Phylogenetic conservatism of mycoparasitism and its contribution to pathogen antagonism.

Closely related species are expected to have similar functional traits due to shared ancestry and phylogenetic inertia. However, few tests of this hypothesis are available for plant-associated fungal symbionts. Fungal leaf endophytes occur in all land plants and can protect their host plant from disease by a variety of mechanisms, including by parasitizing pathogens (e.g., mycoparasitism). Here, we tested whether phylogenetic relatedness among species of Cladosporium, a widespread genus that includes mycoparasitic species, predicts the effect of this endophyte on the severity of leaf rust disease. First, we used congruence among different marker sequences (i.e., genealogical concordance phylogenetic species recognition criterion) to delimit species of Cladosporium. Next, in a controlled experiment, we quantified both mycoparasitism and disease modification for the selected Cladosporium species. We identified 17 species of Cladosporium; all the species reduced rust disease severity in our experiment. Cladosporium phylogeny was a significant predictor of mycoparasitism. However, we did not observe a phylogenetic effect on disease severity overall, indicating that other mechanism/s operating independently of shared ancestry also contributed to endophyte effects on disease severity. Indeed, a second experiment showed that Cladosporium endophyte exudates (no live organism) from divergent species groups equally reduced disease severity. Our results reveal that multiple mechanisms contribute to the protective effects of an endophyte against a plant pathogen, but not all traits underlying these mechanisms are phylogenetically conserved.

Diversity of putative ericoid mycorrhizal fungi increases with soil age and progressive phosphorus limitation across a 4.1-million-year chronosequence.

Ericaceous plants rely on ericoid mycorrhizal fungi for nutrient acquisition. However, the factors that affect the composition and structure of fungal communities associated with the roots of ericaceous plants remain largely unknown. Here, we use a 4.1-million-year (myr) soil chronosequence in Hawaii to test the hypothesis that changes in nutrient availability with soil age determine the diversity and species composition of fungi associated with ericoid roots. We sampled roots of a native Hawaiian plant, Vaccinium calycinum, and used DNA metabarcoding to quantify changes in fungal diversity and community composition. We also used a fertilization experiment at the youngest and oldest sites to assess the importance of nutrient limitation. We found an increase in diversity and a clear pattern of species turnover across the chronosequence, driven largely by putative ericoid mycorrhizal fungi. Fertilization with nitrogen at the youngest site and phosphorus at the oldest site reduced fungal diversity, suggesting a direct role of nutrient limitation. Our results also reveal the presence of novel fungal species associated with Hawaiian Ericaceae and suggest a greater importance of phosphorus availability for communities of ericoid mycorrhizal fungi than is generally assumed.

Greater local diversity under older species pools may arise from enhanced competitive equivalence.

Ecological communities typically contain more species when located within geologically older regions. This pattern is traditionally attributed to the long-term accumulation of species in the regional species pool, with local species interactions playing a minor role. We provide evidence suggesting a more important role of local species interactions than generally assumed. We assembled 320 communities of root-associated fungi under 80 species pools, varying species pool richness and the mean age of the sites from which the fungi were collected across a 4-myr soil chronosequence. We found that local diversity increased more with increasing species pool richness when species were from older sites. We also found that older species pools had lower functional and phylogenetic diversity, indicating greater competitive equivalence among species. Our results suggest that older regions have higher local richness not simply because older pools are more speciose but also because species have evolved traits that allow them to locally co-occur.

Host Genotype and Colonist Arrival Order Jointly Govern Plant Microbiome Composition and Function.

The composition of host-associated microbiomes can have important consequences for host health and fitness [1-3]. Yet we still lack understanding of many fundamental processes that determine microbiome composition [4, 5]. There is mounting evidence that historical contingency during microbiome assembly may overshadow more deterministic processes, such as the selective filters imposed by host traits [6-8]. More specifically, species arrival order has been frequently shown to affect microbiome composition [9-12], a phenomenon known as priority effects [13-15]. However, it is less clear whether priority effects during microbiome assembly are consequential for the host [16] or whether intraspecific variation in host traits can alter the trajectory of microbiome assembly under priority effects. In a greenhouse inoculation experiment using the black cottonwood (Populus trichocarpa) foliar microbiome, we manipulated host genotype and the colonization order of common foliar fungi. We quantified microbiome assembly outcomes using fungal marker gene sequencing and measured susceptibility of the colonized host to a leaf rust pathogen, Melampsora × columbiana. We found that the effect of species arrival order on microbiome composition, and subsequent disease susceptibility, depended on the host genotype. Additionally, we found that microbiome assembly history can affect host disease susceptibility independent of microbiome composition at the time of pathogen exposure, suggesting that the interactive effects of species arrival order and host genotype can decouple community composition and function. Overall, these results highlight the importance of a key process underlying stochasticity in microbiome assembly while also revealing which hosts are most likely to experience these effects.

