Phylogenomics reveals the evolutionary origins of lichenization in chlorophyte algae.

Mutualistic symbioses have contributed to major transitions in the evolution of life. Here, we investigate the evolutionary history and the molecular innovations at the origin of lichens, which are a symbiosis established between fungi and green algae or cyanobacteria. We de novo sequence the genomes or transcriptomes of 12 lichen algal symbiont (LAS) and closely related non-symbiotic algae (NSA) to improve the genomic coverage of Chlorophyte algae. We then perform ancestral state reconstruction and comparative phylogenomics. We identify at least three independent gains of the ability to engage in the lichen symbiosis, one in Trebouxiophyceae and two in Ulvophyceae, confirming the convergent evolution of the lichen symbioses. A carbohydrate-active enzyme from the glycoside hydrolase 8 (GH8) family was identified as a top candidate for the molecular-mechanism underlying lichen symbiosis in Trebouxiophyceae. This GH8 was acquired in lichenizing Trebouxiophyceae by horizontal gene transfer, concomitantly with the ability to associate with lichens fungal symbionts (LFS) and is able to degrade polysaccharides found in the cell wall of LFS. These findings indicate that a combination of gene family expansion and horizontal gene transfer provided the basis for lichenization to evolve in chlorophyte algae.

Biotic interactions outweigh abiotic factors as drivers of bark microbial communities in Central European forests.

Bark surfaces are extensive areas within forest ecosystems, which provide an ideal habitat for microbial communities, through their longevity and seasonal stability. Here we provide a comprehensive account of the bark surface microbiome of living trees in Central European forests, and identify drivers of diversity and community composition. We examine algal, fungal, and bacterial communities and their interactions using metabarcoding on samples from over 750 trees collected in the Biodiversity Exploratories in northern, central, and southern Germany. We show that mutual biotic influence is more important than the abiotic environment with regard to community composition, whereas abiotic conditions and geography are more important for alpha diversity. Important abiotic factors are the relative humidity and light availability, which decrease the algal and bacterial alpha diversity but strongly increase fungal alpha diversity. In addition, temperature is important in shaping the microbial community, with higher temperature leading to homogeneous communities of dominant fungi, but high turnover in bacterial communities. Changes in the community dissimilarity of one organismal group occur in close relation to changes in the other two, suggesting that there are close interactions between the three major groups of the bark surface microbial communities, which may be linked to beneficial exchange. To understand the functioning of the forest microbiome as a whole, we need to further investigate the functionality of interactions within the bark surface microbiome and combine these results with findings from other forest habitats such as soil or canopy.

Gene abundance linked to climate zone: Parallel evolution of gene content along elevation gradients in lichenized fungi.

Introduction: Intraspecific genomic variability affects a species' adaptive potential toward climatic conditions. Variation in gene content across populations and environments may point at genomic adaptations to specific environments. The lichen symbiosis, a stable association of fungal and photobiont partners, offers an excellent system to study environmentally driven gene content variation. Many of these species have remarkable environmental tolerances, and often form populations across different climate zones. Here, we combine comparative and population genomics to assess the presence and absence of genes in high and low elevation genomes of two lichenized fungi of the genus Umbilicaria. Methods: The two species have non-overlapping ranges, but occupy similar climatic niches in North America (U. phaea) and Europe (U. pustulata): high elevation populations are located in the cold temperate zone and low elevation populations in the Mediterranean zone. We assessed gene content variation along replicated elevation gradients in each of the two species, based on a total of 2050 individuals across 26 populations. Specifically, we assessed shared orthologs across species within the same climate zone, and tracked, which genes increase or decrease in abundance within populations along elevation. Results: In total, we found 16 orthogroups with shared orthologous genes in genomes at low elevation and 13 at high elevation. Coverage analysis revealed one ortholog that is exclusive to genomes at low elevation. Conserved domain search revealed domains common to the protein kinase superfamily. We traced the discovered ortholog in populations along five replicated elevation gradients on both continents and found that the number of this protein kinase gene linearly declined in abundance with increasing elevation, and was absent in the highest populations. Discussion: We consider the parallel loss of an ortholog in two species and in two geographic settings a rare find, and a step forward in understanding the genomic underpinnings of climatic tolerances in lichenized fungi. In addition, the tracking of gene content variation provides a widely applicable framework for retrieving biogeographical determinants of gene presence/absence patterns. Our work provides insights into gene content variation of lichenized fungi in relation to climatic gradients, suggesting a new research direction with implications for understanding evolutionary trajectories of complex symbioses in relation to climatic change.

