Fungi are key drivers of leaf litter decomposition in riparian zones, with communities made up of phyllosphere fungi (fungi present on leaves prior to senescence), and fungi that colonize from the environment. While phyllosphere fungi dominate early decomposition in both submerged and terrestrial litter, their contribution to fungal community structure and growth across these environments remains unclear. We used quantitative stable isotope probing to quantify taxon-specific growth rates of phyllosphere fungi and fungi colonizing from the environment, and to differentiate growing fungi from dormant or dead fungi. We quantified the growth rates of phyllosphere and colonizing fungi on two leaf types in aquatic and terrestrial environments over three time points. Growing fungal communities showed substantial overlap between aquatic and terrestrial environments, although this overlap decreased over time. Phyllosphere fungi were more likely to be shared between environments than colonizing fungi. Leaf type exerted a stronger influence on community composition than the environment. Phyllosphere fungi dominated relative abundance and growth during the study, and all fungi grew consistently faster on terrestrial litter. These findings demonstrate the central role of phyllosphere fungi in fungal communities across both aquatic and terrestrial ecosystems, as well as the importance of leaf species.

Abstract Soil protists shape microbial food webs and nutrient cycling, yet methods for measuring their population growth in soil have lagged behind the taxonomic resolution available from 18S rRNA gene sequencing. Microscopy-based approaches can estimate abundance and growth, but with limited taxonomic resolution. Here, we tested whether quantitative stable isotope probing (qSIP) can provide reproducible, sequencing-resolved growth measurements for soil protists. We incubated soil with natural-abundance or ¹ O-labeled water and measured taxon-specific 18 O enrichment in DNA using three common 18S rRNA gene primer sets. 18 O enrichment values were positively correlated across datasets, with relationships closest to 1:1 after poorly resolved taxonomic assignments were excluded, indicating that qSIP provides reproducible population-level growth signals across marker choices. We then compared 18S amplicon profiles from unfractionated DNA with qSIP-derived growth measurements across a soil moisture gradient. Amplicon profiles showed small shifts in relative abundances of major protist groups, whereas qSIP revealed a large moisture response in the growing community: 7 ASVs were growing at 20% field capacity compared with 143 at 80% field capacity, representing 1.6% and 63.3% of total protist relative abundance, respectively. Average growth was <1% day□ 1 in the two driest treatments, increasing to 2.1% day□ 1 at 60% and 5.6% day□ 1 at 80% field capacity; among growing taxa, rates averaged 8.7% and 8.3% day□ 1 in the two wetter treatments. By pairing taxonomic resolution with isotope-based growth estimates, qSIP with 18 O-H 2 O moves soil protist ecology toward quantitative population dynamics: identifying which taxa grow, how fast, and how growth responds to the environment.

Microbial ecological strategies are shaped by a fundamental trade-off: is it better to specialize and thrive in a narrow niche or generalize and persist across diverse environments? In soils, this trade-off is particularly relevant in the rhizosphere and detritusphere, where microorganisms encounter distinct resource inputs from living and decaying roots. Using H ¹ O quantitative stable isotope probing (qSIP), we measured in situ bacterial and fungal growth rates in the rhizosphere, the root detritusphere, and in the combined presence of rhizosphere + root detritus to test whether specialists—microbes growing in a single habitat—grow faster than generalists that persist across multiple environments. Specialists grew consistently faster than generalists, suggesting a trade-off between the breadth of environmental conditions a microorganism can tolerate and its ability to grow quickly in a specific habitat. This cost to broad niche adaptation was apparent for bacteria, but growth rates of fungal saprotrophs varied little between specialists and generalists, reflecting how fundamental differences in life-history strategies between bacteria and fungi can shape microbial responses to resource availability and habitat heterogeneity. Net relatedness and nearest taxon indices indicated total bacterial communities were phylogenetically clustered while specialist and generalist communities were phylogenetically random, suggesting that functional traits, not lineage, best predict ecological strategy. In annual grassland soils, fast-growing specialists may dominate ecosystem processes when resources abound, and slow-growing generalists may sustain element transformations when conditions shift; understanding this interplay is key to predicting soil-carbon trajectories. ### Competing Interest Statement The authors have declared no competing interest.

