Study on endolichenic fungal assemblage in Parmotrema and Heterodermia lichens of Shivamoga, Karnataka.

BACKGROUND: Lichen is a symbiotic association of algae and fungi, recognized as a self-sustaining ecosystem that constitutes an indeterminant number of bacteria, actinomycetes, fungi, and protozoa. We evaluated the endolichenic fungal assemblage given the dearth of knowledge on endolichenic fungi (ELFs), particularly from part of the Central Western Ghats, Karnataka, and conducted a phylogenetic analysis of xylariaceous fungi, the most diversified group of fungi using ITS and ITS+Tub2 gene set. RESULTS: Out of 17 lichen thalli collected from 5 ecoregions, 42 morphospecies recovered, belong to the class Sordariomycetes, Eurotiomycetes, Dothideomycetes, Leotiomycetes, Saccharomycetes. About 19 and 13 ELF genera have been reported from Parmotrema and Heterodermia thallus. Among the ecoregions EC2 showing highest species diversity (Parmotrema (1-D) = 0.9382, (H) = 2.865, Fisher-α = 8.429, Heterodermia (1-D) = 0.8038, H = 1.894, F-α = 4.57) followed the EC3 and EC1. Xylariales are the predominant colonizer reported from at least one thallus from four ecoregions. The morphotypes ELFX04, ELFX05, ELFX08 and ELFX13 show the highest BLAST similarity (> 99%) with Xylaria psidii, X. feejeensis, X. berteri and Hypoxylon fragiforme respectively. Species delimitation and phylogenetic position reveal the closest relation of Xylariaceous ELFs with plant endophytes. CONCLUSIONS: The observation highlights that the deciduous forest harness a high number of endolichenic fungi, a dominant portion of these fungi are non-sporulating and still exist as cryptic. Overall, 8 ELF species recognized based on phylogenetic analysis, including the two newly reported fungi ELFX03 and ELFX06 which are suspected to be new species based on the present evidence. The study proved, that the lichen being rich source to establish fungal diversity and finding new species. Successful amplification of most phylogenetic markers like RPB2, building of comprehensive taxonomic databases and application of multi-omics data are further needed to understand the complex nature of lichen-fungal symbiosis.

Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens from Alpine Areas in Eastern Alps and Equatorial Africa.

The diversity of bacteria associated with alpine lichens was profiled. Lichen samples belonging to the Umbilicariaceae family, commonly known as rock tripe lichens, were gathered from two distinct alpine fellfields: one situated on Mt. Brennkogel located in the Eastern European Alps (Austria), and the other on Mt. Stanley located in the Rwenzori mountains of equatorial Africa (Uganda). The primary aim of this research was to undertake a comparative investigation into the bacterial compositions, and diversities, identifying potential indicators and exploring their potential metabolisms, of these lichen samples. Bulk genomic DNA was extracted from the lichen samples, which was used to amplify the 18S rRNA gene by Sanger sequencing and the V3-V4 region of the 16S rRNA gene by Illumina Miseq sequencing. Examination of the fungal partner was carried out through the analysis of 18S rRNA gene sequences, belonging to the genus Umbilicaria (Ascomycota), and the algal partner affiliated with the lineage Trebouxia (Chlorophyta), constituted the symbiotic components. Analyzing the MiSeq datasets by using bioinformatics methods, operational taxonomic units (OTUs) were established based on a predetermined similarity threshold for the V3-V4 sequences, which were assigned to a total of 26 bacterial phyla that were found in both areas. Eight of the 26 phyla, i.e. Acidobacteriota, Actinomycota, Armatimonadota, Bacteroidota, Chloroflexota, Deinococcota, Planctomycetota, and Pseudomonadota, were consistently present in all samples, each accounting for more than 1% of the total read count. Distinct differences in bacterial composition emerged between lichen samples from Austria and Uganda, with the OTU frequency-based regional indicator phyla, Pseudomonadota and Armatimonadota, respectively. Despite the considerable geographic separation of approximately 5430 km between the two regions, the prediction of potential metabolic pathways based on OTU analysis revealed similar relative abundances. This similarity is possibly influenced by comparable alpine climatic conditions prevailing in both areas.

