IntroductionLichens are poikilohydric organisms whose biochemistry is strongly shaped by environmental conditions, yet their metabolome-wide adaptations across habitats remain unexplored.MethodsHere, we established a continent-scale reference for the in-situ metabolome of the model lichen Ramalina farinacea, from thalli collected across six European regions during winter and summer, using untargeted GC-MS.ResultsIn total, 187 small molecules were annotated, including a compact core of 12 metabolites shared across all sites and seasons. Notably, the most abundant and core metabolites have recognized osmoprotective roles. The metabolome was primarily centered on polyols: arabitol was the predominant metabolite (49.9% of total relative abundance), followed by ribitol, sucrose, sorbitol, and mannitol. Multivariate analyses revealed season, region, and climate-similarity groups as the main drivers of metabolomic dissimilarity. GABA and mannose were consistent summer markers, while linoleic acid and arabitol emerged as the top regional and climatic markers, respectively. Glycerol levels increased towards colder regimes alongside monoglycerides and mono/polyunsaturated fatty acids, reflecting patterns consistent with homeoviscous adaptation. Hierarchical clustering resolved coordinated metabolite modules that distinguish cold-continental from warm-Mediterranean regimes while preserving region-specific chemical fingerprints. Pathway over-representation analyses converged on alanine, aspartate, and glutamate metabolism. This and other differential pathways indicated coupled adjustments in carbon handling and stress physiology.DiscussionThis is the first continent-scale characterization of low-molecular-weight metabolomic variation in a lichen, revealing environmentally linked metabolic remodeling beyond the traditionally studied secondary metabolites and defining new chemoenvironmental markers as the metabolic basis of poikilohydric resilience in lichens.

Acarospora monacensis is revised and recovered from synonymy with Acarospora fuscata. It is reported from the Czech Republic and Germany. Acarospora variegata is revised and reported new for northern Italy based on a historical collection. There are no modern collections. Sarcogyne distans (Arnold) K.Knudsen & Kocourk. is described from historical German collections and modern collections from Austria and Czech Republic. The taxonomic concept hemicryptic hymenial gel reactions to Lugol's (IKI) is defined.

Four days of lichenological excursions in the surroundings of Sajathutte and Eisseehutte (Pragraten, High Tauern) resulted in the list of 428 lichen taxa, 13 lichenicolous fungi and 2 lichen-associated fungi. Five species (Catillaria cf. nigroisidiata, Micarea fennica, Microcalicium loraasii, Placynthium pulvinatum and Rinodina cf. calcigena) are first records for the Alps. In addition to the taxa mentioned, a further ten lichens are new to Austria: Caloplaca approximata, C. fuscorufa, Candelariella aggregata, Halecania spodomela, Lempholemma cladodes, L. isidioides, Porina rosei, Protoparmelia leproloma, Protoparmeliopsis bolcana, and Stereocaulon capitellatum. The area harbours an extraordinarily rich lichen diversity. The combination of high-mountain elements with continental or even xerothermic species is remarkable.

Lichenized algae and cyanobacteria are known to be shared and selected by unrelated lichen-forming fungi coexisting in so-called photobiont-mediated guilds. Life in such a guild could be crucial for the survival of a large group of lichen fungi dependent on horizontal transmission of photobionts. Here, we investigate frequent lichen phycobionts of the genus Trebouxia in rock-dwelling lichen communities. We found intensive and repeated sharing of specific Trebouxia assemblages by co-occurring lichens across distant localities. Rock chemistry, expressed as pH, determined the composition of photobiont pools and separated three saxicolous lichen guilds, sharing environmentally specific photobiont groups. Moreover, unlike the majority of lichen fungi, many Trebouxia photobionts represented opportunists in the choice of general substrate form (soil-rock-tree bark/wood), maintaining their pH preferences. Thus, saxicolous communities form just a part of a complex guild system that is in principle mediated by environmentally conditioned groups of naturally co-occurring photobionts. The complexity of the system is influenced by diverse photobiont life strategies, including also dispersal style. The findings of photobionts strictly or predominantly associated with sexually reproducing fungi stimulated us to emphasize the role of free-dispersing photobionts in the establishment and maintenance of lichen guilds.