Fungi play a crucial role in aquatic leaf litter decomposition. Aquatic fungi have long been thought to spend the majority of their lives in the water. Here, we explore the possibility of an amphibious life cycle, where phyllosphere fungi spend part of their life cycle in aquatic systems. We used internal transcribed spacer (ITS) fungal sequencing to follow phyllosphere fungi onto submerged litter, and quantitative stable isotope probing (qSIP) to differentiate active and inactive fungi. We found that around 30% of fungi active on aquatic litter entered the stream with the leaf and that these phyllosphere fungi were as active, if not more active than, as the fungi colonizing from the water column. These results demonstrate that phyllosphere fungi are an important part of aquatic fungal communities.

Element assimilation rates or the DNA replication rate of microbial taxa can be measured in environmental samples through quantitative DNA-stable isotope probing (DNA-qSIP). Here, we introduce a set of DNA standards that may be used to quantify the density of DNA extracted from environmental samples after isopycnic centrifugation. The standards are approximately 9 Kbp PCR products with either isotopically enriched (98 atom% 15N, 13C) nucleotides or natural abundance nucleotides and have densities that differ by 0.051 g/mL. The internal standards were tested in a DNA-qSIP analysis of bacterial populations in soil exposed to 63 atom% H218O, performed in two different laboratories with different equipment and protocols. While fractionation results, including number of fractions taken and differences in density between adjacent fractions, between the two laboratories were different, the internal standards allowed the two data sets to be compared, and both research groups found similar Excess Atom Fraction (EAF) of oxygen-18 in the DNA of bacterial taxa. These internal DNA standards allow direct comparison of DNA-qSIP results from different experiments regardless of operator, tracer enrichment levels, protocol or equipment used and can support the creation of a large global database that contains qSIP results from many different laboratories. Importance DNA-qSIP is an important technique in microbial ecology that allows the growth and nutrient assimilation rates of microbial taxa, including those that have not yet been cultured, to be measured. Growth and assimilation rates are extremely variable parameters in microbial ecology because most of the microbial community is dormant and inactive, which complicates linking microbial populations to ecosystem processes. DNA-qSIP is increasingly practiced in a range of laboratories, using different equipment, isotopes, substrates, and protocols, making comparison of results among different research groups challenging. Here we describe the development of internal standards, 2 pieces of DNA with different isotopic content and hence buoyant density, that can be added to environmental DNA before isopycnic centrifugation. Our results show the internal DNA standards allow DNA-qSIP results to be compared across laboratories and could contribute to the formation of a large DNA-qSIP database that contains growth or nutrient assimilation rates of microbial taxa from many different experiments. ### Competing Interest Statement The authors have declared no competing interest. U.S. Department of Energy, Office of Biological and Environmental Research, Genomic Science Program, SCW1632 U.S. Department of Energy, DE-AC52-07NA27344

The growth rate of a microorganism is a simple yet profound way to quantify its impact on the world. Microbial fitness in the environment depends on the ability to reproduce quickly when conditions are favorable and adopt a survival physiology when conditions worsen, which cells coordinate by adjusting their growth rate. At the population level, per capita growth rate is a sensitive metric of fitness, linking survival and reproduction to the ecology and evolution of populations. The absolute growth rate of a microbial population reflects rates of resource assimilation, biomass production, and element transformation, some of the many ways that organisms affect Earth’s ecosystems and climate. For soil microorganisms, most of our understanding of growth is based on observations made in culture. This is a crucial limitation given that many soil microbes are not readily cultured and in vitro conditions are unlikely to reflect conditions in the wild. New approaches in ‘omics and stable isotope probing make it possible to sensitively measure growth rates of microbial assemblages and individual taxa in nature, and to couple these measurements to biogeochemical fluxes. Microbial ecologists can now explore how the growth rates of taxa with known traits and evolutionary histories respond to changes in resource availability, environmental conditions, and interactions with other organisms. We anticipate that quantitative and scalable data on the growth rates of soil microorganisms will allow scientists to test and refine ecological theory and advance processbased models of carbon flux, nutrient uptake, and ecosystem productivity. Measurements of in situ microbial growth rates provide insights into the ecology of populations and can be used to quantitatively link microbial diversity to soil biogeochemistry.