The dark side of orange: Multiorganismic continuum dynamics within a lichen of the Atacama Desert.

Over the decades our understanding of lichens has shifted to the fact that they are multiorganismic, symbiotic microecosystems, with their complex interactions coming to the fore due to recent advances in microbiomics. Here, we present a mutualistic-parasitic continuum dynamics scenario between an orange lichen and a lichenicolous fungus from the Atacama Desert leading to the decay of the lichen's photobiont and leaving behind a black lichen thallus. Based on isolation, sequencing, and ecophysiological approaches including metabolic screenings of the symbionts, we depict consequences upon infection with the lichenicolous fungus. This spans from a loss of the lichen's photosynthetic activity and an increased roughness of its surface to an inhibition of the parietin synthesis as a shared pathway between the photobiont and the mycobiont, including a shift of secondary metabolism products. This degree of relations has rarely been documented before, although lichenicolous fungi have been studied for over 200 years, adding an additional level to the view of interactions within lichens.

Genomic analysis of Coccomyxa viridis, a common low-abundance alga associated with lichen symbioses.

Lichen symbiosis is centered around a relationship between a fungus and a photosynthetic microbe, usually a green alga. In addition to their main photosynthetic partner (the photobiont), lichen symbioses can contain additional algae present in low abundance. The biology of these algae and the way they interact with the rest of lichen symbionts remains largely unknown. Here we present the first genome sequence of a non-photobiont lichen-associated alga. Coccomyxa viridis was unexpectedly found in 12% of publicly available lichen metagenomes. With few exceptions, members of the Coccomyxa viridis clade occur in lichens as non-photobionts, potentially growing in thalli endophytically. The 45.7 Mbp genome of C. viridis was assembled into 18 near chromosome-level contigs, making it one of the most contiguous genomic assemblies for any lichen-associated algae. Comparing the C. viridis genome to its close relatives revealed the presence of traits associated with the lichen lifestyle. The genome of C. viridis provides a new resource for exploring the evolution of the lichen symbiosis, and how symbiotic lifestyles shaped evolution in green algae.

Lichen-associated microbial members are prevalent in the snow microbiome of a sub-arctic alpine tundra.

Snow is the largest component of the cryosphere, with its cover and distribution rapidly decreasing over the last decade due to climate warming. It is imperative to characterize the snow (nival) microbial communities to better understand the role of microorganisms inhabiting these rapidly changing environments. Here, we investigated the core nival microbiome, the cultivable microbial members, and the microbial functional diversity of the remote Uapishka mountain range, a massif of alpine sub-arctic tundra and boreal forest. Snow samples were taken over a two-month interval along an altitude gradient with varying degree of anthropogenic traffic and vegetation cover. The core snow alpine tundra/boreal microbiome, which was present across all samples, constituted of Acetobacterales, Rhizobiales and Acidobacteriales bacterial orders, and of Mycosphaerellales and Lecanorales fungal orders, with the dominant fungal taxa being associated with lichens. The snow samples had low active functional diversity, with Richness values ranging from 0 to 19.5. The culture-based viable microbial enumeration ranged from 0 to 8.05 × 103 CFUs/mL. We isolated and whole-genome sequenced five microorganisms which included three fungi, one alga, and one potentially novel bacterium of the Lichenihabitans genus; all of which appear to be part of lichen-associated taxonomic clades.

Biosynthetic gene cluster synteny: Orthologous polyketide synthases in Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus), which together generate a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here, we provide a comparative view of the biosynthetic gene clusters of three lichen mycobionts derived from Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata. In addition, we present a high-quality PacBio metagenome of Parmelia sulcata, from which we extracted the mycobiont bin containing 214 biosynthetic gene clusters. Most biosynthetic gene clusters in these genomes were associated with T1PKSs, followed by NRPSs and terpenes. This study focused on biosynthetic gene clusters related to polyketide synthesis. Based on ketosynthase homology, we identified nine highly syntenic clusters present in all three species. Among the four clusters belonging to nonreducing PKSs, two are putatively linked to lichen substances derived from orsellinic acid (orcinol depsides and depsidones, e.g., lecanoric acid, physodic acid, lobaric acid), one to compounds derived from methylated forms of orsellinic acid (beta orcinol depsides, e.g., atranorin), and one to melanins. Five clusters with orthologs in all three species are linked to reducing PKSs. Our study contributes to sorting and dereplicating the vast PKS diversity found in lichenized fungi. High-quality sequences of biosynthetic gene clusters of these three common species provide a foundation for further exploration into biotechnological applications and the molecular evolution of lichen substances.

