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.

Latitudinal patterns in stabilizing density dependence of forest communities.

Numerous studies have shown reduced performance in plants that are surrounded by neighbours of the same species1,2, a phenomenon known as conspecific negative density dependence (CNDD)3. A long-held ecological hypothesis posits that CNDD is more pronounced in tropical than in temperate forests4,5, which increases community stabilization, species coexistence and the diversity of local tree species6,7. Previous analyses supporting such a latitudinal gradient in CNDD8,9 have suffered from methodological limitations related to the use of static data10-12. Here we present a comprehensive assessment of latitudinal CNDD patterns using dynamic mortality data to estimate species-site-specific CNDD across 23 sites. Averaged across species, we found that stabilizing CNDD was present at all except one site, but that average stabilizing CNDD was not stronger toward the tropics. However, in tropical tree communities, rare and intermediate abundant species experienced stronger stabilizing CNDD than did common species. This pattern was absent in temperate forests, which suggests that CNDD influences species abundances more strongly in tropical forests than it does in temperate ones13. We also found that interspecific variation in CNDD, which might attenuate its stabilizing effect on species diversity14,15, was high but not significantly different across latitudes. Although the consequences of these patterns for latitudinal diversity gradients are difficult to evaluate, we speculate that a more effective regulation of population abundances could translate into greater stabilization of tropical tree communities and thus contribute to the high local diversity of tropical forests.

Mycorrhizal feedbacks influence global forest structure and diversity.

One mechanism proposed to explain high species diversity in tropical systems is strong negative conspecific density dependence (CDD), which reduces recruitment of juveniles in proximity to conspecific adult plants. Although evidence shows that plant-specific soil pathogens can drive negative CDD, trees also form key mutualisms with mycorrhizal fungi, which may counteract these effects. Across 43 large-scale forest plots worldwide, we tested whether ectomycorrhizal tree species exhibit weaker negative CDD than arbuscular mycorrhizal tree species. We further tested for conmycorrhizal density dependence (CMDD) to test for benefit from shared mutualists. We found that the strength of CDD varies systematically with mycorrhizal type, with ectomycorrhizal tree species exhibiting higher sapling densities with increasing adult densities than arbuscular mycorrhizal tree species. Moreover, we found evidence of positive CMDD for tree species of both mycorrhizal types. Collectively, these findings indicate that mycorrhizal interactions likely play a foundational role in global forest diversity patterns and structure.

Masting is uncommon in trees that depend on mutualist dispersers in the context of global climate and fertility gradients.

The benefits of masting (volatile, quasi-synchronous seed production at lagged intervals) include satiation of seed predators, but these benefits come with a cost to mutualist pollen and seed dispersers. If the evolution of masting represents a balance between these benefits and costs, we expect mast avoidance in species that are heavily reliant on mutualist dispersers. These effects play out in the context of variable climate and site fertility among species that vary widely in nutrient demand. Meta-analyses of published data have focused on variation at the population scale, thus omitting periodicity within trees and synchronicity between trees. From raw data on 12 million tree-years worldwide, we quantified three components of masting that have not previously been analysed together: (i) volatility, defined as the frequency-weighted year-to-year variation; (ii) periodicity, representing the lag between high-seed years; and (iii) synchronicity, indicating the tree-to-tree correlation. Results show that mast avoidance (low volatility and low synchronicity) by species dependent on mutualist dispersers explains more variation than any other effect. Nutrient-demanding species have low volatility, and species that are most common on nutrient-rich and warm/wet sites exhibit short periods. The prevalence of masting in cold/dry sites coincides with climatic conditions where dependence on vertebrate dispersers is less common than in the wet tropics. Mutualist dispersers neutralize the benefits of masting for predator satiation, further balancing the effects of climate, site fertility and nutrient demands.

Seed Disinfestation Practices to Control Seed-Borne Fungi and Bacteria in Home Production of Sprouts.

Concern over microbial contamination limits the adoption of home production of sprouts as a nutritious and sustainable food. Simple, accessible approaches to seed disinfection could support safe home seed sprouting. Here, we quantify bacterial and fungal contamination of seeds of 14 plant cultivars sold for home sprout production and test a range of chemical and physical methods for seed disinfestation appropriate for home use. Most seeds are contaminated with a variety of bacteria and fungi, and those microbes are usually limited to the seed surface. Heat treatments are not effective for seed disinfection because the high temperatures needed to effectively reduce microbial contamination also reduce seed germination. Two chlorine-based chemical disinfectants-dilute household bleach (0.6% sodium hypochlorite) and freshly generated hypochlorous acid (800 ppm chlorine)-were the most effective disinfection agents tested (up to a 5-log reduction in bacteria) that also did not harm seed germination.

