Due to their submerged and cryptic lifestyle, the vast majority of fungal species are difficult to observe and describe morphologically, and many remain known to science only from sequences detected in environmental samples. The lack of practices to delimit and name most fungal species is a staggering limitation to communication and interpretation of ecology and evolution in kingdom Fungi. Here, we use environmental sequence data as taxonomical evidence and combine phylogenetic and ecological data to generate and test species hypotheses in the class Archaeorhizomycetes (Taphrinomycotina, Ascomycota). Based on environmental amplicon sequencing from a well-studied Swedish pine forest podzol soil, we generate 68 distinct species hypotheses of Archaeorhizomycetes, of which two correspond to the only described species in the class. Nine of the species hypotheses represent 78% of the sequenced Archaeorhizomycetes community, and are supported by long read data that form the backbone for delimiting species hypothesis based on phylogenetic branch lengths. Soil fungal communities are shaped by environmental filtering and competitive exclusion so that closely related species are less likely to co-occur in a niche if adaptive traits are evolutionarily conserved. In soil profiles, distinct vertical horizons represent a testable niche dimension, and we found significantly differential distribution across samples for a well-supported pair of sister species hypotheses. Based on the combination of phylogenetic and ecological evidence, we identify two novel species for which we provide molecular diagnostics and propose names. While environmental sequences cannot be automatically translated to species, they can be used to generate phylogenetically distinct species hypotheses that can be further tested using sequences as ecological evidence. We conclude that in the case of abundantly and frequently observed species, environmental sequences can support species recognition in the absences of physical specimens, while rare taxa remain uncaptured at our sampling and sequencing intensity.

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large‐scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus's time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic‐dry part that has a species rich grass‐dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%–40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.

Arbuscular mycorrhizal (AM) fungi are ubiquitous mutualistic symbionts of most terrestrial plants and many complete their lifecycles underground. Whole genome analysis of AM fungi has long been restricted to species and strains that can be maintained under controlled conditions that facilitate collection of biological samples. There is some evidence suggesting that AM fungi can adapt to culture resulting in phenotypic and possibly also genotypic changes in the fungi. In this study, we used field isolated spores of AM fungi and identified them as Funneliformis geosporum based on morphology and phylogenetic analyses. We separately assembled the genomes of two representative spores using DNA sequences of 19 and 22 individually amplified nuclei. The genomes were compared with previously published data from other members of Glomeraceae including two strains of F. mosseae. No significant differences were observed among the species in terms of gene content, while the single nucleotide polymorphism density was higher in the strains of F. geosporum than in the strains of F. mosseae. In this study, we demonstrate that it is possible to sequence and assemble genomes from AM fungal spores sampled in the field, which opens up the possibility to include uncultured AM fungi in phylogenomic and comparative genomic analysis and to study genomic variation in natural populations of these important plant symbionts.

Background The colonization of land and the diversification of terrestrial plants is intimately linked to the evolutionary history of their symbiotic fungal partners. Extant representatives of these fungal lineages include mutualistic plant symbionts, the arbuscular mycorrhizal (AM) fungi and related ectomycorrhizal fungi in Endogonales, as well as fungi with saprotrophic, pathogenic and endophytic lifestyles. These fungal groups separate into three monophyletic lineages but their evolutionary relationships remain enigmatic confounding ancestral reconstructions. Their taxonomic ranks are currently fluid. Results In this study, we recognize these three monophyletic linages as phyla, and use a balanced taxon sampling and broad taxonomic representation for phylogenomic analysis that rejects a hard polytomy and resolves Glomeromycota as sister to Mucoromycota and Mortierellomycota. Low copy numbers of genes associated with plant cell wall degradation could not be assigned to the transition to a plant symbiotic lifestyle but appears to be an ancestral phylogenetic signal. Glomeromycota have distinct gene content and the lack of fatty acid synthesis genes is specific to this lineage. Further, gene content analysis demonstrates that many genes previously thought to be missing specifically in Glomeromycota are either missing in all analyzed phyla, or in some cases, are actually present in some of the AM fungal lineages analyzed here, e.g. the phosphorus transporter Pho89. Conclusion Based on a broad taxon sampling of fungal genomes we present a well-supported phylogeny for AM fungi and their sister lineages. We show that both cases of evolutionary transitions to mutualistic plant symbiosis happened in a genomic background profoundly different from that known from the emergence of ectomycorrhizal fungi in Dikarya. These results call for further reevaluation of genomic signatures associated with plant symbiosis.