Identification of a biosynthetic gene cluster for a red pigment cristazarin produced by a lichen-forming fungus Cladonia metacorallifera.

Lichens are known to produce many novel bioactive metabolites. To date, approximately 1,000 secondary metabolites have been discovered, which are predominantly produced by the lichen mycobionts. However, despite the extensive studies on production of lichen secondary metabolites, little is known about the responsible biosynthetic gene clusters (BGCs). Here, we identified a putative BGC that is implicated in production of a red pigment, cristazarin (a naphthazarin derivative), in Cladonia metacorallifera. Previously, cristazarin was shown to be specifically induced in growth media containing fructose as a sole carbon source. Thus, we performed transcriptome analysis of C. metacorallifera growing on different carbon sources including fructose to identify the BGC for cristazarin. Among 39 polyketide synthase (PKS) genes found in the genome of C. metacorallifera, a non-reducing PKS (coined crz7) was highly expressed in growth media containing either fructose or glucose. The borders of a cristazarin gene cluster were delimited by co-expression patterns of neighboring genes of the crz7. BGCs highly conserved to the cristazarin BGC were also found in C. borealis and C. macilenta, indicating that these related species also have metabolic potentials to produce cristazarin. Phylogenetic analysis revealed that the Crz7 is sister to fungal PKSs that biosynthesize an acetylated tetrahydoxynaphthalene as a precursor of melanin pigment. Based on the phylogenetic placement of the Crz7 and putative functions of its neighboring genes, we proposed a plausible biosynthetic route for cristazarin. In this study, we identified a lichen-specific BGC that is likely involved in the biosynthesis of a naphthazarin derivative, cristazarin, and confirmed that transcriptome profiling under inducing and non-inducing conditions is an effective strategy for linking metabolites of interest to biosynthetic genes.

A novel bacteriobiont of the Arctic lichen Flavocetraria nivalis, Lichenifustis flavocetrariae gen. nov, sp. nov. demonstrating hydrolytic properties and containing a full set of the Calvin-Benson-Bassham cycle genes.

A Gram-negative, strictly aerobic, chemoorganotrophic, bacteriochlorophyll a-containing, slow-growing bacterium was isolated from the lichen Flavocetraria nivalis and designated strain BP6-180914 T. Cells of this strain were large nonmotile rods, which reproduced by binary fission. Cells grew under oxic conditions and were able to utilize sugars and several polysaccharides, including starch and pectin. Strain BP6-180914 T was psychrotolerant and moderately acidophilic growing at 4-35 °C (optimum 20-28 °C) and between pH 4.0 and 7.5 (optimum 4.5-5.5). The major fatty acids were C18:1ω7c, C19:0 cyclo, C16:0 and C18:0. The polar lipids were diphosphatidylglycerols, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylcholines, unidentified aminolipids, and a number of glycolipids, the major one being an unidentified glycolipid. The quinone was Q-10. The DNA G + C content was 63.65%. Comparative 16S rRNA gene sequence analysis revealed that strain BP6-180914 T was a member of the order Hyphomicrobiales and belonged to the family Lichenihabitantaceae defined by the lichen-dwelling facultative aerobic chemo-organotroph Lichenihabitans psoromatis (92.7% sequence similarity). The results of phylogenomic and genomic relatedness analyses showed that strain BP6-180914 T could clearly be distinguished from other species in the order Hyphomicrobiales with average nucleotide identity values of < 74.05% and genome-to-genome distance values of < 21.1%. The AAI value of 65.9% between strain BP6-180914 T and L. psoromatis allowed us to assign this strain to the novel genus of the family Lichenihabitantaceae. Therefore, it is proposed that strain BP6-180914 T represents a novel species in a new genus, Lichenifustis flavocetrariae gen. nov., sp. nov.; strain BP6-180914 T (= KCTC 92872 T = VKM B-3641 T = UQM 41506 T) is the type strain.