Measuring the growth rate of a microorganism is a simple yet profound way to quantify its effect on the world. The absolute growth rate of a microbial population reflects rates of resource assimilation, biomass production and element transformation-some of the many ways in which organisms affect Earth's ecosystems and climate. Microbial fitness in the environment depends on the ability to reproduce quickly when conditions are favourable and adopt a survival physiology when conditions worsen, which cells coordinate by adjusting their relative growth rate. At the population level, relative growth rate is a sensitive metric of fitness, linking survival and reproduction to the ecology and evolution of populations. Techniques combining omics and stable isotope probing enable sensitive measurements of the growth rates of microbial assemblages and individual taxa in soil. Microbial ecologists can explore how the growth rates of taxa with known traits and evolutionary histories respond to changes in resource availability, environmental conditions and interactions with other organisms. We anticipate that quantitative and scalable data on the growth rates of soil microorganisms, coupled with measurements of biogeochemical fluxes, will allow scientists to test and refine ecological theory and advance process-based models of carbon flux, nutrient uptake and ecosystem productivity. Measurements of in situ microbial growth rates provide insights into the ecology of populations and can be used to quantitatively link microbial diversity to soil biogeochemistry.

ABSTRACT Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding. IMPORTANCE Ecosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged.

Predicting ecosystem function is critical to assess and mitigate the impacts of climate change. Quantitative predictions of microbially mediated ecosystem processes are typically uninformed by microbial biodiversity. Yet new tools allow the measurement of taxon-specific traits within natural microbial communities. There is mounting evidence of a phylogenetic signal in these traits, which may support prediction and microbiome management frameworks. We investigated phylogeny-based trait prediction using bacterial growth rates from soil communities in Arctic, boreal, temperate, and tropical ecosystems. Here we show that phylogeny predicts growth rates of soil bacteria, explaining an average of 31%, and up to 58%, of the variation within ecosystems. Despite limited overlap in community composition across these ecosystems, shared nodes in the phylogeny enabled ancestral trait reconstruction and cross-ecosystem predictions. Phylogenetic relationships could explain up to 38% (averaging 14%) of the variation in growth rates across the highly disparate ecosystems studied. Our results suggest that shared evolutionary history contributes to similarity in the relative growth rates of related bacteria in the wild, allowing phylogeny-based predictions to explain a substantial amount of the variation in taxon-specific functional traits, within and across ecosystems.

Study of life history strategies may help predict the performance of microorganisms in nature by organizing the complexity of microbial communities into groups of organisms with similar strategies. Here, we tested the extent that one common application of life history theory, the copiotroph-oligotroph framework, could predict the relative population growth rate of bacterial taxa in soils from four different ecosystems. We measured the change of in situ relative growth rate to added glucose and ammonium using both 18O–H2O and 13C quantitative stable isotope probing to test whether bacterial taxa sorted into copiotrophic and oligotrophic groups. We saw considerable overlap in nutrient responses across most bacteria regardless of phyla, with many taxa growing slowly and few taxa that grew quickly. To define plausible life history boundaries based on in situ relative growth rates, we applied Gaussian mixture models to organisms’ joint 18O–13C signatures and found that across experimental replicates, few taxa could consistently be assigned as copiotrophs, despite their potential for fast growth. When life history classifications were assigned based on average relative growth rate at varying taxonomic levels, finer resolutions (e.g., genus level) were significantly more effective in capturing changes in nutrient response than broad taxonomic resolution (e.g., phylum level). Our results demonstrate the difficulty in generalizing bacterial life history strategies to broad lineages, and even to single organisms across a range of soils and experimental conditions. We conclude that there is a continued need for the direct measurement of microbial communities in soil to advance ecologically realistic frameworks.