Vertical foraging shifts in Hawaiian forest birds in response to invasive rat removal.

Worldwide, native species increasingly contend with the interacting stressors of habitat fragmentation and invasive species, yet their combined effects have rarely been examined. Direct negative effects of invasive omnivores are well documented, but the indirect effects of resource competition or those caused by predator avoidance are unknown. Here we isolated and examined the independent and interactive effects of invasive omnivorous Black rats (Rattus rattus) and forest fragment size on the interactions between avian predators and their arthropod prey. Our study examines whether invasive omnivores and ecosystem fragment size impact: 1) the vertical distribution of arthropod species composition and abundance, and 2) the vertical profile of foraging behaviors of five native and two non-native bird species found in our study system. We predicted that the reduced edge effects and greater structural complexity and canopy height of larger fragments would limit the total and proportional habitat space frequented by rats and thus limit their impact on both arthropod biomass and birds' foraging behavior. We experimentally removed invasive omnivorous Black rats across a 100-fold (0.1 to 12 ha) size gradient of forest fragments on Hawai'i Island, and paired foraging observations of forest passerines with arthropod sampling in the 16 rat-removed and 18 control fragments. Rat removal was associated with shifts in the vertical distribution of arthropod biomass, irrespective of fragment size. Bird foraging behavior mirrored this shift, and the impact of rat removal was greater for birds that primarily eat fruit and insects compared with those that consume nectar. Evidence from this model study system indicates that invasive rats indirectly alter the feeding behavior of native birds, and consequently impact multiple trophic levels. This study suggests that native species can modify their foraging behavior in response to invasive species removal and presumably arrival through behavioral plasticity.

Protocols for Investigating the Leaf Mycobiome Using High-Throughput DNA Sequencing.

High-throughput sequencing of taxon-specific loci, or DNA metabarcoding, has become an invaluable tool for investigating the composition of plant-associated fungal communities and for elucidating plant-fungal interactions. While sequencing fungal communities has become routine, there remain numerous potential sources of systematic error that can introduce biases and compromise metabarcoding data. This chapter presents a protocol for DNA metabarcoding of the leaf mycobiome based on current best practices to minimize errors through careful laboratory practices and validation.

Priority effects are interactively regulated by top-down and bottom-up forces: evidence from wood decomposer communities.

Both top-down (grazing) and bottom-up (resource availability) forces can determine the strength of priority effects, or the effects of species arrival history on the structure and function of ecological communities, but their combined influences remain unresolved. To test for such influences, we assembled experimental communities of wood-decomposing fungi using a factorial manipulation of fungivore (Folsomia candida) presence, nitrogen availability, and fungal assembly history. We found interactive effects of all three factors on fungal species composition and wood decomposition 1 year after the fungi were introduced. The strength of priority effects on community structure was affected primarily by nitrogen availability, whereas the strength of priority effects on decomposition rate was interactively regulated by nitrogen and fungivores. These results demonstrate that top-down and bottom-up forces jointly determine how strongly assembly history affects community structure and function.

Forest area and connectivity influence root-associated fungal communities in a fragmented landscape.

Habitat fragmentation is well known to affect plant and animal diversity as a result of reduced habitat area and connectivity, but its effects on microorganisms are poorly understood. Using high-throughput sequencing of two regions of the rRNA gene, we studied the effects of forest area and connectivity on the diversity and composition of fungi associated with the roots of the dominant tree, Metrosideros polymorpha, in a lava-fragmented landscape on the Island of Hawaii. We found that local fungal diversity increased with forest area, whereas fungal species composition was correlated with fragment connectivity. Fragment size and connectivity were significant predictors even when we included environmental covariates, which were also associated with fungal diversity and composition. Fungal species composition was more similar among highly connected fragments than among poorly connected ones. We also identified individual taxa that varied in abundance with connectivity. Taken together, our results show that habitat fragmentation can alter microbial diversity and composition via differential response among fungal phyla and individual taxa to habitat connectivity.