Systemic infection of Bryoria (Lecanoromycetes, Ascomycota) by Athelia (Agaricomycetes, Basidiomycota) in western North America.

Bryoria (Parmeliaceae, Ascomycota) is one of the dominant genera of hair lichens in western North America and is characteristic of high-elevation conifer forest ecosystems. In areas where Bryoria is abundant, it is common to find thalli in which the thalline filaments become conglutinated, forming brittle dead zones. After sampling Bryoria thalli across western Canada and the northwestern United States at different times of the year, we found that this dieback phenomenon is associated with the winter growth of a mold-forming basidiomycete. We report that this fungus belongs to Athelia (Atheliaceae, Basidiomycota), a genus known to contain lichen pathogens, most notably A. arachnoidea. By sequencing a combination of genetic markers-nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), partial nuc 28S rDNA (28S), and partial translation elongation factor 1-α (TEF1)-paired with morphometric analyses, we reveal the involvement of at least three additional lineages of lichen-associated Athelia and describe one as a new species, A. abscondita. Athelia abscondita is morphologically distinguished from other Athelia species by its basidia and basidiospores, was found to frequently infect members of Bryoria sect. Implexae, and was occasionally on other foliose and fruticose species within Parmeliaceae.

Bacterial microbiome in tropical lichens and the effect of the isolation method on culturable lichen-derived actinobacteria.

Ten samples of tropical lichens collected from Doi Inthanon, Thailand, were explored for the diversity of their bacterial microbiomes through 16S rRNA-based metagenomics analysis. The five predominant lichen-associated bacteria belonged to the phyla Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota (12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia contained the highest bacterial species richness. Within the lichens, Ramalina conduplicans and Cladonia rappii showed a distinct bacterial community from the other lichen species. The community of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically active compounds. From the total Operational Taxonomic Units (OTUs) found across the ten different lichen samples, 13.21% were identified as actinobacteria, including the rare actinobacterial genera that are not commonly found, such as Pseudonocardia, Kineosporia, Dactylosporangium, Amycolatopsis, Actinoplanes, and Streptosporangium. Evaluation of the pretreatment method (heat, air-drying, phenol, and flooding) and isolation media used for the culture-dependent actinobacterial isolation revealed that the different pretreatments combined with different isolation media were effective in obtaining several species of actinobacteria. However, metagenomics analyses revealed that there were still several strains, including rare actinobacterial species, that were not isolated. This research strongly suggests that lichens appear to be a promising source for obtaining actinobacteria.

Mitochondrial genomes in the iconic reindeer lichens: Architecture, variation, and synteny across multiple evolutionary scales.

Variation in mitochondrial genome composition across intraspecific, interspecific, and higher taxonomic scales has been little studied in lichen obligate symbioses. Cladonia is one of the most diverse and ecologically important lichen genera, with over 500 species representing an array of unique morphologies and chemical profiles. Here, we assess mitochondrial genome diversity and variation in this flagship genus, with focused sampling of two clades of the "true" reindeer lichens, Cladonia subgenus Cladina, and additional genomes from nine outgroup taxa. We describe composition and architecture at the gene and the genome scale, examining patterns in organellar genome size in larger taxonomic groups in Ascomycota. Mitochondrial genomes of Cladonia, Pilophorus, and Stereocaulon were consistently larger than those of Lepraria and contained more introns, suggesting a selective pressure in asexual morphology in Lepraria driving it toward genomic simplification. Collectively, lichen mitochondrial genomes were larger than most other fungal life strategies, reaffirming the notion that coevolutionary streamlining does not correlate to genome size reductions. Genomes from Cladonia ravenelii and Stereocaulon pileatum exhibited ATP9 duplication, bearing paralogs that may still be functional. Homing endonuclease genes (HEGs), though scarce in Lepraria, were diverse and abundant in Cladonia, exhibiting variable evolutionary histories that were sometimes independent of the mitochondrial evolutionary history. Intraspecific HEG diversity was also high, with C. rangiferina especially bearing a range of HEGs with one unique to the species. This study reveals a rich history of events that have transformed mitochondrial genomes of Cladonia and related genera, allowing future study alongside a wealth of assembled genomes.

