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Dormancy is as an adaptation to living in fluctuating environments. It can also influence species interactions, for example, by providing organisms with a refuge from predators and parasites. Here we test the hypothesis that dormancy generates a seed bank of protected individuals that can modify antagonistic coevolutionary dynamics. We experimentally evolved a spore-forming bacterial host along with a phage parasite that can infect active but not dormant cells. Seed banks buffered population dynamics against phage infection and retained phenotypic diversity that was otherwise lost to directional selection. By storing genetic diversity, seed banks also altered the distribution of host alleles, which contributed to dampened coevolutionary dynamics. Our findings demonstrate that dormancy generates a seed bank that can modify the eco-evolutionary outcomes of host-parasite systems.

By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to tolerate suboptimal conditions that would otherwise reduce their fitness. Dormancy may also benefit bacteria by serving as a refuge from parasitic infections. Here we focus on dormancy in the Firmicutes, where endospore development is transcriptionally regulated by the expression of sigma factors. A disruption of this process could influence the survivorship and reproduction of phages that infect spore-forming hosts with implications for coevolutionary dynamics. Here, we characterized the distribution and diversity of sigma factors in nearly 3,500 phage genomes. Homologs of sporulation-specific sigma factors were identified in phages that infect spore-forming hosts. Unlike sigma factors required for phage reproduction, the sporulation-like sigma factors were non-essential for lytic infection. However, when expressed in the spore-forming Bacillus subtilis, sigma factors from phages activated the bacterial sporulation gene network and reduced spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate a complex and ancient trait in one of the most abundant cell types on Earth.

Fluctuations in the availability of resources constrains the growth and reproduction of individuals, which subsequently effects the evolution of their respective populations. Many organisms contend with such fluctuations by entering a reversible state of reduced metabolic activity, a phenomenon known as dormancy. This pool of dormant individuals (i.e., a seed bank) does not reproduce and is expected to act as an evolutionary buffer, though it is difficult to observe this effect directly over an extended evolutionary timescale. Through genetic manipulation, we analyze the molecular evolutionary dynamics of Bacillus subtilis populations in the presence and absence of a seed bank over 700 days. The ability of these bacteria to enter a dormant state increased the accumulation of genetic diversity over time and altered the trajectory of mutations, findings that were recapitulated using simulations based on a mathematical model of evolutionary dynamics. While the ability to form a seed bank did not alter the degree of negative selection, we found that it consistently altered the direction of molecular evolution across genes. Together, these results show that the ability to form a seed bank can affect the direction and rate of molecular evolution over an extended evolutionary timescale.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life. Here, we report on how a synthetically constructed minimal cell contends with the forces of evolution compared to a non-minimized cell from which it was derived. Genome streamlining was costly, but 80% of fitness was regained in 2000 generations. Although selection acted upon divergent sets of mutations, the rates of adaptation in the minimal and non-minimal cell were equivalent. The only apparent constraint of minimization involved epistatic interactions that inhibited the evolution of cell size. Together, our findings demonstrate the power of natural selection to rapidly optimize fitness in the simplest autonomous organism, with implications for the evolution of cellular complexity.

Microorganisms have the unique ability to survive extended periods of time in environments with extremely low levels of exploitable energy. To determine the extent that energy limitation affects microbial evolution, we examined the molecular evolutionary dynamics of a phylogenetically diverse set of taxa over the course of 1,000 days. We found that periodic exposure to energy limitation affected the rate of molecular evolution, the accumulation of genetic diversity, and the rate of extinction. We then determined the degree that energy limitation affected the spectrum of mutations as well as the direction of evolution at the gene level. Our results suggest that the initial depletion of energy altered the direction and rate of molecular evolution within each taxon, though after the initial depletion the rate and direction did not substantially change. However, this consistent pattern became diminished when comparisons were performed across phylogenetically distant taxa, suggesting that although the dynamics of molecular evolution under energy limitation are highly generalizable across the microbial tree of life, the targets of adaptation are specific to a given taxon.

As the most abundant and diverse form of life on Earth, microorganisms commonly inhabit energy-limited environments where cellular maintenance and growth is highly constrained. To gain insight into how microorganisms persist under such conditions, we derived demographic parameters from a diverse collection of bacteria by censusing 100 populations in a closed system for 1,000 days. All but one taxon survived prolonged resource scarcity, yielding estimated times-to-extinction ranging over four orders of magnitude from 100– 105years. These findings corroborate reports of long-lived bacteria that have been recovered from ancient environmental samples, while providing insight into mechanisms of persistence. Critically, we found that as death rates declined over time, lifespan was extended through the scavenging of dead cells. Although growth and reproduction were dramatically suppressed in the absence of an exogenous resource supply, bacterial populations continued to evolve. Hundreds of mutations were acquired, contributing to genome-wide signatures of negative selection as well as molecular signals of adaptation. Remarkable consistency in the ecological and evolutionary dynamics indicate that distantly related bacteria respond to energy-limitation in a similar and predictable manner, which likely contributes to the stability and robustness of microbial life.

