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.

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.

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.

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.

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.

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.

T-BAS: Tree-Based Alignment Selector toolkit for phylogenetic-based placement, alignment downloads and metadata visualization: an example with the Pezizomycotina tree of life.

Motivation: High-quality phylogenetic placement of sequence data has the potential to greatly accelerate studies of the diversity, systematics, ecology and functional biology of diverse groups. We developed the Tree-Based Alignment Selector (T-BAS) toolkit to allow evolutionary placement and visualization of diverse DNA sequences representing unknown taxa within a robust phylogenetic context, and to permit the downloading of highly curated, single- and multi-locus alignments for specific clades. Results: In its initial form, T-BAS v1.0 uses a core phylogeny of 979 taxa (including 23 outgroup taxa, as well as 61 orders, 175 families and 496 genera) representing all 13 classes of largest subphylum of Fungi-Pezizomycotina (Ascomycota)-based on sequence alignments for six loci (nr5.8S, nrLSU, nrSSU, mtSSU, RPB1, RPB2 ). T-BAS v1.0 has three main uses: (i) Users may download alignments and voucher tables for members of the Pezizomycotina directly from the reference tree, facilitating systematics studies of focal clades. (ii) Users may upload sequence files with reads representing unknown taxa and place these on the phylogeny using either BLAST or phylogeny-based approaches, and then use the displayed tree to select reference taxa to include when downloading alignments. The placement of unknowns can be performed for large numbers of Sanger sequences obtained from fungal cultures and for alignable, short reads of environmental amplicons. (iii) User-customizable metadata can be visualized on the tree. Availability and Implementation: T-BAS Version 1.0 is available online at http://tbas.hpc.ncsu.edu . Registration is required to access the CIPRES Science Gateway and NSF XSEDE's large computational resources. Contact: icarbon@ncsu.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

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.

Contributions of North American endophytes to the phylogeny, ecology, and taxonomy of Xylariaceae (Sordariomycetes, Ascomycota).

The Xylariaceae (Sordariomycetes) comprise one of the largest and most diverse families of Ascomycota, with at least 85 accepted genera and ca. 1343 accepted species. In addition to their frequent occurrence as saprotrophs, members of the family often are found as endophytes in living tissues of phylogenetically diverse plants and lichens. Many of these endophytes remain sterile in culture, precluding identification based on morphological characters. Previous studies indicate that endophytes are highly diverse and represent many xylariaceous genera; however, phylogenetic analyses at the family level generally have not included endophytes, such that their contributions to understanding phylogenetic relationships of Xylariaceae are not well known. Here we use a multi-locus, cumulative supermatrix approach to integrate 92 putative species of fungi isolated from plants and lichens into a phylogenetic framework for Xylariaceae. Our collection spans 1933 isolates from living and senescent tissues in five biomes across the continental United States, and here is analyzed in the context of previously published sequence data from described species and additional taxon sampling of type specimens from culture collections. We found that the majority of strains obtained in our surveys can be classified in the hypoxyloid and xylaroid subfamilies, although many also were found outside of these lineages (as currently circumscribed). Many endophytes were placed in lineages previously not known for endophytism. Most endophytes appear to represent novel species, but inferences are limited by potential gaps in public databases. By linking our data, publicly available sequence data, and records of ascomata, we identify many geographically widespread, host-generalist clades capable of symbiotic associations with diverse photosynthetic partners. Concomitant with such cosmopolitan host use and distributions, many xylariaceous endophytes appear to inhabit both living and non-living plant tissues, with potentially important roles as saprotrophs. Overall, our study reveals major gaps in the availability of multi-locus datasets and metadata for this iconic family, and provides new hypotheses regarding the ecology and evolution of endophytism and other trophic modes across the family Xylariaceae.

Pervasive Effects of Wildfire on Foliar Endophyte Communities in Montane Forest Trees.

Plants in all terrestrial ecosystems form symbioses with endophytic fungi that inhabit their healthy tissues. How these foliar endophytes respond to wildfires has not been studied previously, but is important given the increasing frequency and intensity of severe wildfires in many ecosystems, and because endophytes can influence plant growth and responses to stress. The goal of this study was to examine effects of severe wildfires on endophyte communities in forest trees, with a focus on traditionally fire-dominated, montane ecosystems in the southwestern USA. We evaluated the abundance, diversity, and composition of endophytes in foliage of Juniperus deppeana (Cupressaceae) and Quercus spp. (Fagaceae) collected contemporaneously from areas affected by recent wildfire and paired areas not affected by recent fire. Study sites spanned four mountain ranges in central and southern Arizona. Our results revealed significant effects of fires on endophyte communities, including decreases in isolation frequency, increases in diversity, and shifts in community structure and taxonomic composition among endophytes of trees affected by recent fires. Responses to fire were similar in endophytes of each host in these fire-dominated ecosystems and reflect regional fire-return intervals, with endophytes after fire representing subsets of the regional mycoflora. Together, these findings contribute to an emerging perspective on the responses of diverse communities to severe fire, and highlight the importance of considering fire history when estimating endophyte diversity and community structure for focal biomes.