Metagenomics Shines Light on the Evolution of "Sunscreen" Pigment Metabolism in the Teloschistales (Lichen-Forming Ascomycota).

Fungi produce a vast number of secondary metabolites that shape their interactions with other organisms and the environment. Characterizing the genes underpinning metabolite synthesis is therefore key to understanding fungal evolution and adaptation. Lichenized fungi represent almost one-third of Ascomycota diversity and boast impressive secondary metabolites repertoires. However, most lichen biosynthetic genes have not been linked to their metabolite products. Here we used metagenomic sequencing to survey gene families associated with production of anthraquinones, UV-protectant secondary metabolites present in various fungi, but especially abundant in a diverse order of lichens, the Teloschistales (class Lecanoromycetes, phylum Ascomycota). We successfully assembled 24 new, high-quality lichenized-fungal genomes de novo and combined them with publicly available Lecanoromycetes genomes from taxa with diverse secondary chemistry to produce a whole-genome tree. Secondary metabolite biosynthetic gene cluster (BGC) analysis showed that whilst lichen BGCs are numerous and highly dissimilar, core enzyme genes are generally conserved across taxa. This suggests metabolite diversification occurs via re-shuffling existing enzyme genes with novel accessory genes rather than BGC gains/losses or de novo gene evolution. We identified putative anthraquinone BGCs in our lichen dataset that appear homologous to anthraquinone clusters from non-lichenized fungi, suggesting these genes were present in the common ancestor of the subphylum Pezizomycotina. Finally, we identified unique transporter genes in Teloschistales anthraquinone BGCs that may explain why these metabolites are so abundant and ubiquitous in these lichens. Our results support the importance of metagenomics for understanding the secondary metabolism of non-model fungi such as lichens.

How to build a lichen: from metabolite release to symbiotic interplay.

Exposing their vegetative bodies to the light, lichens are outstanding amongst other fungal symbioses. Not requiring a pre-established host, 'lichenized fungi' build an entirely new structure together with microbial photosynthetic partners that neither can form alone. The signals involved in the transition of a fungus and a compatible photosynthetic partner from a free-living to a symbiotic state culminating in thallus formation, termed 'lichenization', and in the maintenance of the symbiosis, are poorly understood. Here, we synthesise the puzzle pieces of the scarce knowledge available into an updated concept of signalling involved in lichenization, comprising five main stages: (1) the 'pre-contact stage', (2) the 'contact stage', (3) 'envelopment' of algal cells by the fungus, (4) their 'incorporation' into a pre-thallus and (5) 'differentiation' into a complex thallus. Considering the involvement of extracellularly released metabolites in each phase, we propose that compounds such as fungal lectins and algal cyclic peptides elicit early contact between the symbionts-to-be, whereas phytohormone signalling, antioxidant protection and carbon exchange through sugars and sugar alcohols are of continued importance throughout all stages. In the fully formed lichen thallus, secondary lichen metabolites and mineral nutrition are suggested to stabilize the functionalities of the thallus, including the associated microbiota.

Lichen holobionts show compositional structure along elevation.

Holobionts are dynamic ecosystems that may respond to abiotic drivers with compositional changes. Uncovering elevational diversity patterns within these microecosystems can further our understanding of community-environment interactions. Here, we assess how the major components of lichen holobionts-fungal hosts, green algal symbionts, and the bacterial community-collectively respond to an elevational gradient. We analyse populations of two lichen symbioses, Umbilicaria pustulata and U. hispanica, along an elevational gradient spanning 2100 altitudinal metres and covering three major biomes. Our study shows (i) discontinuous genomic variation in fungal hosts with one abrupt genomic differentiation within each of the two host species, (ii) altitudinally structured bacterial communities with pronounced turnover within and between hosts, and (iii) altitude-specific presence of algal symbionts. Alpha diversity of bacterial communities decreased with increasing elevation. A marked turnover in holobiont diversity occurred across two altitudinal belts: at 11°C-13°C average annual temperature (here: 800-1200 m a.s.l.), and at 7°C-9°C average annual temperature (here: 1500-1800 m a.s.l.). The two observed zones mark a clustering of distribution limits and community shifts. The three ensuing altitudinal classes, that is, the most frequent combinations of species in holobionts, approximately correspond to the Mediterranean, cool-temperate, and alpine climate zones. We conclude that multitrophic microecosystems, such as lichen holobionts, respond with concerted compositional changes to climatic factors that also structure communities of macroorganisms, for example, vascular plants.