Climatic factors, soil chemistry and geography are considered as major factors affecting lichen distribution and diversity. To determine how these factors limit or support the associations between the symbiotic partners, we revise the lichen symbiosis as a network of relationships here. More than one thousand thalli of terricolous Cladonia lichens were collected at sites with a wide range of soil chemical properties from seven biogeographical regions of Europe. A total of 18 OTUs of the algal genus Asterochloris and 181 OTUs of Cladonia mycobiont were identified. We displayed all realized pairwise mycobiont-photobiont relationships and performed modularity analysis. It revealed four virtually separated modules of cooperating OTUs. The modules differed in mean annual temperature, isothermality, precipitation, evapotranspiration, soil pH, nitrogen, and carbon contents. Photobiont switching was strictly limited to algae from one module, i.e., algae of similar ecological preferences, and only few mycobionts were able to cooperate with photobionts from different modules. Thus, Cladonia mycobionts generally cannot widen their ecological niches through photobiont switching. The modules also differed in the functional traits of the mycobionts, e.g., sexual reproduction rate, presence of soredia, and thallus type. These traits may represent adaptations to the environmental conditions that drive the differentiation of the modules. In conclusion, the promiscuity in Cladonia mycobionts is strictly limited by climatic factors and soil chemistry.

River gravel bars are dynamic and heterogeneous habitats straddling the transition between aquatic and terrestrial environments. Periodic flooding, low nutrient concentrations, frost, lack of stable sites, drought, and ground surface heat significantly influence the biota of these habitats. Mutualistic symbiosis may be a successful strategy for organisms to survive and proliferate under such harsh conditions. The lichen genus Stereocaulon was selected as a model symbiotic system from among the organisms living on river gravel bars. The goal of the current study was to determine the effect of this dynamic environment on phycobiont (i.e., green eukaryotic photobiont) community structure. We analyzed 147 Stereocaulon specimens collected in the Swiss Alps using Sanger sequencing (fungal internal transcribed spacer (ITS) rDNA, algal ITS rDNA, and algal actin type I gene) and analyzed 8 selected thalli and 12 soil samples using Illumina metabarcoding (ITS2 rDNA). Phytosociological sampling was performed for all 13 study plots. Our analyses of communities of phycobionts, lichens, bryophytes, and vascular plants indicated a gradual change in the phycobiont community along a successional gradient. The particularly large phycobiont diversity associated with Stereocaulon mycobionts included algae, here reported as phycobionts for the first time. Each of the two Stereocaulon mycobiont operational taxonomic units had a distinct pool of predominant phycobionts. The thalli selected for Illumina metabarcoding contained a wide range of additional algae, i.e., they showed algal plurality.

Intrathalline phycobiont diversity was investigated in a rosette-forming lichen, Parmotrema pseudotinctorum, using a combination of Sanger sequencing, 454-pyrosequencing, conventional light and confocal microscopy, and transmission electron microscopy. A total of 39 thalli sampled in five Canary Island populations were investigated. Three novel lineages of lichen phycobionts were detected, all being inferred within the Trebouxia clade G. The most abundant phycobiont lineage, occurring in all lichen populations investigated, is described here as Trebouxia crespoana sp. nov. This species produces spherical to pyriform cells possessing a crenulate chloroplast with lobes elongated at their ends, and one corticola-type pyrenoid with very thin, unbranched tubules of curved profile. Trebouxia crespoana is clearly distinguished from all other Trebouxia species by a characteristic cap-like cell wall thickening produced on one side of vegetative cells, and the larger size of vegetative cells that reach 21(-26) mu m in diameter.

Myrmecia israeliensis has been traditionally considered as a green coccoid free-living microalga.This microalga was previously suggested as the primary phycobiont in the lichens Placidium spp., Heteroplacidium spp., and Psora decipiens.However, due to the absence of ITS rDNA sequences (barcode information) published along with these investigations, the symbiotic nature of M. israeliensis might be confirmed by using the DNA barcoding and different microscopic examinations both in the symbiotic state and in culture.The aim of this study was to settle the presence of M. israeliensis as the primary microalga in squamulose lichens growing in terricolous communities (Psora spp., Placidium spp.and Claviscidium spp.) in 32 localities within European and Canary Island ecosystems by using both molecular and ultrastructural techniques.The lichen-forming fungi were identified using ITS rDNA as a barcode, and in the case of P. decipiens specimens, the mycobiont analyses showed an unexpected variability.Phycobiont phylogenetic analyses were made using both chloroplast (LSU rDNA) and nuclear (ITS rDNA) molecular markers.Our results proved that M. israeliensis is the primary symbiotic microalga in all the chosen and analyzed lichens.In addition, fluorescence microscopy, transmission electron microscopy and scanning electron techniques were used to characterize M. israeliensis.Finally, the presence of this microalga in lichen thalli was verified using different microscopic observations.A combination of different techniques, both molecular and microscopic, allowed for the accurate identification of this symbiotic microalga, beforehand mainly known as free living.Here, we suggest the combination of these techniques to prevent incorrect identification in microalgal lichen studies.

