Insights into the Ecological Diversification of the Hymenochaetales based on Comparative Genomics and Phylogenomics With an Emphasis on Coltricia.

To elucidate the genomic traits of ecological diversification in the Hymenochaetales, we sequenced 15 new genomes, with attention to ectomycorrhizal (EcM) Coltricia species. Together with published data, 32 genomes, including 31 Hymenochaetales and one outgroup, were comparatively analyzed in total. Compared with those of parasitic and saprophytic members, EcM species have significantly reduced number of plant cell wall degrading enzyme genes, and expanded transposable elements, genome sizes, small secreted proteins, and secreted proteases. EcM species still retain some of secreted carbohydrate-active enzymes (CAZymes) and have lost the key secreted CAZymes to degrade lignin and cellulose, while possess a strong capacity to degrade a microbial cell wall containing chitin and peptidoglycan. There were no significant differences in secreted CAZymes between fungi growing on gymnosperms and angiosperms, suggesting that the secreted CAZymes in the Hymenochaetales evolved before differentiation of host trees into gymnosperms and angiosperms. Nevertheless, parasitic and saprophytic species of the Hymenochaetales are very similar in many genome features, which reflect their close phylogenetic relationships both being white rot fungi. Phylogenomic and molecular clock analyses showed that the EcM genus Coltricia formed a clade located at the base of the Hymenochaetaceae and divergence time later than saprophytic species. And Coltricia remains one to two genes of AA2 family. These indicate that the ancestors of Coltricia appear to have originated from saprophytic ancestor with the ability to cause a white rot. This study provides new genomic data for EcM species and insights into the ecological diversification within the Hymenochaetales based on comparative genomics and phylogenomics analyses.

DNA metabarcoding reveals host-specific communities of arthropods residing in fungal fruit bodies.

Biological communities within living organisms are structured by their host's traits. How host traits affect biodiversity and community composition is poorly explored for some associations, such as arthropods within fungal fruit bodies. Using DNA metabarcoding, we characterized the arthropod communities in living fruit bodies of 11 wood-decay fungi from boreal forests and investigated how they were affected by different fungal traits. Arthropod diversity was higher in fruit bodies with a larger surface area-to-volume ratio, suggesting that colonization is crucial to maintain arthropod populations. Diversity was not higher in long-lived fruit bodies, most likely because these fungi invest in physical or chemical defences against arthropods. Arthropod community composition was structured by all measured host traits, namely fruit body size, thickness, surface area, morphology and toughness. Notably, we identified a community gradient where soft and short-lived fruit bodies harboured more true flies, while tougher and long-lived fruit bodies had more oribatid mites and beetles, which might reflect different development times of the arthropods. Ultimately, close to 75% of the arthropods were specific to one or two fungal hosts. Besides revealing surprisingly diverse and host-specific arthropod communities within fungal fruit bodies, our study provided insight into how host traits structure communities.

The Indoor Mycobiomes of Daycare Centers Are Affected by Occupancy and Climate.

Many children spend considerable time in daycare centers and may be influenced by the indoor microorganisms there, including fungi. In this study, we investigate the indoor mycobiomes of 125 daycare centers distributed along strong environmental gradients throughout Norway. Dust samples were collected from doorframes outside and inside buildings using a community science sampling approach. Fungal communities in the dust samples were analyzed using DNA metabarcoding of the internal transcribed spacer 2 (ITS2) region. We observed a marked difference between the outdoor and indoor mycobiomes. The indoor mycobiomes included considerably more yeasts and molds than the outdoor samples, with Saccharomyces, Mucor, Malassezia, and Penicillium being among the most dominant fungal genera. Changes in the indoor fungal richness and composition correlated with numerous variables related to both outdoor and indoor conditions; there was a clear geographic structure in the indoor mycobiome composition that mirrored the outdoor climate, ranging from humid areas in western Norway to drier and colder areas in eastern Norway. Moreover, the number of children in the daycare centers, as well as various building features, influenced the indoor mycobiome composition. We conclude that the indoor mycobiomes in Norwegian daycare centers are structured by multiple factors and are dominated by yeasts and molds. This study exemplifies how community science sampling enables DNA-based analyses of a high number of samples covering wide geographic areas. IMPORTANCE With an alarming increase in chronic diseases like childhood asthma and allergies, there is an increased focus on the exposure of young children to indoor biological and chemical air pollutants. Our study of 125 daycares throughout Norway demonstrates that the indoor mycobiome not only reflects cooccurring outdoor fungi but also includes a high abundance of yeast and mold fungi with an affinity for indoor environments. A multitude of factors influence the indoor mycobiomes in daycares, including the building type, inhabitants, as well as the outdoor environment. Many of the detected yeasts and molds are likely associated with the human body, where some have been coupled with allergies and respiratory problems. Our results call for further studies investigating the potential impact of the identified daycare-associated mycobiomes on children's health.

