Diversity at single nucleotide to pangenome scales among sulfur cycling bacteria in salt marshes.

IMPORTANCE: Salt marshes are known for their significant carbon storage capacity, and sulfur cycling is closely linked with the ecosystem-scale carbon cycling in these ecosystems. Sulfate reducers are key for the decomposition of organic matter, and sulfur oxidizers remove toxic sulfide, supporting the productivity of marsh plants. To date, the complexity of coastal environments, heterogeneity of the rhizosphere, high microbial diversity, and uncultured majority hindered our understanding of the genomic diversity of sulfur-cycling microbes in salt marshes. Here, we use comparative genomics to overcome these challenges and provide an in-depth characterization of sulfur-cycling microbial diversity in salt marshes. We characterize communities across distinct sites and plant species and uncover extensive genomic diversity at the taxon level and specific genomic features present in MAGs affiliated with uncultivated sulfur-cycling lineages. Our work provides insights into the partnerships in salt marshes and a roadmap for multiscale analyses of diversity in complex biological systems.

Parasite manipulation of host phenotypes inferred from transcriptional analyses in a trematode-amphipod system.

Manipulation of host phenotypes by parasites is hypothesized to be an adaptive strategy enhancing parasite transmission across hosts and generations. Characterizing the molecular mechanisms of manipulation is important to advance our understanding of host-parasite coevolution. The trematode (Levinseniella byrdi) is known to alter the colour and behaviour of its amphipod host (Orchestia grillus) presumably increasing predation of amphipods which enhances trematode transmission through its life cycle. We sampled 24 infected and 24 uninfected amphipods from a salt marsh in Massachusetts to perform differential gene expression analysis. In addition, we constructed novel genomic tools for O. grillus including a de novo genome and transcriptome. We discovered that trematode infection results in upregulation of amphipod transcripts associated with pigmentation and detection of external stimuli, and downregulation of multiple amphipod transcripts implicated in invertebrate immune responses, such as vacuolar ATPase genes. We hypothesize that suppression of immune genes and the altered expression of genes associated with coloration and behaviour may allow the trematode to persist in the amphipod and engage in further biochemical manipulation that promotes transmission. The genomic tools and transcriptomic analyses reported provide new opportunities to discover how parasites alter diverse pathways underlying host phenotypic changes in natural populations.

Slip slidin' away: Bristle-driven gliding by Tetradesmus deserticola (Chlorophyta) in microfluidic chambers.

Microalgae within the Scenedesmaceae are often distinguished by spines, bristles, and other wall characteristics. We examined the dynamic production and chemical nature of bristles extruded from the poles of Tetradesmus deserticola previously isolated from microbiotic crust. Rapidly growing cells in a liquid growth medium were established in polydimethylsiloxane microfluidic chambers specially designed to maintain aerobic conditions over time within a chamber 6-12 µm deep. This geometry enabled in-focus imaging of single cells over long periods. Differential interference contrast (DIC) imaging revealed that after multiple fission of mother cells, the newly released, lemon-shaped daughter cells began extruding bristles from each pole. In some instances, the bristles became stuck to either the glass floor or polydimethylsiloxane (PDMS) walls of the chamber, and the force by which the new bristle was extruded was sufficient to propel the cells across the field of view at ~1.2 µm · h-1 . Confocal fluorescence and DIC imaging of cells stained with pontamine fast scarlet and calcofluor, and treated with proteinase K, suggested that bristles are proteinaceous and may also host carbohydrate modifications. The polar bristles extruded by this desert-derived T. deserticola may simply be relics of bristles produced by an aquatic ancestor for flotation or predator deterrence. But, their tendency to attach to glass (silicate) and/or PDMS surfaces suggests a potential role in tethering cells in place or binding soil particles. T. deserticola is closely related to T. obliquus, which is of interest for biofuels development; extruded bristles in T. deserticola may offer tethers for industrial use of these stress-tolerant algae.

Simple Plant and Microbial Exudates Destabilize Mineral-Associated Organic Matter via Multiple Pathways.

