Systemic infection of Bryoria (Lecanoromycetes, Ascomycota) by Athelia (Agaricomycetes, Basidiomycota) in western North America.

Bryoria (Parmeliaceae, Ascomycota) is one of the dominant genera of hair lichens in western North America and is characteristic of high-elevation conifer forest ecosystems. In areas where Bryoria is abundant, it is common to find thalli in which the thalline filaments become conglutinated, forming brittle dead zones. After sampling Bryoria thalli across western Canada and the northwestern United States at different times of the year, we found that this dieback phenomenon is associated with the winter growth of a mold-forming basidiomycete. We report that this fungus belongs to Athelia (Atheliaceae, Basidiomycota), a genus known to contain lichen pathogens, most notably A. arachnoidea. By sequencing a combination of genetic markers-nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), partial nuc 28S rDNA (28S), and partial translation elongation factor 1-α (TEF1)-paired with morphometric analyses, we reveal the involvement of at least three additional lineages of lichen-associated Athelia and describe one as a new species, A. abscondita. Athelia abscondita is morphologically distinguished from other Athelia species by its basidia and basidiospores, was found to frequently infect members of Bryoria sect. Implexae, and was occasionally on other foliose and fruticose species within Parmeliaceae.

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.

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.

In vitro resynthesis of lichenization reveals the genetic background of symbiosis-specific fungal-algal interaction in Usnea hakonensis.

BACKGROUND: Symbiosis is central to ecosystems and has been an important driving force of the diversity of life. Close and long-term interactions are known to develop cooperative molecular mechanisms between the symbiotic partners and have often given them new functions as symbiotic entities. In lichen symbiosis, mutualistic relationships between lichen-forming fungi and algae and/or cyanobacteria produce unique features that make lichens adaptive to a wide range of environments. Although the morphological, physiological, and ecological uniqueness of lichens has been described for more than a century, the genetic mechanisms underlying this symbiosis are still poorly known. RESULTS: This study investigated the fungal-algal interaction specific to the lichen symbiosis using Usnea hakonensis as a model system. The whole genome of U. hakonensis, the fungal partner, was sequenced by using a culture isolated from a natural lichen thallus. Isolated cultures of the fungal and the algal partners were co-cultured in vitro for 3 months, and thalli were successfully resynthesized as visible protrusions. Transcriptomes of resynthesized and natural thalli (symbiotic states) were compared to that of isolated cultures (non-symbiotic state). Sets of fungal and algal genes up-regulated in both symbiotic states were identified as symbiosis-related genes. CONCLUSION: From predicted functions of these genes, we identified genetic association with two key features fundamental to the symbiotic lifestyle in lichens. The first is establishment of a fungal symbiotic interface: (a) modification of cell walls at fungal-algal contact sites; and (b) production of a hydrophobic layer that ensheaths fungal and algal cells;. The second is symbiosis-specific nutrient flow: (a) the algal supply of photosynthetic product to the fungus; and (b) the fungal supply of phosphorous and nitrogen compounds to the alga. Since both features are widespread among lichens, our result may indicate important facets of the genetic basis of the lichen symbiosis.

Complete, high-quality genomes from long-read metagenomic sequencing of two wolf lichen thalli reveals enigmatic genome architecture.

Fungal genomes display incredible levels of complexity and diversity, and are exceptional study systems for genome evolution. Here we used the Oxford Nanopore MinION sequencing platform to generate high-quality fungal genomes from complex metagenomic samples of lichen thalli. We sequenced two wolf lichens using one flow cell per sample, generating 17.1 Gbps for Letharia lupina and 14.3 Gbps for Letharia columbiana. The resulting L. lupina genome is one of the most contiguous lichen genomes available to date, with 49.2 Mbp contained on 31 contigs. The L. columbiana genome, while less contiguous, is still relatively high quality, with 52.3 Mbp on a total of 161 contigs. Each thallus for both species contained multiple distinct haplotypes, a phenomenon that has rarely been empirically demonstrated. The Oxford Nanopore sequencing technologies are robust and effective when applied to complex symbioses, and have the potential to fundamentally transform our understanding of fungal genetics.

Lichen chemistry is concordant with multilocus gene genealogy in the genus Cetrelia (Parmeliaceae, Ascomycota).

The lichen genus Cetrelia represents a taxonomically interesting case where morphologically almost uniform populations differ considerably from each other chemically. Similar variation is not uncommon among lichenized fungi, but it is disputable whether such populations should be considered entities at the species level. Species boundaries in Cetrelia are traditionally delimited either as solely based on morphology or as combinations of morpho- and chemotypes. A dataset of four nuclear markers (ITS, IGS, Mcm7, RPB1) from 62 specimens, representing ten Cetrelia species, was analysed within Bayesian and maximum likelihood frameworks. Analyses recovered a well-resolved phylogeny where the traditional species generally were monophyletic, with the exception of Cetrelia chicitae and Cetrelia pseudolivetorum. Species delimitation analyses supported the distinction of 15 groups within the studied Cetrelia taxa, dividing three traditionally identified species into some species candidates. Chemotypes, distinguished according to the major medullary substance, clearly correlated with clades recovered within Cetrelia, while samples with the same reproductive mode were dispersed throughout the phylogenetic tree. Consequently, delimiting Cetrelia species based only on reproductive morphology is not supported phylogenetically. Character analyses suggest that chemical characters have been more consistent compared to reproductive mode and indicate that metabolite evolution in Cetrelia towards more complex substances is probable.

