Development of a novel mycobiome diagnostic for fungal infection.

BACKGROUND: Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target. METHODS: Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed. RESULTS: The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data. CONCLUSIONS: This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Ascomycetes yeasts: The hidden part of human microbiome.

The fungal component of the microbiota, the mycobiota, has been neglected for a long time due to its poor richness compared to bacteria. Limitations in fungal detection and taxonomic identification arise from using metagenomic approaches, often borrowed from bacteriome analyses. However, the relatively recent discoveries of the ability of fungi to modulate the host immune response and their involvement in human diseases have made mycobiota a fundamental component of the microbial communities inhabiting the human host, deserving some consideration in host-microbe interaction studies and in metagenomics. Here, we reviewed recent data on the identification of yeasts of the Ascomycota phylum across human body districts, focusing on the most representative genera, that is, Saccharomyces and Candida. Then, we explored the key factors involved in shaping the human mycobiota across the lifespan, ranging from host genetics to environment, diet, and lifestyle habits. Finally, we discussed the strengths and weaknesses of culture-dependent and independent methods for mycobiota characterization. Overall, there is still room for some improvements, especially regarding fungal-specific methodological approaches and bioinformatics challenges, which are still critical steps in mycobiota analysis, and to advance our knowledge on the role of the gut mycobiota in human health and disease. This article is categorized under: Immune System Diseases > Genetics/Genomics/Epigenetics Immune System Diseases > Environmental Factors Infectious Diseases > Environmental Factors.

Shotgun metagenomics reveals interkingdom association between intestinal bacteria and fungi involving competition for nutrients.

BACKGROUND: The accuracy of internal-transcribed-spacer (ITS) and shotgun metagenomics has not been robustly evaluated, and the effect of diet on the composition and function of the bacterial and fungal gut microbiome in a longitudinal setting has been poorly investigated. Here we compared two approaches to study the fungal community (ITS and shotgun metagenomics), proposed an enrichment protocol to perform a reliable mycobiome analysis using a comprehensive in-house fungal database, and correlated dietary data with both bacterial and fungal communities. RESULTS: We found that shotgun DNA sequencing after a new enrichment protocol combined with the most comprehensive and novel fungal databases provided a cost-effective approach to perform gut mycobiome profiling at the species level and to integrate bacterial and fungal community analyses in fecal samples. The mycobiome was significantly more variable than the bacterial community at the compositional and functional levels. Notably, we showed that microbial diversity, composition, and functions were associated with habitual diet composition instead of driven by global dietary changes. Our study indicates a potential competitive inter-kingdom interaction between bacteria and fungi for food foraging. CONCLUSION: Together, our present work proposes an efficient workflow to study the human gut microbiome integrating robustly fungal, bacterial, and dietary data. These findings will further advance our knowledge of the interaction between gut bacteria and fungi and pave the way for future investigations in human mycobiome. Video Abstract.

Stable functional structure despite high taxonomic variability across fungal communities in soils of old-growth montane forests.

BACKGROUND: Major advances over the past decade in molecular ecology are providing access to soil fungal diversity in forest ecosystems worldwide, but the diverse functions and metabolic capabilities of this microbial community remain largely elusive. We conducted a field survey in montane old-growth broadleaved and conifer forests, to investigate the relationship between soil fungal diversity and functional genetic traits. To assess the extent to which variation in community composition was associated with dominant tree species (oak, spruce, and fir) and environmental variations in the old-growth forests in the Jade Dragon Snow Mountain in Yunnan Province, we applied rDNA metabarcoding. We also assessed fungal gene expression in soil using mRNA sequencing and specifically assessed the expression of genes related to organic matter decomposition and nutrient acquisition in ectomycorrhizal and saprotrophic fungi. RESULTS: Our taxonomic profiling revealed striking shifts in the composition of the saprotrophic and ectomycorrhizal guilds among the oak-, fir-, and spruce-dominated forests. The core fungal microbiome comprised only ~ 20% of the total OTUs across all soil samples, although the overlap between conifer-associated communities was substantial. In contrast, seasonality and soil layer explained only a small proportion of the variation in community structure. However, despite their highly variable taxonomic composition, fungal guilds exhibited remarkably similar functional traits for growth-related and core metabolic pathways across forest associations, suggesting ecological redundancy. However, we found that the expression profiles of genes related to polysaccharide and protein degradation and nutrient transport notably varied between and within the fungal guilds, suggesting niche adaptation. CONCLUSIONS: Overall, our metatranscriptomic analyses revealed the functional potential of soil fungal communities in montane old-growth forests, including a suite of specialized genes and taxa involved in organic matter decomposition. By linking genes to ecological traits, this study provides insights into fungal adaptation strategies to biotic and environmental factors, and sheds light on the importance of understanding functional gene expression patterns in predicting ecosystem functioning. Video Abstract.