Most mineral-associated organic matter (MAOM) is protected against microbial attack, thereby contributing to long-term carbon storage in soils. However, the extent to which reactive compounds released by plants and microbes may destabilize MAOM and so enhance microbial access, as well as the underlying mechanisms, remain unclear. Here, we tested the ability of functionally distinct model exudates-ligands, reductants, and simple sugars-to promote microbial utilization of monomeric MAOM, bound via outer-sphere complexes to common iron and aluminum (hydr)oxide minerals. The strong ligand oxalic acid induced rapid MAOM mineralization, coinciding with greater sorption to and dissolution of minerals, suggestive of direct MAOM mobilization mechanisms. In contrast, the simple sugar glucose caused slower MAOM mineralization, but stimulated microbial activity and metabolite production, indicating an indirect microbially-mediated mechanism. Catechol, acting as reductant, promoted both mechanisms. While MAOM on ferrihydrite showed the greatest vulnerability to both direct and indirect mechanisms, MAOM on other (hydr)oxides was more susceptible to direct mechanisms. These findings suggest that MAOM persistence, and thus long-term carbon storage within a given soil, is not just a function of mineral reactivity but also depends on the capacity of plant roots and associated microbes to produce reactive compounds capable of triggering specific destabilization mechanisms.

Shared up-regulation and contrasting down-regulation of gene expression distinguish desiccation-tolerant from intolerant green algae.

Among green plants, desiccation tolerance is common in seeds and spores but rare in leaves and other vegetative green tissues. Over the last two decades, genes have been identified whose expression is induced by desiccation in diverse, desiccation-tolerant (DT) taxa, including, e.g., late embryogenesis abundant proteins (LEA) and reactive oxygen species scavengers. This up-regulation is observed in DT resurrection plants, mosses, and green algae most closely related to these Embryophytes. Here we test whether this same suite of protective genes is up-regulated during desiccation in even more distantly related DT green algae, and, importantly, whether that up-regulation is unique to DT algae or also occurs in a desiccation-intolerant relative. We used three closely related aquatic and desert-derived green microalgae in the family Scenedesmaceae and capitalized on extraordinary desiccation tolerance in two of the species, contrasting with desiccation intolerance in the third. We found that during desiccation, all three species increased expression of common protective genes. The feature distinguishing gene expression in DT algae, however, was extensive down-regulation of gene expression associated with diverse metabolic processes during the desiccation time course, suggesting a switch from active growth to energy-saving metabolism. This widespread downshift did not occur in the desiccation-intolerant taxon. These results show that desiccation-induced up-regulation of expression of protective genes may be necessary but is not sufficient to confer desiccation tolerance. The data also suggest that desiccation tolerance may require induced protective mechanisms operating in concert with massive down-regulation of gene expression controlling numerous other aspects of metabolism.

Extraction of high-quality, high-molecular-weight DNA depends heavily on cell homogenization methods in green microalgae.

PREMISE: New sequencing technologies have facilitated genomic studies in green microalgae; however, extracting high-quality DNA is often a bottleneck for long-read sequencing. METHODS AND RESULTS: Here, we present a low-cost, highly transferrable method for the extraction of high-molecular-weight (HMW), high-purity DNA from microalgae. We first determined the effect of sample preparation on DNA quality using three homogenization methods: manual grinding using a mini-pestle, automatic grinding using a vortex adapter, and grinding in liquid nitrogen. We demonstrated the versatility of grinding in liquid nitrogen followed by a modified cetyltrimethylammonium bromide (CTAB) extraction across a suite of aquatic- and desert-evolved algal taxa. Finally, we tested the protocol's robustness by doubling the input material to increase yield, producing per sample up to 20 µg of high-purity DNA longer than 21.2 kbp. CONCLUSIONS: All homogenization methods produced DNA within acceptable parameters for purity, but only liquid nitrogen grinding resulted in HMW DNA. The optimization of cell lysis while minimizing DNA shearing is therefore crucial for the isolation of DNA for long-read genomic sequencing because template DNA length strongly affects read output and length.

Hydraulic redistribution affects modeled carbon cycling via soil microbial activity and suppressed fire.