Population genomics of a forest fungus reveals high gene flow and climate adaptation signatures.

Genome sequencing of spatially distributed individuals sheds light on how evolution structures genetic variation. Populations of Phellopilus nigrolimitatus, a red-listed wood-inhabiting fungus associated with old-growth coniferous forests, have decreased in size over the last century due to a loss of suitable habitats. We assessed the population genetic structure and investigated local adaptation in P. nigrolimitatus, by establishing a reference genome and genotyping 327 individuals sampled from 24 locations in Northern Europe by RAD sequencing. We revealed a shallow population genetic structure, indicating large historical population sizes and high levels of gene flow. Despite this weak substructuring, two genetic groups were recognized; a western group distributed mostly in Norway and an eastern group covering most of Finland, Poland and Russia. This substructuring may reflect coimmigration with the main host, Norway spruce (Picea abies), into Northern Europe after the last ice age. We found evidence of low levels of genetic diversity in southwestern Finland, which has a long history of intensive forestry and urbanization. Numerous loci were significantly associated with one or more environmental factors, indicating adaptation to specific environments. These loci clustered into two groups with different associations with temperature and precipitation. Overall, our findings indicate that the current population genetic structure of P. nigrolimitatus results from a combination of gene flow, genetic drift and selection. The acquisition of similar knowledge especially over broad geographic scales, linking signatures of adaptive genetic variation to evolutionary processes and environmental variation, for other fungal species will undoubtedly be useful for assessment of the combined effects of habitat fragmentation and climate change on fungi strongly bound to old-growth forests.

Spatiotemporal variation of the indoor mycobiome in daycare centers.

BACKGROUND: Children spend considerable time in daycare centers in parts of the world and are exposed to the indoor micro- and mycobiomes of these facilities. The level of exposure to microorganisms varies within and between buildings, depending on occupancy, climate, and season. In order to evaluate indoor air quality, and the effect of usage and seasonality, we investigated the spatiotemporal variation in the indoor mycobiomes of two daycare centers. We collected dust samples from different rooms throughout a year and analyzed their mycobiomes using DNA metabarcoding. RESULTS: The fungal community composition in rooms with limited occupancy (auxiliary rooms) was similar to the outdoor samples, and clearly different from the rooms with higher occupancy (main rooms). The main rooms had higher abundance of Ascomycota, while the auxiliary rooms contained comparably more Basidiomycota. We observed a strong seasonal pattern in the mycobiome composition, mainly structured by the outdoor climate. Most markedly, basidiomycetes of the orders Agaricales and Polyporales, mainly reflecting typical outdoor fungi, were more abundant during summer and fall. In contrast, ascomycetes of the orders Saccharomycetales and Capnodiales were dominant during winter and spring. CONCLUSIONS: Our findings provide clear evidences that the indoor mycobiomes in daycare centers are structured by occupancy as well as outdoor seasonality. We conclude that the temporal variability should be accounted for in indoor mycobiome studies and in the evaluation of indoor air quality of buildings. Video abstract.

Contrasting demographic histories revealed in two invasive populations of the dry rot fungus Serpula lacrymans.

