Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5.

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

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

Microbiomes and metabolomes of dominant coral reef primary producers illustrate a potential role for immunolipids in marine symbioses.

The dominant benthic primary producers in coral reef ecosystems are complex holobionts with diverse microbiomes and metabolomes. In this study, we characterize the tissue metabolomes and microbiomes of corals, macroalgae, and crustose coralline algae via an intensive, replicated synoptic survey of a single coral reef system (Waimea Bay, O'ahu, Hawaii) and use these results to define associations between microbial taxa and metabolites specific to different hosts. Our results quantify and constrain the degree of host specificity of tissue metabolomes and microbiomes at both phylum and genus level. Both microbiome and metabolomes were distinct between calcifiers (corals and CCA) and erect macroalgae. Moreover, our multi-omics investigations highlight common lipid-based immune response pathways across host organisms. In addition, we observed strong covariation among several specific microbial taxa and metabolite classes, suggesting new metabolic roles of symbiosis to further explore.

Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons.

Stable isotope signatures of fungal sporocarps have been instrumental in identifying carbon gains of chlorophyllous orchids from a fungal source. Yet, not all mycorrhizal fungi produce macroscopic sporocarps and frequently fungi of different taxa occur in parallel in orchid roots. To overcome this obstacle, we investigated stable isotope signatures of fungal pelotons extracted from orchid roots and compared these data to the respective orchid and reference plant tissues. Anoectochilus sandvicensis and Epipactis palustris represented specialized or unspecialized rhizoctonia-associated orchids. Epipactis atrorubens and Epipactis leptochila are orchids considered ectomycorrhiza-associated with different preferences for Basidio- and Ascomycota. 13 C enrichment of rhizoctonia pelotons was minor compared with plant tissues and significantly lower than enrichments of pelotons from ectomycorrhizal Epipactis species. 15 N values of pelotons from E. leptochila and E. atrorubens showed similar patterns as known for respective sporocarps of ectomycorrhizal Ascomycota and Basidiomycota, however, with an offset towards lower 15 N enrichments and nitrogen concentrations. Our results suggest an explicit fungal nutrition source of orchids associated with ectomycorrhizal fungi, whereas the low 13 C enrichment in rhizoctonia-associated orchids and fungal pelotons hamper the detection of carbon gains from fungal partners. 15 N isotopic pattern of orchids further suggests a selective transfer of 15 N-enriched protein-nitrogen into orchids.

Isoscapes of remnant and restored Hawaiian montane forests reveal differences in biological nitrogen fixation and carbon inputs.

Deforestation and subsequent land-use conversion has altered ecosystems and led to negative effects on biodiversity. To ameliorate these effects, nitrogen-fixing (N2-fixing) trees are frequently used in the reforestation of degraded landscapes, especially in the tropics; however, their influence on ecosystem properties such as nitrogen (N) availability and carbon (C) stocks are understudied. Here, we use a 30-y old reforestation site of outplanted native N2-fixing trees (Acacia koa) dominated by exotic grass understory, and a neighboring remnant forest dominated by A. koa canopy trees and native understory, to assess whether restoration is leading to similar N and C biogeochemical landscapes and soil and plant properties as a target remnant forest ecosystem. We measured nutrient contents and isotope values (δ15N, δ13C) in soils, A. koa, and non-N2-fixing understory plants (Rubus spp.) and generated δ15N and δ13C isoscapes of the two forests to test for (1) different levels of biological nitrogen fixation (BNF) and its contribution to non-N2-fixing understory plants, and (2) the influence of historic land conversion and more recent afforestation on plant and soil δ13C. In the plantation, A. koa densities were higher and foliar δ15N values for A. koa and Rubus spp. were lower than in the remnant forest. Foliar and soil isoscapes also showed a more homogeneous distribution of low δ15N values in the plantation and greater influence of A. koa on neighboring plants and soil, suggesting greater BNF. Foliar δ13C also indicated higher water use efficiency (WUEi) in the plantation, indicative of differences in plant-water relations or soil water status between the two forest types. Plantation soil δ13C was higher than the remnant forest, consistent with greater contributions of exotic C4-pasture grasses to soil C pools, possibly due to facilitation of non-native grasses by the dense A. koa canopy. These findings are consequential for forest restoration, as they contribute to the mounting evidence that outplanting N2-fixing trees produces different biogeochemical landscapes than those observed in reference ecosystems, thereby influencing plant-soil interactions which can influence restoration outcomes.

A ridge-to-reef ecosystem microbial census reveals environmental reservoirs for animal and plant microbiomes.

