Lichen Symbionts Research Articles

Ultraviolet-induced melanisation in lichens: physiological traits and transcriptome profile.

Lichens are important components of high-latitude boreal and Arctic habitats. While stress tolerant, they are among the most sensitive ecosystem components to climate change, in particular, an increase in ultraviolet light (UV) arising from polar ozone depletion and deforestation. This study is the first to explore the effects of UV-B on gene expression in lichens to predict metabolic pathways involved in tolerance. Using transcriptome profiling and bioinformatic analyses, here we studied the effects of UV-B on gene expression in lichens using Lobaria pulmonaria (L.) Hoff. as a model species. UV-B exposure causes significant browning of the upper cortex of the thallus, which correlates to an increased expression of biosynthetic gene clusters involved in the synthesis of eu- and allomelanins and melanin precursors. Based on transcriptome analyses, we suggest that the biosynthesis of melanins and other secondary metabolites, such as naphthalene derivates, tropolones, anthraquinones, and xanthones, is a trade-off that lichens pay to protect essential metabolic processes such as photosynthesis and respiration. Expression profiles of general stress-associated genes, in particular, related to reactive oxygen species scavenging, protection of proteins, and DNA repair, clearly indicate that the mycobiont is the more UV-B-responsive and susceptible partner in lichen symbiosis. Our findings demonstrate that UV-B stress activates an intricate gene network involved in tolerance mechanisms of lichen symbionts. Knowledge obtained here may enable the prediction of likely effects on lichen biodiversity caused by climate change and pollution.

Symbionts out of sync: Decoupled physiological responses are widespread and ecologically important in lichen associations.

A core vulnerability in symbioses is the need for coordination between the symbiotic partners, which are often assumed to be closely physiologically integrated. We critically re-examine this assumed integration between symbionts in lichen symbioses, recovering a long overlooked yet fundamental physiological asymmetry in carbon balance. We examine the physiological, ecological, and transcriptional basis of this asymmetry in the lichen Evernia mesomorpha. This carbon balance asymmetry depends on hydration source and aligns with climatic range limits. Differences in gene expression across the E. mesomorpha symbiosis suggest that the physiologies of the primary lichen symbionts are decoupled. Furthermore, we use gas exchange data to show that asymmetries in carbon balance are widespread and common across evolutionarily disparate lichen associations. Using carbon balance asymmetry as an example, we provide evidence for the wide-ranging importance of physiological asymmetries in symbioses.

Decoding the evolutionary association among lichen symbionts in Dyplolabia afzelii from the Western Ghats, India

Decoding the evolutionary association among lichen symbionts in Dyplolabia afzelii from the Western Ghats, India

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.

Self potent antimicrobial and antifouling properties of zinc nanoparticles derived from lichen symbionts

AbstractBiofouling poses a significant economic challenge for marine transportation. The use of chemical compounds to eliminate fouling layers beneath the ship's surface, albeit effective, raises concerns due to its potential harm to the aquatic ecosystem. Lichen symbiotic associations, an underexplored research area, show promise in producing antimicrobial and antifouling compounds. These lichen‐associated bacteria engage in various functional activities and have multiple bacterial counterparts. The optimal conditions for cultivating lichen‐associated bacteria for maximum growth were identified as a pH of 7, a temperature of 30°C, and an incubation period of 8 days. Glucose served as the carbon source, while yeast extract served as the nitrogen source. Additionally, zinc nanoparticles were successfully synthesized from the thriving lichen‐associated bacteria. Fourier transform infrared spectroscopy (FTIR) analysis indicated distinctive bands at 3890 and 3103 cm−1, corresponding to the OH stretching of alcohol and acid stretch bands, respectively. Furthermore, the presence of amide in NH banding in the synthesized zinc nanoparticles was represented by a band at 1636 cm−1. SEM images of the produced zinc NPs displayed a spherical shape, ranging in size from 40 to 70 nm. To assess the impact of these nanoparticles from lichen‐associated bacteria on biofouling activity in marine transportation, zebrafish results were analyzed.

Host selection tendency of key microbiota in arid desert lichen crusts.

Lichen genus Endocarpon in biological soil crust form was chosen as a model to investigate the bacterial communities for the first time across four vertically distinct strata. Key bacterial microbiota in lichen thallus were discovered, which were gradually filtered and mainly derived from the crust soil, with clear host selection tendency. The study provided key information to better understand the homeostasis maintenance mechanism of the lichen symbiont and community assembly of desert lichen crust.

A Plastic Biosynthetic Pathway for the Production of Structurally Distinct Microbial Sunscreens.