Noteworthy findings of 24 lichen species are presented. Nine of them are reported as new to Central Europe (Chaenotheca hygrophila, Cladonia krogiana, C. imbricarica, Gyalideopsis muscicola, Lecidea altissima, L. consimilis, Placynthiella hyporhoda, Rinodina stictica, and Waynea giraltiae). Most of further species are largely unrecorded taxa, and new distributional data remarkably extend their known ranges, at the same time being often new country records for Austria, the Czech Republic, Germany, Slovakia or Switzerland. A wider ecological amplitude is pinpointed for three species of Ostropomycetidae (Gyalideopsis helvetica, G. muscicola, Ramonia luteola). The fumarprotocetraric acid strain of Cladonia acuminata is newly reported from Europe. The poorly known taxon Lecidea consimilis is described in detail and lectotypified herein.

Symbiosis plays a fundamental role in nature. Lichens are among the best known, globally distributed symbiotic systems whose ecology is shaped by the requirements of all symbionts forming the holobiont. The widespread lichen-forming fungal genus Stereocaulon provides a suitable model to study the ecology of microscopic green algal symbionts (i.e., phycobionts) within the lichen symbiosis. We analysed 282 Stereocaulon specimens, collected in diverse habitats worldwide, using the algal ITS rDNA and actin gene sequences and fungal ITS rDNA sequences. Phylogenetic analyses revealed a great diversity among the predominant phycobionts. The algal genus Asterochloris (Trebouxiophyceae) was recovered in most sampled thalli, but two additional genera, Vulcanochloris and Chloroidium, were also found. We used variation-partitioning analyses to investigate the effects of climatic conditions, substrate/habitat characteristic, spatial distribution and mycobionts on phycobiont distribution. Based on an analogy, we examined the effects of climate, substrate/habitat, spatial distribution and phycobionts on mycobiont distribution. According to our analyses, the distribution of phycobionts is primarily driven by mycobionts and vice versa. Specificity and selectivity of both partners, as well as their ecological requirements and the width of their niches, vary significantly among the species-level lineages. We demonstrated that species-level lineages, which accept more symbiotic partners, have wider climatic niches, overlapping with the niches of their partners. Furthermore, the survival of lichens on substrates with high concentrations of heavy metals appears to be supported by their association with toxicity-tolerant phycobionts. In general, low specificity towards phycobionts allows the host to associate with ecologically diversified algae, thereby broadening its ecological amplitude.

This paper describes a new genus of lichen photobionts, Vulcanochloris, with three newly proposed species, V. canariensis, V. guanchorum and V. symbiotica. These algae have been discovered as photobionts of lichen Stereocaulon vesuvianum growing on slopes of volcanos and lava fields on La Palma, Canary Islands, Spain. Particular species, as well as the newly proposed genus, are delimited based on ITS rDNA, 18S rDNA and rbcL sequences, chloroplast morphology, and ultrastructural features. Phylogenetic analyses infer the genus Vulcanochloris as a member of Trebouxiophycean order Trebouxiales, in a sister relationship with the genus Asterochloris. Our data point to the similar lifestyle and morphology of these two genera; however, Vulcanochloris can be well distinguished by a unique formation of spherical incisions within the pyrenoid. Mycobiont specificity and geographical distribution of the newly proposed genus is further discussed.

The genus Asterochloris represents one of the most common, widespread, and diverse taxa of lichen photobionts. In this report, we describe and characterize six new species (A. echinata, A. friedlii, A. gaertneri, A. leprarii, A. lobophora, and A. woessiae) that were identified during our recent investigation of photobiont diversity. We found that the species differed genetically, morphologically, ecologically, and with respect to their mycobiont partners. Statistical analyses revealed significant morphological differentiation of all six newly described species, as well as their separation from previously described Asterochloris species. Chloroplast morphology represented the best morphological marker for species delineation. In fact, each species can be recognized by the dominance and unique assemblage of particular chloroplast types. Although genetically well recognized by rapidly evolving internal transcribed spacer rDNA and actin intron markers, all 13 investigated Asterochloris species shared identical small subunit rDNA sequences. We therefore demonstrated that morphologically and ecologically diverse species can frequently be grouped into a single taxonomic unit in whole‐transcriptome sequencing studies, considerably affecting the resulting estimates of species diversity. Finally, we demonstrated the presence of isogamous sexual reproduction in Asterochloris, disputing the current symbiotic dogma of the loss of sexual reproduction in algal symbionts.