Microbes are found in nearly every habitat and organism on the planet, where they are critical to host health, fitness, and metabolism. In most organisms, few microbes are inherited at birth; instead, acquiring microbiomes generally involves complicated interactions between the environment, hosts, and symbionts. Despite the criticality of microbiome acquisition, we know little about where hosts' microbes reside when not in or on hosts of interest. Because microbes span a continuum ranging from generalists associating with multiple hosts and habitats to specialists with narrower host ranges, identifying potential sources of microbial diversity that can contribute to the microbiomes of unrelated hosts is a gap in our understanding of microbiome assembly. Microbial dispersal attenuates with distance, so identifying sources and sinks requires data from microbiomes that are contemporary and near enough for potential microbial transmission. Here, we characterize microbiomes across adjacent terrestrial and aquatic hosts and habitats throughout an entire watershed, showing that the most species-poor microbiomes are partial subsets of the most species-rich and that microbiomes of plants and animals are nested within those of their environments. Furthermore, we show that the host and habitat range of a microbe within a single ecosystem predicts its global distribution, a relationship with implications for global microbial assembly processes. Thus, the tendency for microbes to occupy multiple habitats and unrelated hosts enables persistent microbiomes, even when host populations are disjunct. Our whole-watershed census demonstrates how a nested distribution of microbes, following the trophic hierarchies of hosts, can shape microbial acquisition.

Traits and tradeoffs among non-native ectomycorrhizal fungal symbionts affect pine seedling establishment in a Hawaiian coinvasion landscape.

Pine invasions lead to losses of native biodiversity and ecosystem function, but pine invasion success is often linked to coinvading non-native ectomycorrhizal (EM) fungi. How the community composition, traits, and distributions of these fungi vary over the landscape and how this affects pine success is understudied. A greenhouse bioassay experiment was performed to test the effects of changes in EM fungal community structure from a pine plantation, to an invasion front to currently pine-free areas on percent root colonization and seedling biomass. Soils were also analysed by qPCR to determine changes in inoculum and spore density over distance for a common coinvading EM fungus, Suillus pungens. Percent colonization increased with distance from the plantation, which corresponded with an increase in seedling biomass and stark changes in EM fungal community membership where Suillus spp. dominated currently pine-free areas. However, there was a negative relationship between S. pungens inoculum potential versus root colonization over distance. We conclude that the success of pine invasions is facilitated by specific traits of Suillus spp., but that the success of Suillus is contingent on a lack of competition with other ectomycorrhizal fungi.

Core arbuscular mycorrhizal fungi are predicted by their high abundance-occupancy relationship while host-specific taxa are rare and geographically structured.

Habitat restoration may depend on the recovery of plant microbial symbionts such as arbuscular mycorrhizal (AM) fungi, but this requires a better understanding of the rules that govern their community assembly. We examined the interactions of soil and host-associated AM fungal communities between remnant and restored patches of subtropical montane forests. While AM fungal richness did not differ between habitat types, community membership did and was influenced by geography, habitat and host. These differences were largely driven by rare host-specific AM fungi that displayed near-complete turnover between forest types, while core AM fungal taxa were highly abundant and ubiquitous. The bipartite networks in the remnant forest were more specialized and hosts more specific than in the restored forest. Host-associated AM fungal communities nested within soil communities in both habitats, but only significantly so in the restored forest. Our results provide evidence that restored and remnant forests harbour the same core fungal symbionts, while rare host-specific taxa differ, and that geography, host identity and taxonomic resolution strongly affect the observed distribution patterns of these fungi. We suggest that host-specific interactions with AM fungi, as well as spatial processes, should be explicitly considered to effectively re-establish target host and symbiont communities.

Conceptual Exchanges for Understanding Free-Living and Host-Associated Microbiomes.

Whether a microbe is free-living or associated with a host from across the tree of life, its existence depends on a limited number of elements and electron donors and acceptors. Yet divergent approaches have been used by investigators from different fields. The "environment first" research tradition emphasizes thermodynamics and biogeochemical principles, including the quantification of redox environments and elemental stoichiometry to identify transformations and thus an underlying microbe. The increasingly common "microbe first" research approach benefits from culturing and/or DNA sequencing methods to first identify a microbe and encoded metabolic functions. Here, the microbe itself serves as an indicator for environmental conditions and transformations. We illustrate the application of both approaches to the study of microbiomes and emphasize how both can reveal the selection of microbial metabolisms across diverse environments, anticipate alterations to microbiomes in host health, and understand the implications of a changing climate for microbial function.