Hydraulic redistribution (HR) of water from moist to drier soils, through plant roots, occurs world-wide in seasonally dry ecosystems. Although the influence of HR on landscape hydrology and plant water use has been amply demonstrated, HR's effects on microbe-controlled processes sensitive to soil moisture, including carbon and nutrient cycling at ecosystem scales, remain difficult to observe in the field and have not been integrated into a predictive framework. We incorporated a representation of HR into the Community Land Model (CLM4.5) and found the new model improved predictions of water, energy, and system-scale carbon fluxes observed by eddy covariance at four seasonally dry yet ecologically diverse temperate and tropical AmeriFlux sites. Modeled plant productivity and microbial activities were differentially stimulated by upward HR, resulting at times in increased plant demand outstripping increased nutrient supply. Modeled plant productivity and microbial activities were diminished by downward HR. Overall, inclusion of HR tended to increase modeled annual ecosystem uptake of CO2 (or reduce annual CO2 release to the atmosphere). Moreover, engagement of CLM4.5's ground-truthed fire module indicated that though HR increased modeled fuel load at all four sites, upward HR also moistened surface soil and hydrated vegetation sufficiently to limit the modeled spread of dry season fire and concomitant very large CO2 emissions to the atmosphere. Historically, fire has been a dominant ecological force in many seasonally dry ecosystems, and intensification of soil drought and altered precipitation regimes are expected for seasonally dry ecosystems in the future. HR may play an increasingly important role mitigating development of extreme soil water potential gradients and associated limitations on plant and soil microbial activities, and may inhibit the spread of fire in seasonally dry ecosystems.

A model suite of green algae within the Scenedesmaceae for investigating contrasting desiccation tolerance and morphology.

Microscopic green algae inhabiting desert microbiotic crusts are remarkably diverse phylogenetically, and many desert lineages have independently evolved from aquatic ancestors. Here we worked with five desert and aquatic species within the family Scenedesmaceae to examine mechanisms that underlie desiccation tolerance and release of unicellular versus multicellular progeny. Live cell staining and time-lapse confocal imaging coupled with transmission electron microscopy established that the desert and aquatic species all divide by multiple (rather than binary) fission, although progeny were unicellular in three species and multicellular (joined in a sheet-like coenobium) in two. During division, Golgi complexes were localized near nuclei, and all species exhibited dynamic rotation of the daughter cell mass within the mother cell wall at cytokinesis. Differential desiccation tolerance across the five species, assessed from photosynthetic efficiency during desiccation/rehydration cycles, was accompanied by differential accumulation of intracellular reactive oxygen species (ROS) detected using a dye sensitive to intracellular ROS. Further comparative investigation will aim to understand the genetic, ultrastructural and physiological characteristics supporting unicellular versus multicellular coenobial morphology, and the ability of representatives in the Scenedesmaceae to colonize ecologically diverse, even extreme, habitats.

Toward a Predictive Understanding of Earth's Microbiomes to Address 21st Century Challenges.

Microorganisms have shaped our planet and its inhabitants for over 3.5 billion years. Humankind has had a profound influence on the biosphere, manifested as global climate and land use changes, and extensive urbanization in response to a growing population. The challenges we face to supply food, energy, and clean water while maintaining and improving the health of our population and ecosystems are significant. Given the extensive influence of microorganisms across our biosphere, we propose that a coordinated, cross-disciplinary effort is required to understand, predict, and harness microbiome function. From the parallelization of gene function testing to precision manipulation of genes, communities, and model ecosystems and development of novel analytical and simulation approaches, we outline strategies to move microbiome research into an era of causality. These efforts will improve prediction of ecosystem response and enable the development of new, responsible, microbiome-based solutions to significant challenges of our time.

Functional Responses of Salt Marsh Microbial Communities to Long-Term Nutrient Enrichment.

UNLABELLED: Environmental nutrient enrichment from human agricultural and waste runoff could cause changes to microbial communities that allow them to capitalize on newly available resources. Currently, the response of microbial communities to nutrient enrichment remains poorly understood, and, while some studies have shown no clear changes in community composition in response to heavy nutrient loading, others targeting specific genes have demonstrated clear impacts. In this study, we compared functional metagenomic profiles from sediment samples taken along two salt marsh creeks, one of which was exposed for more than 40 years to treated sewage effluent at its head. We identified strong and consistent increases in the relative abundance of microbial genes related to each of the biochemical steps in the denitrification pathway at enriched sites. Despite fine-scale local increases in the abundance of denitrification-related genes, the overall community structures based on broadly defined functional groups and taxonomic annotations were similar and varied with other environmental factors, such as salinity, which were common to both creeks. Homology-based taxonomic assignments of nitrous oxide reductase sequences in our data show that increases are spread over a broad taxonomic range, thus limiting detection from taxonomic data alone. Together, these results illustrate a functionally targeted yet taxonomically broad response of microbial communities to anthropogenic nutrient loading, indicating some resolution to the apparently conflicting results of existing studies on the impacts of nutrient loading in sediment communities. IMPORTANCE: In this study, we used environmental metagenomics to assess the response of microbial communities in estuarine sediments to long-term, nutrient-rich sewage effluent exposure. Unlike previous studies, which have mainly characterized communities based on taxonomic data or primer-based amplification of specific target genes, our whole-genome metagenomics approach allowed an unbiased assessment of the abundance of denitrification-related genes across the entire community. We identified strong and consistent increases in the relative abundance of gene sequences related to denitrification pathways across a broad phylogenetic range at sites exposed to long-term nutrient addition. While further work is needed to determine the consequences of these community responses in regulating environmental nutrient cycles, the increased abundance of bacteria harboring denitrification genes suggests that such processes may be locally upregulated. In addition, our results illustrate how whole-genome metagenomics combined with targeted hypothesis testing can reveal fine-scale responses of microbial communities to environmental disturbance.