Coexisting species often exhibit negative frequency dependence due to mechanisms that promote population growth and persistence when rare. These stabilizing mechanisms maintain diversity through interspecific niche differences, but also through life-history strategies like dormancy that buffer dynamics in fluctuating environments. However, there are few tests demonstrating how seed banks contribute to long-term community dynamics and the maintenance of diversity. Using multi-year, high-frequency bacterial community data from a north temperate lake, we documented patterns consistent with stabilizing coexistence, including differential responses of bacterial taxa to environmental conditions and the presence of a seed bank. By maintaining rare taxa over winter periods, the seed bank contributed to recurrent seasonal dynamics. Last, strong negative frequency dependence in rare, but metabolically active, taxa suggested biotic interactions promoted coexistence. Together, our results provide field-based evidence that niche differences and seed banks stabilize bacterial community dynamics and contribute to the maintenance of microbial diversity in nature.

The degree that the environment determines what genes contribute towards adaptation is a fundamental question in microbial evolution. Microbial populations are often experimentally passaged in different environments and sequenced in order to identify candidates for adaptation in a particular environment. However, there remains the need to develop an appropriate statistical framework to identify genes that acquired more mutations in one environment over the other (i.e., divergent evolution). Here we demonstrate how the evolutionary outcomes among replicate populations in the same environment, known as parallel evolution, can be leveraged to construct an intuitive statistical test for identifying the genes that contribute towards divergent evolution. To accomplish this task, we examined publicly available evolve-and-resequence experiment datasets and found that the distribution of mutation counts among genes can be predicted using an ensemble of independent Poisson random variables. Building on this result, we propose that the degree of divergent evolution at a given gene between populations from two different environments can be modeled as the difference between two Poisson random variables, known as the Skellam distribution. We then propose and apply a statistical test to identify specific genes that contribute towards divergent evolution. IMPORTANCE: There is currently no existing framework that can be leveraged to identify genes that contribute towards divergent evolution in microbial evolution experiments. To correct for this absence, we investigated the distribution of mutation counts among genes in order to identify an appropriate null model. Our observations suggest that divergent evolution within a given gene can be modeled as the difference in the total number of mutations observed between two environments. This quantity is described by a probability distribution known as the Skellam distribution, providing an appropriate statistical test for researchers seeking to identify the set of genes that contribute towards divergent evolution in evolution experiments.

Tidal freshwater marshes (TFMs) are threatened by seawater intrusion, which can affect microbial communities and alter biogeochemical processes. Here, we report on Seawater Addition Long Term Experiment (SALTEx), a manipulative field experiment that investigated continuous (press) and episodic (pulse, 2 months/yr) inputs of brackish water on microbial communities in a TFM. After 2.5 years, microbial diversity was lower in press treatments than in control (untreated) plots. Sulfate reducers increased in response to both press and pulse treatments whereas methanogens did not differ among treatments. Our results suggest that microbial communities in TFMs are resilient to episodic events, but that continuous seawater intrusion may alter bacterial diversity in ways that affect ecosystem functioning.Scientific Significance StatementSea level rise and seawater intrusion threaten tidal freshwater marshes (TFMs) and the important ecosystem services they provide. Intrusion of seawater in TFMs can occur across a range of timescales, such as episodic events, like storm surges or drought, or continuous intrusion as a result of rising sea level. The effects of these stressors on TFM microbial communities are not well understood. Our multi-year field manipulation of brackish water inputs revealed that microbial communities were resilient to short-term pulses of salinity whereas continuous seawater intrusion led to reduced microbial diversity along with changes in relative abundance of key functional groups. Such alterations may diminish the ability of TFMs to sequester carbon and cycle nutrients.