High-temperature geothermal springs host simplified microbial communities; however, the activities of individual microorganisms and their roles in the carbon cycle in nature are not well understood. Here, quantitative stable isotope probing (qSIP) was used to track the assimilation of 13 C-acetate and 13 C-aspartate into DNA in 74 °C sediments in Gongxiaoshe Hot Spring, Tengchong, China. This revealed a community-wide preference for aspartate and a tight coupling between aspartate incorporation into DNA and the proliferation of aspartate utilizers during labeling. Both 13 C incorporation into DNA and changes in the abundance of taxa during incubations indicated strong resource partitioning and a significant phylogenetic signal for aspartate incorporation. Of the active amplicon sequence variants (ASVs) identified by qSIP, most could be matched with genomes from Gongxiaoshe Hot Spring or nearby springs with an average nucleotide similarity of 99.4%. Genomes corresponding to aspartate primary utilizers were smaller, near-universally encoded polar amino acid ABC transporters, and had codon preferences indicative of faster growth rates. The most active ASVs assimilating both substrates were not abundant, suggesting an important role for the rare biosphere in the community response to organic carbon addition. The broad incorporation of aspartate into DNA over acetate by the hot spring community may reflect dynamic cycling of cell lysis products in situ or substrates delivered during monsoon rains and may reflect N limitation.

Density dependence in an ecological community has been observed in many macro-organismal ecosystems and is hypothesized to maintain biodiversity but is poorly understood in microbial ecosystems. Here, we analyze data from an experiment using quantitative stable isotope probing (qSIP) to estimate per-capita growth and mortality rates of bacterial populations in soils from several ecosystems along an elevation gradient which were subject to nutrient addition of either carbon alone (glucose; C) or carbon with nitrogen (glucose + ammonium-sulfate; C + N). Across all ecosystems, we found that higher population densities, quantified by the abundance of genomes per gram of soil, had lower per-capita growth rates in C + N-amended soils. Similarly, bacterial mortality rates in C + N-amended soils increased at a significantly higher rate with increasing population size than mortality rates in control and C-amended soils. In contrast to the hypothesis that density dependence would promote or maintain diversity, we observed significantly lower bacterial diversity in soils with stronger negative density-dependent growth. Here, density dependence was significantly but weakly responsive to nutrients and was not associated with higher bacterial diversity.

Increases in Arctic temperatures have thawed permafrost and accelerated tundra soil microbial activity, releasing greenhouse gases that amplify climate warming. Warming over time has also accelerated shrub encroachment in the tundra, altering plant input abundance and quality, and causing further changes to soil microbial processes. To better understand the effects of increased temperature and the accumulated effects of climate change on soil bacterial activity, we quantified the growth responses of individual bacterial taxa to short-term warming (3 months) and long-term warming (29 years) in moist acidic tussock tundra. Intact soil was assayed in the field for 30 days using O-18-labeled water, from which taxon-specific rates of O-18 incorporation into DNA were estimated as a proxy for growth. Experimental treatments warmed the soil by approximately 1.5 degrees C. Short-term warming increased average relative growth rates across the assemblage by 36%, and this increase was attributable to emergent growing taxa not detected in other treatments that doubled the diversity of growing bacteria. However, long-term warming increased average relative growth rates by 151%, and this was largely attributable to taxa that co-occurred in the ambient temperature controls. There was also coherence in relative growth rates within broad taxonomic levels with orders tending to have similar growth rates in all treatments. Growth responses tended to be neutral in short-term warming and positive in long-term warming for most taxa and phylogenetic groups co-occurring across treatments regardless of phylogeny. Taken together, growing bacteria responded distinctly to short-term and long-term warming, and taxa growing in each treatment exhibited deep phylogenetic organization.IMPORTANCE Soil carbon stocks in the tundra and underlying permafrost have become increasingly vulnerable to microbial decomposition due to climate change. The microbial responses to Arctic warming must be understood in order to predict the effects of future microbial activity on carbon balance in a warming Arctic. In response to our warming treatments, tundra soil bacteria grew faster, consistent with increased rates of decomposition and carbon flux to the atmosphere. Our findings suggest that bacterial growth rates may continue to increase in the coming decades as faster growth is driven by the accumulated effects of long-term warming. Observed phylogenetic organization of bacterial growth rates may also permit taxonomy-based predictions of bacterial responses to climate change and inclusion into ecosystem models. Soil carbon stocks in the tundra and underlying permafrost have become increasingly vulnerable to microbial decomposition due to climate change. The microbial responses to Arctic warming must be understood in order to predict the effects of future microbial activity on carbon balance in a warming Arctic.