Enterotypes of the human gut mycobiome.

BACKGROUND: The fungal component of the human gut microbiome, also known as the mycobiome, plays a vital role in intestinal ecology and human health. However, the overall structure of the gut mycobiome as well as the inter-individual variations in fungal composition remains largely unknown. In this study, we collected a total of 3363 fungal sequencing samples from 16 cohorts across three continents, including 572 newly profiled samples from China. RESULTS: We identify and characterize four mycobiome enterotypes using ITS profiling of 3363 samples from 16 cohorts. These enterotypes exhibit stability across populations and geographical locations and significant correlation with bacterial enterotypes. Particularly, we notice that fungal enterotypes have a strong age preference, where the enterotype dominated by Candida (i.e., Can_type enterotype) is enriched in the elderly population and confers an increased risk of multiple diseases associated with a compromised intestinal barrier. In addition, bidirectional mediation analysis reveals that the fungi-contributed aerobic respiration pathway associated with the Can_type enterotype might mediate the association between the compromised intestinal barrier and aging. CONCLUSIONS: We show that the human gut mycobiome has stable compositional patterns across individuals and significantly correlates with multiple host factors, such as diseases and host age. Video Abstract.

Quantifying the mycobiome and its major constituents on the skin of 20 normal dogs.

OBJECTIVE: To report the density, and constituents, of the mycobiome on the skin surface of normal dogs. ANIMALS: 20 normal dogs were recruited for this study, with informed consent in all cases. METHODS: Flocked swabs were used to sample the skin surface and to sample the skin surface after superficial scraping with a blunted scapula. Both samples were taken within a brass guide with an internal area of 3.5 cm-2. Next-generation DNA sequencing was used to identify and quantify components of the mycobiome. RESULTS: The median density of the mycobiome was 1.1 X 105 cm-2 (IQR, 27,561, 409,572). Cladosporium spp and Vishniacozyma victoriae were found on all 20 dogs. CLINICAL RELEVANCE: Knowledge of the density and the composition of the cutaneous mycobiome will increase our understanding of skin biology and may have relevance to future therapeutic trials.

Cryptic functional diversity within a grass mycobiome.

Eukaryotic hosts harbor tremendously diverse microbiomes that affect host fitness and response to environmental challenges. Fungal endophytes are prominent members of plant microbiomes, but we lack information on the diversity in functional traits affecting their interactions with their host and environment. We used two culturing approaches to isolate fungal endophytes associated with the widespread, dominant prairie grass Andropogon gerardii and characterized their taxonomic diversity using rDNA barcode sequencing. A randomly chosen subset of fungi representing the diversity of each leaf was then evaluated for their use of different carbon compound resources and growth on those resources. Applying community phylogenetic analyses, we discovered that these fungal endophyte communities are comprised of phylogenetically distinct assemblages of slow- and fast-growing fungi that differ in their use and growth on differing carbon substrates. Our results demonstrate previously undescribed and cryptic functional diversity in carbon resource use and growth in fungal endophyte communities of A. gerardii.

Compartments of roots and mature leaves are key hubs in the connectivity of tea-plant mycobiomes and are influenced by environmental factors and host age.