We present floristic data from 82 localities mainly from eight mountain ranges of the Romanian Carpathians (Fagaras, Parang, Cindrel, Harghita, Hasmas, Bihor, Maramures and Rodna). The main habitats investigated were subalpine and alpine communities, beech and spruce forests, and limestone areas at lower altitudes. During four field expeditions, 364 lichenized and three lichenicolous fungi were recorded. Thirty-seven taxa are new for Romania. Biatora subduplex, Helocarpon pulverulum, Lecanora filamentosa, L. flavoleprosa, Lecidea huxariensis, Leucocarpia biatorella, Myriospora dilatata, Pertusaria borealis, Placynthium dolichoterum, Reichlingia leopoldii, Rhizocarpon carpaticum and Vezdaea stipitata are new for southeastern Europe. Lecanora flowersiana is reported for the first time from Europe. Candelariella commutata, Cetrelia chicitae, Lecanora cinereofusca, Leptogium hildenbrandii, Psoroma tenue var. borealis, Rinodina roscida and Usnea longissima are either phytogeographically remarkable or very rare taxa.

Lichens are symbiotic associations of fungi with green algae or cyanobacteria. They have arisen independently several times within the Ascomycota and Basidiomycota . This symbiosis became with time one of the most successful life forms on Earth. Outside of the symbiotic algae and fungi, there are endophytic fungi, other algae, and lichen-associated bacteria present within lichen thalli. Till now, no lichen-specific pathogens have been reported among bacteria and viruses. Around 15 dsRNA viruses are known from Eurotiomycetes and another dsRNA and reverse transcribed ssRNA viruses from Dothideomycetes containing some lichenized fungal lineages. Algal viruses have been identified from less than 1 % of known eukaryotic algal species but no virus has been found in Trebouxia or in Trentepohlia (Chlorophyta, Pleurastrophyceae, Pleurastrales), the most common green lichen photobionts. On the other hand, dsDNA viruses infecting related Chlorella algae are well known from freshwater phytoplankton. However, high-molecular weight dsRNA isolated from different lichen thalli indicated to us presence of ss or dsRNA viruses. A PCR-based search for viruses with genus-specific and species-specific primers resulted in amplification of genome segments highly identical with those of plant cytorhabdoviruses and with Apple mosaic virus (ApMV). The nucleotide sequence of the putative lichen cytorhabdovirus showed high identity (98 %) with Ivy latent cytorhabdovirus. The nucleotide sequences of six Apple mosaic virus isolates from lichens showed high similarity with ApMV isolates from apple and pear hosts. The lichen ApMV isolates were mechanically transmitted to an herbaceous host and detected positive in ELISA 14 days thereafter, which support its infectivity on plants. We prepared axenic cultures of photobionts identified as Trebouxia sp. from this ApMV-positive lichen samples. All these cultures were positive for ApMV in RT-PCR test. We suggest that lichens as a whole (or their photobionts, more specifically) could serve as reservoirs for viruses, despite the fact that the way of transmission between different organisms is not clear. We showed that lichens could harbour several viruses simultaneously, as the plant cytorhabdovirus and ApMV were detected in the same host, also.

The development of characteristic thallus structures in lichen-forming fungi requires the association with suitable photoautotrophic partners. Previous work suggests that fungi have a specific range of compatible photobionts and that selected algal strains are also correlated with the habitat conditions. We selected the rock-inhabiting crust lichen Protoparmeliopsis muralis, which exhibits high flexibility in algal associations. We present a geographically extended and detailed analysis of algal association patterns including thalli which host superficial algal colonies. We sampled 17 localities in Europe, and investigated the photobiont genotypic diversity within and between thalli and compared the diversity of intrathalline photobionts and externally associate algal communities between washed and unwashed thalli by single-strand conformation polymorphism analyses and ITS sequence data. The results show that (1) photobiont population within the lichen thalli is homogeneous; (2) multiple photobiont genotypes occur within single areoles and lobes of individual lichens; and (3) algal communities which superficially colonize the lichen thalli host taxa known as photobionts in unrelated lichens. Photobiont association patterns are extremely flexible in this ecologically versatile crust-forming lichen. We suggest that lichen surfaces represent a potential temporary niche for free-living stages of lichen photobionts, which could facilitate the establishment of further lichens in the proximal area.