Virus diversity and activity is driven by snowmelt and host dynamics in a high-altitude watershed soil ecosystem.

BACKGROUND: Viruses impact nearly all organisms on Earth, including microbial communities and their associated biogeochemical processes. In soils, highly diverse viral communities have been identified, with a global distribution seemingly driven by multiple biotic and abiotic factors, especially soil temperature and moisture. However, our current understanding of the stability of soil viral communities across time and their response to strong seasonal changes in environmental parameters remains limited. Here, we investigated the diversity and activity of environmental soil DNA and RNA viruses, focusing especially on bacteriophages, across dynamics' seasonal changes in a snow-dominated mountainous watershed by examining paired metagenomes and metatranscriptomes. RESULTS: We identified a large number of DNA and RNA viruses taxonomically divergent from existing environmental viruses, including a significant proportion of fungal RNA viruses, and a large and unsuspected diversity of positive single-stranded RNA phages (Leviviricetes), highlighting the under-characterization of the global soil virosphere. Among these, we were able to distinguish subsets of active DNA and RNA phages that changed across seasons, consistent with a "seed-bank" viral community structure in which new phage activity, for example, replication and host lysis, is sequentially triggered by changes in environmental conditions. At the population level, we further identified virus-host dynamics matching two existing ecological models: "Kill-The-Winner" which proposes that lytic phages are actively infecting abundant bacteria, and "Piggyback-The-Persistent" which argues that when the host is growing slowly, it is more beneficial to remain in a dormant state. The former was associated with summer months of high and rapid microbial activity, and the latter with winter months of limited and slow host growth. CONCLUSION: Taken together, these results suggest that the high diversity of viruses in soils is likely associated with a broad range of host interaction types each adapted to specific host ecological strategies and environmental conditions. As our understanding of how environmental and host factors drive viral activity in soil ecosystems progresses, integrating these viral impacts in complex natural microbiome models will be key to accurately predict ecosystem biogeochemistry. Video Abstract.

Differential microbial assembly processes and co-occurrence networks in the soil-root continuum along an environmental gradient.

Microorganisms of the soil-root continuum play key roles in ecosystem function. The Loess Plateau is well known for its severe soil erosion and thick loess worldwide, where mean annual precipitation (MAP) and soil nutrients decrease from the southeast to the northwest. However, the relative influence of environmental factors on the microbial community in four microhabitats (bulk soil, rhizosphere, rhizoplane, and endosphere) in the soil-root continuum along the environmental gradient in the Loess Plateau remains unclear. In this study, we investigated 82 field sites from warm-temperate to desert grasslands across the Loess Plateau, China, to assess the bacterial diversity, composition, community assembly, and co-occurrence networks in the soil-root continuum along an environmental gradient using bacterial 16S recombinant DNA amplicon sequencing. We discovered that the microhabitats explained the largest source of variations in the bacterial diversity and community composition in this region. Environmental factors (e.g., MAP, soil organic carbon, and pH) impacted the soil, rhizosphere, and rhizoplane bacterial communities, but their effects on the bacterial community decreased with increased proximity to roots from the soil to the rhizoplane, and the MAP enlarged the dissimilarity of microbial communities from the rhizosphere and rhizoplane to bulk soil. Additionally, stochastic assembly processes drove the endosphere communities, whereas the soil, rhizosphere, and rhizoplane communities were governed primarily by the variable selection of deterministic processes, which showed increased importance from warm-temperate to desert grasslands. Moreover, the properties of the microbial networks in the rhizoplane community indicate more stable networks in desert grasslands, likely conferring the resistance of microbial communities in higher stress environments. Collectively, our results showed that the bacterial communities in the soil-root continuum had different sensitivities and assembly mechanisms along an environmental gradient. These patterns are shaped simultaneously by the intertwined dimensions of proximity to roots and environmental stress change in the Loess Plateau.

Measurement of Volatile Compounds for Real-Time Analysis of Soil Microbial Metabolic Response to Simulated Snowmelt.