Climate influences soil microbial composition and function, but the relative importance of a site's historic climate versus its more immediate environmental conditions is unclear. Using quantitative stable isotope probing (qSIP), we characterized actively growing soil microbial communities and soil properties in three California annual grasslands that span a rainfall gradient and have developed on similar parent material. The soils were assayed in the wet winter season, when environmental conditions are most similar across sites. Since growing populations might be expected to be most responsive to contemporary environmental conditions, we hypothesized that the structure of growing microbial communities would be more similar across the gradient than that of total communities (i.e., including non-growing populations). In addition, we hypothesized that population growth rates would be slowest in the driest site, reflecting a legacy effect of low soil moisture on microbial growth. Soils along the rainfall gradient differed in pH, texture, and cation exchange capacity, but not in total C, C:N or dominant minerals. The radiocarbon (14C) age of soil C (reflecting turnover time) increased with mean annual precipitation but soil respiration was uniformly modern, reflecting microbial reliance on recent C inputs across the sites. The structure of both total and growing microbial communities differed across sites. Across major microbial phyla, including the Actinobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Proteobacteria, bacterial growth rates were consistently lower in the site with the lowest mean annual precipitation. Taxa that were growing at the dry site alone grew more slowly than taxa that grew at multiple sites. These results reflect the influence of climate history and point to the role of environmental filtering at the driest site in shaping its slower growing microbial community, possibly reflecting adaptation to repeated exposure to water stress. Lastly, across taxa, the growth rate of a taxon at one site was correlated with its growth rate in the other sites. This growth rate coherence is likely a consequence of genetically determined physiological traits and is consistent with the idea that evolutionary history constrains growth rate.

Earth system models project altered precipitation regimes across much of the globe. Soil microorganisms in Mediterranean climates must withstand both direct physiological stress during prolonged periods of low soil moisture and be able to compete for resources when seasonal rains return and plant growth resumes5. However, we do not have a mechanistic understanding of how altered soil moisture regimes affect microbial population dynamics and in turn how this will affect soil carbon (C) persistence. We used quantitative stable isotope probing (qSIP) to compare total and growing soil microbial communities across three California annual grassland ecosystems with Mediterranean climates that span a rainfall gradient and have developed from similar parent material. Sampling was conducted during the wet season, when environmental conditions were most similar across the sites. We assessed multiple edaphic variables, including the radiocarbon (14C) age of soil C. We hypothesized that the long-term legacy effect of soil water limitation would be reflected in lower community growth capacity at the driest site. We also predicted that actively growing communities would be more compositionally similar across the gradient than the total (active + inactive) microbiome. Community and phylum mean bacterial growth rates increased from the driest site to the intermediate site, and rates were similar at the intermediate and wettest sites. These differences were persistent across major phyla, including the Actinobacteria, Bacteroidetes, and Proteobacteria. Additionally, soil C at the driest site was younger than the wet or intermediate sites. The microbial families that grew fastest at the driest site include taxa that have been described as having traits that are advantageous for surviving dry spells, such as spore formation, polyhydroxyalkanoate accumulation, carotenoid biosynthesis, extracellular polymeric substances production, and trehalose synthesis. Microbial communities at the driest site displayed phylogenetic clustering, suggesting environmental filtering for slow-growing microbial taxa that can withstand water stress at this site. Taxonomic identity was a strong predictor of growth, such that the growth rates of a taxon at one site predicted its growth rates at the others. We think this finding reflects the influence of genetic and physiological constraints on growth which appear to persist across rainfall gradients, edaphic properties, and biological communities. Lastly, we found that actively growing taxa represented (28-58%) of the taxa comprising total communities and that the composition of growing and total communities were similar. The finding that the growing communities were just a subset of the total microbiome, despite environmental conditions being favorable for growth, raises questions about the mechanisms maintaining soil microbial diversity in ecosystems with Mediterannean-type climates.