Habitat and tree species identity shape aboveground and belowground fungal communities in central European forests.

Introduction: Trees interact with fungi in mutualistic, saprotrophic, and pathogenic relationships. With their extensive aboveground and belowground structures, trees provide diverse habitats for fungi. Thus, tree species identity is an important driver of fungal community composition in forests. Methods: Here we investigate how forest habitat (bark surface vs. soil) and tree species identity (deciduous vs. coniferous) affect fungal communities in two Central European forests. We assess differences and interactions between fungal communities associated with bark surfaces and soil, in forest plots dominated either by Fagus sylvatica, Picea abies, or Pinus sylvestris in two study regions in southwestern and northeastern Germany. Results: ITS metabarcoding yielded 3,357 fungal amplicon sequence variants (ASVs) in the northern and 6,088 in the southern region. Overall, soil communities were 4.7 times more diverse than bark communities. Habitat type explained 48-69% of the variation in alpha diversity, while tree species identity explained >1-3%. NMDS ordinations showed that habitat type and host tree species structured the fungal communities. Overall, few fungal taxa were shared between habitats, or between tree species, but the shared taxa were highly abundant. Network analyses, based on co-occurrence patterns, indicate that aboveground and belowground communities form distinct subnetworks. Discussion: Our study suggests that habitat (bark versus soil) and tree species identity are important factors structuring fungal communities in temperate European forests. The aboveground (bark-associated) fungal community is currently poorly known, including a high proportion of reads assigned to "unknown Ascomycota" or "unknown Dothideomycetes." The role of bark as a habitat and reservoir of unique fungal diversity in forests has been underestimated.

Physiological Plasticity as a Strategy to Cope with Harsh Climatic Conditions: Ecophysiological Meta-Analysis of the Cosmopolitan Moss Ceratodon purpureus in the Southern Hemisphere.

Determining the physiological tolerance ranges of species is necessary to comprehend the limits of their responsiveness under strong abiotic pressures. For this purpose, the cosmopolitan moss Ceratodon purpureus (Hedw.) Brid. is a good model due to its wide geographical distribution throughout different biomes and habitats. In order to disentangle how this species copes with stresses such as extreme temperatures and high radiation, we designed a meta-analysis by including the main photosynthetic traits obtained by gas exchange measurements in three contrasting habitats from the Southern Hemisphere. Our findings highlight that traits such as respiration homeostasis, modulation of the photosynthetic efficiency, adjustment of the optimal temperature, and switching between shade and sun-adapted forms, which are crucial in determining the responsiveness of this species. In fact, these ecophysiological traits are in concordance with the climatic particularities of each habitat. Furthermore, the photosynthetic trends found in our study point out how different Livingston Island (Maritime Antarctica) and Granite Harbour (Continental Antarctica) are for plant life, while the population from the Succulent Karoo Desert (South Africa) shares traits with both Antarctic regions. Altogether, the study highlights the high resilience of C. purpureus under abrupt climate changes and opens new perspectives about the wide spectrum of physiological responses of cryptogams to cope with climate change scenarios.

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.

Metatranscriptomics reveals contrasting effects of elevation on the activity of bacteria and bacterial viruses in soil.