A major goal of metacommunity ecology is to infer the local- and regional-scale processes that underlie community assembly. In dendritic ecological networks (e.g., stream metacommunities), branching and directional dispersal connectivity can alter the balance between local and regional factors during assembly. However, the implications of vertical habitat structure (e.g., planktonic versus benthic sediments) in dendritic metacommunities remain unclear. In this study, we analyzed the bacterial metacommunity of a fifth-order mountain stream network to assess habitat differences in the (1) dominant community assembly processes, (2) spatial scaling of community assembly processes, and (3) longitudinal variation in community assembly. Using taxonomic and phylogenetic null modeling approaches, we found habitat-specific spatial patterns of community assembly across the dendritic network. Compositional differences between planktonic and benthic communities were maintained by divergent species sorting, but stochasticity influenced assembly at local scales. Planktonic communities showed scale-dependent assembly, transitioning from convergent sorting at local scales to divergent sorting at regional scales, while sediment community assembly was less scale dependent (convergent sorting remained important across all scales). While divergent sorting structured headwaters in both habitat types, sediment communities converged in structure downstream. Taken together, our results show that vertical habitat structure regulates the scale-dependent processes of community assembly across the dendritic metacommunity.

The movement of organisms across habitat boundaries has important consequences for populations, communities, and ecosystems. However, because most species are not well adapted to all habitat types, dispersal into suboptimal habitats could induce physiological changes associated with persistence strategies that influence community assembly. For example, high rates of cross-boundary dispersal are thought to maintain sink populations of terrestrial bacteria in aquatic habitats, but these bacteria may also persist by lowering their metabolic activity, introducing metabolic heterogeneity that buffers the population against niche selection. To differentiate between these assembly processes, we analyzed bacterial composition along a hydrological flow path from terrestrial soils through an aquatic reservoir by sequencing the active and total (active + inactive) portions of the community. When metabolic heterogeneity was ignored, our data were consistent with views that cross-boundary dispersal is important for structuring aquatic bacterial communities. In contrast, we found evidence for strong niche selection when metabolic heterogeneity was explicitly considered, suggesting that, relative to persistence strategies, dispersal may have a weaker effect on aquatic community assembly than previously thought. By accounting for metabolic heterogeneity in complex communities, our findings clarify the roles of local- and regional-scale assembly processes in terrestrial-aquatic meta-ecosystems.

Much of Earth’s biodiversity has the capacity to engage in dormancy whereby individuals enter a reversible state of reduced metabolic activity. By increasing resilience to unfavorable conditions, dormancy leads to the accumulation of “seed banks” that should diminish the influence of environmental filtering, while allowing passive dispersers to colonize new habitats. Although prevalent among single-celled organisms, evidence that dormancy influences patterns of microbial biodiversity and biogeography is lacking. We constructed geographical and environmental distance-decay relationships (DDRs) using 16S rRNA sequencing to characterize the total (DNA) and the active (RNA) bacterial communities in a regional survey of 49 forested ponds. As expected, the total community harbored greater diversity and exhibited weaker DDRs than the active portion of the community. These empirical observations were robust to different measures of community similarity and random resampling tests. Furthermore, findings from the field survey were reproduced by models that included aspects of dormancy along with the geographical coordinates and environmental characteristics of our study system. In addition to maintaining local diversity, our results support recent theoretical predictions that dormancy shapes geographical patterns of biodiversity.

Climate change is accelerating global soil respiration, which could in turn accelerate climate change. The biological mechanisms through which soil carbon (C) responds to climate are not well understood, limiting our ability to predict future global soil respiration rates. As part of a climate manipulation experiment, we tested whether differences in soil heterotrophic respiration driven by season or climate treatment (RH) are linked to 1) relative abundances of microbes in active and dormant metabolic states, 2) net changes in microbial biomass and/or 3) changes in the relative abundances of microbial groups with different C-use strategies. We used a flow-cytometric single-cell metabolic assay to quantify the abundance of active and dormant microbes, and the phospholipid fatty acid (PLFA) method to determine microbial biomass and ratios of fungi:bacteria and Gram-positive:Gram-negative bacteria. RH did not respond to climate treatments but was greater in the warm and dry summer than in the cool and less-dry fall. These dynamics were better explained when microbial data were taken into account compared to when only physical data (temperature and moisture) were used. Overall, our results suggest that RH responses to temperature are stronger when soil contains more active microbes, and that seasonal patterns of RH can be better explained by shifts in microbial activity than by shifts in the relative abundances of fungi and Gram-positive and Gram-negative bacteria. These findings contribute to our understanding of how and under which conditions microbes influence soil C responses to climate.

