Herbarium specimens reveal that mycorrhizal type does not mediate declining temperate tree nitrogen status over a century of environmental change.

Rising atmospheric carbon dioxide concentrations (CO2 ) and atmospheric nitrogen (N) deposition have contrasting effects on ectomycorrhizal (EM) and arbuscular mycorrhizal (AM) symbioses, potentially mediating forest responses to environmental change. In this study, we evaluated the cumulative effects of historical environmental change on N concentrations and δ15 N values in AM plants, EM plants, EM fungi, and saprotrophic fungi using herbarium specimens collected in Minnesota, USA from 1871 to 2016. To better understand mycorrhizal mediation of foliar δ15 N, we also analyzed a subset of previously published foliar δ15 N values from across the United States to parse the effects of N deposition and CO2 rise. Over the last century in Minnesota, N concentrations declined among all groups except saprotrophic fungi. δ15 N also declined among all groups of plants and fungi; however, foliar δ15 N declined less in EM plants than in AM plants. In the analysis of previously published foliar δ15 N values, this slope difference between EM and AM plants was better explained by nitrogen deposition than by CO2 rise. Mycorrhizal type did not explain trajectories of plant N concentrations. Instead, plants and EM fungi exhibited similar declines in N concentrations, consistent with declining forest N status despite moderate levels of N deposition.

Long-term elevated precipitation induces grassland soil carbon loss via microbe-plant-soil interplay.

Global climate models predict that the frequency and intensity of precipitation events will increase in many regions across the world. However, the biosphere-climate feedback to elevated precipitation (eP) remains elusive. Here, we report a study on one of the longest field experiments assessing the effects of eP, alone or in combination with other climate change drivers such as elevated CO2 (eCO2 ), warming and nitrogen deposition. Soil total carbon (C) decreased after a decade of eP treatment, while plant root production decreased after 2 years. To explain this asynchrony, we found that the relative abundances of fungal genes associated with chitin and protein degradation increased and were positively correlated with bacteriophage genes, suggesting a potential viral shunt in C degradation. In addition, eP increased the relative abundances of microbial stress tolerance genes, which are essential for coping with environmental stressors. Microbial responses to eP were phylogenetically conserved. The effects of eP on soil total C, root production, and microbes were interactively affected by eCO2 . Collectively, we demonstrate that long-term eP induces soil C loss, owing to changes in microbial community composition, functional traits, root production, and soil moisture. Our study unveils an important, previously unknown biosphere-climate feedback in Mediterranean-type water-limited ecosystems, namely how eP induces soil C loss via microbe-plant-soil interplay.

How do soil microbial communities respond to fire in the intermediate term? Investigating direct and indirect effects associated with fire occurrence and burn severity.

Fires transform soil microbial communities directly via heat-induced mortality and indirectly by altering plant and soil characteristics. Emerging evidence suggests the magnitude of changes to some plant and soil properties increases with burn severity, but the persistence of changes varies among plant and soil characteristics, ranging from months to years post-fire. Thus, which environmental attributes shape microbial communities at intermediate time points during ecosystem recovery, and how these characteristics vary with severity, remains poorly understood. We identified the network of properties that influence microbial communities three years after fire, along a burn severity gradient in Sierra Nevada mixed-conifer forest. We used phospholipid fatty acid (PLFA) analysis and bacterial 16S-rDNA amplicon sequencing to characterize the microbial community in mineral soil. Using structural equation modelling, we applied a systems approach to identifying the interconnected relationships among severity, vegetation, soil, and microbial communities. Dead tree basal area, soil pH, and extractable phosphorus increased with severity, whereas live tree basal area, forest floor mass, and the proportion of the ≥53 µm soil fraction decreased. Forest floor loss was associated with decreased soil moisture across the severity gradient, decreased live tree basal area was associated with increased shrub coverage, and increased dead tree basal area was associated with increases in total and inorganic soil nitrogen. Soil fungal abundance decreased across the severity gradient, despite a slightly positive response of fungi to lower soil moisture in high severity areas. Bacterial phylogenetic diversity was negatively related to severity and was driven by differences in nutrients and soil texture. The abundance of Bacteroidetes increased and the abundance of Acidobacteria decreased across the severity gradient due to differences in soil pH. Overall, we found that the effects of burn severity on vegetation and soil physicochemical characteristics interact to shape microbial communities at an intermediate time point in ecosystem recovery.