Soil microbial diversity affects ecosystem functioning and global biogeochemical cycles. Soil bacterial communities catalyse a diversity of biogeochemical reactions and have thus sparked considerable scientific interest. One driver of bacterial community dynamics in natural ecosystems has so far been largely neglected: the predator-prey interactions between bacterial viruses (bacteriophages) and bacteria. To generate ground level knowledge on environmental drivers of these particular predator-prey dynamics, we propose an activity-based ecological framework to simultaneous capture community dynamics of bacteria and bacteriophages in soils. An ecological framework and specifically the analyses of community dynamics across latitudinal and elevational gradients have been widely used in ecology to understand community-wide responses of innumerable taxa to environmental change, in particular to climate. Here, we tested the hypothesis that the activity of bacteria and bacteriophages codeclines across an elevational gradient. We used metatranscriptomics to investigate bacterial and bacteriophage activity patterns at five sites across 400 elevational metres in the Swiss Alps in 2015 and 2017. We found that metabolic activity (transcription levels) of bacteria declined significantly with increasing elevation, but activity of bacteriophages did not. We showed that bacteriophages are consistently active in soil along the entire gradient, making bacteriophage activity patterns divergent from that of their putative bacterial prey. Future efforts will be necessary to link the environment-activity relationship to predator-prey dynamics, and to understand the magnitude of viral contributions to carbon, nitrogen and phosphorus cycling when infection causes bacterial cell death, a process that may represent an overlooked component of soil biogeochemical cycles.

Fungal Host Affects Photosynthesis in a Lichen Holobiont.

Corals and lichens are iconic examples of photosynthetic holobionts, i.e., ecological and evolutionary units resulting from the tightly integrated association of algae and prokaryotic microbiota with animal or fungal hosts, respectively. While the role of the coral host in modulating photosynthesis has been clarified to a large extent in coral holobionts, the role of the fungal host in this regard is far less understood. Here, we address this question by taking advantage of the recent discovery of highly specific fungal-algal pairings corresponding to climatically adapted ecotypes of the lichen-forming genus Umbilicaria. Specifically, we compared chlorophyll a fluorescence kinetics among lichen thalli consisting of different fungal-algal combinations. We show that photosynthetic performance in these lichens is not only driven by algal genotype, but also by fungal host species identity and intra-host genotype. These findings shed new light on the closely intertwined physiological processes of fungal and algal partners in the lichen symbiosis. Indeed, the specific combinations of fungal and algal genotypes within a lichen individual-and the resulting combined functional phenotype-can be regarded as a response to the environment. Our findings suggest that characterizing the genetic composition of both eukaryotic partners is an important complimentary step to understand and predict the lichen holobiont's responses to environmental change.

Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi.

Lichen-forming fungi establish stable symbioses with green algae or cyanobacteria. Many species have broad distributions, both in geographic and ecological space, making them ideal subjects to study organism-environment interactions. However, little is known about the specific mechanisms that contribute to environmental adaptation in lichen-forming fungi. The circadian clock provides a well-described mechanism that contributes to regional adaptation across a variety of species, including fungi. Here, we identify the putative circadian clock components in phylogenetically divergent lichen-forming fungi. The core circadian genes (frq, wc-1, wc-2, frh) are present across the Fungi, including 31 lichen-forming species, and their evolutionary trajectories mirror overall fungal evolution. Comparative analyses of the clock genes indicate conserved domain architecture among lichen- and non-lichen-forming taxa. We used RT-qPCR to examine the core circadian loop of two unrelated lichen-forming fungi, Umbilicaria pustulata (Lecanoromycetes) and Dermatocarpon miniatum (Eurotiomycetes), to determine that the putative frq gene is activated in a light-dependent manner similar to the model fungus Neurospora crassa. Together, these results demonstrate that lichen-forming fungi retain functional light-responsive mechanisms, including a functioning circadian clock. Our findings provide a stepping stone into investigating the circadian clock in the lichen symbiosis, e.g. its role in adaptation, and in synchronizing the symbiotic interaction.

A Candidate Gene Cluster for the Bioactive Natural Product Gyrophoric Acid in Lichen-Forming Fungi.