Microbial diversity is strongly affected by the bottom-up effects of resource availability. However, because resource pools often exist as heterogeneous mixtures of distinct molecules, resource heterogeneity may also affect community diversity. To test this hypothesis, we surveyed bacterial communities in lakes that varied in resource concentration. In addition, we characterized resource heterogeneity in these lakes using an ecosystem metabolomics approach. Overall, resource concentration and resource heterogeneity affected bacterial resource-diversity relationships. We found strong relationships between bacterial alpha-diversity (richness and evenness) and resource concentration and richness, but richness and evenness responded in different ways. Likewise, we found associations between the composition of the bacterial community and both resource concentration and composition, but the relationship with resource composition was stronger. Last, in the surveyed communities the presence of resource generalists may have reduced the effect of resource heterogeneity on community composition. These results have implications for understanding the interactions between bacteria and organic matter and suggest that changes in organic matter composition may alter the structure and function of bacterial communities.

Extracellular or “relic” DNA is one of the largest pools of nucleic acids in the mbiosphere1,2. Relic DNA can influence a number of important ecological and evolutionary processes, but it may also bias estimates of microbial abundance and diversity, which has implications for understanding environmental, engineered, and host-associated ecosystems. We developed models capturing the fundamental processes that regulate the size and composition of the relic DNA pools to identify scenarios leading to biased estimates of biodiversity. Our models predict that bias increases with relic DNA pool size, but only when the species abundance distributions (SAD) of relic and intact DNA are distinct from one another. We evaluated our model predictions by quantifying relic DNA and assessing its contribution to bacterial diversity using 16S rRNA gene sequences collected from different ecosystem types, including soil, sediment, water, and the mammalian gut. On average, relic DNA made up 33 % of the total bacterial DNA pool, but exceeded 80 % in some samples. Despite its abundance, relic DNA had no effect on estimates of taxonomic and phylogenetic diversity, even in ecosystems where processes such as the physical protection of relic DNA are common and predicted by our models to generate bias. Rather, our findings are consistent with the expectation that relic DNA sequences degrade in proportion to their abundance and therefore may contribute minimally to estimates of microbial diversity.

Translating the ever-increasing wealth of information on microbiomes (environment, host, or built environment) to advance the understanding of system-level processes is proving to be an exceptional research challenge. One reason for this challenge is that relationships between characteristics of microbiomes and the system-level processes they influence are often evaluated in the absence of a robust conceptual framework and reported without elucidating the underlying causal mechanisms. The reliance on correlative approaches limits the potential to expand the inference of a single relationship to additional systems and advance the field. We propose that research focused on how microbiomes influence the systems they inhabit should work within a common framework and target known microbial processes that contribute to the system-level processes of interest. Here we identify three distinct categories of microbiome characteristics (microbial processes, microbial community properties, and microbial membership) and propose a framework to empirically link each of these categories to each other and the broader system level processes they affect. We posit that it is particularly important to distinguish microbial community properties that can be predicted from constituent taxa (community aggregated traits) from and those properties that are currently unable to be predicted from constituent taxa (emergent properties). Existing methods in microbial ecology can be applied to more explicitly elucidate properties within each of these categories and connect these three categories of microbial characteristics with each other. We view this proposed framework, gleaned from a breadth of research on environmental microbiomes and ecosystem processes, as a promising pathway with the potential to advance discovery and understanding across a broad range of microbiome science.

Sources and sinks of methane (CH4 ) are critical for understanding global biogeochemical cycles and their role in climate change. A growing number of studies have reported that CH4 concentrations in cave ecosystems are depleted, leading to the notion that these subterranean environments may act as sinks for atmospheric CH4 . Recently, it was hypothesized that this CH4 depletion may be caused by radiolysis, an abiotic process whereby CH4 is oxidized via interactions with ionizing radiation derived from radioactive decay. An alternate explanation is that the depletion of CH4 concentrations in caves could be due to biological processes, specifically oxidation by methanotrophic bacteria. We theoretically explored the radiolysis hypothesis and conclude that it is a kinetically constrained process that is unlikely to lead to the rapid loss of CH4 in subterranean environments. We present results from a controlled laboratory experiment to support this claim. We then tested the microbial oxidation hypothesis with a set of mesocosm experiments that were conducted in two Vietnamese caves. Our results reveal that methanotrophic bacteria associated with cave rocks consume CH4 at a rate of 1.3-2.7 mg CH4 · m-2 · d-1 . These CH4 oxidation rates equal or exceed what has been reported in other habitats, including agricultural systems, grasslands, deciduous forests, and Arctic tundra. Together, our results suggest that depleted concentrations of CH4 in caves are most likely due to microbial activity, not radiolysis as has been recently claimed. Microbial methanotrophy has the potential to oxidize CH4 not only in caves, but also in smaller-size open subterranean spaces, such as cracks, fissures, and other pores that are connected to and rapidly exchange with the atmosphere. Future studies are needed to understand how subterranean CH4 oxidation scales up to affect local, regional, and global CH4 cycling.