Understanding the tripartite consortium of crop, mycobiome, and environment is necessary to advance smart farming. Owing to their life cycle of hundreds of years, tea plants are excellent models for studying these entwined relationships; however, observations on this globally important cash crop with numerous health benefits are still rudimentary. Here, the fungal taxa along the soil-tea plant continuum in tea gardens of different ages in famous high-quality tea-growing regions in China were characterized using DNA metabarcoding. Using machine learning, we dissected the spatiotemporal distribution, co-occurrence patterns, assembly, and their associations in different compartments of tea-plant mycobiomes, and further explored how these potential interactions were driven by environmental factors and tree age, and how they influenced the market prices of tea. The results revealed that Compartment niche differentiation was the key driving force behind variation in the tea-plant mycobiome. The mycobiome of roots had the highest specific proportion and convergence and almost did not overlap with the soil. The enrichment ratio of developing leaves to root mycobiome increased with increasing tree age, while mature leaves showed the highest value in the Laobanzhang (LBZ) tea garden with top market prices and displayed the strongest depletion effect on mycobiome association along the soil-tea plant continuum. The balance between determinism and stochasticity in the assembly process was co-driven by compartment niches and life cycle variation. Fungal guild analysis showed that altitude indirectly affected market prices of tea by mediating the abundance of the plant pathogen. The relative importance of plant pathogen and ectomycorrhizae could be used to assess the age of tea. Biomarkers were mainly distributed in soil compartments, and Clavulinopsis miyabeana, Mortierella longata, and Saitozyma sp. may affect the spatiotemporal dynamics of tea-plant mycobiomes and their ecosystem services. Soil properties (mainly total potassium) and tree age indirectly affected the developing leaves via positively influencing the mycobiome of mature leaves. In contrast, the climate directly and significantly drove the mycobiome composition of the developing leaves. Moreover, the proportion of negative correlations in the co-occurrence network positively regulated tea-plant mycobiome assembly, which significantly affected the market prices of tea in the structural equation model with network complexity as hub. These findings indicate that mycobiome signatures play pivotal roles in the adaptive evolution and fungal disease control of tea plants and can help develop better agricultural practices that focus on both plant health and financial profits, and provide a new strategy for assessing tea quality grade and age.

Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

The structure of root-associated fungal communities is related to the long-term effects of plant diversity on productivity.

Root-associated fungi could play a role in determining both the positive relationship between plant diversity and productivity in experimental grasslands, and its strengthening over time. This hypothesis assumes that specialized pathogenic and mutualistic fungal communities gradually assemble over time, enhancing plant growth more in species-rich than in species-poor plots. To test this hypothesis, we used high-throughput amplicon sequencing to characterize root-associated fungal communities in experimental grasslands of 1 and 15 years of age with varying levels of plant species richness. Specifically, we tested whether the relationship between fungal communities and plant richness and productivity becomes stronger with the age of the experimental plots. Our results showed that fungal diversity increased with plant diversity, but this relationship weakened rather than strengthened over the two time points. Contrastingly, fungal community composition showed increasing associations with plant diversity over time, suggesting a gradual build-up of specific fungal assemblages. Analyses of different fungal guilds showed that these changes were particularly marked in pathogenic fungi, whose shifts in relative abundance are consistent with the pathogen dilution hypothesis in diverse plant communities. Our results suggest that root-associated fungal pathogens play more specific roles in determining the diversity-productivity relationship than other root-associated plant symbionts.

Porcine fungal mock community analyses: Implications for mycobiome investigations.

