Environmental drivers and cryptic biodiversity hotspots define endophytes in Earth's largest terrestrial biome.

Understanding how symbiotic associations differ across environmental gradients is key to predicting the fate of symbioses as environments change, and it is vital for detecting global reservoirs of symbiont biodiversity in a changing world.1,2,3 However, sampling of symbiotic partners at the full-biome scale is difficult and rare. As Earth's largest terrestrial biome, boreal forests influence carbon dynamics and climate regulation at a planetary scale. Plants and lichens in this biome host the highest known phylogenetic diversity of fungal endophytes, which occur within healthy photosynthetic tissues and can influence hosts' resilience to stress.4,5 We examined how communities of endophytes are structured across the climate gradient of the boreal biome, focusing on the dominant plant and lichen species occurring across the entire south-to-north span of the boreal zone in eastern North America. Although often invoked for understanding the distribution of biodiversity, neither a latitudinal gradient nor mid-domain effect5,6,7 can explain variation in endophyte diversity at this trans-biome scale. Instead, analyses considering shifts in forest characteristics, Picea biomass and age, and nutrients in host tissues from 46° to 58° N reveal strong and distinctive signatures of climate in defining endophyte assemblages in each host lineage. Host breadth of endophytes varies with climate factors, and biodiversity hotspots can be identified at plant-community transitions across the boreal zone at a global scale. Placed against a backdrop of global circumboreal sampling,4 our study reveals the sensitivity of endophytic fungi, their reservoirs of biodiversity, and their important symbiotic associations, to climate.

Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi.

Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism's genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

Coniochaeta elegans sp. nov., Coniochaeta montana sp. nov. and Coniochaeta nivea sp. nov., three new species of endophytes with distinctive morphology and functional traits.

A growing interest in fungi that occur within symptom-less plants and lichens (endophytes) has uncovered previously uncharacterized species in diverse biomes worldwide. In many temperate and boreal forests, endophytic Coniochaeta (Sacc.) Cooke (Coniochaetaceae, Coniochaetales, Sordariomycetes, Ascomycota) are commonly isolated on standard media, but rarely are characterized. We examined 26 isolates of Coniochaeta housed at the Gilbertson Mycological Herbarium. The isolates were collected from healthy photosynthetic tissues of conifers, angiosperms, mosses and lichens in Canada, Sweden and the United States. Their barcode sequences (nuclear ribosomal internal transcribed spacer and 5.8S; ITS rDNA) were ≤97% similar to any documented species available through GenBank. Phylogenetic analyses based on two loci (ITS rDNA and translation elongation factor 1-alpha) indicated that two isolates represented Coniochaeta cymbiformispora, broadening the ecological niche and geographic range of a species known previously from burned soil in Japan. The remaining 24 endophytes represented three previously undescribed species that we characterize here: Coniochaeta elegans sp. nov., Coniochaeta montana sp. nov. and Coniochaeta nivea sp. nov. Each has a wide host range, including lichens, bryophytes and vascular plants. C. elegans sp. nov. and C. nivea sp. nov. have wide geographic ranges. C. montana sp. nov. occurs in the Madrean biome of Arizona (USA), where it is sympatric with the other species described here. All three species display protease, chitinase and cellulase activity in vitro. Overall, this study provides insight into the ecological and evolutionary diversity of Coniochaeta and suggests that these strains may be amenable for studies of traits relevant to a horizontally transmitted, symbiotic lifestyle.

Strobiloscyphones A-F, 6-Isopentylsphaeropsidones and Other Metabolites from Strobiloscypha sp. AZ0266, a Leaf-Associated Fungus of Douglas Fir.