Snowmelt dynamics are a significant determinant of microbial metabolism in soil and regulate global biogeochemical cycles of carbon and nutrients by creating seasonal variations in soil redox and nutrient pools. With an increasing concern that climate change accelerates both snowmelt timing and rate, obtaining an accurate characterization of microbial response to snowmelt is important for understanding biogeochemical cycles intertwined with soil. However, observing microbial metabolism and its dynamics non-destructively remains a major challenge for systems such as soil. Microbial volatile compounds (mVCs) emitted from soil represent information-dense signatures and when assayed non-destructively using state-of-the-art instrumentation such as Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-TOF-MS) provide time resolved insights into the metabolism of active microbiomes. In this study, we used PTR-TOF-MS to investigate the metabolic trajectory of microbiomes from a subalpine forest soil, and their response to a simulated wet-up event akin to snowmelt. Using an information theory approach based on the partitioning of mutual information, we identified mVC metabolite pairs with robust interactions, including those that were non-linear and with time lags. The biological context for these mVC interactions was evaluated by projecting the connections onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) network of known metabolic pathways. Simulated snowmelt resulted in a rapid increase in the production of trimethylamine (TMA) suggesting that anaerobic degradation of quaternary amine osmo/cryoprotectants, such as glycine betaine, may be important contributors to this resource pulse. Unique and synergistic connections between intermediates of methylotrophic pathways such as dimethylamine, formaldehyde and methanol were observed upon wet-up and indicate that the initial pulse of TMA was likely transformed into these intermediates by methylotrophs. Increases in ammonia oxidation signatures (transformation of hydroxylamine to nitrite) were observed in parallel, and while the relative role of nitrifiers or methylotrophs cannot be confirmed, the inferred connection to TMA oxidation suggests either a direct or indirect coupling between these processes. Overall, it appears that such mVC time-series from PTR-TOF-MS combined with causal inference represents an attractive approach to non-destructively observe soil microbial metabolism and its response to environmental perturbation.

Growing-season warming and winter soil freeze/thaw cycles increase transpiration in a northern hardwood forest.

Climate models project higher growing-season temperatures and a decline in the depth and duration of winter snowpack throughout many north temperate ecosystems over the next century. A smaller snowpack is projected to induce more frequent soil freeze/thaw cycles in winter in northern hardwood forests of the northeastern United States. We measured the combined effects of warmer growing-season soil temperatures and increased winter freeze/thaw cycles on rates of leaf-level photosynthesis and transpiration (sap flow) of red maple (Acer rubrum) trees in a northern hardwood forest at the Climate Change Across Seasons Experiment at Hubbard Brook Experimental Forest in New Hampshire. Soil temperatures were warmed 5°C above ambient temperatures during the growing season and soil freeze/thaw cycles were induced in winter to mimic the projected changes in soil temperature over the next century. Relative to reference plots, growing-season soil warming increased rates of leaf-level photosynthesis by up to 85.32 ± 4.33%, but these gains were completely offset by soil freeze/thaw cycles in winter, suggesting that increased freeze/thaw cycles in winter over the next 100 yr will reduce the effect of warming on leaf-level carbon gains. Soil warming in the growing season increased rates of transpiration per kilopascal of vapor pressure deficit (VPD) by up to 727.39 ± 0.28%, even when trees were exposed to increased frequency of soil freeze/thaw cycles in the previous winter, which could influence regional hydrology in the future. Using climate projections downscaled from the Coupled Model Intercomparison Project, we project increased rates of whole-season transpiration in these forests over the next century by 42-61%. We also project 52-77 additional days when daily air temperatures will be above the long-term average daily maximum during the growing season at Hubbard Brook. Together, these results show that projected changes in climate across both the growing season and winter are likely to cause greater rates of water uptake and have no effect on rates of leaf-level carbon uptake by trees, with potential ecosystem consequences for hydrology and carbon cycling in northern hardwood forests.

Soil Microbes Trade-Off Biogeochemical Cycling for Stress Tolerance Traits in Response to Year-Round Climate Change.

Winter air temperatures are rising faster than summer air temperatures in high-latitude forests, increasing the frequency of soil freeze/thaw events in winter. To determine how climate warming and soil freeze/thaw cycles affect soil microbial communities and the ecosystem processes they drive, we leveraged the Climate Change across Seasons Experiment (CCASE) at the Hubbard Brook Experimental Forest in the northeastern United States, where replicate field plots receive one of three climate treatments: warming (+5°C above ambient in the growing season), warming in the growing season + winter freeze/thaw cycles (+5°C above ambient +4 freeze/thaw cycles during winter), and no treatment. Soil samples were taken from plots at six time points throughout the growing season and subjected to amplicon (rDNA) and metagenome sequencing. We found that soil fungal and bacterial community composition were affected by changes in soil temperature, where the taxonomic composition of microbial communities shifted more with the combination of growing-season warming and increased frequency of soil freeze/thaw cycles in winter than with warming alone. Warming increased the relative abundance of brown rot fungi and plant pathogens but decreased that of arbuscular mycorrhizal fungi, all of which recovered under combined growing-season warming and soil freeze/thaw cycles in winter. The abundance of animal parasites increased significantly under combined warming and freeze/thaw cycles. We also found that warming and soil freeze/thaw cycles suppressed bacterial taxa with the genetic potential for carbon (i.e., cellulose) decomposition and soil nitrogen cycling, such as N fixation and the final steps of denitrification. These new soil communities had higher genetic capacity for stress tolerance and lower genetic capacity to grow or reproduce, relative to the communities exposed to warming in the growing season alone. Our observations suggest that initial suppression of biogeochemical cycling with year-round climate change may be linked to the emergence of taxa that trade-off growth for stress tolerance traits.