Introduction: The gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. Next generation sequencing has improved our understanding of fungi in the gut, but methodological challenges remain. Biases are introduced during DNA isolation, primer design and choice, polymerase selection, sequencing platform selection, and data analyses, as fungal reference databases are often incomplete or contain erroneous sequences. Methods: Here, we compared the accuracy of taxonomic identifications and abundances from mycobiome analyses which vary among three commonly selected target gene regions (18S, ITS1, or ITS2) and the reference database (UNITE - ITS1, ITS2 and SILVA - 18S). We analyze multiple communities including individual fungal isolates, a mixed mock community created from five common fungal isolates found in weanling piglet feces, a purchased commercial fungal mock community, and piglet fecal samples. In addition, we calculated gene copy numbers for the 18S, ITS1, and ITS2 regions of each of the five isolates from the piglet fecal mock community to determine whether copy number affects abundance estimates. Finally, we determined the abundance of taxa from several iterations of our in-house fecal community to assess the effects of community composition on taxon abundance. Results: Overall, no marker-database combination consistently outperformed the others. Internal transcribed space markers were slightly superior to 18S in the identification of species in tested communities, but Lichtheimia corymbifera, a common member of piglet gut communities, was not amplified by ITS1 and ITS2 primers. Thus, ITS based abundance estimates of taxa in piglet mock communities were skewed while 18S marker profiles were more accurate. Kazachstania slooffiae displayed the most stable copy numbers (83-85) while L. corymbifera displayed significant variability (90-144) across gene regions. Discussion: This study underscores the importance of preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest and raises questions regarding the validity of fungal abundance estimates.

Metatranscriptomics and metabarcoding reveal spatiotemporal shifts in fungal communities and their activities in Chinese coastal waters.

Fungal communities are diverse and abundant in coastal waters, yet, their ecological roles and adaptations remain largely unknown. To address these gaps, ITS2 metabarcoding and metatranscriptomic analyses were used to capture the whole suite of fungal diversity and their metabolic potential in water column and sediments in the Yellow Sea during August and October 2019. ITS2 metabarcoding described successfully the abundance of Dikarya during August and October at the different examined habitats, but strongly underrepresented or failed to identify other fungal taxa, including zoosporic and early-diverging lineages, that were abundant in the mycobiome as uncovered by metatranscriptomes. Metatranscriptomics also revealed enriched expression of genes annotated to zoosporic fungi (e.g., chytrids) mainly in the surface water column in October. This enriched expression was correlated with the two-fold increase in chlorophyll-a intensity attributed to phytoplanktonic species which are known to be parasitized by chytrids. The concurrent high expression of genes related to calcium signalling and GTPase activity suggested that these metabolic traits facilitate the parasitic lifestyle of chytrids. Similarly, elevated expression of phagosome genes annotated to Rozellomycota, an early-diverging fungal phylum not fully detected with ITS2 metabarcoding, suggested that this taxon utilizes a suite of feeding modes, including phagotrophy in this coastal setting. Our data highlight the necessity of using combined approaches to accurately describe the community structure of coastal mycobiome. We also provide in-depth insights into the fungal ecological roles in coastal waters, and report potential metabolic mechanisms utilized by fungi to cope with environmental stresses that occur during distinct seasonal months in coastal ecosystems.

Impact of diet and host genetics on the murine intestinal mycobiome.

The mammalian gut is home to a diverse microbial ecosystem, whose composition affects various physiological traits of the host. Next-generation sequencing-based metagenomic approaches demonstrated how the interplay of host genetics, bacteria, and environmental factors shape complex traits and clinical outcomes. However, the role of fungi in these complex interactions remains understudied. Here, using 228 males and 363 females from an advanced-intercross mouse line, we provide evidence that fungi are regulated by host genetics. In addition, we map quantitative trait loci associated with various fungal species to single genes in mice using whole genome sequencing and genotyping. Moreover, we show that diet and its' interaction with host genetics alter the composition of fungi in outbred mice, and identify fungal indicator species associated with different dietary regimes. Collectively, in this work, we uncover an association of the intestinal fungal community with host genetics and a regulatory role of diet in this ecological niche.

Sapwood mycobiome varies across host, plant compartment and environments in Nothofagus forests from Northern Patagonia.

