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A candidate gene cluster for the bioactive natural product gyrophoric acid in lichen-forming fungi
(2022)
Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Despite this, they a have limited implementation in industry, because the corresponding genes remain unknown for most of the natural products. Here we implement a long-read sequencing and bioinformatic approach to identify the 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 cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria, with only three genes that are conserved across species, including the PKS gene. In addition, our results suggest that the same cluster codes for different but structurally similar NPs, i.e., GA, umbilicaric acid 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. We show that bioinformatics approaches are useful in linking genes and potentially associated natural products. Genome analyses help unlocking the pharmaceutical potential of organisms such as lichens, which are biosynthetically diverse but slow growing, and difficult to cultivate due to their symbiotic nature.
High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here, we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (1) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding; (2) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments; and (3) is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus giving the user maximum control over data treatment and avoiding a black-box approach. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.
Biosynthetic gene content of the "Perfume Lichens" Evernia prunastri and Pseudevernia furfuracea
(2019)
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.
Species recognition in lichen-forming fungi has been a challenge because of unsettled species concepts, few taxonomically relevant traits, and limitations of traditionally used morphological and chemical characters for identifying closely related species. Here we analyze species diversity in the cosmopolitan genus Protoparmelia s.l. The ~25 described species in this group occur across diverse habitats from the boreal -arctic/alpine to the tropics, but their relationship to each other remains unexplored. In this study, we inferred the phylogeny of 18 species currently assigned to this genus based on 160 specimens and six markers: mtSSU, nuLSU, ITS, RPB1, MCM7, and TSR1. We assessed the circumscription of species-level lineages in Protoparmelia s. str. using two coalescent-based species delimitation methods – BP&P and spedeSTEM. Our results suggest the presence of a tropical and an extra-tropical lineage, and eleven previously unrecognized distinct species-level lineages in Protoparmelia s. str. Several cryptic lineages were discovered as compared to phenotype-based species delimitation. Many of the putative species are supported by geographic evidence.
The implementation of HTS (high-throughput sequencing) approaches is rapidly changing our understanding of the lichen symbiosis, by uncovering high bacterial and fungal diversity, which is often host-specific. Recently, HTS methods revealed the presence of multiple photobionts inside a single thallus in several lichen species. This differs from Sanger technology, which typically yields a single, unambiguous algal sequence per individual. Here we compared HTS and Sanger methods for estimating the diversity of green algal symbionts within lichen thalli using 240 lichen individuals belonging to two species of lichen-forming fungi. According to HTS data, Sanger technology consistently yielded the most abundant photobiont sequence in the sample. However, if the second most abundant photobiont exceeded 30% of the total HTS reads in a sample, Sanger sequencing generally failed. Our results suggest that most lichen individuals in the two analyzed species, Lasallia hispanica and L. pustulata, indeed contain a single, predominant green algal photobiont. We conclude that Sanger sequencing is a valid approach to detect the dominant photobionts in lichen individuals and populations. We discuss which research areas in lichen ecology and evolution will continue to benefit from Sanger sequencing, and which areas will profit from HTS approaches to assessing symbiont diversity.
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.
Keystone mutualisms, such as corals, lichens or mycorrhizae, sustain fundamental ecosystem functions. Range dynamics of these symbioses are, however, inherently difficult to predict because host species may switch between different symbiont partners in different environments, thereby altering the range of the mutualism as a functional unit. Biogeographic models of mutualisms thus have to consider both the ecological amplitudes of various symbiont partners and the abiotic conditions that trigger symbiont replacement. To address this challenge, we here investigate 'symbiont turnover zones'--defined as demarcated regions where symbiont replacement is most likely to occur, as indicated by overlapping abundances of symbiont ecotypes. Mapping the distribution of algal symbionts from two species of lichen-forming fungi along four independent altitudinal gradients, we detected an abrupt and consistent β-diversity turnover suggesting parallel niche partitioning. Modelling contrasting environmental response functions obtained from latitudinal distributions of algal ecotypes consistently predicted a confined altitudinal turnover zone. In all gradients this symbiont turnover zone is characterized by approximately 12°C average annual temperature and approximately 5°C mean temperature of the coldest quarter, marking the transition from Mediterranean to cool temperate bioregions. Integrating the conditions of symbiont turnover into biogeographic models of mutualisms is an important step towards a comprehensive understanding of biodiversity dynamics under ongoing environmental change.