Six new 6-isopentylsphaeropsidones, strobiloscyphones A-F (1-6), and a new hexadecanoic acid, (2Z,4E,6E)-8,9-dihydroxy-10-oxohexadeca-2,4,6-trienoic acid (7), together with sphaeropsidone (8) and its known synthetic analogue 5-dehydrosphaeropsidone (9) were isolated from Strobiloscypha sp. AZ0266, a fungus inhabiting the leaf litter of Douglas fir (Pseudotsuga menziesii). The structures of 1-7 were established on the basis of their high-resolution mass and 1D and 2D NMR spectroscopic data, and their relative and/or absolute configurations were determined by NOE, comparison of experimental and calculated ECD spectra, and application of the modified Mosher's ester method. Of these, strobiloscyphone F (6) contains a novel highly oxygenated tetracyclic oxireno-octahydrodibenzofuran ring system. Natural products 1, 6, and 9 and the semisynthetic analogue 12 derived from 8 exhibited cytotoxic activity, whereas 9 and 12 showed antimicrobial activity. Possible biosynthetic pathways to 1-6, 8, and 9 are proposed.

Climate and seasonality drive the richness and composition of tropical fungal endophytes at a landscape scale.

Understanding how species-rich communities persist is a foundational question in ecology. In tropical forests, tree diversity is structured by edaphic factors, climate, and biotic interactions, with seasonality playing an essential role at landscape scales: wetter and less seasonal forests typically harbor higher tree diversity than more seasonal forests. We posited that the abiotic factors shaping tree diversity extend to hyperdiverse symbionts in leaves-fungal endophytes-that influence plant health, function, and resilience to stress. Through surveys in forests across Panama that considered climate, seasonality, and covarying biotic factors, we demonstrate that endophyte richness varies negatively with temperature seasonality. Endophyte community structure and taxonomic composition reflect both temperature seasonality and climate (mean annual temperature and precipitation). Overall our findings highlight the vital role of climate-related factors in shaping the hyperdiversity of these important and little-known symbionts of the trees that, in turn, form the foundations of tropical forest biodiversity.

Methodological Approaches Frame Insights into Endophyte Richness and Community Composition.

Isolating microbes is vital to study microbiomes, but insights into microbial diversity and ecology can be constrained by recalcitrant or unculturable strains. Culture-free methods (e.g., next-generation sequencing, NGS) have become popular in part because they detect greater richness than culturing alone. Both approaches are used widely to characterize microfungi within healthy leaves (foliar endophytes), but methodological differences among studies can constrain large-scale insights into endophyte ecology. We examined endophytes in a temperate plant community to quantify how certain methodological factors, such as the choice of cultivation media for culturing and storage period after leaf collection, affect inferences regarding endophyte communities; how such effects vary among plant taxa; and how complementary culturing and NGS can be when subsets of the same plant tissue are used for each. We found that endophyte richness and composition from culturing were consistent across five media types. Insights from culturing and NGS were largely robust to differences in storage period (1, 5, and 10 days). Although endophyte richness, composition, and taxonomic diversity identified via culturing vs. NGS differed markedly, both methods revealed host-structured communities. Studies differing only in cultivation media or storage period thus can be compared to estimate endophyte richness, composition, and turnover at scales larger than those of individual studies alone. Our data show that it is likely more important to sample more host species, rather than sampling fewer species more intensively, to quantify endophyte diversity in given locations, with the richest insights into endophyte ecology emerging when culturing and NGS are paired.

Cyanolichen microbiome contains novel viruses that encode genes to promote microbial metabolism.