Boreal peatlands are important global carbon reservoirs that are vulnerable to increasing CO2 and associated warming. Soil microbes regulate the balance of carbon that is stored in peat or remineralized to CO2; so characterizing microbial responses to warming and rising CO2 is critical to predicting how peatlands will feed back to ongoing climate change. To address microbiome responses to changing climate, we examined taxon-specific bacterial growth under elevated CO2 and across a warming gradient in a peatland using 18O-water quantitative stable isotope probing. Using in situ temperatures, we clustered the responses of bacterial taxa according to excess atom fraction 18O of their genomes, a proxy for growth. Many taxa that showed little to no growth across the temperature range under ambient CO2 grew rapidly at certain temperatures under elevated CO2, highlighting a strong interplay between warming and CO2 concentrations. The temperature of maximum growth for Proteobacteria shifted higher under elevated CO2, while that of Acidobacteria shifted lower. We found support for phylogenetic conservation of growth patterns among Acidobacteria and Proteobacteria under ambient, but not elevated CO2. Our results suggest that certain taxa may be predisposed for growth under altered climate conditions, with a disproportionate influence on carbon cycling and peatland feedbacks to climate change.

Biological soil crusts (biocrusts) are critical components of dryland and other ecosystems worldwide, and are increasingly recognized as novel model ecosystems from which more general principles of ecology can be elucidated. Biocrusts are often diverse communities, comprised of both eukaryotic and prokaryotic organisms with a range of metabolic lifestyles that enable the fixation of atmospheric carbon and nitrogen. However, how the function of these biocrust communities varies with succession is incompletely characterized, especially in comparison to more familiar terrestrial ecosystem types such as forests. We conducted a greenhouse experiment to investigate how community composition and soil-atmosphere trace gas fluxes of CO2, CH4, and N2O varied from early-successional light cyanobacterial biocrusts to mid-successional dark cyanobacteria biocrusts and late-successional moss-lichen biocrusts and as biocrusts of each successional stage matured. Cover type richness increased as biocrusts developed, and richness was generally highest in the late-successional moss-lichen biocrusts. Microbial community composition varied in relation to successional stage, but microbial diversity did not differ significantly among stages. Net photosynthetic uptake of CO2 by each biocrust type also increased as biocrusts developed but tended to be moderately greater (by up to ≈25%) for the mid-successional dark cyanobacteria biocrusts than the light cyanobacterial biocrusts or the moss-lichen biocrusts. Rates of soil C accumulation were highest for the dark cyanobacteria biocrusts and light cyanobacteria biocrusts, and lowest for the moss-lichen biocrusts and bare soil controls. Biocrust CH4 and N2O fluxes were not consistently distinguishable from the same fluxes measured from bare soil controls; the measured rates were also substantially lower than have been reported in previous biocrust studies. Our experiment, which uniquely used greenhouse-grown biocrusts to manipulate community composition and accelerate biocrust development, shows how biocrust function varies along a dynamic gradient of biocrust successional stages.