Hawaiian Fungal Amplicon Sequence Variants Reveal Otherwise Hidden Biogeography.

To study biogeography and other ecological patterns of microorganisms, including fungi, scientists have been using operational taxonomic units (OTUs) as representations of species or species hypotheses. However, when defined by 97% sequence similarity cutoff at an accepted barcode locus such as 16S in bacteria or ITS in fungi, these OTUs can obscure biogeographic patterns, mask taxonomic diversity, and hinder meta-analyses. Amplicon sequence variants (ASVs) have been proposed to alleviate all of these issues and have been shown to do so in bacteria. Analyzing ASVs is just emerging as a common practice among fungal studies, and it is unclear whether the benefits found in bacterial studies of using such an approach carryover to fungi. Here, we conducted a meta-analysis of Hawaiian fungi by analyzing ITS1 amplicon sequencing data as ASVs and exploring ecological patterns. These surveys spanned three island groups and five ecosystems combined into the first comprehensive Hawaiian Mycobiome ASV Database. Our results show that ASVs can be used to combine fungal ITS surveys, increase reproducibility, and maintain the broad ecological patterns observed with OTUs, including diversity orderings. Additionally, the ASVs that comprise some of the most common OTUs in our database reveals some island specialists, indicating that traditional OTU clustering can obscure important biogeographic patterns. We recommend that future fungal studies, especially those aimed at assessing biogeography, analyze ASVs rather than OTUs. We conclude that similar to bacterial studies, ASVs improve reproducibility and data sharing for fungal studies.

Hawaiian Island endemic and indigenous plant species have higher mycorrhizal incidence than the global average.

PREMISE: Prior efforts have shown that continents harbor a greater proportion of mycorrhizal hosts than on islands. However, in the Hawaiian Islands, estimates of the proportion of mycorrhizal plant species are higher than on continents (>90%), but there are few studies to support this claim. Concurrently, Hawaii's flora faces some of the greatest global risks of extinction, and significant efforts are aimed at restoring native vegetation. Arbuscular mycorrhizal (AM) fungi have been shown to improve plant restoration success, but little work has been done in Hawaii to understand the extent of mycorrhizal associations among native plant populations. METHODS: We surveyed 35 native Hawaiian plant species in the wild, focusing on plant species that are reared for reintroduction. Roots from wild individuals were collected from 10 sites on Oahu to determine degree of mycorrhizal fungal colonization and how this varies across host populations. RESULTS: Of the species surveyed, 97% had evidence of mycorrhizal colonization, including 25 endemic and nine indigenous species from 23 families. The mycorrhizal status of 22 of the species surveyed was unknown before this study. For four species, the degree of colonization by AM fungi differed among sites, and these differences corresponded with variations in precipitation and temperature. CONCLUSIONS: The high incidence of mycorrhizal colonization provides evidence that island flora can actually harbor more mycorrhizal hosts than species on mainlands and that future reintroduction projects should consider the potentially important roles of AM fungi for success of these hosts in the wild.

Three decades post-reforestation has not led to the reassembly of arbuscular mycorrhizal fungal communities associated with remnant primary forests.

The negative effects of deforestation can potentially be ameliorated through ecological restoration. However, reforestation alone may not reassemble the same ecological communities or functions as primary forests. In part, this failure may be owed to forest ecosystems inherently involving complex interactions among guilds of organisms. Plants, which structure forest food webs, rely on intimate associations with symbiotic microbes such as root-inhabiting mycorrhizal fungi. Here, we leverage a large-scale reforestation project on Hawai'i Island underway for over three decades to assess whether arbuscular mycorrhizal (AM) fungal communities have concurrently been restored. The reference ecosystem for this restoration project is a remnant montane native Hawaiian forest that provides critical habitat for endangered birds. We sampled soils from 12 plots within remnant and restored forest patches and characterized AM fungal communities using high-throughput amplicon sequencing. While some AM fungal community metrics were comparable between remnant and restored forest (e.g. species richness), other key characteristics were not. Specifically, community membership and the identity of AM fungal keystone species differed between the two habitat types, as well as the primary environmental factors influencing community composition. Remnant forest AM fungal communities were strongly associated with soil chemical properties, especially pH, while restored forest communities were influenced by the spatial proximity to remnant forests. We posit that combined, these differences in soil AM fungal communities could be negatively affecting the recruitment of native plant hosts and that future restoration efforts should consider plant-microbe interactions as an important facet of forest health.

Fungal aerobiota are not affected by time nor environment over a 13-y time series at the Mauna Loa Observatory.

