Lichen-forming fungi produce a vast number of unique natural products with a wide variety of biological activities and human uses. Although lichens have remarkable potential in natural product research and industry, the molecular mechanisms underlying the biosynthesis of lichen metabolites are poorly understood. Here we use genome mining and comparative genomics to assess biosynthetic gene clusters and their putative regulators in the genomes of two lichen-forming fungi, which have substantial commercial value in the perfume industry, Evernia prunastri and Pseudevernia furfuracea. We report a total of 80 biosynthetic gene clusters (polyketide synthases (PKS), non-ribosomal peptide synthetases and terpene synthases) in E. prunastri and 51 in P. furfuracea. We present an in-depth comparison of 11 clusters, which show high homology between the two species. A ketosynthase (KS) phylogeny shows that biosynthetic gene clusters from E. prunastri and P. furfuracea are widespread across the Fungi. The phylogeny includes 15 genomes of lichenized fungi and all fungal PKSs with known functions from the MIBiG database. Phylogenetically closely related KS domains predict not only similar PKS architecture but also similar cluster architecture. Our study highlights the untapped biosynthetic richness of lichen-forming fungi, provides new insights into lichen biosynthetic pathways and facilitates heterologous expression of lichen biosynthetic gene clusters.

Summary: Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen‐forming fungus Umbilicaria pustulata . We collected a total of 600 individuals from the Mediterranean and cold‐temperate climates. Population genomic analyses indicate that U . pustulata contains three clusters that are highly differentiated between the Mediterranean and cold‐temperate populations. One entire cluster is exclusively present in cold‐temperate populations, and a second cluster is putatively dysfunctional in all cold‐temperate populations. In the third cluster variation is fixed in all cold‐temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate‐associated BGCs and BGC variations in lichen‐forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus). They are known to produce 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 comprehensive view of the biosynthetic gene clusters of all organisms comprising a lichen thallus: fungi, green algae, and bacteria. We present two high-quality PacBio metagenomes, in which we identified a total of 460 biosynthetic gene clusters. Lichen mycobionts yielded 73–114 clusters, other lichen associated ascomycetes 8–40, green algae of the genus Trebouxia 14–19, and lichen-associated bacteria 101–105 clusters. The mycobionts contained mainly T1PKSs, followed by NRPSs, and terpenes; Trebouxia reads harbored mainly clusters linked to terpenes, followed by NRPSs and T3PKSs. Other lichen-associated ascomycetes and bacteria contained a mix of diverse biosynthetic gene clusters. In this study, we identified for the first time the biosynthetic gene clusters of entire lichen holobionts. The yet untapped biosynthetic potential of two species of the genus Hypogymnia is made accessible for further research.

The green algal genus Trebouxia is the most frequently encountered photobiont of the lichen symbiosis. The single-celled symbionts have a worldwide distribution, including all continents and climate zones. The vast, largely undescribed, diversity of Trebouxia lineages is currently grouped into four phylogenetic clades (A, C, I, and S), based on a multilocus phylogeny. Genomes are still scarce, however, and it is unclear how the phylogenetic diversity, the broad ecological tolerances, and the ability to form symbioses with many different fungal host species are reflected in genome-wide differences. Here, we generated PacBio-based de novo genomes of six Trebouxia lineages belonging to the Clades A and S, isolated from lichen individuals of the genus Umbilicaria. Sequences belonging to Clade S have been reported in a previous study, but were reassembled and reanalyzed here. Genome sizes ranged between 63.08 and 73.88 Mb. Repeat content accounted for 9% to 16% of the genome sequences. Based on RNA evidence, we predicted 14,109 to 16,701 gene models per genome, of which 5,203 belonged to a core set of gene families shared by all 6 lineages. Between 121 and 454, gene families are specific to each lineage. About 53% of the genes could be functionally annotated. The presence of biosynthetic gene clusters (6 to 17 per genome) suggests that Trebouxia algae are able to synthesize alkaloids, saccharides, terpenes, NRPSs, and T3PKSs. Phylogenomic comparisons of the six strains indicate prevalent gene gain during Trebouxia evolution. Some of the gene families that exhibited significant evolutionary changes (i.e. gene expansion and contraction) are associated with metabolic processes linked to protein phosphorylation, which is known to have a role in photosynthesis regulation, particularly under changing light conditions. Overall, there is substantial genomic divergence within the algal genus Trebouxia, which may contribute to the genus’ large ecological amplitude concerning fungal host diversity and climatic niches.

Transposable elements (TEs) are an important source of genome plasticity across the tree of life. Drift and natural selection are important forces shaping TE distribution and accumulation. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the host’s ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold temperate. We trace the occurrence of the newly identified TEs in populations along three elevation gradients using a Pool-Seq approach to identify TE insertions of potential adaptive significance. We found that TEs cover 21.26% of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. We identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function. This pioneering study of the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution.

Lichens are composite organisms that produce a wide variety of secondary metabolites; many of the compounds have a high potential as bioactive compounds. The major limitations of using bioactive compounds from lichens is their slow growth rate and the damage to environmental populations caused by massive collection. The alternative to the massive collection of lichens in the field is their culture under laboratory conditions. We chose two related lichen species of Parmeliaceae that produce similar metabolites and isolated from spores in cultures placed under axenic conditions for over 550 days. From these cultures, we sampled 35 mg of each species from different culture media at two sampling times. The samples were analyzed using high-performance liquid chromatography (HPLC) to detect and identify major compounds. We found no differences in the metabolites produced within the species in comparisons between different culture media. Our results show that the mycobiont cultures produced different secondary metabolites than those found in natural lichen thalli. Moreover, different secondary metabolites between species and different metabolites over time were observed. We conclude that mycobiont cultures are a promising alternative for determining bioactive compounds and enhancing the efficiency of growth and production. These could be a good option for eco-friendly metabolite production.