Earth system models project altered precipitation regimes across much of the globe; in California, the winter wet season is predicted to extend into spring, and the summer dry period to lengthen. How these precipitation trends will affect microbial traits and soil carbon (C) cycling is a key knowledge gap. Specifically, we do not have a mechanistic understanding of the linkages between soil moisture legacy effects, microbial population dynamics and soil C persistence. Using quantitative stable isotope probing (qSIP), we compared total and growing soil microbial communities across three California annual grasslands that span a rainfall gradient yet developed on similar parent material. We also assessed multiple edaphic variables, including the radiocarbon ( 14 C) age of soil C, and found soil C turnover time increased with annual precipitation, but that soil microbes respired recently-fixed C regardless of site rainfall history. Samples were assayed in the wet season, when we expected environmental conditions would be most similar across sites. We hypothesized that growing communities would be more compositionally similar across the gradient than the total background microbiome. We also predicted that the long-term legacy effect of soil water limitation would be reflected in a lower community growth capacity at the driest site. We found that the proportion of the total community that was detected as growing was 28%, 48% and 58% at the wet, intermediate and dry sites, respectively. The composition of growing communities strongly resembled that of total communities, and growing communities were no more similar across the gradient than total communities, indicating a strong effect of climate of the structure of growing microbial communities. Members of three phyla, Acidobacteria, Actinobacteria, and Proteobacteria, were responsible for ∼79% of the cumulative 18 O assimilation and 80% of all taxa that we defined as ‘growers’. Bacterial growth rates were low at the driest site relative to the intermediate and wettest sites. Reduced growth at the driest site was observed across major phyla, including the Actinobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Proteobacteria. Microbial communities at the driest site displayed phylogenetic clustering, suggesting that climate history impacts microbial growth through environmental filtering for slow growing taxa. Taxonomic identity was a strong predictor of growth, such that the growth rates of a taxon at one site predicted its growth rates at the others. This cross-site coherence in growth is likely a consequence of genetically determined physiological traits, and is consistent with the idea that evolutionary history influences growth rate.

Secondary minerals (clays and metal oxides) are important components of the soil matrix. Clay minerals affect soil carbon persistence and cycling, and they also select for distinct microbial communities. Here we show that soil mineral assemblages—particularly short-range order minerals—affect both bacterial community composition and taxon-specific growth. Three soils with different parent material and presence of short-range order minerals were collected from ecosystems with similar vegetation and climate. These three soils were provided with 18O-labeled water and incubated with or without artificial root exudates or pine needle litter. Quantitative stable isotope probing was used to determine taxon-specific growth. We found that the growth of bacteria varied among soils of different mineral assemblages but found the trend of growth suppression in the presence of short-range order minerals. Relative growth of bacteria declined with increasing concentration of short-range order minerals between 25–36% of taxa present in all soils. Carbon addition in the form of plant litter or root exudates weakly affected relative growth of taxa (p = 0.09) compared to the soil type (p < 0.01). However, both exudate and litter carbon stimulated growth for at least 34% of families in the soils with the most and least short-range order minerals. In the intermediate short-range order soil, fresh carbon reduced growth for more bacterial families than were stimulated. These results highlight how bacterial-mineral-substrate interactions are critical to soil organic carbon processing, and how growth variation in bacterial taxa in these interactions may contribute to soil carbon persistence and loss.

The carbon stored in soil exceeds that of plant biomass and atmospheric carbon and its stability can impact global climate. Growth of decomposer microorganisms mediates both the accrual and loss of soil carbon. Growth is sensitive to temperature and given the vast biological diversity of soil microorganisms, the response of decomposer growth rates to warming may be strongly idiosyncratic, varying among taxa, making ecosystem predictions difficult. Here, we show that 15 years of warming by transplanting plant-soil mesocosms down in elevation, strongly reduced the growth rates of soil microorganisms, measured in the field using undisturbed soil. The magnitude of the response to warming varied among microbial taxa. However, the direction of the response-reduced growth-was universal and warming explained twofold more variation than did the sum of taxonomic identity and its interaction with warming. For this ecosystem, most of the growth responses to warming could be explained without taxon-specific information, suggesting that in some cases microbial responses measured in aggregate may be adequate for climate modeling. Long-term experimental warming also reduced soil carbon content, likely a consequence of a warming-induced increase in decomposition, as warming-induced changes in plant productivity were negligible. The loss of soil carbon and decreased microbial biomass with warming may explain the reduced growth of the microbial community, more than the direct effects of temperature on growth. These findings show that direct and indirect effects of long-term warming can reduce growth rates of soil microbes, which may have important feedbacks to global warming.