Symbiont-specific responses to environmental cues in a threesome lichen symbiosis.

Photosymbiodemes are a special case of lichen symbiosis where one lichenized fungus engages in symbiosis with two different photosynthetic partners, a cyanobacterium and a green alga, to develop two distinctly looking photomorphs. We compared gene expression of thallus sectors of the photosymbiodeme-forming lichen Peltigera britannica containing cyanobacterial photobionts with thallus sectors with both green algal and cyanobacterial photobionts and investigated differential gene expression at different temperatures representing mild and putatively stressful conditions. First, we quantified photobiont-mediated differences in fungal gene expression. Second, because of known ecological differences between photomorphs, we investigated symbiont-specific responses in gene expression to temperature increases. Photobiont-mediated differences in fungal gene expression could be identified, with upregulation of distinct biological processes in the different morphs, showing that interaction with specific symbiosis partners profoundly impacts fungal gene expression. Furthermore, high temperatures expectedly led to an upregulation of genes involved in heat shock responses in all organisms in whole transcriptome data and to an increased expression of genes involved in photosynthesis in both photobiont types at 15 and 25°C. The fungus and the cyanobacteria exhibited thermal stress responses already at 15°C, the green algae mainly at 25°C, demonstrating symbiont-specific responses to environmental cues and symbiont-specific ecological optima.

Recent advances in research for potential utilization of unexplored lichen metabolites.

Several research studies have shown that lichens are productive organisms for the synthesis of a broad range of secondary metabolites. Lichens are a self-sustainable stable microbial ecosystem comprising an exhabitant fungal partner (mycobiont) and at least one or more photosynthetic partners (photobiont). The successful symbiosis is responsible for their persistence throughout time and allows all the partners (holobionts) to thrive in many extreme habitats, where without the synergistic relationship they would be rare or non-existent. The ability to survive in harsh conditions can be directly correlated with the production of some unique metabolites. Despite the potential applications, these unique metabolites have been underutilised by pharmaceutical and agrochemical industries due to their slow growth, low biomass availability and technical challenges involved in their artificial cultivation. However, recent development of biotechnological tools such as molecular phylogenetics, modern tissue culture techniques, metabolomics and molecular engineering are opening up a new opportunity to exploit these compounds within the lichen holobiome for industrial applications. This review also highlights the recent advances in culturing the symbionts and the computational and molecular genetics approaches of lichen gene regulation recognized for the enhanced production of target metabolites. The recent development of multi-omics novel biodiscovery strategies aided by synthetic biology in order to study the heterologous expressed lichen-derived biosynthetic gene clusters in a cultivatable host offers a promising means for a sustainable supply of specialized metabolites.

Unraveling usnic acid: a comparison of biosynthetic gene clusters between two reindeer lichen (Cladonia rangiferina and C. uncialis).

Lichenized fungi are known for their production of a diversity of secondary metabolites, many of which have broad biological and pharmacological applications. By far the most well-studied of these metabolites is usnic acid. While this metabolite has been well-known and researched for decades, the gene cluster responsible for its production was only recently identified from the species Cladonia uncialis. Usnic acid production varies considerably in the genus Cladonia, even among closely related taxa, and many species, such as C. rangiferina, have been inferred to be incapable of producing the metabolite based on analysis by thin-layer chromatography (TLC). We sequenced and examined the usnic acid biosynthetic gene clusters, or lack thereof, from four closely related Cladonia species (C. oricola, C. rangiferina, C. stygia, and C. subtenuis), and compare them against those of C. uncialis. We complement this comparison with tiered chemical profile analyses to confirm the presence or absence of usnic acid in select samples, using both HPLC and LC-MS. Despite long-standing reporting that C. rangiferina lacks the ability to produce usnic acid, we observed functional gene clusters from the species and detected usnic acid when extracts were examined by LC-MS. By contrast, C. stygia and C. oricola, have been previously described as lacking the ability to produce usnic acid, lacked the gene cluster entirely, and no usnic acid could be detected in C. oricola extracts via HPLC or LC-MS. This work suggests that chemical profiles attained through inexpensive and low-sensitivity methods like TLC may fail to detect low abundance metabolites that can be taxonomically informative. This study also bolsters understanding of the usnic acid gene cluster in lichens, revealing differences among domains of the polyketide synthase which may explain observed differences in expression. These results reinforce the need for comprehensive characterization of lichen secondary metabolite profiles with sensitive LC-MS methods.