Lichen thalli are formed through the symbiotic association of a filamentous fungus and photosynthetic green alga and/or cyanobacterium. Recent studies have revealed lichens also host highly diverse communities of secondary fungal and bacterial symbionts, yet few studies have examined the viral component within these complex symbioses. Here, we describe viral biodiversity and functions in cyanolichens collected from across North America and Europe. As current machine-learning viral-detection tools are not trained on complex eukaryotic metagenomes, we first developed efficient methods to remove eukaryotic reads prior to viral detection and a custom pipeline to validate viral contigs predicted with three machine-learning methods. Our resulting high-quality viral data illustrate that every cyanolichen thallus contains diverse viruses that are distinct from viruses in other terrestrial ecosystems. In addition to cyanobacteria, predicted viral hosts include other lichen-associated bacterial lineages and algae, although a large fraction of viral contigs had no host prediction. Functional annotation of cyanolichen viral sequences predicts numerous viral-encoded auxiliary metabolic genes (AMGs) involved in amino acid, nucleotide, and carbohydrate metabolism, including AMGs for secondary metabolism (antibiotics and antimicrobials) and fatty acid biosynthesis. Overall, the diversity of cyanolichen AMGs suggests that viruses may alter microbial interactions within these complex symbiotic assemblages.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Teratopyrones A-C, Dimeric Naphtho-γ-Pyrones and Other Metabolites from Teratosphaeria sp. AK1128, a Fungal Endophyte of Equisetum arvense.

Bioassay-guided fractionation of a cytotoxic extract derived from a solid potato dextrose agar (PDA) culture of Teratosphaeria sp. AK1128, a fungal endophyte of Equisetum arvense, afforded three new naphtho-γ-pyrone dimers, teratopyrones A-C (1-3), together with five known naphtho-γ-pyrones, aurasperone B (4), aurasperone C (5), aurasperone F (6), nigerasperone A (7), and fonsecin B (8), and two known diketopiperazines, asperazine (9) and isorugulosuvine (10). The structures of 1-3 were determined on the basis of their spectroscopic data. Cytotoxicity assay revealed that nigerasperone A (7) was moderately active against the cancer cell lines PC-3M (human metastatic prostate cancer), NCI-H460 (human non-small cell lung cancer), SF-268 (human CNS glioma), and MCF-7 (human breast cancer), with IC50s ranging from 2.37 to 4.12 µM while other metabolites exhibited no cytotoxic activity up to a concentration of 5.0 µM.

Marine mammal skin microbiotas are influenced by host phylogeny.

Skin-associated microorganisms have been shown to play a role in immune function and disease of humans, but are understudied in marine mammals, a diverse animal group that serve as sentinels of ocean health. We examined the microbiota associated with 75 epidermal samples opportunistically collected from nine species within four marine mammal families, including: Balaenopteridae (sei and fin whales), Phocidae (harbour seal), Physeteridae (sperm whales) and Delphinidae (bottlenose dolphins, pantropical spotted dolphins, rough-toothed dolphins, short-finned pilot whales and melon-headed whales). The skin was sampled from free-ranging animals in Hawai'i (Pacific Ocean) and off the east coast of the United States (Atlantic Ocean), and the composition of the bacterial community was examined using the sequencing of partial small subunit (SSU) ribosomal RNA genes. Skin microbiotas were significantly different among host species and taxonomic families, and microbial community distance was positively correlated with mitochondrial-based host genetic divergence. The oceanic location could play a role in skin microbiota variation, but skin from species sampled in both locations is necessary to determine this influence. These data suggest that a phylosymbiotic relationship may exist between microbiota and their marine mammal hosts, potentially providing specific health and immune-related functions that contribute to the success of these animals in diverse ocean ecosystems.

Host availability drives distributions of fungal endophytes in the imperilled boreal realm.

Boreal forests represent the world's largest terrestrial biome and provide ecosystem services of global importance. Highly imperilled by climate change, these forests host Earth's greatest phylogenetic diversity of endophytes, a hyperdiverse group of symbionts that are defined by their occurrence within living, symptomless plant and lichen tissues. Endophytes shape the ecological and evolutionary trajectories of plants and are therefore key to the function and resilience of terrestrial ecosystems. A critical step in linking the ecological functions of endophytes with those of their hosts is to understand the distributions of these symbionts at the global scale; however, turnover in host taxa with geography and climate can confound insights into endophyte biogeography. As a result, global drivers of endophyte diversity and distributions are not known. Here, we leverage sampling from phylogenetically diverse boreal plants and lichens across North America and Eurasia to show that host filtering in distinctive environments, rather than turnover with geographical or environmental distance, is the main determinant of the community composition and diversity of endophytes. We reveal the distinctiveness of boreal endophytes relative to soil fungi worldwide and endophytes from diverse temperate biomes, highlighting a high degree of global endemism. Overall, the distributions of endophytes are directly linked to the availability of compatible hosts, highlighting the role of biotic interactions in shaping fungal communities across large spatial scales, and the threat that climate change poses to biological diversity and function in the imperilled boreal realm.