Tools for the Microbiome: Nano and Beyond.

The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.

Rhizosphere heterogeneity shapes abundance and activity of sulfur-oxidizing bacteria in vegetated salt marsh sediments.

Salt marshes are highly productive ecosystems hosting an intense sulfur (S) cycle, yet little is known about S-oxidizing microorganisms in these ecosystems. Here, we studied the diversity and transcriptional activity of S-oxidizers in salt marsh sediments colonized by the plant Spartina alterniflora, and assessed variations with sediment depth and small-scale compartments within the rhizosphere. We combined next-generation amplicon sequencing of 16S rDNA and rRNA libraries with phylogenetic analyses of marker genes for two S-oxidation pathways (soxB and rdsrAB). Gene and transcript numbers of soxB and rdsrAB phylotypes were quantified simultaneously, using newly designed (RT)-qPCR assays. We identified a diverse assemblage of S-oxidizers, with Chromatiales and Thiotrichales being dominant. The detection of transcripts from S-oxidizers was mostly confined to the upper 5 cm sediments, following the expected distribution of root biomass. A common pool of species dominated by Gammaproteobacteria transcribed S-oxidation genes across roots, rhizosphere, and surrounding sediment compartments, with rdsrAB transcripts prevailing over soxB. However, the root environment fine-tuned the abundance and transcriptional activity of the S-oxidizing community. In particular, the global transcription of soxB was higher on the roots compared to mix and rhizosphere samples. Furthermore, the contribution of Epsilonproteobacteria-related S-oxidizers tended to increase on Spartina roots compared to surrounding sediments. These data shed light on the under-studied oxidative part of the sulfur cycle in salt marsh sediments and indicate small-scale heterogeneities are important factors shaping abundance and potential activity of S-oxidizers in the rhizosphere.

Modelled hydraulic redistribution by sunflower (Helianthus annuus†L.) matches observed data only after including night-time transpiration.

The movement of water from moist to dry soil layers through the root systems of plants, referred to as hydraulic redistribution (HR), occurs throughout the world and is thought to influence carbon and water budgets and ecosystem functioning. The realized hydrologic, biogeochemical and ecological consequences of HR depend on the amount of redistributed water, whereas the ability to assess these impacts requires models that correctly capture HR magnitude and timing. Using several soil types and two ecotypes of sunflower (Helianthus annuus L.) in split-pot experiments, we examined how well the widely used HR modelling formulation developed by Ryel et al. matched experimental determination of HR across a range of water potential driving gradients. H. annuus carries out extensive night-time transpiration, and although over the last decade it has become more widely recognized that night-time transpiration occurs in multiple species and many ecosystems, the original Ryel et al. formulation does not include the effect of night-time transpiration on HR. We developed and added a representation of night-time transpiration into the formulation, and only then was the model able to capture the dynamics and magnitude of HR we observed as soils dried and night-time stomatal behaviour changed, both influencing HR.

Sagebrush carrying out hydraulic lift enhances surface soil nitrogen cycling and nitrogen uptake into inflorescences.