Coexistence is stabilized by conspecific negative density dependence via fungal pathogens more than oomycete pathogens.

Plant pathogens are often hypothesized to promote species coexistence by generating conspecific negative density dependence (CNDD). However, the relative importance of fungal versus oomycete pathogens in maintaining plant species coexistence and community composition remains unresolved, despite their recognized effects on plant performance. Here, we use fungicide application to investigate how fungal versus oomycete pathogens affect plant species coexistence in an alpine meadow. We found that the severity of foliar fungal disease was density-dependent at both intra- and interspecific levels. Fungal pathogen-exclusion treatment successfully decreased the severity of foliar fungal diseases, with no detectable effects on root colonization by arbuscular mycorrhizal fungi or on soil chemical properties. Fungal pathogens were important factors shaping CNDD across 25 coexisting plant species. Exclusion of fungal pathogens significantly reduced plant species richness and Shannon's evenness. Treatments that excluded fungal pathogens also led to significant shifts in plant community composition toward more Poaceae and Cyperaceae. These results indicate that fungal pathogens, especially those affecting aboveground plant parts, may play a larger role in maintaining species coexistence and shaping community composition than has been previously recognized.

Limits to reproduction and seed size-number trade-offs that shape forest dominance and future recovery.

The relationships that control seed production in trees are fundamental to understanding the evolution of forest species and their capacity to recover from increasing losses to drought, fire, and harvest. A synthesis of fecundity data from 714 species worldwide allowed us to examine hypotheses that are central to quantifying reproduction, a foundation for assessing fitness in forest trees. Four major findings emerged. First, seed production is not constrained by a strict trade-off between seed size and numbers. Instead, seed numbers vary over ten orders of magnitude, with species that invest in large seeds producing more seeds than expected from the 1:1 trade-off. Second, gymnosperms have lower seed production than angiosperms, potentially due to their extra investments in protective woody cones. Third, nutrient-demanding species, indicated by high foliar phosphorus concentrations, have low seed production. Finally, sensitivity of individual species to soil fertility varies widely, limiting the response of community seed production to fertility gradients. In combination, these findings can inform models of forest response that need to incorporate reproductive potential.

Globally, tree fecundity exceeds productivity gradients.

Lack of tree fecundity data across climatic gradients precludes the analysis of how seed supply contributes to global variation in forest regeneration and biotic interactions responsible for biodiversity. A global synthesis of raw seedproduction data shows a 250-fold increase in seed abundance from cold-dry to warm-wet climates, driven primarily by a 100-fold increase in seed production for a given tree size. The modest (threefold) increase in forest productivity across the same climate gradient cannot explain the magnitudes of these trends. The increase in seeds per tree can arise from adaptive evolution driven by intense species interactions or from the direct effects of a warm, moist climate on tree fecundity. Either way, the massive differences in seed supply ramify through food webs potentially explaining a disproportionate role for species interactions in the wet tropics.

North American tree migration paced by climate in the West, lagging in the East.

Tree fecundity and recruitment have not yet been quantified at scales needed to anticipate biogeographic shifts in response to climate change. By separating their responses, this study shows coherence across species and communities, offering the strongest support to date that migration is in progress with regional limitations on rates. The southeastern continent emerges as a fecundity hotspot, but it is situated south of population centers where high seed production could contribute to poleward population spread. By contrast, seedling success is highest in the West and North, serving to partially offset limited seed production near poleward frontiers. The evidence of fecundity and recruitment control on tree migration can inform conservation planning for the expected long-term disequilibrium between climate and forest distribution.

Is there tree senescence? The fecundity evidence.

Despite its importance for forest regeneration, food webs, and human economies, changes in tree fecundity with tree size and age remain largely unknown. The allometric increase with tree diameter assumed in ecological models would substantially overestimate seed contributions from large trees if fecundity eventually declines with size. Current estimates are dominated by overrepresentation of small trees in regression models. We combined global fecundity data, including a substantial representation of large trees. We compared size-fecundity relationships against traditional allometric scaling with diameter and two models based on crown architecture. All allometric models fail to describe the declining rate of increase in fecundity with diameter found for 80% of 597 species in our analysis. The strong evidence of declining fecundity, beyond what can be explained by crown architectural change, is consistent with physiological decline. A downward revision of projected fecundity of large trees can improve the next generation of forest dynamic models.

Parasitism to mutualism continuum for Joshua trees inoculated with different communities of arbuscular mycorrhizal fungi from a desert elevation gradient.

