Should we hail the Red King? Evolutionary consequences of a mutualistic lifestyle in genomes of lichenized ascomycetes.

The Red Queen dynamic is often brought into play for antagonistic relationships. However, the coevolutionary effects of mutualistic interactions, which predict slower evolution for interacting organisms (Red King), have been investigated to a lesser extent. Lichens are a stable, mutualistic relationship of fungi and cyanobacteria and/or algae, which originated several times independently during the evolution of fungi. Therefore, they represent a suitable system to investigate the coevolutionary effect of mutualism on the fungal genome. We measured substitution rates and selective pressure of about 2000 protein-coding genes (plus the rDNA region) in two different classes of Ascomycota, each consisting of closely related lineages of lichenized and non-lichenized fungi. Our results show that independent lichenized clades are characterized by significantly slower rates for both synonymous and non-synonymous substitutions. We hypothesize that this evolutionary pattern is connected to the lichen life cycle (longer generation time of lichenized fungi) rather than a result of different selection strengths, which is described as the main driver for the Red Kind dynamic. This first empirical evidence of slower evolution in lichens provides an important insight on how biotic cooperative interactions are able to shape the evolution of symbiotic organisms.

An Overview of Genomics, Phylogenomics and Proteomics Approaches in Ascomycota.

Fungi are among the most successful eukaryotes on Earth: they have evolved strategies to survive in the most diverse environments and stressful conditions and have been selected and exploited for multiple aims by humans. The characteristic features intrinsic of Fungi have required evolutionary changes and adaptations at deep molecular levels. Omics approaches, nowadays including genomics, metagenomics, phylogenomics, transcriptomics, metabolomics, and proteomics have enormously advanced the way to understand fungal diversity at diverse taxonomic levels, under changeable conditions and in still under-investigated environments. These approaches can be applied both on environmental communities and on individual organisms, either in nature or in axenic culture and have led the traditional morphology-based fungal systematic to increasingly implement molecular-based approaches. The advent of next-generation sequencing technologies was key to boost advances in fungal genomics and proteomics research. Much effort has also been directed towards the development of methodologies for optimal genomic DNA and protein extraction and separation. To date, the amount of proteomics investigations in Ascomycetes exceeds those carried out in any other fungal group. This is primarily due to the preponderance of their involvement in plant and animal diseases and multiple industrial applications, and therefore the need to understand the biological basis of the infectious process to develop mechanisms for biologic control, as well as to detect key proteins with roles in stress survival. Here we chose to present an overview as much comprehensive as possible of the major advances, mainly of the past decade, in the fields of genomics (including phylogenomics) and proteomics of Ascomycota, focusing particularly on those reporting on opportunistic pathogenic, extremophilic, polyextremotolerant and lichenized fungi. We also present a review of the mostly used genome sequencing technologies and methods for DNA sequence and protein analyses applied so far for fungi.

Environmental DNA assessment of airborne plant and fungal seasonal diversity.

Environmental DNA (eDNA) metabarcoding and metagenomics analyses can improve taxonomic resolution in biodiversity studies. Only recently, these techniques have been applied in aerobiology, to target bacteria, fungi and plants in airborne samples. Here, we present a nine-month aerobiological study applying eDNA metabarcoding in which we analyzed simultaneously airborne diversity and variation of fungi and plants across five locations in North and Central Italy. We correlated species composition with the ecological characteristics of the sites and the seasons. The most abundant taxa among all sites and seasons were the fungal genera Cladosporium, Alternaria, and Epicoccum and the plant genera Brassica, Corylus, Cupressus and Linum, the latter being much more variable among sites. PERMANOVA and indicator species analyses showed that the plant diversity from air samples is significantly correlated with seasons, while that of fungi varied according to the interaction between seasons and sites. The results consolidate the performance of a new eDNA metabarcoding pipeline for the simultaneous amplification and analysis of airborne plant and fungal particles. They also highlight the promising complementarity of this approach with more traditional biomonitoring frameworks and routine reports of air quality provided by environmental agencies.

PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding.

DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called 'better clustering for QIIME' (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering. Database URL: https://github.com/apallavicini/bc4q; https://github.com/apallavicini/PLANiTS.

Phylogenetic relationships of rock-inhabiting black fungi belonging to the widespread genera Lichenothelia and Saxomyces.

Rock-inhabiting fungi (RIF) are adapted to thrive in oligotrophic environments and to survive under conditions of abiotic stress. Under these circumstances, they form biocoenoses with other tolerant organisms, such as lichens, or with less specific phototrophic consortia of aerial algae or cyanobacteria. RIF are phylogenetically diverse, and their plastic morphological characters hamper the straightforward species delimitation of many taxa. Here, we present a phylogenetic study of two RIF genera, Lichenothelia and Saxomyces. Representatives of both genera inhabit rather similar niches on rocks, but their phylogenetic relationships are unknown so far. The cosmopolitan genus Lichenothelia is recognized by characters of fertile ascomata and includes species with different life strategies. In contrast, Saxomyces species were described exclusively by mycelial characters found in cultured isolates from rock samples collected at high alpine elevations. Here, we use an extended taxon sampling of Dothideomycetes to study the phylogenetic relationships of both Lichenothelia and Saxomyces. We consider environmental samples, type species, and cultured isolates of both genera and demonstrate their paraphyly, as well as the occurrence of teleomorphs in Saxomyces. We applied three species delimitation methods to improve species recognition based on molecular data. We show the distinctiveness of the two main lineages of Lichenothelia (Lichenotheliales s. str.) and Saxomyces and discuss differences in species delimitation depending on molecular markers or methods. We revise the taxonomy of the two genera and describe three new taxa, Lichenothelia papilliformis, L. muriformis, and Saxomyces americanus, and the teleomorph of S. penninicus.

Genome-scale data resolve ancestral rock-inhabiting lifestyle in Dothideomycetes (Ascomycota).

Dothideomycetes is the most diverse fungal class in Ascomycota and includes species with a wide range of lifestyles. Previous multilocus studies have investigated the taxonomic and evolutionary relationships of these taxa but often failed to resolve early diverging nodes and frequently generated inconsistent placements of some clades. Here, we use a phylogenomic approach to resolve relationships in Dothideomycetes, focusing on two genera of melanized, extremotolerant rock-inhabiting fungi, Lichenothelia and Saxomyces, that have been suggested to be early diverging lineages. We assembled phylogenomic datasets from newly sequenced (4) and previously available genomes (238) of 242 taxa. We explored the influence of tree inference methods, supermatrix vs. coalescent-based species tree, and the impact of varying amounts of genomic data. Overall, our phylogenetic reconstructions provide consistent and well-supported topologies for Dothideomycetes, recovering Lichenothelia and Saxomyces among the earliest diverging lineages in the class. In addition, many of the major lineages within Dothideomycetes are recovered as monophyletic, and the phylogenomic approach implemented strongly supports their relationships. Ancestral character state reconstruction suggest that the rock-inhabiting lifestyle is ancestral within the class.