The dielectric response of hydrated water as a structural signature of nanoconfined lichen melanins.

Lichens are unique symbiotic organisms from a mutually beneficial alliance of fungi and algae/cyanobacteria that successfully survive extreme temperatures and drought conditions. Most probably such extraordinary vitality of lichens is underlain by melanins, one of the main structural and chemical lichen components, and their mutual relationship with residual water. In this paper, we propose mechanisms, which allow lichens to store up the extra water in their structure. Melanins that are constituents of the cortical lichen layer and presumably contribute to unique water-lichen interactions are chosen for physical experiments in a wide temperature domain. Two melanin pigments extracted from different lichens are studied here - eumelanin from Lobaria pulmonaria and allomelanin from Cetraria islandica. To investigate the inner melanin structure and water-melanin interactions, FTIR and BDS techniques are applied. The BDS technique was used in a wide temperature region of 123-293 K for melanins with various hydration levels. The relaxation processes related to the confinement of supercooled water - in melanins are observed and discussed in details. At medium and high hydration levels, the relaxation process in two melanins of different chemical compositions and supramolecular structures exhibits a well-known crossover that was already observed in many types of confinements. The analysis of FTIR and BDS results helps to clarify the lichen-water interaction processes.

The yeast lichenosphere: high diversity of basidiomycetes from the lichens Tephromela atra and Rhizoplaca melanophthalma.

Lichens are well-known examples of complex symbiotic associations between organisms from different Kingdoms. Microfungi in particular, establish diverse associations with the hosting lichen thallus, as species-specific parasites or transient co-inhabitants. The whole community of lichen-associated fungi constitute the 'lichen mycobiome' comprising both ascomycetes and basidiomycetes, including filamentous and yeast taxa. Metabarcoding results and microscopy analyses show that in some thalli, basidiomycetes are frequent lichen-associated fungi but still only a few species could be axenically isolated and morphologically characterized. Within a broad project aiming at characterizing the mycobiome diversity by culture-dependent and independent approaches in two lichen species selected as reference models - Rhizoplaca melanophthalma and Tephromela atra, we succeed in isolating and culturing 76 new strains of basidiomycetous yeasts. The lichen thalli were collected in different mountain regions worldwide and at relatively high elevation. The yeast strains were isolated on different growth media and were studied for their morphological and genetic diversity. Nuclear internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequence analyses identified them to belong to ten families within the orders Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes. The yeasts here detected showed patterns of host-preference in a few cases and they are potentially related to the ecological conditions.

Microbial community associated with the crustose lichen Rhizocarpon geographicum L. (DC.) living on oceanic seashore: A large source of diversity revealed by using multiple isolation methods.

Recently, the study of the interactions within a microcosm between hosts and their associated microbial communities drew an unprecedented interest arising from the holobiont concept. Lichens, a symbiotic association between a fungus and an alga, are redefined as complex ecosystems considering the tremendous array of associated microorganisms that satisfy this concept. The present study focuses on the diversity of the microbiota associated with the seashore located lichen Rhizocarpon geographicum, recovered by different culture-dependent methods. Samples harvested from two sites allowed the isolation and the molecular identification of 68 fungal isolates distributed in 43 phylogenetic groups, 15 bacterial isolates distributed in five taxonomic groups and three microalgae belonging to two species. Moreover, for 12 fungal isolates belonging to 10 different taxa, the genus was not described in GenBank. These fungal species have never been sequenced or described and therefore non-studied. All these findings highlight the novel and high diversity of the microflora associated with R. geographicum. While many species disappear every day, this work suggests that coastal and wild environments still contain an unrevealed variety to offer and that lichens constitute a great reservoir of new microbial taxa which can be recovered by multiplying the culture-dependent techniques.