Global Spore Sampling Project: A global, standardized dataset of airborne fungal DNA.

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.

A case study on the application of spore sampling for the monitoring of macrofungi.

Fungi play a vital role in ecosystem functioning, yet significant knowledge gaps persist in understanding their diversity and distribution leading to uncertainties about their threat status and extinction risk. This is partly owed to the difficulty of monitoring fungi using traditional fruiting body surveys. The present study evaluates airborne environmental DNA (eDNA) sampling as a monitoring tool with a focus on grassland macrofungi. We applied active and passive air sampling methods, complemented by extensive field surveys of waxcap and clavarioid fungi-species groups of high relevance for conservation. Twenty-nine species were recorded during the field surveys, 19 of which were also detectable by ITS2 metabarcoding of the collected samples. An additional 12 species from the studied genera were identified exclusively in air eDNA. We found that the patterns of species detection and read abundance in air samples reflected the abundance and occurrence of fruiting bodies on the field. Dispersal kernels fitted for the three dominant species predicted rapidly decreasing spore concentrations with increasing distance from fruitbodies. Airborne assemblages were dominated by a high diversity of common species, while rare and threatened red-listed species were under-represented, which underscores the difficulty in detecting rare species, not only in conventional surveys. Considering the benefits and drawbacks of air sampling and fruitbody surveys, we conclude that air sampling serves as a cost- and time-efficient tool to characterize local macrofungal communities, providing the potential to facilitate and improve future fungal monitoring efforts.

Detection of a Conspecific Mycovirus in Two Closely Related Native and Introduced Fungal Hosts and Evidence for Interspecific Virus Transmission.

Hymenoscyphus albidus is a native fungus in Europe where it behaves as a harmless decomposer of leaves of common ash. Its close relative Hymenoscyphus fraxineus was introduced into Europe from Asia and currently threatens ash (Fraxinus sp.) stands all across the continent causing ash dieback. H. fraxineus isolates from Europe were previously shown to harbor a mycovirus named Hymenoscyphus fraxineus Mitovirus 1 (HfMV1). In the present study, we describe a conspecific mycovirus that we detected in H. albidus. HfMV1 was consistently identified in H. albidus isolates (mean prevalence: 49.3%) which were collected in the sampling areas before the arrival of ash dieback. HfMV1 strains in both fungal hosts contain a single ORF of identical length (717 AA) for which a mean pairwise identity of 94.5% was revealed. The occurrence of a conspecific mitovirus in H. albidus and H. fraxineus is most likely the result of parallel virus evolution in the two fungal hosts. HfMV1 sequences from H. albidus showed a higher nucleotide diversity and a higher number of mutations compared to those from H. fraxineus, probably due to a bottleneck caused by the introduction of H. fraxineus in Europe. Our data also points to multiple interspecific virus transfers from H. albidus to H. fraxineus, which could have contributed to the intraspecific virus diversity found in H. fraxineus.

MAT--gene structure and mating behavior of Hymenoscyphus fraxineus and Hymenoscyphus albidus.

Recently, different reproductive modes were proposed between the emerging forest pathogen Hymenoscyphus fraxineus and its closely related avirulent sister species, Hymenoscyphus albidus. In the present study, inter- and intraspecific crosses were performed to experimentally assess the reproduction barriers between the two species and to verify H. albidus' putative haploid-selfing reproductive mode. By means of H. fraxineus-specific microsatellite markers, no hybridization was observed in 29 apothecia that emerged from inter-specific crosses, suggesting reproduction barriers are well-established. In a similar experimental setup, we used two newly developed polymorphic H. albidus-specific microsatellites to show that haploid-selfing is H. albidus' only reproductive mode (N=17 apothecia). Further to this, the reproductive modes of both species were investigated under natural conditions. Microsatellite allele-segregation studies of H. fraxineus' single-spore progeny of apothecia (N=31) from field samples suggest that often more than two paternal nuclei are involved in mating. In contrast, analysis of single-spore progeny of field-collected H. albidus apothecia (N=21) confirmed the solely haploid-selfing reproductive mode detected in vitro. Furthermore, we present the complete mating type 1-1 locus of H. fraxineus and report the finding of three additional genes within this region; the as yet unobserved typical mating type gene MAT1-1-1, a DNA polymerase zeta catalytic subunit-like gene and a pre-mRNA-splicing factor SLU7-like gene. The same genes were also detected in the homothallic mating type locus of H. albidus. Further analysis confirmed the expression of all typical mating type genes (MAT1-2-1, MAT1-1-3, MAT1-1-1) in both species. Interestingly, the MAT1-1-3 gene of homothallic H. albidus is expressed despite three stop codons interrupting the coding sequence. Overall, our findings highlight vital differences in the reproduction systems of the two species and suggest that interspecific hybridization is not possible.

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus.

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemic's front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (GST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.

Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback.

The ascomycete Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) causes a lethal disease known as ash dieback on Fraxinus excelsior and Fraxinus angustifolia in Europe. The pathogen was probably introduced from East Asia and the disease emerged in Poland in the early 1990s; the subsequent epidemic is spreading to the entire native distribution range of the host trees. This pathogen profile represents a comprehensive review of the state of research from the discovery of the pathogen and points out knowledge gaps and research needs. TAXONOMY: Members of the genus Hymenoscyphus (Helotiales, Leotiomycetidae, Leotiomycetes, Ascomycota) are small discomycetes which form their ascomata on dead plant material. A phylogeny based on the internal transcribed spacers (ITSs) of the rDNA indicated the avirulent Hymenoscyphus albidus, a species native to Europe, as the closest relative of H. pseudoalbidus. SYMPTOMS: Hymenoscyphus pseudoalbidus causes necrotic lesions on leaves, twigs and stems, eventually leading to wilting and dieback of girdled shoots. Bark lesions are characterized by a typical dark- to cinnamon-brown discoloration. LIFE CYCLE: Hymenoscyphus pseudoalbidus is heterothallic and reproduces sexually on ash petioles in the litter once a year. Ascospores are wind dispersed and infect ash leaves during the summer. The asexual spores only serve as spermatia. TOOLS AND TECHNIQUES: The most important techniques for fungal handling, such as detection, isolation, culturing, storage, crossing and ascocarp production, are briefly described. MANAGEMENT: Once the disease is established, management is hardly possible. The occurrence of a small fraction of partially tolerant trees constitutes hope for resistance breeding in the future. Healthy-looking trees should be preserved.