Fire affects the taxonomic and functional composition of soil microbial communities, with cascading effects on grassland ecosystem functioning.

Fire is a crucial event regulating the structure and functioning of many ecosystems. Yet few studies have focused on how fire affects taxonomic and functional diversities of soil microbial communities, along with changes in plant communities and soil carbon (C) and nitrogen (N) dynamics. Here, we analyze these effects in a grassland ecosystem 9 months after an experimental fire at the Jasper Ridge Global Change Experiment site in California, USA. Fire altered soil microbial communities considerably, with community assembly process analysis showing that environmental selection pressure was higher in burned sites. However, a small subset of highly connected taxa was able to withstand the disturbance. In addition, fire decreased the relative abundances of most functional genes associated with C degradation and N cycling, implicating a slowdown of microbial processes linked to soil C and N dynamics. In contrast, fire stimulated above- and belowground plant growth, likely enhancing plant-microbe competition for soil inorganic N, which was reduced by a factor of about 2. To synthesize those findings, we performed structural equation modeling, which showed that plants but not microbial communities were responsible for significantly higher soil respiration rates in burned sites. Together, our results demonstrate that fire 'reboots' the grassland ecosystem by differentially regulating plant and soil microbial communities, leading to significant changes in soil C and N dynamics.

Impacts of Sampling Design on Estimates of Microbial Community Diversity and Composition in Agricultural Soils.

Soil microbiota play important and diverse roles in agricultural crop nutrition and productivity. Yet, despite increasing efforts to characterize soil bacterial and fungal assemblages, it is challenging to disentangle the influences of sampling design on assessments of communities. Here, we sought to determine whether composite samples-often analyzed as a low cost and effort alternative to replicated individual samples-provide representative summary estimates of microbial communities. At three Minnesota agricultural research sites planted with an oat cover crop, we conducted amplicon sequencing for soil bacterial and fungal communities (16SV4 and ITS2) of replicated individual or homogenized composite soil samples. We compared soil microbiota from within and among plots and then among agricultural sites using both sampling strategies. Results indicated that single or multiple replicated individual samples, or a composite sample from each plot, were sufficient for distinguishing broad site-level macroecological differences among bacterial and fungal communities. Analysis of a single sample per plot captured only a small fraction of the distinct OTUs, diversity, and compositional variability detected in the analysis of multiple individual samples or a single composite sample. Likewise, composite samples captured only a fraction of the diversity represented by the six individual samples from which they were formed, and, on average, analysis of two or three individual samples offered greater compositional coverage (i.e., greater number of OTUs) than a single composite sample. We conclude that sampling design significantly impacts estimates of bacterial and fungal communities even in homogeneously managed agricultural soils, and our findings indicate that while either strategy may be sufficient for broad macroecological investigations, composites may be a poor substitute for replicated samples at finer spatial scales.

Soil microbial restoration strategies for promoting climate-ready prairie ecosystems.