Fungi are ubiquitous and often abundant components of virtually all ecosystems on Earth, serving a diversity of functions. While there is clear evidence that fungal-mediated processes can influence environmental conditions, and in turn select for specific fungi, it is less clear how fungi respond to environmental fluxes over relatively long time frames. Here we set out to examine changes in airborne fungi collected over the course of 13 y, which is the longest sampling time to date. Air filter samples were collected from the Mauna Loa Observatory (MLO) on Hawaii Island, and analyzed using Illumina amplicon sequencing. As a study site, MLO is unique because of its geographic isolation and high elevation, making it an ideal place to capture global trends in climate and aerobiota. We found that the fungal aerobiota sampled at MLO had high species turnover, but compositional similarity did not decrease as a function of time between samples. We attribute these patterns to neutral processes such as idiosyncratic dispersal timing and trajectories. Furthermore, the composition of fungi at any given point was not significantly influenced by any local or global environmental variables we examined. This, and our additional finding of a core set of persistent fungi during our entire sampling period, indicates some degree of stability among fungi in the face of natural environmental fluctuations and human-associated global change. We conclude that the movement of fungi through the atmosphere is a relatively stochastic process.

A Developing Symbiosis: Enabling Cross-Talk Between Ecologists and Microbiome Scientists.

Like all interactions, the success of cross-discipline collaborations relies on effective communication. Ecology offers theoretical frameworks and lexicons to study microbiomes. Yet some of the terms and concepts borrowed from ecology are being used discordantly by microbiome studies from their traditional definitions. Here we define some of the ecological terms and concepts as they are used in ecology and the study of microbiomes. Where applicable, we have provided the historical context of the terms, highlighted examples from microbiome studies, and considered the research methods involved. We divided these concepts into four sections: Biomes, Diversity, Symbiosis, and Succession. Biomes encompass the interactions within the biotic and abiotic features of an environment. This extends to the term "microbiome," derived from "biome," and includes an environment and all the microbes within it. Diversity encompasses patterns of species richness, abundance, and biogeography, all of which are important to understanding the distribution of microbiomes. Symbiosis emphasizes the relationships between organisms within a community. Symbioses are often misunderstood to be synonymous with mutualism. We discard that implication, in favor of a broader, more historically accurate definition which spans the continuum from parasitism to mutualism. Succession includes classical succession, alternative stable states, community assembly frameworks, and r/K-selection. Our hope is that as microbiome researchers continue to apply ecological terms, and as ecologists continue to gain interest in microbiomes, each will do so in a way that enables cross-talk between them. We recommend initiating these collaborations by using a common lexicon, from which new concepts can emerge.

Remote tropical island colonization does not preclude symbiotic specialists: new evidence of mycorrhizal specificity across the geographic distribution of the Hawaiian endemic orchid Anoectochilus sandvicensis.

BACKGROUND AND AIMS: For symbiotic organisms, their colonization and spread across remote oceanic islands should favour generalists. Plants that form obligate symbiotic associations with microbes dominate island ecosystems, but the relationship between island inhabitance and symbiotic specificity is unclear, especially in the tropics. To fill this gap, we examined the mycorrhizal specificity of the Hawaiian endemic orchid Anoectochilus sandvicensis across multiple populations encompassing its entire geographic distribution. METHODS: By molecular phylogenetic approaches we identified the mycorrhizal fungi associated with A. sandvicensis across its entire geographic distribution and determined the relationship of these fungi to others found elsewhere around the globe. With richness estimators, we assessed the mycorrhizal specificity of A. sandvicensis within and among islands. We then tested whether geographic proximity of orchid populations was a significant predictor for the presence of particular mycorrhizal fungi and their community composition. KEY RESULTS: We found that each population of A. sandvicensis forms specific associations with one of three fungi in the genus Ceratobasidium and that the closest relatives of these fungi are globally widespread. Based on diversity indices, A. sandvicensis populations were estimated to partner with one to four mycorrhizal taxa with an estimated total of four compatible mycorrhizal fungi across its entire distribution. However, the geographic proximity of orchid populations was not a significant predictor of mycorrhizal fungal community composition. CONCLUSIONS: Our findings indicate that the colonization and survival of plant species on even the most remote oceanic islands is not restricted to symbiotic generalists, and that partnering with few, but cosmopolitan microbial symbionts is an alternative means for successful island establishment. We suggest that the spatial distribution and abundance of symbionts in addition to island age, size and isolation should also be taken into consideration for predictions of island biodiversity.

Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research.