Mycosporine-like amino acids (MAAs) are small, colorless, and water-soluble secondary metabolites. They have high molar extinction coefficients and a unique UV radiation absorption mechanism that make them effective sunscreens. Here we report the discovery of two structurally distinct MAAs from the lichen symbiont strain Nostoc sp. UHCC 0926. We identified these MAAs as aplysiapalythine E (C23H38N2O15) and tricore B (C34H53N4O15) using a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy. We obtained a 8.3 Mb complete genome sequence of Nostoc sp. UHCC 0926 to gain insights into the genetic basis for the biosynthesis of these two structural distinct MAAs. We identified MAA biosynthetic genes encoded in three separate locations of the genome. The organization of biosynthetic enzymes in Nostoc sp. UHCC 0926 necessitates a branched biosynthetic pathway to produce two structurally distinct MAAs. We detected the presence of such discontiguous MAA biosynthetic gene clusters in 12% of the publicly available complete cyanobacterial genomes. Bioinformatic analysis of public MAA biosynthetic gene clusters suggests that they are subject to rapid evolutionary processes resulting in highly plastic biosynthetic pathways that are responsible for the chemical diversity in this family of microbial sunscreens.

The Tripartite Lichen Ricasolia virens: Involvement of Cyanobacteria and Bacteria in Its Morphogenesis.

Ricasolia virens is an epiphytic lichen-forming fungus mainly distributed in Western Europe and Macaronesia in well-structured forests with ecological continuity that lack eutrophication. It is considered to be threatened or extinct in many territories in Europe (IUCN). Despite its biological and ecological relevance, studies on this taxon are scarce. The thalli are tripartite, and the mycobiont has a simultaneous symbiotic relationship with cyanobacteria and green microalgae, which represent interesting models to analyse the strategies and adaptations resulting from the interactions of lichen symbionts. The present study was designed to contribute to a better understanding of this taxon, which has shown a clear decline over the last century. The symbionts were identified by molecular analysis. The phycobiont is Symbiochloris reticulata, and the cyanobionts (Nostoc) are embedded in internal cephalodia. Light, transmission electron and low-temperature scanning microscopy techniques were used to investigate the thallus anatomy, ultrastructure of microalgae and ontogeny of pycnidia and cephalodia. The thalli are very similar to its closest relative, Ricasolia quercizans. The cellular ultrastructure of S. reticulata by TEM is provided. Non-photosynthetic bacteria located outside the upper cortex are introduced through migratory channels into the subcortical zone by the splitting of fungal hyphae. Cephalodia were very abundant, but never as external photosymbiodemes.

Composition, structure and robustness of Lichen guilds

Symbiosis is a major engine of evolutionary innovation underlying many extant complex organisms. Lichens are a paradigmatic example that offers a unique perspective on the role of symbiosis in ecological success and evolutionary diversification. Lichen studies have produced a wealth of information regarding the importance of symbiosis, but they frequently focus on a few species, limiting our understanding of large-scale phenomena such as guilds. Guilds are groupings of lichens that assist each other’s proliferation and are intimately linked by a shared set of photobionts, constituting an extensive network of relationships. To characterize the network of lichen symbionts, we used a large data set (n=206 publications) of natural photobiont-mycobiont associations. The entire lichen network was found to be modular, but this organization does not directly match taxonomic information in the data set, prompting a reconsideration of lichen guild structure and composition. The multiscale nature of this network reveals that the major lichen guilds are better represented as clusters with several substructures rather than as monolithic communities. Heterogeneous guild structure fosters robustness, with keystone species functioning as bridges between guilds and whose extinction would endanger global stability.

Genomic and Metabolite Profiling Reveal a Novel Streptomyces Strain, QHH-9511, from the Qinghai-Tibet Plateau.