Plant roots serve as conduits for water flow not only from soil to leaves but also from wetter to drier soil. This hydraulic redistribution through root systems occurs in soils worldwide and can enhance stomatal opening, transpiration, and plant carbon gain. For decades, upward hydraulic lift (HL) of deep water through roots into dry, litter-rich, surface soil also has been hypothesized to enhance nutrient availability to plants by stimulating microbially controlled nutrient cycling. This link has not been demonstrated in the field. Working in sagebrush-steppe, where water and nitrogen limit plant growth and reproduction and where HL occurs naturally during summer drought, we slightly augmented deep soil water availability to 14 HL+ treatment plants throughout the summer growing season. The HL+ sagebrush lifted greater amounts of water than control plants and had slightly less negative predawn and midday leaf water potentials. Soil respiration was also augmented under HL+ plants. At summer's end, application of a gas-based (15)N isotopic labeling technique revealed increased rates of nitrogen cycling in surface soil layers around HL+ plants and increased uptake of nitrogen into HL+ plants' inflorescences as sagebrush set seed. These treatment effects persisted even though unexpected monsoon rainstorms arrived during assays and increased surface soil moisture around all plants. Simulation models from ecosystem to global scales have just begun to include effects of hydraulic redistribution on water and surface energy fluxes. Results from this field study indicate that plants carrying out HL can also substantially enhance decomposition and nitrogen cycling in surface soils.

Better to light a candle than curse the darkness: illuminating spatial localization and temporal dynamics of rapid microbial growth in the rhizosphere.

The rhizosphere is a hotbed of microbial activity in ecosystems, fueled by carbon compounds from plant roots. Basic questions about the location and dynamics of plant-spurred microbial growth in the rhizosphere are difficult to answer with standard, destructive soil assays mixing a multitude of microbe-scale microenvironments in a single, often sieved, sample. Soil microbial biosensors designed with the luxCDABE reporter genes fused to a promoter of interest enable continuous imaging of the microbial perception of (and response to) environmental conditions in soil. We used the common soil bacterium Pseudomonas putida KT2440 as host to plasmid pZKH2 containing a fusion between the strong constitutive promoter nptII and luxCDABE (coding for light-emitting proteins) from Vibrio fischeri. Experiments in liquid media demonstrated that high light production by KT2440/pZKH2 was associated with rapid microbial growth supported by high carbon availability. We applied the biosensors in microcosms filled with non-sterile soil in which corn (Zea mays L.), black poplar (Populus nigra L.), or tomato (Solanum lycopersicum L.) was growing. We detected minimal light production from microbiosensors in the bulk soil, but biosensors reported continuously from around roots for as long as six days. For corn, peaks of luminescence were detected 1-4 and 20-35 mm along the root axis behind growing root tips, with the location of maximum light production moving farther back from the tip as root growth rate increased. For poplar, luminescence around mature roots increased and decreased on a coordinated diel rhythm, but was not bright near root tips. For tomato, luminescence was dynamic, but did not exhibit a diel rhythm, appearing in acropetal waves along roots. KT2440/pZKH2 revealed that root tips are not always the only, or even the dominant, hotspots for rhizosphere microbial growth, and carbon availability is highly variable in space and time around roots.

The xanthophyll cycle and NPQ in diverse desert and aquatic green algae.

It has long been suspected that photoprotective mechanisms in green algae are similar to those in seed plants. However, exceptions have recently surfaced among aquatic and marine green algae in several taxonomic classes. Green algae are highly diverse genetically, falling into 13 named classes, and they are diverse ecologically, with many lineages including members from freshwater, marine, and terrestrial habitats. Genetically similar species living in dramatically different environments are potentially a rich source of information about variations in photoprotective function. Using aquatic and desert-derived species from three classes of green algae, we examined the induction of photoprotection under high light, exploring the relationship between nonphotochemical quenching and the xanthophyll cycle. In liquid culture, behavior of aquatic Entransia fimbriata (Klebsormidiophyceae) generally matched patterns observed in seed plants. Nonphotochemical quenching was lowest after overnight dark adaptation, increased with light intensity, and the extent of nonphotochemical quenching correlated with the extent of deepoxidation of xanthophyll cycle pigments. In contrast, overnight dark adaptation did not minimize nonphotochemical quenching in the other species studied: desert Klebsormidium sp. (Klebsormidiophyceae), desert and aquatic Cylindrocystis sp. (Zygnematophyceae), and desert Stichococcus sp. (Trebouxiophyceae). Instead, exposure to low light reduced nonphotochemical quenching below dark-adapted levels. De-epoxidation of xanthophyll cycle pigments paralleled light-induced changes in nonphotochemical quenching for species within Klebsormidiophyceae and Trebouxiophyceae, but not Zygnematophyceae. Inhibition of violaxanthin-zeaxanthin conversion by dithiothreitol reduced high-light-associated nonphotochemical quenching in all species (Zygnematophyceae the least), indicating that zeaxanthin can contribute to photoprotection as in seed plants but to different extents depending on taxon or lineage.