Identification of a putative polyketide synthase gene involved in usnic acid biosynthesis in the lichen Nephromopsis pallescens.

Usnic acid is a unique polyketide produced by lichens. To characterize usnic acid biosynthesis, the transcriptome of the usnic-acid-producing lichen-forming fungus Nephromopsis pallescens was sequenced using Illumina NextSeq technology. Seven complete non-reducing polyketide synthase genes and nine highly-reducing polyketide synthase genes were obtained through transcriptome analysis. Gene expression results obtained by qPCR and usnic acid detection with LCMS-IT-TOF showed that Nppks7 is probably involved in usnic acid biosynthesis in N. pallescens. Nppks7 is a non-reducing polyketide synthase with a MeT domain that also possesses beta-ketoacyl-ACP synthase, acyl transferase, product template, acyl carrier protein, C-methyltransferase, and Claisen cyclase domains. Phylogenetic analysis shows that Nppks7and other polyketide synthases from lichens form a unique monophyletic clade. Taken together, our data indicate that Nppks7 is a novel PKS in N. pallescens that is likely involved in usnic acid biosynthesis.

Fungal communities associated with Evernia prunastri, Ramalina fastigiata and Pleurosticta acetabulum: Three epiphytic lichens potentially active against Candida biofilms.

Fungal communities associated to three epiphytic lichens active against Candida, were investigated using culture-based methods We hypothetized that associated fungi would contribute to lichens activities. The ability of specific fungi to grow inside or outside lichens was investigated. To detect biogenesis pathways involved in the production of secondary metabolites, genes coding for nonribosomal peptide synthetase (NRPS) and polyketide synthase I (PKS I) were screened by PCR from fungal DNA extracts. Both endo and epilichenic communities were isolated from two fructicose (Evernia prunastri and Ramalina fastigiata) and one foliose (Pleurosticta acetabulum) lichens. A total of 86 endolichenic and 114 epilichenic isolates were obtained, corresponding to 18 and 24 phylogenetic groups respectively suggesting a wide diversity of fungi. The communities and the species richness were distinct between the three lichens which hosted potentially new fungal species. Additionally, the endo- and epilichenic communities differed in their composition: Sordariomycetes were particularly abundant among endolichenic fungi and Dothideomycetes among epilichenic fungi. Only a few fungi colonized both habitats, such as S. fimicola, Cladosporium sp1 and Botrytis cinerea. Interestingly, Nemania serpens (with several genotypes) was the most abundant endolichenic fungus (53% of isolates) and was shared by the three lichens. Finally, 12 out of 36 phylogenetic groups revealed the presence of genes coding for nonribosomal peptide synthetase (NRPs) and polyketide synthase I (PKS I). This study shows that common lichens are reservoirs of diverse fungal communities, which could potentially contribute to global activity of the lichen and, therefore, deserve to be isolated for further chemical studies.

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding.

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials.

A temporal banding approach for consistent taxonomic ranking above the species level.

Comparable taxonomic ranks within clades can facilitate more consistent classifications and objective comparisons among taxa. Here we use a temporal approach to identify taxonomic ranks. This is an extension of the temporal banding approach including a Temporal Error Score that finds an objective cut-off for each taxonomic rank using information for the current classification. We illustrate this method using a data set of the lichenized fungal family Parmeliaceae. To assess its performance, we simulated the effect of taxon sampling and compared our method with the other temporal banding method. For our sampled phylogeny, 11 of the 12 included families remained intact and 55 genera were confirmed, whereas 32 genera were lumped and 15 genera were split. Taxon sampling impacted the method at the genus level, whereas yielded only insignificant changes at the family level. The other available temporal approach also gives a similar cutoff point to our method. Our approach to identify taxonomic ranks enables taxonomists to revise and propose classifications on an objective basis, changing ranks of clades only when inconsistent with most taxa in a phylogenetic tree. An R script to find the time point with the minimal temporal error is provided.

Panmixia and dispersal from the Mediterranean Basin to Macaronesian Islands of a macrolichen species.

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.

Limitations of Species Delimitation Based on Phylogenetic Analyses: A Case Study in the Hypogymnia hypotrypa Group (Parmeliaceae, Ascomycota).