Global forests are increasingly being threatened by altered climatic conditions and increased attacks by pests and pathogens. The complex ecological interactions among pathogens, microbial communities, tree hosts and the environment are important drivers of forest dynamics. Little is known about the ecology of forest pathology and related microbial communities in temperate forests of the southern hemisphere. In this study, we used next-generation sequencing to characterize sapwood-inhabiting fungal communities in North Patagonian Nothofagus forests and assessed patterns of diversity of taxa and ecological guilds across climatic, site and host variables (health condition and compartment) as a contribution to Nothofagus autecology. The diversity patterns inferred through the metabarcoding analysis were similar to those obtained through culture-dependent approaches. However, we detected additional heterogeneity and greater richness with culture-free methods. Host species was the strongest driver of fungal community structure and composition, while host health status was the weakest. The relative impacts of site, season, plant compartment and health status were different for each tree species; these differences can be interpreted as a matter of water availability. For Nothofagus dombeyi, which is distributed across a wide range of climatic conditions, site was the strongest driver of community composition. The microbiome of N. pumilio varied more with season and temperature, a relevant factor for forest conservation in the present climate change scenario. Both species carry a number of potential fungal pathogens in their sapwood, whether they exhibit symptoms or not. Our results provide insight into the diversity of fungi associated with the complex pathobiome of the dominant Nothofagus species in southern South America.

Los bosques del mundo están cada vez más amenazados por las condiciones climáticas alteradas y el aumento de los ataques de plagas y patógenos. Las complejas interacciones ecológicas entre los patógenos, las comunidades microbianas, los árboles hospedantes y el medio ambiente son impulsores importantes de la dinámica forestal. Poco se sabe sobre la ecología de la patología forestal y las comunidades microbianas relacionadas en los bosques templados del hemisferio sur. En este estudio, utilizamos la secuenciación Illumina para caracterizar las comunidades de hongos que habitan en la albura en los bosques de Nothofagus de la Patagonia Norte y evaluamos los patrones de diversidad de taxones y gremios ecológicos a través de variables climáticas, de sitio y de hospedante (identidad, condición de salud y compartimento) como una contribución a la autoecología de los Nothofagus. Los patrones de diversidad inferidos a través del análisis metabarcoding fueron similares a los obtenidos a través de enfoques dependientes de cultivo. Sin embargo, detectamos mayor heterogeneidad y mayor riqueza con métodos independientes de cultivo. La especie hospedante fue el modelador más fuerte de la estructura y composición de la comunidad fúngica, mientras que el estado de salud del hospedante fue el más débil. El impacto relativo del sitio, la estación, el compartimento y el estado de salud fueron diferentes para cada especie de árbol; estas diferencias pueden interpretarse en clave de disponibilidad de agua. Para N. dombeyi, que se distribuye a lo largo de una amplia gama de condiciones climáticas, el sitio fue el principal modelador de la composición de la comunidad. El micobioma de Nothofagus pumilio varió más con la estación y la temperatura, un factor relevante para la conservación de los bosques en el escenario actual de cambio climático. Ambas especies portan una serie de patógenos fúngicos potenciales en su albura, ya sea que muestren síntomas o no. Nuestros resultados brindan una idea de la diversidad de hongos asociados con el complejo patobioma de las especies dominantes de Nothofagus en el sur de América del Sur.

Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

Fungal species participate in vast numbers of processes in the landscape around us. However, their cryptic mycelial growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enables identification and relative quantification of fungal community members across well-replicated experimental settings.Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, i.e., the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon pool to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile.

Ecological stoichiometry and fungal community turnover reveal variation among mycorrhizal partners in their responses to warming and drought.

Symbiotic fungi mediate important energy and nutrient transfers in terrestrial ecosystems. Environmental change can lead to shifts in communities of symbiotic fungi, but the consequences of these shifts for nutrient dynamics among symbiotic partners are poorly understood. Here, we assessed variation in carbon (C), nitrogen (N) and phosphorus (P) in tissues of arbuscular mycorrhizal (AM) fungi and a host plant (Medicago sativa) in response to experimental warming and drought. We linked compositional shifts in AM fungal communities in roots and soil to variation in hyphal chemistry by using high-throughput DNA sequencing and joint species distribution modelling. Compared to plants, AM hyphae was 43% lower in (C) and 24% lower in (N) but more than nine times higher in (P), with significantly lower C:N, C:P and N:P ratios. Warming and drought resulted in increases in (P) and reduced C:P and N:P ratios in all tissues, indicating fungal P accumulation was exacerbated by climate-associated stress. Warming and drought modified the composition of AM fungal communities, and many of the AM fungal genera that were linked to shifts in mycelial chemistry were also negatively impacted by climate variation. Our study offers a unified framework to link climate change, fungal community composition, and community-level functional traits. Thus, our study provides insight into how environmental change can alter ecosystem functions via the promotion or reduction of fungal taxa with different stoichiometric characteristics and responses.