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Despite this, they have limited implementation in industry mostly because the corresponding genes are unknown for most of their natural products. Here, we implement a long-read sequencing and bioinformatic approach to identify the putative biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate the cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria, and only three genes are conserved across species, including the polyketide synthase (PKS) gene. In addition, our results suggest that the same cluster codes for different, but structurally similar compounds, namely, GA, umbilicaric-, and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster and, thus, provides essential information to open new avenues for biotechnological approaches to producing and modifying GA and similar lichen-derived compounds. GA PKS is the first tridepside PKS to be identified. IMPORTANCE The implementation of natural products in the pharmaceutical industry relies on the possibility of modifying the natural product (NP) pathway to optimize yields and pharmacological effects. Characterization of genes and pathways underlying natural product biosynthesis is a major bottleneck for exploiting the medicinal properties of the natural products. Genome mining is a promising and relatively cost- and time-effective approach to utilize unexplored NP resources for drug discovery. In this study, we identify the most likely gene cluster for the lichen-forming fungal depside gyrophoric acid in nine Umbilicaria species. This compound shows cytotoxic and antiproliferative properties against several cancer cell lines and is also a broad-spectrum antimicrobial agent. This information paves the way for generating GA analogs with modified properties by selective activation/deactivation of genes.

Climate change leads to higher NPP at the end of the century in the Antarctic Tundra: Response patterns through the lens of lichens.

Poikilohydric autotrophs are the main colonizers of the permanent ice-free areas in the Antarctic tundra biome. Global climate warming and the small human footprint in this ecosystem make it especially vulnerable to abrupt changes. Elucidating the effects of climate change on the Antarctic ecosystem is challenging because it mainly comprises poikilohydric species, which are greatly influenced by microtopographic factors. In the present study, we investigated the potential effects of climate change on the metabolic activity and net primary photosynthesis (NPP) in the widespread lichen species Usnea aurantiaco-atra. Long-term monitoring of chlorophyll a fluorescence in the field was combined with photosynthetic performance measurements in laboratory experiments in order to establish the daily response patterns under biotic and abiotic factors at micro- and macro-scales. Our findings suggest that macroclimate is a poor predictor of NPP, thereby indicating that microclimate is the main driver due to the strong effects of microtopographic factors on cryptogams. Metabolic activity is also crucial for estimating the NPP, which is highly dependent on the type, distribution, and duration of the hydration sources available throughout the year. Under RCP 4.5 and RCP 8.5, metabolic activity will increase slightly compared with that at present due to the increased precipitation events predicted in MIROC5. Temperature is highlighted as the main driver for NPP projections, and thus climate warming will lead to an average increase in NPP of 167-171% at the end of the century. However, small changes in other drivers such as light and relative humidity may strongly modify the metabolic activity patterns of poikilohydric autotrophs, and thus their NPP. Species with similar physiological response ranges to the species investigated in the present study are expected to behave in a similar manner provided that liquid water is available.

Genome mining as a biotechnological tool for the discovery of novel biosynthetic genes in lichens.

Natural products (NPs) and their derivatives are a major contributor to modern medicine. Historically, microorganisms such as bacteria and fungi have been instrumental in generating drugs and lead compounds because of the ease of culturing and genetically manipulating them. However, the ever-increasing demand for novel drugs highlights the need to bioprospect previously unexplored taxa for their biosynthetic potential. Next-generation sequencing technologies have expanded the range of organisms that can be explored for their biosynthetic content, as these technologies can provide a glimpse of an organism's entire biosynthetic landscape, without the need for cultivation. The entirety of biosynthetic genes can be compared to the genes of known function to identify the gene clusters potentially coding for novel products. In this study, we mine the genomes of nine lichen-forming fungal species of the genus Umbilicaria for biosynthetic genes, and categorize the biosynthetic gene clusters (BGCs) as "associated product structurally known" or "associated product putatively novel". Although lichen-forming fungi have been suggested to be a rich source of NPs, it is not known how their biosynthetic diversity compares to that of bacteria and non-lichenized fungi. We found that 25%-30% of biosynthetic genes are divergent as compared to the global database of BGCs, which comprises 1,200,000 characterized biosynthetic genes from plants, bacteria, and fungi. Out of 217 BGCs, 43 were highly divergant suggesting that they potentially encode structurally and functionally novel NPs. Clusters encoding the putatively novel metabolic diversity comprise polyketide synthases (30), non-ribosomal peptide synthetases (12), and terpenes (1). Our study emphasizes the utility of genomic data in bioprospecting microorganisms for their biosynthetic potential and in advancing the industrial application of unexplored taxa. We highlight the untapped structural metabolic diversity encoded in the lichenized fungal genomes. To the best of our knowledge, this is the first investigation identifying genes coding for NPs with potentially novel properties in lichenized fungi.

