A database of high-resolution MS/MS spectra for lichen metabolites.

While analytical techniques in natural products research massively shifted to liquid chromatography-mass spectrometry, lichen chemistry remains reliant on limited analytical methods, Thin Layer Chromatography being the gold standard. To meet the modern standards of metabolomics within lichenochemistry, we announce the publication of an open access MS/MS library with 250 metabolites, coined LDB for Lichen DataBase, providing a comprehensive coverage of lichen chemodiversity. These were donated by the Berlin Garden and Botanical Museum from the collection of Siegfried Huneck to be analyzed by LC-MS/MS. Spectra at individual collision energies were submitted to MetaboLights (https://www.ebi.ac.uk/metabolights/MTBLS999) while merged spectra were uploaded to the GNPS platform (CCMSLIB00004751209 to CCMSLIB00004751517). Technical validation was achieved by dereplicating three lichen extracts using a Molecular Networking approach, revealing the detection of eleven unique molecules that would have been missed without LDB implementation to the GNPS. From a chemist's viewpoint, this database should help streamlining the isolation of formerly unreported metabolites. From a taxonomist perspective, the LDB offers a versatile tool for the chemical profiling of newly reported species.

Discovering cryptic species in the Aspiciliella intermutans complex (Megasporaceae, Ascomycota) - First results using gene concatenation and coalescent-based species tree approaches.

Taxonomic identifications in some groups of lichen-forming fungi have been challenge largely due to the scarcity of taxonomically relevant features and limitations of morphological and chemical characters traditionally used to distinguish closely related taxa. Delineating species boundaries in closely related species or species complexes often requires a range of multisource data sets and comprehensive analytical methods. Here we aim to examine species boundaries in a group of saxicolous lichen forming fungi, the Aspiciliella intermutans complex (Megasporaceae), widespread mainly in the Mediterranean. We gathered DNA sequences of the nuclear ribosomal internal transcribed spacer (nuITS), the nuclear large subunit (nuLSU), the mitochondrial small subunit (mtSSU) ribosomal DNA, and the DNA replication licensing factor MCM7 from 80 samples mostly from Iran, Caucasia, Greece and eastern Europe. We used a combination of phylogenetic strategies and a variety of empirical, sequence-based species delimitation approaches to infer species boundaries in this group. The latter included: the automatic barcode gap discovery (ABGD), the multispecies coalescent approach *BEAST and Bayesian Phylogenetics and Phylogeography (BPP) program. Different species delimitation scenarios were compared using Bayes factors species delimitation analysis. Furthermore, morphological, chemical, ecological and geographical features of the sampled specimens were examined. Our study uncovered cryptic species diversity in A. intermutans and showed that morphology-based taxonomy may be unreliable, underestimating species diversity in this group of lichens. We identified a total of six species-level lineages in the A. intermutans complex using inferences from multiple empirical operational criteria. We found little corroboration between morphological and ecological features with our proposed candidate species, while secondary metabolite data do not corroborate tree topology. The present study on the A. intermutans species-complex indicates that the genus Aspiciliella, as currently circumscribed, is more diverse in Eurasia than previously expected.

Relict high-Andean ecosystems challenge our concepts of naturalness and human impact.

What would current ecosystems be like without the impact of mankind? This question, which is critical for ecosystem management, has long remained unanswered due to a lack of present-day data from truly undisturbed ecosystems. Using mountaineering techniques, we accessed pristine relict ecosystems in the Peruvian Andes to provide this baseline data and compared it with the surrounding accessible and disturbed landscape. We show that natural ecosystems and human impact in the high Andes are radically different from preconceived ideas. Vegetation of these 'lost worlds' was dominated by plant species previously unknown to science that have become extinct in nearby human-affected ecosystems. Furthermore, natural vegetation had greater plant biomass with potentially as much as ten times more forest, but lower plant diversity. Contrary to our expectations, soils showed relatively little degradation when compared within a vegetation type, but differed mainly between forest and grassland ecosystems. At the landscape level, a presumed large-scale forest reduction resulted in a nowadays more acidic soilscape with higher carbon storage, partly ameliorating carbon loss through deforestation. Human impact in the high Andes, thus, had mixed effects on biodiversity, while soils and carbon stocks would have been mainly indirectly affected through a suggested large-scale vegetation change.

From one to six: unrecognized species diversity in the genus Acantholichen (lichenized Basidiomycota: Hygrophoraceae).

We present a taxonomic revision of the lichenized basidiomycete genus Acantholichen, species of which produce a characteristic blue-gray, microsquamulose thallus with spiny apical hyphal cells known as acanthohyphidia. Since its discovery, the genus was thought to be monospecific, only including the generic type, A. pannarioides. However, a detailed morphological and anatomical study of recently collected specimens from the Galápagos, Costa Rica, Brazil and Colombia, combined with a molecular phylogenetic analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and 28S of the nuc rDNA and RPB2 sequences, revealed a much more diverse and widespread species assemblage. Based on the results of these analyses, we describe five new species in the genus: A. albomarginatus, A. campestris, A. galapagoensis, A. sorediatus and A. variabilis. We also provide an identification key to all species, anatomical and morphological descriptions, photographs and a table comparing main characters of each species.