The prevalence of superbugs, represented by methicillin-resistant Staphylococcus aureus (MRSA), has become a serious clinical and public safety concern with rising incidence in hospitals. Polyketides with diverse chemical structures harbor many antimicrobial activities, including those of rifampin and rapamycin against MRSA. Streptomyces sp. QHH-9511 was isolated from a niche habitat in the Qinghai-Tibet Plateau and used to produce antibacterial metabolites. Herein, an integrated approach combining genome mining and metabolic analysis were employed to decipher the chemical origin of the antibacterial components with pigmented properties in strain QHH-9511, a novel Streptomyces species from a lichen symbiont on the Qinghai-Tibet Plateau. Genomic phylogeny assembled at the chromosome level revealed its unique evolutionary state. Further genome mining uncovered 36 candidate gene clusters, most of which were uncharacterized. Meanwhile, based on liquid chromatography coupled to diode array detection mass spectrometry, a series of granaticins, BSMs, chromones, phaeochromycins, and related molecules were discovered by using the Global Natural Product Social molecular networking platform. Subsequently, several pigment compounds were isolated and identified by high-resolution mass spectrometry and/or nuclear magnetic resonance, among which the structure-activity relationships of seven aromatic polyketides showed that the fused lactone ring of the C-2 carboxyl group could increase antibacterial activity. Genetic experiments indicated that all seven aromatic polyketides are a series of metabolic shunts produced by a single type II polyketide synthase (PKS) cluster. Comparative genomic analysis of granaticin producers showed that the granaticin gene cluster is widely distributed. This study provides an efficient method to combine genome mining and metabolic profiling techniques to uncover bioactive metabolites derived from specific habitats, while deepening our understanding of aromatic polyketide biosynthesis. IMPORTANCE Undescribed microorganisms from special habitats are being screened for anti-superbug drug molecules. In a project to screen actinomycetes for anti-MRSA activity, we isolated a Streptomyces strain from Qinghai Lake lichens. The phylogeny based on the genome assembled at the chromosome level revealed this strain's unique evolutionary state. The chemical origins of the antibacterial components with pigment properties in strain QHH-9511 were determined using an integrated approach combining genome mining and metabolic analysis. Further genome mining uncovered 36 secondary metabolite gene clusters, the majority of which were previously unknown. A series of aromatic compounds were discovered using molecular network analysis, separation, and extraction. Genetic experiments revealed that all seven aromatic polyketides are a series of metabolic shunts produced by a single cluster of type II PKSs. This study describes a method for identifying novel Streptomyces from specific habitats by combining genome mining with metabolic profiling techniques.

Opening the Gap: Rare Lichens With Rare Cyanobionts - Unexpected Cyanobiont Diversity in Cyanobacterial Lichens of the Order Lichinales.

The last decades of research led to a change in understanding of lichens that are now seen as self-sustaining micro-ecosystems, harboring diverse microbial organisms in tight but yet not fully understood relationships. Among the diverse interdependencies, the relationship between the myco- and photobiont is the most crucial, determining the shape, and ecophysiological properties of the symbiotic consortium. Roughly 10% of lichens associate with cyanobacteria as their primary photobiont, termed cyanolichens. Up to now, the diversity of cyanobionts of bipartite lichens resolved by modern phylogenetic approaches is restricted to the filamentous and heterocytous genera of the order Nostocales. Unicellular photobionts were placed in the orders Chroococcales, Pleurocapsales, and Chroococcidiopsidales. However, especially the phylogeny and taxonomy of the Chroococcidiopsidales genera remained rather unclear. Here we present new data on the identity and phylogeny of photobionts from cyanolichens of the genera Gonohymenia, Lichinella, Peccania, and Peltula from a broad geographical range. A polyphasic approach was used, combining morphological and cultivation-depending characteristics (microscopy, staining techniques, life cycle observation, baeocyte motility, and nitrogen fixation test) with phylogenetic analyses of the 16S rRNA and 16S–23S ITS gene region. We found an unexpectedly high cyanobiont diversity in the cyanobacterial lichens of the order Lichinales, including two new genera and seven new species, all of which were not previously perceived as lichen symbionts. As a result, we describe the novel unicellular Chroococcidiopsidales genera Pseudocyanosarcina gen. nov. with the species Pseudocyanosarcina phycocyania sp. nov. (from Peltula clavata, Australia) and Compactococcus gen. nov. with the species Compactococcus sarcinoides sp. nov. (from Gonohymenia sp., Australia) and the new Chroococcidiopsidales species Aliterella compacta sp. nov. (from Peltula clavata, Australia), Aliterella gigantea sp. nov. (from Peltula capensis; South Africa), Sinocapsa ellipsoidea sp. nov. (from Peccania cerebriformis, Austria), as well as the two new Nostocales species Komarekiella gloeocapsoidea sp. nov. (from Gonohymenia sp., Czechia) and Komarekiella globosa sp. nov. (from Lichinella cribellifera, Canary Islands, Spain). Our study highlights the role of cyanolichens acting as a key in untangling cyanobacterial taxonomy and diversity. With this study, we hope to stimulate further research on photobionts, especially of rare cyanolichens.

Lichen algae: the photosynthetic partners in lichen symbioses

AbstractA 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).

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products.

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies.

Thallus Growth Stage and Geographic Origin Shape Microalgal Diversity in Ramalina farinacea Lichen Holobionts.