Virus diversity in metagenomes of a lichen symbiosis (Umbilicaria phaea): complete viral genomes, putative hosts and elevational distributions.

Viruses can play critical roles in symbioses by initiating horizontal gene transfer, affecting host phenotypes, or expanding their host's ecological niche. However, knowledge of viral diversity and distribution in symbiotic organisms remains elusive. Here we use deep-sequenced metagenomic DNA (PacBio Sequel II; two individuals), paired with a population genomics approach (Pool-seq; 11 populations, 550 individuals) to understand viral distributions in the lichen Umbilicaria phaea. We assess (i) viral diversity in lichen thalli, (ii) putative viral hosts (fungi, algae, bacteria) and (iii) viral distributions along two replicated elevation gradients. We identified five novel viruses, showing 28%-40% amino acid identity to known viruses. They tentatively belong to the families Caulimoviridae, Myoviridae, Podoviridae and Siphoviridae. Our analysis suggests that the Caulimovirus is associated with green algal photobionts (Trebouxia) of the lichen, and the remaining viruses with bacterial hosts. We did not detect viral sequences in the mycobiont. Caulimovirus abundance decreased with increasing elevation, a pattern reflected by a specific algal lineage hosting this virus. Bacteriophages showed population-specific patterns. Our work provides the first comprehensive insights into viruses associated with a lichen holobiont and suggests an interplay of viral hosts and environment in structuring viral distributions.

Depside and Depsidone Synthesis in Lichenized Fungi Comes into Focus through a Genome-Wide Comparison of the Olivetoric Acid and Physodic Acid Chemotypes of Pseudevernia furfuracea.

Primary biosynthetic enzymes involved in the synthesis of lichen polyphenolic compounds depsides and depsidones are non-reducing polyketide synthases (NR-PKSs), and cytochrome P450s. However, for most depsides and depsidones the corresponding PKSs are unknown. Additionally, in non-lichenized fungi specific fatty acid synthases (FASs) provide starters to the PKSs. Yet, the presence of such FASs in lichenized fungi remains to be investigated. Here we implement comparative genomics and metatranscriptomics to identify the most likely PKS and FASs for olivetoric acid and physodic acid biosynthesis, the primary depside and depsidone defining the two chemotypes of the lichen Pseudevernia furfuracea. We propose that the gene cluster PF33-1_006185, found in both chemotypes, is the most likely candidate for the olivetoric acid and physodic acid biosynthesis. This is the first study to identify the gene cluster and the FAS likely responsible for olivetoric acid and physodic acid biosynthesis in a lichenized fungus. Our findings suggest that gene regulation and other epigenetic factors determine whether the mycobiont produces the depside or the depsidone, providing the first direct indication that chemotype diversity in lichens can arise through regulatory and not only through genetic diversity. Combining these results and existing literature, we propose a detailed scheme for depside/depsidone synthesis.

Climate-specific biosynthetic gene clusters in populations of a lichen-forming fungus.

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.

Transposable Elements in the Genome of the Lichen-Forming Fungus Umbilicaria pustulata and Their Distribution in Different Climate Zones along Elevation.

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.

Genome-Wide Analysis of Biosynthetic Gene Cluster Reveals Correlated Gene Loss with Absence of Usnic Acid in Lichen-Forming Fungi.

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster-homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)-varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.

What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen.

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis-the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)-were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

A Glimpse into Genetic Diversity and Symbiont Interaction Patterns in Lichen Communities from Areas with Different Disturbance Histories in Bialowieza Forest, Poland.

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 Bialowieza 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.