Anthropogenic disturbances can have strong impacts on lichen communities, as well as on individual species of lichenized fungi. Traditionally, lichen monitoring studies are based on the presence and abundance of fungal morphospecies. However, the photobionts, as well photobiont mycobiont interactions also contribute to the structure, composition, and resilience of lichen communities. Here we assess the genetic diversity and interaction patterns of algal and fungal partners in lichen communities along an anthropogenic disturbance gradient in Białowieża Forest (Poland). We sampled a total of 224 lichen thalli in a protected, a managed, and a disturbed area of the forest, and sequenced internal transcribed spacer (ITS) ribosomal DNA (rDNA) of both, fungal and algal partners. Sequence clustering using a 97% similarity threshold resulted in 46 fungal and 23 green algal operational taxonomic units (OTUs). Most of the recovered photobiont OTUs (14 out of 23) had no similar hit in the NCBI-BLAST search, suggesting that even in well studied regions, such as central Europe, a lot of photobiont diversity is yet undiscovered. If a mycobiont was present at more than one site, it was typically associated with the same photobiont OTU(s). Generalist species, i.e., taxa that associate with multiple symbiont partners, occurred in all three disturbance regimes, suggesting that such taxa have few limitations in colonizing or persisting in disturbed areas. Trebouxia jamesii associated with 53% of the fungal OTUs, and was generally the most common photobiont OTU in all areas, implying that lichens that associate with this symbiont are not limited by the availability of compatible photobionts in Central European forests, regardless of land use intensity.

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.

Members of the common lichen photobiont genus Trebouxia occur from the arctic to tropical terrestrial habitats however, the mechanisms of environmental stress tolerance in Trebouxia are little understood. Here we studied six species, which belong to the S-clade and A-clade, and were isolated from the lichen-forming fungi Umbilicaria pustulata and U. phaea. These species have demonstrated extensive genomic divergence, particularly in genome regions associated with photosynthesis. Therefore, we hypothesized that they will exhibit differential performance under varying light conditions. We assessed their physiological and transcriptomic responses to short and prolonged exposure to high light (HL). Average levels of Fv/Fm and NPQ showed significant reduction following HL exposure, but this varied among species. Further, only a few differentially expressed genes (DEGs) were found for specific species following exposure to 1 h of HL. On the other hand, there are more DEGs found for those exposed to prolonged HL, particularly photoprotection-associated genes related to NPQ, photosystem II repair, oxygen evolving assembly and biosynthesis of photoprotective pigments. Overall, our findings show that in Trebouxia, the capacity to withstand high light conditions is highly species-specific, and not driven by phylogenetic relatedness or climatic niche preference.

In the lichen symbiosis, the fungal and algal partners constitute a closely integrated system. The combination of fungal and algal partners changes along climate gradients in many species, and is expected to be adaptive. However, the functional mechanisms behind this symbiosis-mediated environmental adaptation are unknown. We investigated which transcriptional profiles are associated with specific fungal–algal symbiont pairings found in lichens from high-elevation (Lower Supratemperate) and low-elevation (Lower Mesomediterranean) sites at two extremes of a climatic gradient on Mount Limbara, Sardinia. Using laboratory-acclimatized thalli, we found that lichen fungal and algal symbionts show variable expression profiles between high- and low-elevation individuals: circadian- and temperature-associated genes for fungi and light-responsive genes for algae show climate-specific patterns. High- and low-elevation individuals differentially express sugar transporters in both symbionts, pointing to symmetrical and climate-dependent sugar transport mechanisms between them. A light pulse treatment identified asymmetries between fungal and algal light responses, with high- and low-elevation fungal symbionts but only low-elevation algal symbionts showing a response. Together, these results tie previously observed genomic variation along climatic gradients in a lichen species to functional differences in transcription for the fungal and algal symbionts, contributing to our understanding of environmental specialization and niche-specific partner combinations in lichens.

The green-algal genus Trebouxia (Trebouxiophyceae, Chlorophyta) is the most common photosynthetic symbiont of lichens, displaying high phylogenetic diversity, and worldwide distribution across all climate zones. These single-celled terrestrial algae are valuable systems to study diversification, environmental adaptation and species interactions, yet genomic resources remain limited. We present over 30 new chloroplast and mitochondrial genomes of Trebouxia species, extracted from PacBio metagenomes of diverse Umbilicaria lichens from multiple climate zones. The genomes represent previously identified operational taxonomic units (OTUs) Trebouxia jamesii (A03), T. sp. (A04), T. incrustata (A06), T. vagua (A10), T. sp. (S02), T. sp. (S03), T. sp. (S04), T. suecica (S05), T. sp. (S08), T. angustilobata (S09), T. simplex (S10), T. sp. (S20) and T. barrenoae (S28); a newly designated OTU T. sp. (A57), and several Single-Occurrence Sequences (SOS) from clades A, I, and S. Up to four Trebouxia OTUs were found within a single thallus. Organelle genomes vary considerably in size and structure. The consensus phylogenies from chloroplast (77 genes) and mitochondrial (32 genes) genes are largely congruent with the nuclear ITS tree, differing mainly in the derived clade S sections. All genes are under purifying selection, with mitochondrial genes exhibiting higher nucleotide diversity and hence phylogenetic resolution than chloroplast genes. Certain gene and protein features correlate with temperature variability, and some (such as GC content, arginine, and valine content) mirror findings in mycobiont nuclear genomes from the same samples and highlight shared signatures of environmental adaptation. We designed primers for new, variable phylogenetic markers, including chloroplast genes ftsH and rpoC1, and mitochondrial genes ATP1, ATP6, and ND6. Overall, this study advances our understanding of organelle genome evolution in Trebouxia and provides valuable resources for future ecological and evolutionary research.