Interaction type influences ecological network structure more than local abiotic conditions: evidence from endophytic and endolichenic fungi at a continental scale.

Understanding the factors that shape community assembly remains one of the most enduring and important questions in modern ecology. Network theory can reveal rules of community assembly within and across study systems and suggest novel hypotheses regarding the formation and stability of communities. However, such studies generally face the challenge of disentangling the relative influence of factors such as interaction type and environmental conditions on shaping communities and associated networks. Endophytic and endolichenic symbioses, characterized by microbial species that occur within healthy plants and lichen thalli, represent some of the most ubiquitous interactions in nature. Fungi that engage in these symbioses are hyperdiverse, often horizontally transmitted, and functionally beneficial in many cases, and they represent the diversification of multiple phylogenetic groups. We evaluated six measures of ecological network structure for >4100 isolates of endophytic and endolichenic fungi collected systematically from five sites across North America. Our comparison of these co-occurring interactions in biomes ranging from tundra to subtropical forest showed that the type of interactions (i.e., endophytic vs. endolichenic) had a much more pronounced influence on network structure than did environmental conditions. In particular, endophytic networks were less nested, less connected, and more modular than endolichenic networks in all sites. The consistency of the network structure within each interaction type, independent of site, is encouraging for current efforts devoted to gathering metadata on ecological network structure at a global scale. We discuss several mechanisms potentially responsible for such patterns and draw attention to knowledge gaps in our understanding of networks for diverse interaction types.

Oxaspirol B with p97 Inhibitory Activity and Other Oxaspirols from Lecythophora sp. FL1375 and FL1031, Endolichenic Fungi Inhabiting Parmotrema tinctorum and Cladonia evansii.

A new metabolite, oxaspirol D (4), together with oxaspirols B (2) and C (3) were isolated from Lecythophora sp. FL1375, an endolichenic fungus isolated from Parmotrema tinctorum, whereas Lecythophora sp. FL1031 inhabiting the lichen Cladonia evansii afforded oxaspirols A (1), B (2), and C (3). Of these, oxaspirol B (2) showed moderate p97 ATPase inhibitory activity. A detailed characterization of all oxaspirols was undertaken because structures proposed for known oxaspirols have involved incomplete assignments of NMR spectroscopic data leading only to their planar structures. Thus, the naturally occurring isomeric mixture (2a and 2b) of oxaspirol B was separated as their diacetates (5a and 5b) and the structures and absolute configurations of 1, 2a, 2b, 3, and 4 were determined by the application of spectroscopic techniques including two-dimensional NMR and the modified Mosher's ester method. Oxaspirol B (2) and its diacetates 5a and 5b were evaluated for their ATPase inhibitory activities of p97, p97 mutants, and other ATP-utilizing enzymes, and only 2 was found to be active, indicating the requirement of some structural features in oxaspirols for their activity. Additional biochemical and cellular assays suggested that 2 was a reversible, non-ATP competitive, and specific inhibitor of p97.

Phylogenetic analyses of eurotiomycetous endophytes reveal their close affinities to Chaetothyriales, Eurotiales, and a new order - Phaeomoniellales.

Symbiotic fungi living in plants as endophytes, and in lichens as endolichenic fungi, cause no apparent symptoms to their hosts. They are ubiquitous, ecologically important, hyperdiverse, and represent a rich source of secondary compounds for new pharmaceutical and biocontrol products. Due in part to the lack of visible reproductive structures and other distinctive phenotypic traits for many species, the diversity and phylogenetic affiliations of these cryptic fungi are often poorly known. The goal of this study was to determine the phylogenetic placement of representative endophytes within the Eurotiomycetes (Pezizomycotina, Ascomycota), one of the most diverse and evolutionarily dynamic fungal classes, and to use that information to infer processes of macroevolution in trophic modes. Sequences of a single locus marker spanning the nuclear ribosomal internal transcribed spacer region (nrITS) and 600 base pairs at the 5' end of the nuclear ribosomal large subunit (nrLSU) were obtained from previous studies of >6000 endophytic and endolichenic fungi from diverse biogeographic locations and hosts. We conducted phylum-wide phylogenetic searches using this marker to determine which fungal strains belonged to Eurotiomycetes and the results were used as the basis for a class-wide, seven-locus phylogenetic study focusing on endophytic and endolichenic Eurotiomycetes. Our cumulative supermatrix-based analyses revealed that representative endophytes within Eurotiomycetes are distributed in three main clades: Eurotiales, Chaetothyriales and Phaeomoniellales ord. nov., a clade that had not yet been described formally. This new order, described herein, is sister to the clade including Verrucariales and Chaetothyriales. It appears to consist mainly of endophytes and plant pathogens. Morphological characters of endophytic Phaeomoniellales resemble those of the pathogenic genus Phaeomoniella. This study highlights the capacity of endophytic and endolichenic fungi to expand our understanding of the ecological modes associated with particular clades, and provides a first estimation of their phylogenetic relationships in the Eurotiomycetes.