Lichen symbioses are microecosystems hosting many other living organisms besides the two major lichen symbionts (i.e., lichenized fungi [the mycobiont] and green microalgae or cyanobacteria [the photobiont]). Recent investigations evidenced that other fungi, non-photosynthetic bacteria, and microalgae co-inhabit within the lichen thalli, but their diversity and their roles are still underinvestigated. Here we present an ad hoc stratified sampling design and in-depth Illumina paired-end metabarcoding approach to explore microalgal diversity in lichen thalli of the model species Ramalina farinacea from different ecologies. Lichen thalli were surveyed according to three different sizes, and different thallus parts were considered for molecular, bioinformatics, and community diversity analyses. The results revealed that microalgal diversity strongly depends on the growth stage of the thalli, the geographic area, and the habitat type. The results also show that microalgal diversity does not vary along the thallus branches (lacinias)-that is, it does not correlate with the apical growth and founder effects-and that there is no balanced co-presence of two main photobionts as previously established in R. farinacea. The sampling design performed here minimizes bias in the assessment of photobiont diversity in lichens and is proposed to be reliable and applicable to further study microalgal diversity in lichen symbioses.

Predicted Input of Uncultured Fungal Symbionts to a Lichen Symbiosis from Metagenome-Assembled Genomes.

Basidiomycete yeasts have recently been reported as stably associated secondary fungal symbionts of many lichens, but their role in the symbiosis remains unknown. Attempts to sequence their genomes have been hampered both by the inability to culture them and their low abundance in the lichen thallus alongside two dominant eukaryotes (an ascomycete fungus and chlorophyte alga). Using the lichen Alectoria sarmentosa, we selectively dissolved the cortex layer in which secondary fungal symbionts are embedded to enrich yeast cell abundance and sequenced DNA from the resulting slurries as well as bulk lichen thallus. In addition to yielding a near-complete genome of the filamentous ascomycete using both methods, metagenomes from cortex slurries yielded a 36- to 84-fold increase in coverage and near-complete genomes for two basidiomycete species, members of the classes Cystobasidiomycetes and Tremellomycetes. The ascomycete possesses the largest gene repertoire of the three. It is enriched in proteases often associated with pathogenicity and harbors the majority of predicted secondary metabolite clusters. The basidiomycete genomes possess ∼35% fewer predicted genes than the ascomycete and have reduced secretomes even compared with close relatives, while exhibiting signs of nutrient limitation and scavenging. Furthermore, both basidiomycetes are enriched in genes coding for enzymes producing secreted acidic polysaccharides, representing a potential contribution to the shared extracellular matrix. All three fungi retain genes involved in dimorphic switching, despite the ascomycete not being known to possess a yeast stage. The basidiomycete genomes are an important new resource for exploration of lifestyle and function in fungal–fungal interactions in lichen symbioses.

Antimicrobial activity and biosynthetic potential of cultivable actinomycetes associated with Lichen symbiosis from Qinghai-Tibet Plateau

Actinobacteria that inhabit lichen symbionts are considered a promising yet previously underexplored source of novel compounds. Here, for the first time, we conducted a comprehensive investigation with regard to strain isolation and identification of lichen-associated actinobacteria from Tibet Plateau, antimicrobial activity screening, biosynthetic genes detection, bioactive metabolites identification and activity prediction. A large number of culturable actinomycetes were isolated from lichens around Qinghai Lake, in Qinghai-Tibet Plateau. Twenty-seven strains with distinct morphological characteristics were preliminarily studied. 16S rRNA gene identification showed that 13 strains were new species. The PCR-screening of specific biosynthetic genes indicated that these 27 isolates had abundant intrinsic biosynthetic potential. The antimicrobial activity experiment screened out some potential biological control antagonistic bacteria. The metabolites of 13 strains of Streptomyces with antibacterial activity were analyzed by LC-HRMS, and further 18 compounds were identified by NMR and / or LC-HRMS. The identified compounds were mainly pyrrolidine and indole derivatives, as well as anthracyclines. Seven compounds were identified with less biological activity, then predicted and evaluated their biological activity. The predicted results showed that compound 2 had excellent inhibitory activity on HIV-1 reverse transcriptase. Overall, the results indicate actinobacteria isolated from unexploited plateau lichen are promising sources of biological active metabolite, which could provide important bioactive compounds as potential antibiotic drugs.

