Lauren C. Cline

Soil microbial communities are shaped by plant-driven changes in resource availability during secondary succession

Although we understand the ecological processes eliciting changes in plant community composition during secondary succession, we do not understand whether co-occurring changes in plant detritus shape saprotrophic microbial communities in soil. In this study, we investigated soil microbial composition and function across an old-field chronosequence ranging from 16 to 86 years following agricultural abandonment, as well as three forests representing potential late-successional ecosystems. Fungal and bacterial community composition was quantified from ribosomal DNA, and insight into the functional potential of the microbial community to decay plant litter was gained from shotgun metagenomics and extracellular enzyme assays. Accumulation of soil organic matter across the chronosequence exerted a positive and significant effect on fungal phylogenetic β-diversity and the activity of extracellular enzymes with lignocellulolytic activity. In addition, the increasing abundance of lignin-rich C4 grasses was positively related to the composition of fungal genes with lignocellulolytic function, thereby linking plant community composition, litter biochemistry, and microbial community function. However, edaphic properties were the primary agent shaping bacterial communities, as bacterial β-diversity and variation in functional gene composition displayed a significant and positive relationship to soil pH across the chronosequence. The late-successional forests were compositionally distinct from the oldest old fields, indicating that substantial changes occur in soil microbial communities as old fields give way to forests. Taken together, our observations demonstrate that plants govern the turnover of soil fungal communities and functional characteristics during secondary succession, due to the continual input of detritus and differences in litter biochemistry among plant species.

Dispersal limitation structures fungal community assembly in a long‐term glacial chronosequence

Microbial communities in soil mediate biogeochemical processes; however, understanding forces shaping their composition and function remains a gap in our ecological knowledge. We investigated phylogenetic turnover and functional gene composition of saprotrophic fungi along a 4000-year glacial chronosequence. A direct relationship between β-diversity and geographic distance, a proxy for time since deglaciation, indicated that dispersal limitation shapes saprotrophic fungal communities. Further, we infer that dispersal limitation may also influence fungal functional properties as metabolic potential and functional richness increased with site age. Despite attempts to minimize environmental variation, a direct relationship between β-diversity and biogeochemical differences across sites indicated that environmental filtering further shapes fungal community composition. However, environmental filtering was overshadowed by the effect of dispersal limitation when tested by multiple regression. Fungal β-diversity and composition of functional genes involved in plant litter decay were unrelated, suggesting that functional traits are not phylogenetically conserved across this chronosequence. Our study suggests that dispersal limitation operates in structuring present-day fungal community composition and functional potential. Further, we demonstrate the need to integrate functional and phylogenetic approaches to more accurately portray microbial communities and their functional capacities.

Anthropogenic N Deposition Slows Decay by Favoring Bacterial Metabolism: Insights from Metagenomic Analyses

Litter decomposition is an enzymatically-complex process that is mediated by a diverse assemblage of saprophytic microorganisms. It is a globally important biogeochemical process that can be suppressed by anthropogenic N deposition. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. Here, we paired extracellular enzyme assays with shotgun metagenomics to assess if anthropogenic N deposition has altered the functional potential of microbial communities inhabiting decaying forest floor. Experimental N deposition significantly reduced the activity of extracellular enzymes mediating plant cell wall decay, which occurred concurrently with changes in the relative abundance of metagenomic functional gene pathways mediating the metabolism of carbohydrates, aromatic compounds, as well as microbial respiration. Moreover, experimental N deposition increased the relative abundance of 50 of the 60 gene pathways, the majority of which were associated with saprotrophic bacteria. Conversely, the relative abundance and composition of fungal genes mediating the metabolism of plant litter was not affected by experimental N deposition. Future rates of atmospheric N deposition have favored saprotrophic soil bacteria, whereas the metabolic potential of saprotrophic fungi appears resilient to this agent of environmental change. Results presented here provide evidence that changes in the functional capacity of saprotrophic soil microorganisms mediate how anthropogenic N deposition increases C storage in soil.