Nanopore sequencing of full rRNA operon improves resolution in mycobiome analysis and reveals high diversity in both human gut and environments.

High-throughput sequencing has substantially improved our understanding of fungal diversity. However, the short read (<500 bp) length of current second-generation sequencing approaches provides limited taxonomic and phylogenetic resolution for species discrimination. Longer sequences containing more information are highly desired to provide greater taxonomic resolution. Here, we amplified full-length rRNA operons (~5.5 kb) and established a corresponding fungal rRNA operon database for ONT sequences (FRODO), which contains ONT sequences representing eight phyla, 41 classes, 109 orders, 256 families, 524 genera and 1116 species. We also benchmarked the optimal method for sequence classification and determined that the RDP classifier based on our FRODO database was capable of improving the classification of ONT reads, with an average of 98%-99% reads correctly classified at the genus or species level. We investigated the applicability of our approach in three representative mycobiomes, namely, the soil, marine and human gut mycobiomes, and found that the gut contains the largest number of unknown species (over 90%), followed by the marine (42%) and soil (33.8%) mycobiomes. We also observed a distinct difference in the composition of the marine and soil mycobiomes, with the highest richness and diversity detected in soils. Overall, our study provides a systematic approach for mycobiome studies and revealed that the previous methods might have underestimated the diversity of mycobiome species. Future application of this method will lead to a better understanding of the taxonomic and functional diversity of fungi in environmental and health-related mycobiomes.

Assessing the causes and consequences of gut mycobiome variation in a wild population of the Seychelles warbler.

BACKGROUND: Considerable research has focussed on the importance of bacterial communities within the vertebrate gut microbiome (GM). However, studies investigating the significance of other microbial kingdoms, such as fungi, are notably lacking, despite their potential to influence host processes. Here, we characterise the fungal GM of individuals living in a natural population of Seychelles warblers (Acrocephalus sechellensis). We evaluate the extent to which fungal GM structure is shaped by environment and host factors, including genome-wide heterozygosity and variation at key immune genes (major histocompatibility complex (MHC) and Toll-like receptor (TLR)). Importantly, we also explore the relationship between fungal GM differences and subsequent host survival. To our knowledge, this is the first time that the genetic drivers and fitness consequences of fungal GM variation have been characterised for a wild vertebrate population. RESULTS: Environmental factors, including season and territory quality, explain the largest proportion of variance in the fungal GM. In contrast, neither host age, sex, genome-wide heterozygosity, nor TLR3 genotype was associated with fungal GM differences in Seychelles warblers. However, the presence of four MHC-I alleles and one MHC-II allele was associated with changes in fungal GM alpha diversity. Changes in fungal richness ranged from between 1 and 10 sequencing variants lost or gained; in some cases, this accounted for 20% of the fungal variants carried by an individual. In addition to this, overall MHC-I allelic diversity was associated with small, but potentially important, changes in fungal GM composition. This is evidenced by the fact that fungal GM composition differed between individuals that survived or died within 7 months of being sampled. CONCLUSIONS: Our results suggest that environmental factors play a primary role in shaping the fungal GM, but that components of the host immune system-specifically the MHC-may also contribute to the variation in fungal communities across individuals within wild populations. Furthermore, variation in the fungal GM can be associated with differential survival in the wild. Further work is needed to establish the causality of such relationships and, thus, the extent to which components of the GM may impact host evolution. Video Abstract.