A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66 families.

The Lecanoromycetes is the largest class of lichenized Fungi, and one of the most species-rich classes in the kingdom. Here we provide a multigene phylogenetic synthesis (using three ribosomal RNA-coding and two protein-coding genes) of the Lecanoromycetes based on 642 newly generated and 3329 publicly available sequences representing 1139 taxa, 317 genera, 66 families, 17 orders and five subclasses (four currently recognized: Acarosporomycetidae, Lecanoromycetidae, Ostropomycetidae, Umbilicariomycetidae; and one provisionarily recognized, 'Candelariomycetidae'). Maximum likelihood phylogenetic analyses on four multigene datasets assembled using a cumulative supermatrix approach with a progressively higher number of species and missing data (5-gene, 5+4-gene, 5+4+3-gene and 5+4+3+2-gene datasets) show that the current classification includes non-monophyletic taxa at various ranks, which need to be recircumscribed and require revisionary treatments based on denser taxon sampling and more loci. Two newly circumscribed orders (Arctomiales and Hymeneliales in the Ostropomycetidae) and three families (Ramboldiaceae and Psilolechiaceae in the Lecanorales, and Strangosporaceae in the Lecanoromycetes inc. sed.) are introduced. The potential resurrection of the families Eigleraceae and Lopadiaceae is considered here to alleviate phylogenetic and classification disparities. An overview of the photobionts associated with the main fungal lineages in the Lecanoromycetes based on available published records is provided. A revised schematic classification at the family level in the phylogenetic context of widely accepted and newly revealed relationships across Lecanoromycetes is included. The cumulative addition of taxa with an increasing amount of missing data (i.e., a cumulative supermatrix approach, starting with taxa for which sequences were available for all five targeted genes and ending with the addition of taxa for which only two genes have been sequenced) revealed relatively stable relationships for many families and orders. However, the increasing number of taxa without the addition of more loci also resulted in an expected substantial loss of phylogenetic resolving power and support (especially for deep phylogenetic relationships), potentially including the misplacements of several taxa. Future phylogenetic analyses should include additional single copy protein-coding markers in order to improve the tree of the Lecanoromycetes. As part of this study, a new module ("Hypha") of the freely available Mesquite software was developed to compare and display the internodal support values derived from this cumulative supermatrix approach.

Implementing a cumulative supermatrix approach for a comprehensive phylogenetic study of the Teloschistales (Pezizomycotina, Ascomycota).

The resolution of the phylogenetic relationships within the order Teloschistales (Ascomycota, lichen-forming-fungi), with nearly 2000 known species and outstanding phenotypic diversity, has been hindered by the limitation in the resolving power that single-locus or two-locus phylogenetic studies have provided to date. In this context, an extensive taxon sampling within the Teloschistales with more loci (especially nuclear protein-coding genes) was needed to confront the current taxonomic delimitations and to understand evolutionary trends within this order. Comprehensive maximum likelihood and bayesian analyses were performed based on seven loci using a cumulative supermatrix approach, including protein-coding genes RPB1 and RPB2 in addition to nuclear and mitochondrial ribosomal RNA-coding genes. We included 167 taxa representing 12 of the 15 genera recognized within the currently accepted Teloschistineae, 22 of the 43 genera within the Physciineae, 49 genera of the closely related orders Lecanorales, Lecideales, and Peltigerales, and the dubiously placed family Brigantiaeaceae and genus Sipmaniella. Although the progressive addition of taxa (cumulative supermatrix approach) with increasing amounts of missing data did not dramatically affect the loss of support and resolution, the monophyly of the Teloschistales in the current sense was inconsistent, depending on the loci-taxa combination analyzed. Therefore, we propose a new, but provisional, classification for the re-circumscribed orders Caliciales and Teloschistales (previously referred to as Physciineae and Teloschistineae, respectively). We report here that the family Brigantiaeaceae, previously regarded as incertae sedis within the subclass Lecanoromycetidae, and Sipmaniella, are members of the Teloschistales in a strict sense. Within this order, one lineage led to the diversification of the mostly epiphytic crustose Brigantiaeaceae and Letrouitiaceae, with a circumpacific center of diversity and found mostly in the tropics. The other main lineage led to another epiphytic crustose family, mostly tropical, and with an Australasian center of diversity--the Megalosporaceae--which is sister to the mainly rock-inhabiting, cosmopolitan, and species rich Teloschistaceae, with a diversity of growth habits ranging from crustose to fruticose. Our results confirm the use of a cumulative supermatrix approach as a viable method to generate comprehensive phylogenies summarizing relationships of taxa with multi-locus to single locus data.