Lessons from culturing lichen soredia

Vegetative propagules play various important roles in lichen biology. We cultured soredia of Cladonia lichens in vitro and obtained three noteworthy results. Firstly, soredia are a beneficial source for the isolation of lichen symbionts. The mycobiont was obtained from 66% and the photobiont from 67% of the cultured soredia that were not contaminated. Secondly, the development of soredia followed a previously recognized pattern, arachnoid stage – soredium field – primordium, but a thalline structure was not achieved. We suggest that thallus formation in vitro is a question of favourable environmental factors, not partners compatibility. Finally, we discovered that fungi, other than the mycobiont, as well as airborne contaminants are dispersed together with lichen soredia. This is the first-ever report of such a phenomenon. The possible ecological consequences are discussed. Cystobasidiomycete yeasts were found among these fungi. We isolated representatives of three different lineages from a single thallus suggesting a low specificity for this association.

Myco- and photobiont associations in crustose lichens in the McMurdo Dry Valleys (Antarctica) reveal high differentiation along an elevational gradient

Climatically extreme regions such as the polar deserts of the McMurdo Dry Valleys (78° S) in Continental Antarctica are key areas for a better understanding of changes in ecosystems. Therefore, it is particularly important to analyze and communicate current patterns of biodiversity in these sensitive areas, where precipitation mostly occurs in form of snow and liquid water is rare. Humidity provided by dew, clouds, and fog are the main water sources, especially for rock-dwelling crustose lichens as one of the most common vegetation-forming organisms. We investigated the diversity and interaction specificity of myco-/photobiont associations of 232 crustose lichen specimens, collected along an elevational gradient (171–959 m a.s.l.) within the McMurdo Dry Valleys. The mycobiont species and photobiont OTUs were identified by using three markers each (nrITS, mtSSU, RPB1, and nrITS, psbJ-L, COX2). Elevation, positively associated with water availability, turned out to be the key factor explaining most of the distribution patterns of the mycobionts. Pairwise comparisons showed Lecidea cancriformis and Rhizoplaca macleanii to be significantly more common at higher elevations and Carbonea vorticosa and Lecidea polypycnidophora at lower elevations. Lichen photobionts were dominated by the globally distributed Trebouxia OTU, Tr_A02 which occurred at all habitats. Network specialization resulting from myco-/photobiont bipartite network structure varied with elevation and associated abiotic factors. Along an elevational gradient, the spatial distribution, diversity, and genetic variability of the lichen symbionts appear to be mainly influenced by improved water relations at higher altitudes.

Metaproteomics reveals the structural and functional diversity of Dermatocarpon miniatum (L.) W. Mann. Microbiota

Metaproteomics is a strategy to understand the taxonomy, functionality and metabolic pathways of the microbial communities. The relationship among the symbiotic microbiota in the entire lichen thallus, Dermatocarpon miniatum, was evaluated using the metaproteomic approach. Proteomic profiling using one-dimensional SDS-PAGE followed by LC-MS/MS analysis resulted in a total of 138 identified proteins via Mascot search against UniRef100 and Swiss-Prot databases. In addition to the fungal and algal partners, D. miniatum proteome encompasses proteins from prokaryotes, which is a multifarious community mainly dominated by cyanobacteria and proteobacteria. While proteins assigned to fungus were the most abundant (55 %), followed by protists (16 %), bacterial (13 %), plant (11 %), and viral (1 %) origin, whereas 4 % remained undefined. Various proteins were assigned to the different lichen symbionts by using Gene Ontology (GO) terms, e.g. fungal proteins involved in the oxidation-reduction process, protein folding and glycolytic process, while protists and bacterial proteins were involved in photosynthetic electron transport in photosystem II (PS II), ATP synthesis coupled proton transport, and carbon fixation. The presence of bacterial communities extended the traditional concept of fungal-algal lichen symbiotic interaction.

Phylogenetic diversity of the lichenized algal genusTrebouxia(Trebouxiophyceae, Chlorophyta): a new lineage and novel insights from fungal-algal association patterns of Icelandic cetrarioid lichens (Parmeliaceae, Ascomycota)

AbstractLichens have high tolerance to harsh environmental conditions, where lichen symbiont interactions (e.g. myco- and photobionts) may play a crucial role. The characterization of fungal-algal association patterns is essential to understand their symbiotic interactions. This study investigated fungal-algal association patterns in Icelandic cetrarioid lichens using a multi-locus phylogenetic framework, including fungal nrITS, MCM7, mtSSU, RPB1 and RPB2 and algal nrITS, nrLSU, rbcL and mtCOXII data. Most Icelandic cetrarioid lichenized fungi were found to be specifically associated to the known Trebouxia clade “S” (Trebouxia simplex/suecica group), whereas the lichen-forming fungus Cetrariella delisei forms a symbiosis with a previously unrecognized lineage of Trebouxia, provisionally named as the “D” clade. This new Trebouxia lineage is supported by maximum likelihood and Bayesian phylogenetic analyses using all four included algal loci.