The Snowmelt Niche Differentiates Three Microbial Life Strategies That Influence Soil Nitrogen Availability During and After Winter.

Soil microbial biomass can reach its annual maximum pool size beneath the winter snowpack and is known to decline abruptly following snowmelt in seasonally snow-covered ecosystems. Observed differences in winter versus summer microbial taxonomic composition also suggests that phylogenetically conserved traits may permit winter- versus summer-adapted microorganisms to occupy distinct niches. In this study, we sought to identify archaea, bacteria, and fungi that are associated with the soil microbial bloom overwinter and the subsequent biomass collapse following snowmelt at a high-altitude watershed in central Colorado, United States. Archaea, bacteria, and fungi were categorized into three life strategies (Winter-Adapted, Snowmelt-Specialist, Spring-Adapted) based upon changes in abundance during winter, the snowmelt period, and after snowmelt in spring. We calculated indices of phylogenetic relatedness (archaea and bacteria) or assigned functional attributes (fungi) to organisms within life strategies to infer whether phylogenetically conserved traits differentiate Winter-Adapted, Snowmelt-Specialist, and Spring-Adapted groups. We observed that the soil microbial bloom was correlated in time with a pulse of snowmelt infiltration, which commenced 65 days prior to soils becoming snow-free. A pulse of nitrogen (N, as nitrate) occurred after snowmelt, along with a collapse in the microbial biomass pool size, and an increased abundance of nitrifying archaea and bacteria (e.g., Thaumarchaeota, Nitrospirae). Winter- and Spring-Adapted archaea and bacteria were phylogenetically clustered, suggesting that phylogenetically conserved traits allow Winter- and Spring-Adapted archaea and bacteria to occupy distinct niches. In contrast, Snowmelt-Specialist archaea and bacteria were phylogenetically overdispersed, suggesting that the key mechanism(s) of the microbial biomass crash are likely to be density-dependent (e.g., trophic interactions, competitive exclusion) and affect organisms across a broad phylogenetic spectrum. Saprotrophic fungi were the dominant functional group across fungal life strategies, however, ectomycorrhizal fungi experienced a large increase in abundance in spring. If well-coupled plant-mycorrhizal phenology currently buffers ecosystem N losses in spring, then changes in snowmelt timing may alter ecosystem N retention potential. Overall, we observed that snowmelt separates three distinct soil niches that are occupied by ecologically distinct groups of microorganisms. This ecological differentiation is of biogeochemical importance, particularly with respect to the mobilization of nitrogen during winter, before and after snowmelt.

Roots Mediate the Effects of Snowpack Decline on Soil Bacteria, Fungi, and Nitrogen Cycling in a Northern Hardwood Forest.