Abstract Due to their submerged and cryptic lifestyle the vast majority of fungal species are difficult to observe and describe morphologically and many remains known to science only from sequences detected in environmental samples. The lack of rules to delimit and name most fungal species is a staggering limitation to communication and interpretation of ecology and evolution in kingdom Fungi. Here we use environmental sequence data as taxonomical evidence and take an integrated taxonomic approach by combining phylogenetic and ecological data to generate and test species hypothesis in the class Archaeorhizomycetes (Taphrinomycotina, Ascomycota). Based on environmental amplicon sequencing we recognize 68 distinct phylogenetic species hypotheses (PSHs) of Archaeorhizomycetes, including the two described species in the class, in a well-studied Swedish pine forest podzol soil. Nine of the species hypotheses, including the more abundant ones, are supported by long read data and represent 78% of the sequenced Archaeorhizomycetes community. Among well supported sister PSHs, significantly differential distribution in the soil profile provide additional ecological evidence supporting the identification of two novel species for which we provide molecular diagnostics and propose names. Our analysis indicates that frequent and abundant taxa can be phylogenetically resolved in environmental samples, while rare taxa remain un-captured at our sampling and sequencing intensity. While environmental sequences cannot be automatically translated to species hypothesis, they can be used to generate phylogenetic and ecological evidence supporting species recognition of abundant species without physical specimens.

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communites of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing and evaluated the performance of three different methods for generation of operational taxonomic units (OTUs). The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus’s time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic-dry part that has a species rich grass-dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigare how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30-40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.

Background With the expanding ash dieback epidemic that has spread across the European continent, an improved functional understanding of the disease development in afflicted hosts is needed. The study investigated whether differences in necrosis extension between common ash (Fraxinus excelsior) trees with different levels of susceptibility to the fungus Hymenoscyphus fraxineus are associated with, and can be explained by, the differences in gene expression patterns. We inoculated seemingly healthy branches of each of two resistant and susceptible ash genotypes with H. fraxineus grown in a common garden. Results Ten months after the inoculation, the length of necrosis on the resistant genotypes were shorter than on the susceptible genotypes. RNA sequencing of bark samples collected at the border of necrotic lesions and from healthy tissues distal to the lesion revealed relatively limited differences in gene expression patterns between susceptible and resistant genotypes. At the necrosis front, only 138 transcripts were differentially expressed between the genotype categories while 1082 were differentially expressed in distal, non-symptomatic tissues. Among these differentially expressed genes, several genes in the mevalonate (MVA) and iridoid pathways were found to be co-regulated, possibly indicating increased fluxes through these pathways in response to H. fraxineus. Comparison of transcriptional responses of symptomatic and non-symptomatic ash in a controlled greenhouse experiment revealed a relatively small set of genes that were differentially and concordantly expressed in both studies. This gene-set included the rate-limiting enzyme in the MVA pathway and a number of transcription factors. Furthermore, several of the concordantly expressed candidate genes show significant similarity to genes encoding players in the abscisic acid- or Jasmonate-signalling pathways. Conclusions A set of candidate genes, concordantly expressed between field and greenhouse experiments, was identified. The candidates are associated with hormone signalling and specialized metabolite biosynthesis pathways indicating the involvement of these pathways in the response of the host to infection by H. fraxineus.