Fungal populations that reproduce sexually are likely to be genetically more diverse and have a higher adaptive potential than asexually reproducing populations. Mating systems of fungal species can be self-incompatible, requiring the presence of isolates of different mating-type genes for sexual reproduction to occur, or self-compatible, requiring only one. Understanding the distribution of mating-type genes in populations can help to assess the potential of self-incompatible species to reproduce sexually. In the locally threatened epiphytic lichen-forming fungus Lobaria pulmonaria (L.) Hoffm., low frequency of sexual reproduction is likely to limit the potential of populations to adapt to changing environmental conditions. Our study provides direct evidence of self-incompatibility (heterothallism) in L. pulmonaria. It can thus be hypothesized that sexual reproduction in small populations might be limited by an unbalanced distribution of mating-type genes. We therefore assessed neutral genetic diversity (using microsatellites) and mating-type ratio in 27 lichen populations (933 individuals). We found significant differences in the frequency of the two mating types in 13 populations, indicating a lower likelihood of sexual reproduction in these populations. This suggests that conservation translocation activities aiming at maximizing genetic heterogeneity in threatened and declining populations should take into account not only presence of fruiting bodies in transplanted individuals, but also the identity and balanced representation of mating-type genes.
Genome mining as a biotechnological tool for the discovery of novel biosynthetic genes in lichens
(2022)
The ever-increasing demand for novel drugs highlights the need for bioprospecting unexplored taxa for their biosynthetic potential. Lichen-forming fungi (LFF) are a rich source of natural products but their implementation in pharmaceutical industry is limited, mostly because the genes corresponding to a majority of their natural products is unknown. Furthermore, it is not known to what extent these genes encode structurally novel molecules. Advance in next-generation sequencing technologies has expanded the range of organisms that could be exploited for their biosynthetic potential. In this study, we mine the genomes of nine lichen-forming fungal species of the genus Umbilicaria for biosynthetic genes, and categorize the BGCs as “associated product structurally known”, and “associated product putatively novel”. We found that about 25-30% of the biosynthetic genes are divergent when compared to the global database of BGCs comprising of 1,200,000 characterized biosynthetic genes from planta, bacteria and fungi. Out of 217 total BGCs, 43 were only distantly related to known BGCs, suggesting they encode structurally and functionally unknown natural products. Clusters encoding the putatively novel metabolic diversity comprise PKSs (30), NRPSs (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 therapeutic properties in LFF.
Foliar fungal communities of plants are diverse and ubiquitous. In grasses endophytes may increase host fitness; in trees, their ecological roles are poorly understood. We investigated whether the genotype of the host tree influences community structure of foliar fungi. We sampled leaves from genotyped balsam poplars from across the species' range, and applied 454 amplicon sequencing to characterize foliar fungal communities. At the time of the sampling the poplars had been growing in a common garden for two years. We found diverse fungal communities associated with the poplar leaves. Linear discriminant analysis and generalized linear models showed that host genotypes had a structuring effect on the composition of foliar fungal communities. The observed patterns may be explained by a filtering mechanism which allows the trees to selectively recruit fungal strains from the environment. Alternatively, host genotype-specific fungal communities may be present in the tree systemically, and persist in the host even after two clonal reproductions. Both scenarios are consistent with host tree adaptation to specific foliar fungal communities and suggest that there is a functional basis for the strong biotic interaction.
In the last decades, natural products from lichens have gained more interest for pharmaceutical application due to the broad range of their biological activity. However, isolation of the compounds of interest directly from the lichen is neither feasible nor sustainable due to slow growth of many lichens. In order to develop a pipeline for heterologous expression of lichen biosynthesis gene clusters and thus the sustainable production of their bioactive compounds we have identified and characterized the phosphopantheteinyl transferase (PPTase) EppA from the lichen Evernia prunastri. The Sfp-type PPTase EppA was functionally characterized through heterologous expression in E. coli using the production of the blue pigment indigoidine as readout and by complementation of a lys5 deletion in S. cerevisiae.