Tractable practices for soil microbial restoration in tallgrass prairies reclaimed from agriculture are a critical gap in traditional ecological restoration. Long-term fertilization and tilling permanently alter soil bacterial and fungal communities, requiring microbe-targeted restoration methods to improve belowground ecosystem services and carbon storage in newly restored prairies. These techniques are particularly important when restoring for climate-ready ecosystems, adapted to altered temperature regimes. To approach these issues, we conducted a multi-factorial greenhouse experiment to test the effects of plant species richness, soil amendment and elevated temperature on soil microbial diversity, growth, and function. Treatments consisted of three seedlings of one plant species (Andropogon gerardii) or one seedling each of three plant species (A. gerardii, Echinacea pallida, Coreopsis lanceolata). Soil amendments included cellulose addition, inoculation with a microbial community collected from an undisturbed remnant prairie, and a control. We assessed microbial communities using extracellular enzyme assays, Illumina sequencing of the bacterial 16S rRNA gene, predicted bacterial metabolic pathways from sequence data and phospholipid fatty acid analysis (PLFA), which includes both bacterial and fungal lipid abundances. Our results indicate that addition of cellulose selects for slow-growing bacterial taxa (Verrucomicrobia) and fungi at ambient temperature. However, at elevated temperature, selection for slow-growing bacterial taxa is enhanced, while selection for fungi is lost, indicating temperature sensitivity among fungi. Cellulose addition was a more effective means of altering soil community composition than addition of microbial communities harvested from a remnant prairie. Soil water content was typically higher in the A. gerardii treatment alone, regardless of temperature, but at ambient temperature only, predicted metagenomics pathways for bacterial carbon metabolism were more abundant with A. gerardii. In summary, these results from a mesocosm test case indicate that adding cellulose to newly restored soil and increasing the abundance of C4 grasses, such as A. gerardii, can select for microbial communities adapted for slow growth and carbon storage. Further testing is required to determine if these approaches yield the same results in a field-level experiment.

Impacts of species richness on productivity in a large-scale subtropical forest experiment.

Biodiversity experiments have shown that species loss reduces ecosystem functioning in grassland. To test whether this result can be extrapolated to forests, the main contributors to terrestrial primary productivity, requires large-scale experiments. We manipulated tree species richness by planting more than 150,000 trees in plots with 1 to 16 species. Simulating multiple extinction scenarios, we found that richness strongly increased stand-level productivity. After 8 years, 16-species mixtures had accumulated over twice the amount of carbon found in average monocultures and similar amounts as those of two commercial monocultures. Species richness effects were strongly associated with functional and phylogenetic diversity. A shrub addition treatment reduced tree productivity, but this reduction was smaller at high shrub species richness. Our results encourage multispecies afforestation strategies to restore biodiversity and mitigate climate change.

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition. An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

Key Edaphic Properties Largely Explain Temporal and Geographic Variation in Soil Microbial Communities across Four Biomes.

Soil microbial communities play a critical role in nutrient transformation and storage in all ecosystems. Quantifying the seasonal and long-term temporal extent of genetic and functional variation of soil microorganisms in response to biotic and abiotic changes within and across ecosystems will inform our understanding of the effect of climate change on these processes. We examined spatial and seasonal variation in microbial communities based on 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) composition across four biomes: a tropical broadleaf forest (Hawaii), taiga (Alaska), semiarid grassland-shrubland (Utah), and a subtropical coniferous forest (Florida). In this study, we used a team-based instructional approach leveraging the iPlant Collaborative to examine publicly available National Ecological Observatory Network (NEON) 16S gene and PLFA measurements that quantify microbial diversity, composition, and growth. Both profiling techniques revealed that microbial communities grouped strongly by ecosystem and were predominately influenced by three edaphic factors: pH, soil water content, and cation exchange capacity. Temporal variability of microbial communities differed by profiling technique; 16S-based community measurements showed significant temporal variability only in the subtropical coniferous forest communities, specifically through changes within subgroups of Acidobacteria. Conversely, PLFA-based community measurements showed seasonal shifts in taiga and tropical broadleaf forest systems. These differences may be due to the premise that 16S-based measurements are predominantly influenced by large shifts in the abiotic soil environment, while PLFA-based analyses reflect the metabolically active fraction of the microbial community, which is more sensitive to local disturbances and biotic interactions. To address the technical issue of the response of soil microbial communities to sample storage temperature, we compared 16S-based community structure in soils stored at -80°C and -20°C and found no significant differences in community composition based on storage temperature. Free, open access datasets and data sharing platforms are powerful tools for integrating research and teaching in undergraduate and graduate student classrooms. They are a valuable resource for fostering interdisciplinary collaborations, testing ecological theory, model development and validation, and generating novel hypotheses. Training in data analysis and interpretation of large datasets in university classrooms through project-based learning improves the learning experience for students and enables their use of these significant resources throughout their careers.