Despite increasing acknowledgment that microorganisms underpin the healthy functioning of basically all multicellular life, few cross-disciplinary teams address the diversity and function of microbiota across organisms and ecosystems. Our newly formed consortium of junior faculty spanning fields such as ecology and geoscience to mathematics and molecular biology from the University of Hawai'i at Manoa aims to fill this gap. We are united in our mutual interest in advancing a new paradigm for biology that incorporates our modern understanding of the importance of microorganisms. As our first concerted research effort, we will assess the diversity and function of microbes across an entire watershed on the island of Oahu, Hawai'i. Due to its high ecological diversity across tractable areas of land and sea, Hawai'i provides a model system for the study of complex microbial communities and the processes they mediate. Owing to our diverse expertise, we will leverage this study system to advance the field of biology.

Biological invasions increase the richness of arbuscular mycorrhizal fungi from a Hawaiian subtropical ecosystem.

Biological invasions can have various impacts on the diversity of important microbial mutualists such as mycorrhizal fungi, but few studies have tested whether the effects of invasions on mycorrhizal diversity are consistent across spatial gradients. Furthermore, few of these studies have taken place in tropical ecosystems that experience an inordinate rate of invasions into native habitats. Here, we examined the effects of plant invasions dominated by non-native tree species on the diversity of arbuscular mycorrhizal (AM) fungi in Hawaii. To test the hypothesis that invasions result in consistent changes in AM fungal diversity across spatial gradients relative to native forest habitats, we sampled soil in paired native and invaded sites from three watersheds and used amplicon sequencing to characterize AM fungal communities. Whether our analyses considered phylogenetic relatedness or not, we found that invasions consistently increased the richness of AM fungi. However, AM fungal species composition was not related to invasion status of the vegetation nor local environment, but stratified by watershed. Our results suggest that while invasions can lead to an overall increase in the diversity of microbial mutualists, the effects of plant host identity or geographic structuring potentially outweigh those of invasive species in determining the community membership of AM fungi. Thus, host specificity and spatial factors such as dispersal need to be taken into consideration when examining the effects of biological invasions on symbiotic microbes.

Uncovering unseen fungal diversity from plant DNA banks.

Throughout the world DNA banks are used as storage repositories for genetic diversity of organisms ranging from plants to insects to mammals. Designed to preserve the genetic information for organisms of interest, these banks also indirectly preserve organisms' associated microbiomes, including fungi associated with plant tissues. Studies of fungal biodiversity lag far behind those of macroorganisms, such as plants, and estimates of global fungal richness are still widely debated. Utilizing previously collected specimens to study patterns of fungal diversity could significantly increase our understanding of overall patterns of biodiversity from snapshots in time. Here, we investigated the fungi inhabiting the phylloplane among species of the endemic Hawaiian plant genus, Clermontia (Campanulaceae). Utilizing next generation DNA amplicon sequencing, we uncovered approximately 1,780 fungal operational taxonomic units from just 20 DNA bank samples collected throughout the main Hawaiian Islands. Using these historical samples, we tested the macroecological pattern of decreasing community similarity with decreasing geographic proximity. We found a significant distance decay pattern among Clermontia associated fungal communities. This study provides the first insights into elucidating patterns of microbial diversity through the use of DNA bank repository samples.

The carbon and nitrogen ecophysiologies of two endemic tropical orchids mirrors those of their temperate relatives and the local environment.

Orchids are one of the most widely distributed plant families. However, current research on the ecophysiology of terrestrial orchids is biased towards temperate species. Thus, it is currently unknown whether tropical terrestrial orchids belong to similar trophic guilds as their temperate relatives. To examine the ecophysiologies of two tropical terrestrial orchids, I analysed the carbon and nitrogen stable isotope compositions and nitrogen concentrations of the Hawaiian endemics Anoectochilus sandvicensis and Liparis hawaiensis. I compared these values with those of surrounding vegetation and their temperate relatives. I found that A. sandvicensis was consistently enriched in the heavy isotope of nitrogen (15N) and had higher nitrogen (N) concentrations than surrounding vegetation, and these values were even higher than those of its temperate relatives. Carbon stable isotope composition among populations of A. sandvicensis varied by island. These results point to local environment and evolutionary history determining the ecophysiology of this species. Whereas L.hawaiensis was also enriched in 15N and had on average higher N concentrations than surrounding vegetation, these values were not significantly different from temperate relatives, indicating that evolutionary history may be a stronger predictor of this orchid species' ecophysiology than environment. I suggest that both Hawaiian species are potentially partially mycoheterotrophic.