The movement of resources between terrestrial and aquatic habitats has strong effects on ecological processes in recipient ecosystems. Allochthonous inputs modify the quality and quantity of the available resource pool in ways that may alter the composition and stability of recipient communities. Inputs of terrestrial dissolved organic carbon (tDOC) into aquatic ecosystems represent a large influx of resources that has the potential to affect local communities, especially microorganisms. To evaluate the effects terrestrial inputs on aquatic microbial community composition and stability, we manipulated the supply rate of tDOC to a set of experimental ponds. Along the tDOC supply gradient, we measured changes in diversity and taxon-specific changes in abundance and activity. We then determined community stability by perturbing each pond using a pulse of inorganic nutrients and measuring changes in composition and activity (i.e., responsiveness) along the gradient. Terrestrial DOC supply significantly altered the composition of the active microbial community. The composition of the active bacterial community changed via decreases in richness and evenness as well as taxon-specific changes in abundance and activity indicating species sorting along the gradient. Likewise, the responsiveness of the active bacterial community decreased along the gradient, which led to a more stable active community. We did not, however, observe these changes in diversity and stability in the total community (i.e., active and inactive organisms), which suggests that tDOC supply modifies microbial community stability through functional not structural changes. Together, these results show that altered aquatic terrestrial linkages can have profound effects on the activity and stability of the base of the food web and thus can alter ecosystem functioning.

Microbiomes can aid in the protection of hosts from infection and disease, but the mechanisms underpinning these functions in complex environmental systems remain unresolved. Soils contain microbiomes that influence plant performance, including their susceptibility to disease. For example, some soil microorganisms produce antimicrobial compounds that suppress the growth of plant pathogens, which can provide benefits for sustainable agricultural management. Evidence shows that crop rotations increase soil fertility and tend to promote microbial diversity, and it has been hypothesized that crop rotations can enhance disease suppressive capacity, either through the influence of plant diversity impacting soil bacterial composition or through the increased abundance of disease suppressive microorganisms. In this study, we used a long-term field experiment to test the effects of crop diversity through time (i.e., rotations) on soil microbial diversity and disease suppressive capacity. We sampled soil from seven treatments along a crop diversity gradient (from monoculture to five crop species rotation) and a spring fallow (non-crop) treatment to examine crop diversity influence on soil microbiomes including bacteria that are capable of producing antifungal compounds. Crop diversity significantly influenced bacterial community composition, where the most diverse cropping systems with cover crops and fallow differed from bacterial communities in the 1-3 crop species diversity treatments. While soil bacterial diversity was about 4% lower in the most diverse crop rotation (corn-soy-wheat + 2 cover crops) compared to monoculture corn, crop diversity increased disease suppressive functional group prnD gene abundance in the more diverse rotation by about 9% compared to monocultures. Identifying patterns in microbial diversity and ecosystem function relationships can provide insight into microbiome management, which will require manipulating soil nutrients and resources mediated through plant diversity.

Marine viruses are critical drivers of ocean biogeochemistry and their abundances vary spatiotem- porally in the global oceans, with upper estimates exceeding 10 8 per ml. Over many years, a con- sensus has emerged that virus abundances are typically 10-fold higher than prokaryote abundances. The use of a fixed-ratio suggests that the relationship between virus and prokaryote abundances is both predictable and linear. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5671 prokaryote and virus abundance estimates from 25 distinct marine surveys to characterize the relationship between virus and prokaryote abundances. We find that the median virus-to-prokaryote ratio (VPR) is 10:1 and 16:1 in the near- and sub-surface oceans, respectively. Nonetheless, we observe substantial variation in the VPR and find either no or limited explanatory power using fixed-ratio models. Instead, virus abundances are better described as nonlinear, power-law functions of prokaryote abundances - par- ticularly when considering relationships within distinct marine surveys. Estimated power-laws have scaling exponents that are typically less than 1, signifying that the VPR decreases with prokaryote density, rather than remaining fixed. The emergence of power-law scaling presents a challenge for mechanistic models seeking to understand the ecological causes and consequences of marine virus- microbe interactions. Such power-law scaling also implies that efforts to average viral effects on microbial mortality and biogeochemical cycles using “representative” abundances or abundance- ratios need to be refined if they are to be utilized to make quantitative predictions at regional or global ocean scales.