Initial colonization, community assembly and ecosystem function: fungal colonist traits and litter biochemistry mediate decay rate

Priority effects are an important ecological force shaping biotic communities and ecosystem processes, in which the establishment of early colonists alters the colonization success of later‐arriving organisms via competitive exclusion and habitat modification. However, we do not understand which biotic and abiotic conditions lead to strong priority effects and lasting historical contingencies. Using saprotrophic fungi in a model leaf decomposition system, we investigated whether compositional and functional consequences of initial colonization were dependent on initial colonizer traits, resource availability or a combination thereof. To test these ideas, we factorially manipulated leaf litter biochemistry and initial fungal colonist identity, quantifying subsequent community composition, using neutral genetic markers, and community functional characteristics, including enzyme potential and leaf decay rates. During the first 3 months, initial colonist respiration rate and physiological capacity to degrade plant detritus were significant determinants of fungal community composition and leaf decay, indicating that rapid growth and lignolytic potential of early colonists contributed to altered trajectories of community assembly. Further, initial colonization on oak leaves generated increasingly divergent trajectories of fungal community composition and enzyme potential, indicating stronger initial colonizer effects on energy‐poor substrates. Together, these observations provide evidence that initial colonization effects, and subsequent consequences on litter decay, are dependent upon substrate biochemistry and physiological traits within a regional species pool. Because microbial decay of plant detritus is important to global C storage, our results demonstrate that understanding the mechanisms by which initial conditions alter priority effects during community assembly may be key to understanding the drivers of ecosystem‐level processes.

Moving beyond de novo clustering in fungal community ecology

High throughput sequencing (HTS) has rapidly become the de facto tool for characterizing microbial community structure in a wide variety of habitats (Caporaso et al., 2011; Peay et al., 2016; Truong et al., 2017). Accompanying the expanding use of HTS to quantify microbial diversity is the need to delineate species, the ecological unit traditionally used to compare the richness and composition of communities across treatments, locations or habitats (Magurran, 2005). Due to the challenges in identifying microbial species using morphology or biology alone, designations are typically made by ‘binning’ DNA sequences that meet a similarity threshold into operational taxonomic units (OTUs; Blaxter et al., 2005). Currently, the most widely employed approach for defining fungal OTUs is done according to similarities among sequences within the dataset (Supporting Information Fig. S1). Commonly referred to as de novo clustering (Bik et al., 2012), this approach requires no input database as a reference, which is advantageous when characterizing communities with little a priori knowledge. Despite this benefit, the ecological insights gleaned from de novo clustering can be limited by the challenge of directly comparing OTU identity across different studies (€ Opik et al., 2014), and the coarse phylogenetic resolution of many taxonomic assignments (Halwachs et al., 2017). One alternative to de novo clustering is the closed reference approach, where OTUs are binned according to sequence similarity of those in a reference database. With this approach, both OTU clustering and taxonomic designations occur simultaneously. Although the use of closed reference clustering in fungal ecology has been scarce (Fig. S1), it has become increasingly common in the molecular characterization of arbuscular mycorrhizal (AM) fungal communities as well as in many bacterial ‘microbiome’ studies (€ Opik et al., 2014; Kelly et al., 2016). The relatively low taxonomic and phylogenetic diversity of AM fungal communities (Stajich et al., 2009; Redecker et al., 2013; Davison et al., 2015), combined with a curated database (€ Opik et al., 2010) and increasingly wide usage of the 18S rRNA gene for molecular characterization ( € Opik et al., 2014), may explain why AM fungal community ecologists (relative to other fungal ecologists) have readily embraced closed reference clustering. Notably, the closed reference clustering approach has contributed significant new ecological understanding to patterns of AM community assembly by tracking OTUs (referred to as VT, € Opik et al., 2010) across studies with both contrasting habitat types and a wide variety of spatial scales (Davison et al., 2015; Garc ıa de Le on et al., 2016). A second alternative to de novo clustering is an open reference approach, which first clusters sequences to a reference database, followed by de novo clustering of the remaining unmatched sequences. This hybrid approach can combine the advantages of the two aforementioned clustering approaches (Rideout et al., 2014; He et al., 2015), but its interpretation can be problematic if the OTU definitions between closed reference and de novo approaches differ. Although open reference clustering is the least commonly used in fungal community ecology analyses to date (Fig. S1), it has been employed in studies of both arbuscular mycorrhizal and ectomycorrhizal fungal communities (Dumbrell et al., 2010; Jarvis et al., 2015). The increasingly widespread adoption of reference-based clustering inmanymicrobial analyses raises the question: should fungal ecologists re-consider their default use of de novo clustering? In particular, it seems that reference-based clusteringmay represent an increasingly useful approach to fungal community analyses as databases such as UNITE (K~oljalg et al., 2013) grow in size and a greater diversity of fungal habitats are molecularly characterized. Recent studies have suggested that reference-based clustering can increase OTU stability and taxonomic accuracy relative to de novo clustering (He et al., 2015; Halwachs et al., 2017), although how this clustering approach influences fungal community analyses across diverse habitats is currently unclear. To assess this gap in knowledge, we compared the relative performance of de novo, closed reference, and open reference clustering approaches on a mock community, as well as samples from four ecologically distinct habitats. These habitats varied in the degree to which fungal composition was captured by the UNITE database, providing an opportunity to investigate the importance of a priori habitat characterization on clustering approach performance. Using dead wood, live wood, live leaf and forest soil samples, we quantified fungal species assignments, OTU richness and community composition from ITS1 amplicon libraries sequenced on the IlluminaMiSeq platform.We compared two de novo clustering algorithms (CD-HIT and USEARCH; Li & Godzik, 2006; Edgar, 2010), two closed reference clustering algorithms (BLAST and NINJA-OPS; Altschul et al., 1990; Al-Ghalith et al., 2016), as well as two open reference clustering scenarios (NINJA/USEARCH; BLAST/ CD-HIT) applying a 97% sequence similarity cutoff for OTU clustering aswell as taxonomy assignments (Table S1). For the open reference clustering, sequences were first clustered by a closed reference algorithm (i.e. NINJA or BLAST); the remaining sequences that failed to cluster were then clustered by a de novo clustering approach (i.e. USEARCH or CD-HIT), and the OTU tables were combined (sensu Rideout et al., 2014). The UNITE database (v.7.0) was used for reference-based clustering as well as for designating