Most desert plants form symbiotic relationships with arbuscular mycorrhizal fungi (AMF), yet fungal identity and impacts on host plants remain largely unknown. Despite widespread recognition of the importance of AMF relationships for plant functioning, we do not know how fungal community structure changes across a desert climate gradient, nor the impacts of different fungal communities on host plant species. Because climate change can shape the distribution of species through effects on species interactions, knowing how the ranges of symbiotic partners are geographically structured and the outcomes of those species interactions informs theory and improves management recommendations. Here we used high throughput sequencing to examine the AMF community of Joshua trees along a climate gradient in Joshua Tree National Park. We then used a range of performance measures and abiotic factors to evaluate how different AMF communities may affect Joshua tree fitness. We found that fungal communities change with elevation resulting in a spectrum of interaction outcomes from mutualism to parasitism that changed with the developmental stage of the plant. Nutrient accumulation and the mycorrhizal growth response of Joshua tree seedlings inoculated with fungi from the lowest (warmest) elevations was first negative, but after 9 months had surpassed that of plants with other fungal treatments. This indicates that low elevation fungi are costly for the plant to initiate symbiosis, yet confer benefits over time. The strong relationship between AMF community and plant growth suggests that variation in AMF community may have long term consequences for plant populations along an elevation gradient.

Arbuscular mycorrhizal trees influence the latitudinal beta-diversity gradient of tree communities in forests worldwide.

Arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) associations are critical for host-tree performance. However, how mycorrhizal associations correlate with the latitudinal tree beta-diversity remains untested. Using a global dataset of 45 forest plots representing 2,804,270 trees across 3840 species, we test how AM and EcM trees contribute to total beta-diversity and its components (turnover and nestedness) of all trees. We find AM rather than EcM trees predominantly contribute to decreasing total beta-diversity and turnover and increasing nestedness with increasing latitude, probably because wide distributions of EcM trees do not generate strong compositional differences among localities. Environmental variables, especially temperature and precipitation, are strongly correlated with beta-diversity patterns for both AM trees and all trees rather than EcM trees. Results support our hypotheses that latitudinal beta-diversity patterns and environmental effects on these patterns are highly dependent on mycorrhizal types. Our findings highlight the importance of AM-dominated forests for conserving global forest biodiversity.

Host evolutionary relationships explain tree mortality caused by a generalist pest-pathogen complex.

The phylogenetic signal of transmissibility (competence) and attack severity among hosts of generalist pests is poorly understood. In this study, we examined the phylogenetic effects on hosts differentially affected by an emergent generalist beetle-pathogen complex in California and South Africa. Host types (non-competent, competent and killed-competent) are based on nested types of outcomes of interactions between host plants, the beetles and the fungal pathogens. Phylogenetic dispersion analysis of each host type revealed that the phylogenetic preferences of beetle attack and fungal growth were a nonrandom subset of all available tree and shrub species. Competent hosts were phylogenetically narrower by 62 Myr than the set of all potential hosts, and those with devastating impacts were the most constrained by 107 Myr. Our results show a strong phylogenetic signal in the relative effects of a generalist pest-pathogen complex on host species, demonstrating that the strength of multi-host pest impacts in plants can be predicted by host evolutionary relationships. This study presents a unifying theoretical approach to identifying likely disease outcomes across multiple host-pest combinations.

Continent-wide tree fecundity driven by indirect climate effects.

Indirect climate effects on tree fecundity that come through variation in size and growth (climate-condition interactions) are not currently part of models used to predict future forests. Trends in species abundances predicted from meta-analyses and species distribution models will be misleading if they depend on the conditions of individuals. Here we find from a synthesis of tree species in North America that climate-condition interactions dominate responses through two pathways, i) effects of growth that depend on climate, and ii) effects of climate that depend on tree size. Because tree fecundity first increases and then declines with size, climate change that stimulates growth promotes a shift of small trees to more fecund sizes, but the opposite can be true for large sizes. Change the depresses growth also affects fecundity. We find a biogeographic divide, with these interactions reducing fecundity in the West and increasing it in the East. Continental-scale responses of these forests are thus driven largely by indirect effects, recommending management for climate change that considers multiple demographic rates.

Mycorrhizal type influences plant density dependence and species richness across 15 temperate forests.

