration cycles, to avoid photooxidative damage. Analysis of chlorophyll a fluorescence induction curves in the lichen microalgae of the Trebouxiophyceae Asterochloris erici and in Trebouxia jamesii (TR1) and Trebouxia sp. (TR9) phycobionts, isolated from the lichen Ramalina farinacea, shows differences with higher plants. In the presence of the photosynthetic electron transport inhibitor DCMU, the kinetics of Q A reduction is related to variable fluorescence by a sigmoidal function that approaches a horizontal asymptote. An excellent fit to these curves was obtained by applying a model based on the following assumptions: (1) after closure, the reaction centers (RCs) can be converted into “energy sink” centers (sRCs); (2) the probability of energy leaving the sRCs is very low or zero and (3) energy is not transferred from the antenna of PSII units with sRCs to other PSII units. The formation of sRCs units is also induced by repetitive light saturating pulses or at the transition from dark to light and probably requires the accumulation of reduced Q A, as well as structural changes in the reaction centers of PSII. This type of energy sink would provide a very efficient way to protect symbiotic microalgae against abrupt changes in light intensity.

An understanding of how biotic interactions shape species’ distributions is central to predicting host–symbiont responses under climate change. Switches to locally adapted algae have been proposed to be an adaptive strategy of lichen-forming fungi to cope with environmental change. However, it is unclear how lichen photobionts respond to environmental gradients, and whether they play a role in determining the fungal host’s upper and lower elevational limits. Deep-coverage Illumina DNA metabarcoding was used to track changes in the community composition of Trebouxia algae associated with two phylogenetically closely related, but ecologically divergent fungal hosts along a steep altitudinal gradient in the Mediterranean region. We detected the presence of multiple Trebouxia species in the majority of thalli. Both altitude and host genetic identity were strong predictors of photobiont community assembly in these two species. The predominantly clonally dispersing fungus showed stronger altitudinal structuring of photobiont communities than the sexually reproducing host. Elevation ranges of the host were not limited by the lack of compatible photobionts. Our study sheds light on the processes guiding the formation and distribution of specific fungal–algal combinations in the lichen symbiosis. The effect of environmental filtering acting on both symbiotic partners appears to shape the distribution of lichens.

• The epiphyte Evernia mesomorpha forms a lichen association with green algae in the genus Trebouxia. Little is known about the population structure of E. mesomorpha. Here, population structure of the algal and fungal symbionts was examined for 290 lichen thalli on 29 jack pine (Pinus banksiana) trees in Manitoba. • Through phylogenetic analysis of internal transcribed spacer (ITS) nuclear ribosomal DNA (rDNA) sequences, five algal genotypes were detected that were nested within T. jamesii. Two fungal genotypes were detected that formed a clade with two other Evernia species. The genus Evernia was paraphyletic with E. prunastri, sister to Parmelia saxatilis. Restriction fragment length polymorphism (RFLP) of ITS rDNA showed multiple algal genotypes in 45% of the 290 lichen thalli collected, whereas all thalli only contained one fungal genotype. • Low population subdivision of algal and fungal genotypes among trees suggested that the algal symbiont was being dispersed in the lichen soredium. • Low fungal specificity for multiple algal genotypes and a hypothesized algal switch may be important life history strategies for E. mesomorpha to adapt to changing environmental conditions.