Probing promise versus performance in longer read fungal metabarcoding

In the rapidly evolving world of methodologies to study fungi and other microorganisms, there has been growing interest in the adoption of so-called ‘third-generation’ technologies for high throughput amplicon sequencing (also known as metabarcoding). This interest is largely based on the capacity for longer sequence read lengths (> 500 bp), which have the potential to provide more accurate phylogenetic inference than current ‘second generation’ technologies (James et al., 2016; Schloss et al., 2016; Singer et al., 2016). Despite this attraction, higher error rates and the high cost per base pair have inhibited the widespread adoption of this ‘next’ in microbial metabarcoding. Given the challenges in adopting any new technology, careful benchmarking tests are needed to determine whether significantly greater insights can be gleaned, or if currently establishedmethods remain sufficient. In this issue of New Phytologist, Tedersoo et al. (2018; pp. 1370–1385) conduct the first of these benchmarking tests for fungi and other eukaryotes, directly comparing Illumina MiSeq (i.e. second generation) and PacBio datasets (i.e. third generation) generated from the same soil samples and analyzed for taxonomic richness and composition. While their collective analyses indicate that longer amplicons can be successfully generated with relatively low error rates, they also demonstrate many technical issues with PacBio-based data, which require careful attention.

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater β-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.

Elk, sagebrush, and saprotrophs: indirect top‐down control on microbial community composition and function

Saprotrophic microbial communities in soil are primarily structured by the availability of growth-limiting resources (i.e., plant detritus), a bottom-up ecological force. However, foraging by native ungulates can alter plant community composition and the nature of detritus entering soil, plausibly exerting an indirect, top-down ecological force that shapes both the composition and function of soil microbial communities. To test this idea, we used physiological assays and molecular approaches to quantify microbial community composition and function inside and outside of replicate, long-term (60-80 yr) winter-foraging exclosures in sagebrush steppe of Wyoming, USA. Winter foraging exclusion substantially increased shrub biomass (2146 g/m2 vs. 87 g/m2), which, in turn, increased the abundance of bacterial and fungal genes with lignocellulolytic function; microbial respiration (+50%) and net N mineralization (+70%) also were greater in the absence of winter foraging. Our results reveal that winter foraging by native, migratory ungulates in sagebrush steppe exerts an indirect, top-down ecological force that shapes the composition and function of soil microbial communities. Because approximately 25% of the Earths land surface is influenced by grazing animals, this indirect top-down ecological force could function to broadly shape the community membership and physiological capacity of saprotrophic microbial communities in shrub steppe.