Recent studies suggest that the mycorrhizal type associated with tree species is an important trait influencing ecological processes such as response to environmental conditions and conspecific negative density dependence (CNDD). However, we lack a general understanding of how tree mycorrhizal type influences CNDD strength and the resulting patterns of species abundance and richness at larger spatial scales. We assessed 305 species across 15 large, stem-mapped, temperate forest dynamics plots in Northeastern China and North America to explore the relationships between tree mycorrhizal type and CNDD, species abundance, and species richness at a regional scale. Tree species associated with arbuscular mycorrhizal (AM) fungi showed a stronger CNDD and a more positive relationship with species abundance than did tree species associated with ectomycorrhizal (ECM) fungi. For each plot, both basal area and stem abundance of AM tree species was lower than that of ECM tree species, suggesting that AM tree species were rarer than ECM tree species. Finally, ECM tree dominance showed a negative effect on plant richness across plots. These results provide evidence that tree mycorrhizal type plays an important role in influencing CNDD and species richness, highlighting this trait as an important factor in structuring plant communities in temperate forests.

Soil microbes drive phylogenetic diversity-productivity relationships in a subtropical forest.

The relationship between plant diversity and productivity and the mechanisms underpinning that relationship remain poorly resolved in species-rich forests. We combined extensive field observations and experimental manipulations in a subtropical forest to test how species richness (SR) and phylogenetic diversity (PD) interact with putative root-associated pathogens and how these interactions mediate diversity-productivity relationships. We show that (i) both SR and PD were positively correlated with biomass for both adult trees and seedlings across multiple spatial scales, but productivity was best predicted by PD; (ii) significant positive relationships between PD and productivity were observed in nonsterile soil only; and (iii) root fungal diversity was positively correlated with plant PD and SR, while the relative abundance of putative pathogens was negatively related to plant PD. Our findings highlight the key role of soil pathogenic fungi in tree diversity-productivity relationships and suggest that increasing PD may counteract negative effects of plant-soil feedback.

Linking Aboveground Traits to Root Traits and Local Environment: Implications of the Plant Economics Spectrum.

The plant economics spectrum proposes that ecological traits are functionally coordinated and adapt along environmental gradients. However, empirical evidence is mixed about whether aboveground and root traits are consistently linked and which environmental factors drive functional responses. Here we measure the strength of relationships between aboveground and root traits, and examine whether community-weighted mean trait values are adapted along gradients of light and soil fertility, based on the seedling censuses of 57 species in a subtropical forest. We found that aboveground traits were good predictors of root traits; specific leaf area, dry matter, nitrogen and phosphorus content were strongly correlated with root tissue density and specific root length. Traits showed patterns of adaptation along the gradients of soil fertility and light; species with fast resource-acquisitive strategies were more strongly associated with high soil phosphorus, potassium, openness, and with low nitrogen, organic matter conditions. This demonstrates the potential to estimate belowground traits from known aboveground traits in seedling communities, and suggests that soil fertility is one of the main factors driving functional responses. Our results extend our understanding of how ecological strategies shape potential responses of plant communities to environmental change.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Ectomycorrhizas and tree seedling establishment are strongly influenced by forest edge proximity but not soil inoculum.

Reforestation is challenging when timber harvested areas have been degraded, invaded by nonnative species, or are of marginal suitability to begin with. Conifers form mutualistic partnerships with ectomycorrhizal fungi (EMF) to obtain greater access to soil resources, and these partnerships may be especially important in degraded areas. However, timber harvest can impact mycorrhizal fungi by removing or compacting topsoil, removing host plants, and warming and drying the soil. We used a field experiment to evaluate the role of EMF in Douglas-fir reforestation in clearcuts invaded by Cytisus scoparius (Scotch broom) where traditional reforestation approaches have repeatedly failed. We tested how planting distance from intact Douglas-fir forest edges influenced reforestation success and whether inoculation with forest soils can be used to restore EMF relationships. We used an Illumina DNA sequencing approach to measure the abundance, richness and composition of ectomycorrhizal fungi on Douglas-fir roots, and assessed differences in Douglas-fir seedling survival and growth near to and far from forest edges with and without forest soil inoculum. Planting Douglas-fir seedlings near forest edges increased seedling survival, growth, and EMF root colonization. Edge proximity had no effect on EMF richness but did change fungal community composition. Inoculations with forest soil did not increase EMF abundance or richness or change community composition, nor did it improve seedling establishment. With Illumina sequencing, we identified two to three times greater species richness than described in previous edge effects studies. Of the 95 EMF species we identified, 40% of the species occurred on less than 5% of the seedlings. The ability to detect fungi at low abundance may explain why we did not detect differences in EMF richness with distance to hosts as previous studies. Our findings suggest that forest edges are suitable for reforestation, even when the interiors of deforested areas are not. We advocate for timber harvest designs that maximize edge habitat where ectomycorrhizal fungi contribute to tree establishment. However, this study does not support the use of inoculation with forest soil as a simple method to enhance EMF and seedling survival.