Delimiting species boundaries among closely related lineages often requires a range of independent data sets and analytical approaches. Similar to other organismal groups, robust species circumscriptions in fungi are increasingly investigated within an empirical framework. Here we attempt to delimit species boundaries in a closely related clade of lichen-forming fungi endemic to Asia, the Hypogymnia hypotrypa group (Parmeliaceae). In the current classification, the Hypogymnia hypotrypa group includes two species: H. hypotrypa and H. flavida, which are separated based on distinctive reproductive modes, the former producing soredia but absent in the latter. We reexamined the relationship between these two species using phenotypic characters and molecular sequence data (ITS, GPD, and MCM7 sequences) to address species boundaries in this group. In addition to morphological investigations, we used Bayesian clustering to identify potential genetic groups in the H. hypotrypa/H. flavida clade. We also used a variety of empirical, sequence-based species delimitation approaches, including: the "Automatic Barcode Gap Discovery" (ABGD), the Poisson tree process model (PTP), the General Mixed Yule Coalescent (GMYC), and the multispecies coalescent approach BPP. Different species delimitation scenarios were compared using Bayes factors delimitation analysis, in addition to comparisons of pairwise genetic distances, pairwise fixation indices (FST). The majority of the species delimitation analyses implemented in this study failed to support H. hypotrypa and H. flavida as distinct lineages, as did the Bayesian clustering analysis. However, strong support for the evolutionary independence of H. hypotrypa and H. flavida was inferred using BPP and further supported by Bayes factor delimitation. In spite of rigorous morphological comparisons and a wide range of sequence-based approaches to delimit species, species boundaries in the H. hypotrypa group remain uncertain. This study reveals the potential limitations of relying on distinct reproductive strategies as diagnostic taxonomic characters for Hypogymnia and also the challenges of using popular sequence-based species delimitation methods in groups with recent diversification histories.

An Integrative Approach for Understanding Diversity in the Punctelia rudecta Species Complex (Parmeliaceae, Ascomycota).

High levels of cryptic diversity have been documented in lichenized fungi, especially in Parmeliaceae, and integrating various lines of evidence, including coalescent-based species delimitation approaches, help establish more robust species circumscriptions. In this study, we used an integrative taxonomic approach to delimit species in the lichen-forming fungal genus Punctelia (Parmeliaceae), with a particular focus on the cosmopolitan species P. rudecta. Nuclear, mitochondrial ribosomal DNA and protein-coding DNA sequences were analyzed in phylogenetic and coalescence-based frameworks. Additionally, morphological, ecological and geographical features of the sampled specimens were evaluated. Five major strongly supported monophyletic clades were recognized in the genus Punctelia, and each clade could be characterized by distinct patterns in medullary chemistry. Punctelia rudecta as currently circumscribed was shown to be polyphyletic. A variety of empirical species delimitation methods provide evidence for a minimum of four geographically isolated species within the nominal taxon Punctelia rudecta, including a newly described saxicolous species, P. guanchica, and three corticolous species. In order to facilitate reliable sample identification for biodiversity, conservation, and air quality bio-monitoring research, these three species have been epitypified, in addition to the description of a new species.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

Who's getting around? Assessing species diversity and phylogeography in the widely distributed lichen-forming fungal genus Montanelia (Parmeliaceae, Ascomycota).

Brown parmelioid lichens comprise a number of distinct genera in one of the most species-rich families of lichen-forming fungi, Parmeliaceae (Ascomycota). In spite of their superficial similarity, a number of studies of brown parmelioids have provided important insight into diversification in lichen-forming fungi with cosmopolitan distributions. In this study we assess species diversity, biogeography and diversification of the genus Montanelia, which includes alpine to temperate saxicolous species. We sampled each of the five known species, four of which are known from broad, intercontinental distributions. In order to identify potential biogeographical patterns, each broadly distributed species was represented by individuals collected across their intercontinental distributions. Molecular sequence data were generated for six loci, including three nuclear protein-coding markers (MCM7, RPB1, and RPB2), two nuclear ribosomal markers (ITS and nrLSU), and a fragment of the mitochondrial small subunit. We used three sequence-based species delimitations methods to validate traditional, phenotype-based species and circumscribe previously unrecognized species-level lineages in Montanelia. Relationships among putative lineages and divergence times were estimated within a coalescent-based multi-locus species tree framework. Based on the results of the species delimitation analyses, we propose that the genus Montanelia is likely comprised of six to nine species-level lineages, including previously unrecognized species-level diversity in the nominal taxa M. panniformis and M. tominii. In contrast, molecular sequence data suggest that M. predisjuncta may be conspecific with the widespread taxon M. disjuncta in spite of distinct morphological differences. The rate-based age estimation of the most recent common ancestor of Montanelia (ca. 23.1Ma) was similar to previous estimates based on the fossil record. Furthermore, our data suggest that diversification in Montanelia occurred largely during the Neogene. At least three Montanelia species are broadly distributed throughout Asia, Europe, and North America with no evidence of phylogeographic substructure. In contrast to broadly distributed Montanelia species, our study suggests Pleistocene-dominated diversification and complex biogeographic history in the M. tominii group. Our analyses provide additional insight for understanding diversification and uncovering cryptic diversity in cosmopolitan species of lichen-forming fungi.