Rising winter air temperature will reduce snow depth and duration over the next century in northern hardwood forests. Reductions in snow depth may affect soil bacteria and fungi directly, but also affect soil microbes indirectly through effects of snowpack loss on plant roots. We incubated root exclusion and root ingrowth cores across a winter climate-elevation gradient in a northern hardwood forest for 29 months to identify direct (i.e., winter snow-mediated) and indirect (i.e., root-mediated) effects of winter snowpack decline on soil bacterial and fungal communities, as well as on potential nitrification and net N mineralization rates. Both winter snowpack decline and root exclusion increased bacterial richness and phylogenetic diversity. Variation in bacterial community composition was best explained by differences in winter snow depth or soil frost across elevation. Root ingrowth had a positive effect on the relative abundance of several bacterial taxonomic orders (e.g., Acidobacterales and Actinomycetales). Nominally saprotrophic (e.g., Saccharomycetales and Mucorales) or mycorrhizal (e.g., Helotiales, Russalales, Thelephorales) fungal taxonomic orders were also affected by both root ingrowth and snow depth variation. However, when grouped together, the relative abundance of saprotrophic fungi, arbuscular mycorrhizal fungi, and ectomycorrhizal fungi were not affected by root ingrowth or snow depth, suggesting that traits in addition to trophic mode will mediate fungal community responses to snowpack decline in northern hardwood forests. Potential soil nitrification rates were positively related to ammonia-oxidizing bacteria and archaea abundance (e.g., Nitrospirales, Nitrosomondales, Nitrosphaerales). Rates of N mineralization were positively and negatively correlated with ectomycorrhizal and saprotrophic fungi, respectively, and these relationships were mediated by root exclusion. The results from this study suggest that a declining winter snowpack and its effect on plant roots each have direct effects on the diversity and abundance of soil bacteria and fungal communities that interact to determine rates of soil N cycling in northern hardwood forests.

Climate Change Across Seasons Experiment (CCASE): A new method for simulating future climate in seasonally snow-covered ecosystems.

Climate models project an increase in mean annual air temperatures and a reduction in the depth and duration of winter snowpack for many mid and high latitude and high elevation seasonally snow-covered ecosystems over the next century. The combined effects of these changes in climate will lead to warmer soils in the growing season and increased frequency of soil freeze-thaw cycles (FTCs) in winter due to the loss of a continuous, insulating snowpack. Previous experiments have warmed soils or removed snow via shoveling or with shelters to mimic projected declines in the winter snowpack. To our knowledge, no experiment has examined the interactive effects of declining snowpack and increased frequency of soil FTCs, combined with soil warming in the snow-free season on terrestrial ecosystems. In addition, none have mimicked directly the projected increase in soil FTC frequency in tall statured forests that is expected as a result of a loss of insulating snow in winter. We established the Climate Change Across Seasons Experiment (CCASE) at Hubbard Brook Experimental Forest in the White Mountains of New Hampshire in 2012 to assess the combined effects of these changes in climate on a variety of pedoclimate conditions, biogeochemical processes, and ecology of northern hardwood forests. This paper demonstrates the feasibility of creating soil FTC events in a tall statured ecosystem in winter to simulate the projected increase in soil FTC frequency over the next century and combines this projected change in winter climate with ecosystem warming throughout the snow-free season. Together, this experiment provides a new and more comprehensive approach for climate change experiments that can be adopted in other seasonally snow-covered ecosystems to simulate expected changes resulting from global air temperature rise.

Reduced snow cover alters root-microbe interactions and decreases nitrification rates in a northern hardwood forest.

Snow cover is projected to decline during the next century in many ecosystems that currently experience a seasonal snowpack. Because snow insulates soils from frigid winter air temperatures, soils are expected to become colder and experience more winter soil freeze-thaw cycles as snow cover continues to decline. Tree roots are adversely affected by snowpack reduction, but whether loss of snow will affect root-microbe interactions remains largely unknown. The objective of this study was to distinguish and attribute direct (e.g., winter snow- and/or soil frost-mediated) vs. indirect (e.g., root-mediated) effects of winter climate change on microbial biomass, the potential activity of microbial exoenzymes, and net N mineralization and nitrification rates. Soil cores were incubated in situ in nylon mesh that either allowed roots to grow into the soil core (2 mm pore size) or excluded root ingrowth (50 µm pore size) for up to 29 months along a natural winter climate gradient at Hubbard Brook Experimental Forest, NH (USA). Microbial biomass did not differ among ingrowth or exclusion cores. Across sampling dates, the potential activities of cellobiohydrolase, phenol oxidase, and peroxidase, and net N mineralization rates were more strongly related to soil volumetric water content (P < 0.05; R2  = 0.25-0.46) than to root biomass, snow or soil frost, or winter soil temperature (R2  < 0.10). Root ingrowth was positively related to soil frost (P < 0.01; R2  = 0.28), suggesting that trees compensate for overwinter root mortality caused by soil freezing by re-allocating resources towards root production. At the sites with the deepest snow cover, root ingrowth reduced nitrification rates by 30% (P < 0.01), showing that tree roots exert significant influence over nitrification, which declines with reduced snow cover. If soil freezing intensifies over time, then greater compensatory root growth may reduce nitrification rates directly via plant-microbe N competition and indirectly through a negative feedback on soil moisture, resulting in lower N availability to trees in northern hardwood forests.