T-BAS Version 2.1: Tree-Based Alignment Selector Toolkit for Evolutionary Placement of DNA Sequences and Viewing Alignments and Specimen Metadata on Curated and Custom Trees.

The Tree-Based Alignment Selector (T-BAS) toolkit combines phylogenetic-based placement of DNA sequences with alignment and specimen metadata visualization tools in an integrative pipeline for analyzing microbial biodiversity. The release of T-BAS version 2.1 makes available reference phylogenies, supports multilocus sequence placements and permits uploading and downloading trees, alignments, and specimen metadata.

Age-related variation in the oral microbiome of urban Cooper's hawks (Accipiter cooperii).

BACKGROUND: Bird species worldwide are affected by trichomoniasis caused by the protist Trichomonas gallinae. In avivorous raptors such as Cooper's hawks (Accipiter cooperii), nestlings are more susceptible than fledglings and adults. Previous research suggested a link between oral pH and susceptibility: the oral pH of fledgling and adult hawks is more than seven times more acidic than that of nestlings. We speculated that this age-specific difference in pH would correspond to age-specific differences in the oral microbiota of Cooper's hawks. We examined the oral microbiomes of 31 healthy, wild Cooper's hawks in Tucson, Arizona (USA). Individuals represented three age classes (nestlings, fledglings, and adults). We designed our study with multiple controls, replicated sampling, mock communities, and stringent quality-controls to address challenges that can limit the inferential quality of microbiome data sets. RESULTS: Richness of bacterial communities in oral cavities of Cooper's hawks differed as a function of age but not as a function of sex, sampling date, or sampling location. Bacterial communities in oral cavities of nestlings differed from those of fledglings and adults, whereas communities in fledglings and adults did not differ from each other. Communities were similar in males and females and did not differ over the sampling season. Prevalence of acid-producing bacteria in fledgling and adults vs. nestlings is consistent with previous reports of age-specific variation in oral pH, but further research is needed to establish a causal link to pH levels or susceptibility to disease. Analyses of mock communities demonstrated high repeatability and showed that operon number and read abundance were highly correlated. CONCLUSIONS: The oral microbiota of wild Cooper's hawks differs between nestlings and older birds. Variation in the oral microbiome is consistent with differences in oral pH between nestlings and older individuals. Overall our study provides a first perspective on bacterial communities associated with oral cavities of a wild raptor.

Using collections data to infer biogeographic, environmental, and host structure in communities of endophytic fungi.

Biodiversity collections contain a wealth of information encapsulated both in specimens and in their metadata, providing the foundation for diverse studies in fields such as ecology. Yet biodiversity repositories can present a challenge for ecological inferences because collections rarely are structured with ecological questions in mind: collections may be opportunistic in space or time, may focus on particular taxonomic groups, may reflect different collection strategies in different places or times, or may not be exhaustive in terms of retaining every specimen or having similar metadata for each record. In addition to its primary holdings, the Robert L. Gilbertson Mycological Herbarium at the University of Arizona holds a collection of living specimens of fungi isolated from the interior of healthy plants and lichens (i.e., endophytic and endolichenic fungi). Over the past decade, more than 7000 isolates from the southwestern United States were accessioned, including strains from diverse hosts in more than 50 localities across the biotically rich state of Arizona. This collection is distinctive in that metadata and barcode sequences are available for each specimen, many localities have been sampled with consistent methods, and all isolates obtained in surveys have been retained. Here, we use this herbarium collection to examine endophyte community structure in an ecological and evolutionary context. We then artificially restructure the collection to resemble collections more typical of biodiversity repositories, providing a case study for ecological insights that can be gleaned from collections that were not structured explicitly to address ecological questions. Overall, our analyses highlight the relevance of biogeography, climate, hosts, and geographic separation in endophyte community composition. This study showcases the importance of extensive metadata in collections and highlights the utility of biodiversity collections that can yield emergent insights from many surveys to answer ecological questions in mycology, ultimately providing information for understanding and conserving fungal biodiversity.