The magnitude of hydraulic redistribution by plant roots: a review and synthesis of empirical and modeling studies.

Hydraulic redistribution (HR) - the movement of water from moist to dry soil through plant roots - occurs worldwide within a range of different ecosystems and plant species. The proposed ecological and hydrologic impacts of HR include increasing dry-season transpiration and photosynthetic rates, prolonging the life span of fine roots and maintaining root-soil contact in dry soils, and moving rainwater down into deeper soil layers where it does not evaporate. In this review, we compile estimates of the magnitude of HR from ecosystems around the world, using representative empirical and modeling studies from which we could extract amounts of water redistributed by plant root systems. The reported average magnitude of HR varies by nearly two orders of magnitude across ecosystems, from 0.04 to 1.3 mm H(2)O d(-1) in the empirical literature, and from 0.1 to 3.23 mm H(2)O d(-1) in the modeling literature. Using these synthesized data, along with other published studies, we examine this variation in the magnitude of upward and downward HR, considering effects of plant, soil and ecosystem characteristics, as well as effects of methodological details (in both empirical and modeling studies) on estimates of HR. We take both ecological and hydrologic perspectives.

Extended local similarity analysis (eLSA) of microbial community and other time series data with replicates.

BACKGROUND: The increasing availability of time series microbial community data from metagenomics and other molecular biological studies has enabled the analysis of large-scale microbial co-occurrence and association networks. Among the many analytical techniques available, the Local Similarity Analysis (LSA) method is unique in that it captures local and potentially time-delayed co-occurrence and association patterns in time series data that cannot otherwise be identified by ordinary correlation analysis. However LSA, as originally developed, does not consider time series data with replicates, which hinders the full exploitation of available information. With replicates, it is possible to understand the variability of local similarity (LS) score and to obtain its confidence interval. RESULTS: We extended our LSA technique to time series data with replicates and termed it extended LSA, or eLSA. Simulations showed the capability of eLSA to capture subinterval and time-delayed associations. We implemented the eLSA technique into an easy-to-use analytic software package. The software pipeline integrates data normalization, statistical correlation calculation, statistical significance evaluation, and association network construction steps. We applied the eLSA technique to microbial community and gene expression datasets, where unique time-dependent associations were identified. CONCLUSIONS: The extended LSA analysis technique was demonstrated to reveal statistically significant local and potentially time-delayed association patterns in replicated time series data beyond that of ordinary correlation analysis. These statistically significant associations can provide insights to the real dynamics of biological systems. The newly designed eLSA software efficiently streamlines the analysis and is freely available from the eLSA homepage, which can be accessed at http://meta.usc.edu/softs/lsa.

Nitrogen deposition, competition and the decline of a regionally threatened legume, Desmodium cuspidatum.

Increased nitrogen (N) deposition, resulting from the combustion of fossil fuels, production of synthetic fertilizers, growth of N(2)-fixing crops and high-intensity agriculture, is one of the anthropogenic factors most likely to cause global biodiversity changes over the next century. This influence may be especially large in temperate zone forests, which are highly N limited and occur in regions with the highest levels of N deposition. Within these ecosystems, N(2)-fixing plants, including legumes, may be more sensitive to N deposition than other plant species. Though it has long been recognized that the competitive edge conferred by N(2)-fixation diminishes with increasing soil N availability, the conservation implications of increased N deposition on native N(2)-fixers have received less attention. We focus on Desmodium cuspidatum, which has experienced dramatic population losses in the last 30-40 years in the northeastern United States. We explore competition between this regionally threatened legume and a common non-N(2)-fixing neighbor, Solidago canadensis, across a gradient of N deposition. Our data show that increased N deposition may be detrimental to N(2)-fixers such as D. cuspidatum in two ways: (1) biomass accumulation in the non-N(2)-fixer, S. canadensis, responds more strongly to increasing N deposition, and (2) S. canadensis competes strongly for available mineral nitrogen and can assimilate N previously fixed by D. cuspidatum, resulting in D. cuspidatum relying more heavily on energetically expensive N(2)-fixation when grown with S. canadensis. N deposition may thus reduce or eliminate the competitive advantage of N(2)-fixing species growing in N-limited ecosystems.