Globalization and international trade have impacted organisms around the world leading to a considerable number of species establishing in new geographic areas. Many organisms have taken advantage of human-made environments, including buildings. One such species is the dry rot fungus Serpula lacrymans, which is the most aggressive wood-decay fungus in indoor environments in temperate regions. Using population genomic analyses of 36 full genome sequenced isolates, we demonstrated that European and Japanese isolates are highly divergent and the populations split 3000-19,000 generations ago, probably predating human influence. Approximately 250 generations ago, the European population went through a tight bottleneck, probably corresponding to the fungus colonization of the built environment in Europe. The demographic history of these populations, probably lead to low adaptive potential. Only two loci under selection were identified using a Fst outlier approach, and selective sweep analyses identified three loci with extended haplotype homozygosity. The selective sweep analyses found signals in genes possibly related to decay of various substrates in Japan and in genes involved DNA replication and protein modification in Europe. Our results suggest that the dry rot fungus independently established in indoor environments in Europe and Japan and that invasive species can potentially establish large populations in new habitats based on a few colonizing individuals.

Analysing indoor mycobiomes through a large-scale citizen science study in Norway.

In the built environment, fungi can cause important deterioration of building materials and have adverse health effects on occupants. Increased knowledge about indoor mycobiomes from different regions of the world, and their main environmental determinants, will enable improved indoor air quality management and identification of health risks. This is the first citizen science study of indoor mycobiomes at a large geographical scale in Europe, including 271 houses from Norway and 807 dust samples from three house compartments: outside of the building, living room and bathroom. The fungal community composition determined by DNA metabarcoding was clearly different between indoor and outdoor samples, but there were no significant differences between the two indoor compartments. The 32 selected variables, related to the outdoor environment, building features and occupant characteristics, accounted for 15% of the overall variation in community composition, with the house compartment as the key factor (7.6%). Next, climate was the main driver of the dust mycobiomes (4.2%), while building and occupant variables had significant but minor influences (1.4% and 1.1%, respectively). The house-dust mycobiomes were dominated by ascomycetes (⁓70%) with Capnodiales and Eurotiales as the most abundant orders. Compared to the outdoor samples, the indoor mycobiomes showed higher species richness, which is probably due to the mixture of fungi from outdoor and indoor sources. The main indoor indicator fungi belonged to two ecological groups with allergenic potential: xerophilic moulds and skin-associated yeasts. Our results suggest that citizen science is a successful approach for unravelling the built microbiome at large geographical scales.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

The influence of intraspecific sequence variation during DNA metabarcoding: A case study of eleven fungal species.

DNA metabarcoding has become a powerful approach for analysing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers - like the fungal ITS region, may include considerable intraspecific variation, which can lead to oversplitting of species during DNA metabarcoding analyses. Here we assessed the effects of intraspecific sequence variation in DNA metabarcoding by analysing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS) coupled with error correction with the software dada2. All the eleven species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, typically occurring in low frequencies, were probably due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS-based fungal community analyses, but an extra clustering step is needed to approach species-level resolution.

Fungal sporocarps house diverse and host-specific communities of fungicolous fungi.

Sporocarps (fruit bodies) are the sexual reproductive stage in the life cycle of many fungi. They are highly nutritious and consequently vulnerable to grazing by birds and small mammals, and invertebrates, and can be infected by microbial and fungal parasites and pathogens. The complexity of communities thriving inside sporocarps is largely unknown. In this study, we revealed the diversity, taxonomic composition and host preference of fungicolous fungi (i.e., fungi that feed on other fungi) in sporocarps. We carried out DNA metabarcoding of the ITS2 region from 176 sporocarps of 11 wood-decay fungal host species, all collected within a forest in northeast Finland. We assessed the influence of sporocarp traits, such as lifespan, morphology and size, on the fungicolous fungal community. The level of colonisation by fungicolous fungi, measured as the proportion of non-host ITS2 reads, varied between 2.8-39.8% across the 11 host species and was largely dominated by Ascomycota. Host species was the major determinant of the community composition and diversity of fungicolous fungi, suggesting that host adaptation is important for many fungicolous fungi. Furthermore, the alpha diversity was consistently higher in short-lived and resupinate sporocarps compared to long-lived and pileate ones, perhaps due to a more hostile environment for fungal growth in the latter too. The fungicolous fungi represented numerous lineages in the fungal tree of life, among which a significant portion was poorly represented with reference sequences in databases.

Population genomics of an outbreak of the potato late blight pathogen, Phytophthora infestans, reveals both clonality and high genotypic diversity.