Cytotoxic and Noncytotoxic Metabolites from Teratosphaeria sp. FL2137, a Fungus Associated with Pinus clausa.

A new naphthoquinone, teratosphaerone A (1), four new naphthalenones, namely, teratosphaerone B (2), structurally related to 1, iso-balticol B (3), iso-balticol B-4,9-acetonide (4), and (+)-balticol C (5), a new furanonaphthalenone, (3a S,9 R,9a S)-1(9a),3(3a),9-hexahydromonosporascone (6), and the known metabolite monosporascone (7) were isolated from Teratosphaeria sp. FL2137, a fungal strain inhabiting the internal tissue of recently dead but undecomposed foliage of Pinus clausa. The structures of 1-6 were elucidated on the basis of their spectroscopic data including 2D NMR, and absolute configurations of 2, 3, and 6 were determined by the modified Mosher's ester method. When evaluated in a panel of five tumor cell lines, metabolites 1 and 7 isolated from a cytotoxic fraction of the extract exhibited moderate selectivity for metastatic breast adenocarcinoma cell line MDA-MB-231. Of these, 1 showed cytotoxicity to this cell line with an IC50 of 1.2 ± 0.1 µM.

Phage hunters: Computational strategies for finding phages in large-scale 'omics datasets.

A plethora of tools exist for identifying phage sequences in bacterial genomes, single cell amplified genomes, and host-associated and environmental metagenomes. Yet because the genetics of phages and their hosts are closely intertwined, distinguishing viral from bacterial signal remains an ongoing challenge. Further the size, quantity and fragmentary nature of modern 'omics datasets ushers in a new set of computational challenges. Here, we detail the promises and pitfalls of using currently available gene-centric or k-mer based tools for identifying prophage sequences in genomes and prophage and viral contigs in metagenomes. Each of these methods offers a unique piece of the puzzle to elucidating the intriguing signatures of phage-host coevolution.

Soilborne fungi have host affinity and host-specific effects on seed germination and survival in a lowland tropical forest.

The Janzen-Connell (JC) hypothesis provides a conceptual framework for explaining the maintenance of tree diversity in tropical forests. Its central tenet-that recruits experience high mortality near conspecifics and at high densities-assumes a degree of host specialization in interactions between plants and natural enemies. Studies confirming JC effects have focused primarily on spatial distributions of seedlings and saplings, leaving major knowledge gaps regarding the fate of seeds in soil and the specificity of the soilborne fungi that are their most important antagonists. Here we use a common garden experiment in a lowland tropical forest in Panama to show that communities of seed-infecting fungi are structured predominantly by plant species, with only minor influences of factors such as local soil type, forest characteristics, or time in soil (1-12 months). Inoculation experiments confirmed that fungi affected seed viability and germination in a host-specific manner and that effects on seed viability preceded seedling emergence. Seeds are critical components of reproduction for tropical trees, and the factors influencing their persistence, survival, and germination shape the populations of seedlings and saplings on which current perspectives regarding forest dynamics are based. Together these findings bring seed dynamics to light in the context of the JC hypothesis, implicating them directly in the processes that have emerged as critical for diversity maintenance in species-rich tropical forests.

An Endohyphal Bacterium (Chitinophaga, Bacteroidetes) Alters Carbon Source Use by Fusarium keratoplasticum (F. solani Species Complex, Nectriaceae).

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog® phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant- and seed-fungus interactions, such as D-trehalose, myo-inositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusarium species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus-bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.