High concentration of basidiolichens in a single family of agaricoid mushrooms (Basidiomycota: Agaricales: Hygrophoraceae).

The Agaricales is the largest and most diverse order of mushroom-forming Basidiomycota, with over 100 natural groups recognized in recent Fungal Tree of Life studies. Most agarics are either saprotrophic or ectomycorrhizal fungi, but the family Hygrophoraceae is in part characterized by a unique and remarkable diversity of lichenized forms. The most familiar of these is the chlorolichen genus Lichenomphalia, whose phylogenetic position in the Agaricales has been established. Recent limited evidence suggested that Hygrophoraceae also contains cyanolichens in the genus Dictyonema, which indicates a remarkable concentration and diversity of lichen-formers in a single family of agarics. To demonstrate the relationships of lichen-formers to other fungi in the family, we assembled ribosomal sequences from 52 species representing recognized groups within the Hygrophoraceae, among them new sequences representing Acantholichen and most species and forms of Dictyonema. The molecular data were evaluated using parsimony, likelihood, Bayesian, and distance analyses, including coding of ambiguous regions by means of INAASE and ARC, all of which indicate that Dictyonema and Acantholichen form a monophyletic clade derived from the primarily bryophilous genus Arrhenia and sister to the enigmatic Athelia pyriformis, a species unrelated to the Atheliales for which we are proposing a new genus name Eonema. The chlorolichen genus Lichenomphalia may be polyphyletic. Fungi in the Dictyonema-Acantholichen clade are typically tropical, entirely lichenized, and associate with cyanobacterial photobionts. Our data indicate a transition from agaricoid-omphalinoid basidiomes observed in Arrhenia to stereoid-corticioid forms in Dictyonema, and also support a previous suggestion of a connection between loss of clamp connections and lichenization. The diverse basidiome and thallus morphologies and nutritional ecologies of these fungi indicate a remarkable evolutionary flexibility that appears to have developed in part as a consequence of symbiosis.

Do lichens domesticate photobionts like farmers domesticate crops? Evidence from a previously unrecognized lineage of filamentous cyanobacteria.

Phylogenetic diversity of lichen photobionts is low compared to that of fungal counterparts. Most lichen fungi are thought to be associated with just four photobiont genera, among them the cyanobacteria Nostoc and Scytonema, two of the most important nitrogen fixers in humid ecosystems. Although many Nostoc photobionts have been identified using isolated cultures and sequences, the identity of Scytonema photobionts has never been confirmed by culturing or sequencing. We investigated the phylogenetic placement of presumed Scytonema photobionts and unicellular morphotypes previously assigned to Chroococcus, from tropical Dictyonema, Acantholichen, Coccocarpia, and Stereocaulon lichens. While we confirm that filamentous and unicellular photobiont morphotypes belong to a single clade, this clade does not cluster with Scytonema but represents a novel, previously unrecognized, highly diverse, exclusively lichenized lineage, for which the name Rhizonema is available. The phylogenetic structure observed in this novel lineage suggests absence of coevolution with associated mycobionts at the species or clade level. Instead, highly efficient photobiont strains appear to have evolved through photobiont sharing between unrelated, but ecologically similar, coexisting lineages of lichenized fungi ("lichen guilds"), via the selection of particular photobiont strains through and subsequent horizontal transfer among unrelated mycobionts, a phenomenon not unlike crop domestication.

The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

A five-gene phylogeny of Pezizomycotina.

Pezizomycotina is the largest subphylum of Ascomycota and includes the vast majority of filamentous, ascoma-producing species. Here we report the results from weighted parsimony, maximum likelihood and Bayesian phylogenetic analyses of five nuclear loci (SSU rDNA, LSU rDNA, RPB1, RPB2 and EF-lalpha) from 191 taxa. Nine of the 10 Pezizomycotina classes currently recognized were represented in the sampling. These data strongly supported the monophyly of Pezizomycotina, Arthoniomycetes, Eurotiomycetes, Orbiliomycetes and Sordariomycetes. Pezizomycetes and Dothideomycetes also were resolved as monophyletic but not strongly supported by the data. Lecanoromycetes was resolved as paraphyletic in parsimony analyses but monophyletic in maximum likelihood and Bayesian analyses. Leotiomycetes was polyphyletic due to exclusion of Geoglossaceae. The two most basal classes of Pezizomycotina were Orbiliomycetes and Pezizomycetes, both of which comprise species that produce apothecial ascomata. The seven remaining classes formed a monophyletic group that corresponds to Leotiomyceta. Within Leotiomyceta, the supraclass clades of Leotiomycetes s.s. plus Sordariomycetes and Arthoniomycetes plus Dothideomycetes were resolved with moderate support.

Assembling the fungal tree of life: progress, classification, and evolution of subcellular traits.

Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.