An outbreak of the potato late blight pathogen Phytophthora infestans in Denmark was characterized in order to resolve the population structure and determine to what extent sexual reproduction was occurring. A standard set of microsatellite simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers generated using restriction site-associated DNA sequencing (RAD-seq) were employed in parallel. A total of 83 individuals, isolated from seven different potato fields in 2014, were analysed together with five Danish whole-genome sequenced isolates, as well as two Mexican individuals used as an outgroup. From a filtered dataset of 55 288 SNPs, population genomics analyses revealed no sign of recombination, implying clonality. In spite of this, multilocus genotypes were unique to individual potato fields, with little evidence of gene flow between fields. Ploidy analysis performed on the SNPs dataset indicated that the majority of isolates were diploid. These contradictory results with clonality and high genotypic diversity may suggest that rare sexual events likely still contribute to the population. Comparison of the results generated by SSRs vs SNPs data indicated that large marker sets, generated by RAD-seq, may be advised going forward, as it provides a higher level of genetic discrimination than SSRs.

Population structure of Serpula lacrymans in Europe with an outlook to the French population.

In this study the genetic variation and population structure in a French population of the dry rot fungus S. lacrymans was investigated using 14 microsatellites markers and compared to the rest of Europe. In that comparison the French population possessed the same allelic diversity as rest of Europe. A weak geographic structuring of the genetic variation was observed across Europe, where the French isolates to some extent separated from the rest of Europe, indicating that weak barriers to gene flow exists. Eighty percent of the isolates had unique multilocus microsatellite genotypes, which corresponds to high recombination and dispersal by sexual spores. Deviations from Hardy-Weinberg expectations were observed in multiple loci. In most loci there was an excess of heterozygotes, which could be due to either non-random mating, presence of more than two nuclei in the secondary mycelia or another unrecognized process. A total of six vegetative compatibility (VC) groups were present in Europe, out of which four were sampled in France. One VC group was over-represented in France while two others were underrepresented, as compared to the rest of Europe.

Molecular characterization of sexual diversity in a population of Serpula lacrymans, a tetrapolar basidiomycete.

Different mating systems have evolved in the fungal kingdom, including a tetrapolar multiallelic mating system in many basidiomycetes. In tetrapolar species, the presence of different alleles at two mating loci (MAT A and MAT B) is necessary for mating to occur. The tetrapolar fungus Serpula lacrymans causes wood-decay in buildings in temperate regions worldwide and is present in Europe with a genetically homogeneous founder population. Using genome sequence data, we annotated the two mating type loci for S. lacrymans and found the expected synteny with other basidiomycetes, except for a retrotransposon being present in one locus (MAT A). We developed markers linked to the MAT A and MAT B regions and used these to investigate the mating type diversity in the European population. Moreover, we found a good match between the genetic markers and functional mating types as revealed by segregation and mating studies. A low diversity of mating types is present in the European S. lacrymans population caused by the founder event where a limited number of genotypes were introduced. This finding contrasts the situation in natural fungal populations where a high diversity of mating types is normally present. Although S. lacrymans has a large and viable population in Europe, we argue that the low mating type diversity restrains the dispersal and establishment of the fungus.

Improved molecular methods to characterise Serpula lacrymans and other Basidiomycetes involved in wood decay.

Early detection of indoor wood-decay fungi is crucial to prevent building deterioration and thereby avoid considerable economic loss. Due to their increased sensitivity, two reliable DNA-based fingerprinting techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) and denaturing high-performance liquid chromatography (DHPLC), were used to identify Serpula lacrymans and to profile wood-rot Basidiomycetes in the built environment. Molecular fungal diversity was assessed on 74 environmental samples, collected from 2003 to 2009 from infected buildings in France. S. lacrymans, the most widespread, indoor wood-decay fungus accounted for 64% of total wood-rot Basidiomycetes. A number of other common wood-rot fungi such as Coniophora puteana, Trametes versicolor and Donkioporia expansa were identified. Other Basidiomycetes such as Phlebiopsis gigantea and Scleroderma verrucosum were detected for the first time in the built environment. Reliable diagnostic tools were developed using two PCR-based molecular typing techniques, one for routine diagnosis and another one for community inventories. Together they provided useful data for characterising the complexity of wood-decay ecosystems and helped reveal the coexistence of different wood-decay fungi within the same microbiotope.