Panmixia and dispersal from the Mediterranean Basin to Macaronesian Islands of a macrolichen species
(2017)
The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, represents a center of diversification for many organisms. The genetic structure and connectivity of mainland and island microbial populations has been poorly explored, in particular in the case of symbiotic fungi. Here we investigated genetic diversity and spatial structure of the obligate outcrossing lichen-forming fungus Parmelina carporrhizans in the Mediterranean region. Using eight microsatellite and mating-type markers we showed that fungal populations are highly diverse but lack spatial structure. This is likely due to high connectivity and long distance dispersal of fungal spores. Consistent with low levels of linkage disequilibrium and lack of clonality, we detected both mating-type idiomorphs in all populations. Furthermore we showed that the Macaronesian Islands are the result of colonization from the Mediterranean Basin. The unidirectional gene flow, though, seemed not to be sufficient to counterbalance the effects of drift, resulting in comparatively allelic poor peripheral populations. Our study is the first to shed light on the high connectivity and lack of population structure in natural populations of a strictly sexual lichen fungus. Our data further support the view of the Macaronesian Islands as the end of the colonization road for this symbiotic ascomycete.
Intraspecific genomic variability affects a species’ adaptive potential towards 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 species have remarkable environmental tolerances, and often form populations in different climate zones. Here we combine comparative and population genomics to assess the presence and absence of genes in high elevation and low elevation genomes of two lichenized fungi of the genus Umbilicaria. 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. 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 kinases (PKs) superfamily. We traced the discovered ortholog in populations along five replicated elevation gradients on both continents. The protein kinase gene linearly declined in abundance with increasing elevation, and was absent in the highest populations. 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.
Background Transposable elements (TEs) are an important source of genome plasticity across the tree of life. Accumulating evidence suggests that TEs may not be randomly distributed in the genome. Drift and natural selection are important forces shaping TE distribution and accumulation, acting directly on the TE element or indirectly on the host species. 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 replicated elevation gradients using a Pool-Seq approach, to identify TE insertions of potential adaptive significance.
Results We found that TEs cover 21.26 % of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. Out of a total of 182 TE copies 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.
Conclusions This pioneering study into the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution. Particularly, it may serve as a foundation for assessing the impact of TE dynamics on fungal adaptation to the abiotic environment, and the impact of TE activity on the evolution and maintenance of a symbiotic lifestyle.
Tree bark constitutes ideal habitat for microbial communities, because it is a stable substrate, rich in micro-niches. Bacteria, fungi, and terrestrial microalgae together form microbial communities, which in turn support more bark-associated organisms, such as mosses, lichens, and invertebrates, thus contributing to forest biodiversity. We have a limited understanding of the diversity and biotic interactions of the bark-associated microbiome, as investigations have mainly focussed on agriculturally relevant systems and on single taxonomic groups. Here we implemented a multi-kingdom metabarcoding approach to analyse diversity and community structure of the green algal, bacterial, and fungal components of the bark-associated microbial communities of beech, the most common broadleaved tree of Central European forests. We identified the most abundant taxa, hub taxa, and co-occurring taxa. We found that tree size (as a proxy for age) is an important driver of community assembly, suggesting that environmental filtering leads to less diverse fungal and algal communities over time. Conversely, forest management intensity had negligible effects on microbial communities on bark. Our study suggests the presence of undescribed, yet ecologically meaningful taxa, especially in the fungi, and highlights the importance of bark surfaces as a reservoir of microbial diversity. Our results constitute a first, essential step towards an integrated framework for understanding microbial community assembly processes on bark surfaces, an understudied habitat and neglected component of terrestrial biodiversity. Finally, we propose a cost-effective sampling strategy to study bark-associated microbial communities across large spatial or environmental scales.