Microbial lipid and amino sugar responses to long-term simulated global environmental changes in a California annual grassland.

Global environmental change is predicted to have major consequences for carbon cycling and the functioning of soil ecosystems. However, we have limited knowledge about its impacts on the microorganisms, which act as a "valve" between carbon sequestered in soils versus released into the atmosphere. In this study we examined microbial response to continuous 9-years manipulation of three global change factors (elevated CO2, warming, and nitrogen deposition), singly and in combination using two methods: lipid and amino sugar biomarkers at the Jasper Ridge Global Change Experiment (JRGCE). The two methods yielded important distinctions. There were limited microbial lipid differences, but many significant effects for microbial amino sugars. We found that CO2 was not a direct factor influencing soil carbon and major amino sugar pools, but had a positive impact on bacterial-derived muramic acid. Likewise, warming and nitrogen deposition appeared to enrich residues specific to bacteria despite an overall depletion in total amino sugars. The results indicate that elevated CO2, warming, and nitrogen deposition all appeared to increase bacterial-derived residues, but this accumulation effect was far offset by a corresponding decline in fungal residues. The sensitivity of microbial residue biomarker amino sugars to warming and nitrogen deposition may have implications for our predictions of global change impacts on soil stored carbon.

Uncoupling of microbial community structure and function in decomposing litter across beech forest ecosystems in Central Europe.

The widespread paradigm in ecology that community structure determines function has recently been challenged by the high complexity of microbial communities. Here, we investigate the patterns of and connections between microbial community structure and microbially-mediated ecological function across different forest management practices and temporal changes in leaf litter across beech forest ecosystems in Central Europe. Our results clearly indicate distinct pattern of microbial community structure in response to forest management and time. However, those patterns were not reflected when potential enzymatic activities of microbes were measured. We postulate that in our forest ecosystems, a disconnect between microbial community structure and function may be present due to differences between the drivers of microbial growth and those of microbial function.

Soil microbial responses to warming and increased precipitation and their implications for ecosystem C cycling.

A better understanding of soil microbial ecology is critical to gaining an understanding of terrestrial carbon (C) cycle-climate change feedbacks. However, current knowledge limits our ability to predict microbial community dynamics in the face of multiple global change drivers and their implications for respiratory loss of soil carbon. Whether microorganisms will acclimate to climate warming and ameliorate predicted respiratory C losses is still debated. It also remains unclear how precipitation, another important climate change driver, will interact with warming to affect microorganisms and their regulation of respiratory C loss. We explore the dynamics of microorganisms and their contributions to respiratory C loss using a 4-year (2006-2009) field experiment in a semi-arid grassland with increased temperature and precipitation in a full factorial design. We found no response of mass-specific (per unit microbial biomass C) heterotrophic respiration to warming, suggesting that respiratory C loss is directly from microbial growth rather than total physiological respiratory responses to warming. Increased precipitation did stimulate both microbial biomass and mass-specific respiration, both of which make large contributions to respiratory loss of soil carbon. Taken together, these results suggest that, in semi-arid grasslands, soil moisture and related substrate availability may inhibit physiological respiratory responses to warming (where soil moisture was significantly lower), while they are not inhibited under elevated precipitation. Although we found no total physiological response to warming, warming increased bacterial C utilization (measured by BIOLOG EcoPlates) and increased bacterial oxidation of carbohydrates and phenols. Non-metric multidimensional scaling analysis as well as ANOVA testing showed that warming or increased precipitation did not change microbial community structure, which could suggest that microbial communities in semi-arid grasslands are already adapted to fluctuating climatic conditions. In summary, our results support the idea that microbial responses to climate change are multifaceted and, even with no large shifts in community structure, microbial mediation of soil carbon loss could still occur under future climate scenarios.