Fungal endophytes in aboveground tissues of desert plants: infrequent in culture, but highly diverse and distinctive symbionts.

In hot deserts, plants cope with aridity, high temperatures, and nutrient-poor soils with morphological and biochemical adaptations that encompass intimate microbial symbioses. Whereas the root microbiomes of arid-land plants have received increasing attention, factors influencing assemblages of symbionts in aboveground tissues have not been evaluated for many woody plants that flourish in desert environments. We evaluated the diversity, host affiliations, and distributions of endophytic fungi associated with photosynthetic tissues of desert trees and shrubs, focusing on nonsucculent woody plants in the species-rich Sonoran Desert. To inform our strength of inference, we evaluated the effects of two different nutrient media, incubation temperatures, and collection seasons on the apparent structure of endophyte assemblages. Analysis of >22,000 tissue segments revealed that endophytes were isolated four times more frequently from photosynthetic stems than leaves. Isolation frequency was lower than expected given the latitude of the study region and varied among species a function of sampling site and abiotic factors. However, endophytes were very species-rich and phylogenetically diverse, consistent with less arid sites of a similar latitudinal position. Community composition differed among host species, but not as a function of tissue type, sampling site, sampling month, or exposure. Estimates of abundance, diversity, and composition were not influenced by isolation medium or incubation temperature. Phylogenetic analyses of the most commonly isolated genus (Preussia) revealed multiple evolutionary origins of desert-plant endophytism and little phylogenetic structure with regard to seasonality, tissue preference, or optimal temperatures and nutrients for growth in vitro. Together, these results provide insight into endophytic symbioses in desert-plant communities and can be used to optimize strategies for capturing endophyte biodiversity at regional scales.

Anteaglonialides A-F and Palmarumycins CE(1)-CE(3) from Anteaglonium sp. FL0768, a Fungal Endophyte of the Spikemoss Selaginella arenicola.

Anteaglonialides A-F (1-6), bearing a spiro[6-(tetrahydro-7-furanyl)cyclohexane-1,2'-naphtho[1,8-de][1,3]-dioxin]-10-one skeleton, three new spirobisnaphthalenes, palmarumycins CE1-CE3 (7-9), nine known palmarumycin analogues, palmarumycins CP5 (10), CP4a (11), CP3 (12), CP17 (13), CP2 (14), and CP1 (15), CJ-12,371 (16), 4-O-methyl CJ-12,371 (17), and CP4 (18), together with a possible artifact, 4a(5)-anhydropalmarumycin CE2 (8a), and four known metabolites, O-methylherbarin (19), herbarin (20), herbaridine B (21), and hyalopyrone (22), were encountered in a cytotoxic extract of a potato dextrose agar culture of Anteaglonium sp. FL0768, an endophytic fungus of the sand spikemoss, Selaginella arenicola. The planar structures and relative configurations of the new metabolites 1-9 were elucidated by analysis of extensive spectroscopic data, and the absolute configuration of 1 was determined by the modified Mosher's ester method. Application of the modified Mosher's ester method combined with the NOESY data resulted in revision of the absolute configuration previously proposed for 10. Co-occurrence of 1-6 and 7-18 in this fungus led to the proposal that the anteagloniolides may be biogenetically derived from palmarumycins. Among the metabolites encountered, anteaglonialide F (6) and known palmarumycins CP3 (12) and CP1 (15) exhibited strong cytotoxic activity against the human Ewing's sarcoma cell line CHP-100, with IC50 values of 1.4, 0.5, and 1.6 µM, respectively.

Genetic variation in horizontally transmitted fungal endophytes of pine needles reveals population structure in cryptic species.

UNLABELLED: • PREMISE OF THE STUDY: Fungal endophytes comprise one of the most ubiquitous groups of plant symbionts, inhabiting healthy leaves and stems of all major lineages of plants. Together, they comprise immense species richness, but little is known about the fundamental processes that generate their diversity. Exploration of their population structure is needed, especially with regard to geographic distributions and host affiliations.• METHODS: We take a multilocus approach to examine genetic variation within and among populations of Lophodermium australe, an endophytic fungus commonly associated with healthy foliage of pines in the southeastern United States. Sampling focused on two pine species ranging from montane to coastal regions of North Carolina and Virginia.• KEY RESULTS: Our sampling revealed two genetically distinct groups within Lophodermium australe. Our analysis detected less than one migrant per generation between them, indicating that they are distinct species. The species comprising the majority of isolates (major species) demonstrated a panmictic structure, whereas the species comprising the minority of isolates (cryptic species) demonstrated isolation by distance. Distantly related pine species hosted the same Lophodermium species, and host species did not influence genetic structure.• CONCLUSIONS: We present the first evidence for isolation by distance in a foliar fungal endophyte that is horizontally transmitted. Cryptic species may be common among microbial symbionts and are important to delimit when exploring their genetic structure and microevolutionary processes. The hyperdiversity of endophytic fungi may be explained in part by cryptic species without apparent ecological and morphological differences as well as genetic diversification within rare fungal species across large spatial scales.

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.