Nitrogen cycling in desert biological soil crusts across biogeographic regions in the Southwestern United States

Biological soil crusts (BSCs) are thought to be important in the fertility of arid lands as gateways for carbon (C) and nitrogen (N). Studies on the Colorado Plateau have shown that an incomplete internal N cycle operates in BSCs that results in significant exports of dissolved organic N, ammonia and nitrate into the bulk soil through percolating water, thus mechanistically explaining their role as a N gateway. It is not known if this pattern is found in other arid regions. To examine this, we measured rates of major biogeochemical N-transformations in a variety of BSCs collected from the Colorado Plateau and the Mojave, Sonoran and Chihuahuan Deserts. Dinitrogen fixation and aerobic ammonia oxidation were prominent transformations at all sites. We found anaerobic ammonia oxidation (anammox) rates to be below the detection limit in all cases, and at least 50-fold smaller than rates of N₂-fixation, making it an irrelevant process for these BSCs. Heterotrophic denitrification was also of little consequence for the flow of N, with rates at least an order of magnitude smaller than those of N₂-fixation. Thus we could confirm that despite the demonstrable differences in microbial community composition and soil material, BSCs from major biogeographic regions in arid North America displayed a remarkably consistent pattern of internal N cycling. The implications for arid land fertility drawn from previous studies in the Colorado Plateau appear applicable to BSCs across other arid regions of the Southwestern United States.     

The effects of snowpack properties and plant strategies on litter decomposition during winter in subalpine meadows

Background and aims

Approximately 50 % of belowground organic carbon is present in the northern permafrost region and due to changes in climate there are concerns that this carbon will be rapidly released to the atmosphere. The release of carbon in arctic soils is thought to be intimately linked to the N cycle through the N cycle’s influence on microbial activity. The majority of new N input into arctic systems occurs through N₂-fixation; therefore, N₂-fixation may be the key driver of greenhouse gases from these ecosystems.

Methods

At Alexandra Fjord lowland, Ellesmere Island, Canada concurrent measurements of N₂-fixation, N mineralization and nitrification rates, dissolved organic soil N (DON) and C, inorganic soil N and surface greenhouse gas fluxes (CO₂, N₂O and CH₄) were taken in two ecosystem types (Wet Sedge Meadow and Dryas Heath) over the 2009 growing season (June-August). Using Structural Equation Modelling we evaluated the hypothesis that CO₂, CH₄ and N₂O flux are linked to N₂-fixation via the N cycle.

Results

The soil N cycle was linked to CO₂ flux in the Dryas Heath ecosystem via DON concentrations, but there was no link between the soil N cycle and CO₂ flux in the Wet Sedge Meadow. Methane flux was also not linked to the soil N cycle, nor surface soil temperature or moisture in either ecosystem. The soil N cycle was closely linked to N₂O emissions but via nitrification in the Wet Sedge Meadow and inorganic N in the Dryas Heath, indicating the important role of nitrification in net N₂O flux from arctic ecosystems.

Conclusions

Our results should be interpreted with caution given the high variability in both the rates of the N cycling processes and greenhouse gas flux found in both ecosystems over the growing season. However, while N₂-fixation and other N cycling processes may play a more limited role in instantaneous CO₂ emissions, these processes clearly play an important role in controlling N₂O emissions.     

Microscale heterogeneity of the soil nitrogen cycling microbial functional structure and potential metabolism

Soil aggregates, with complex spatial and nutritional heterogeneity, are clearly important for regulating microbial community ecology and biogeochemistry in soils. However, how the taxonomic composition and functional attributes of N-cycling-microbes within different soil particle-size fractions under a long-term fertilization treatment remains largely unknown. Here, we examined the composition and metabolic potential for urease activity, nitrification, N₂O production and reduction of the microbial communities attached to different sized soil particles (2000–250, 250–53 and <53 μm) using a functional gene microarray (GeoChip) and functional assays. We found that urease activity and nitrification were higher in <53 μm fractions, whereas N₂O production and reduction rates were greater in 2000–250 and 250–53 μm across different fertilizer regimes. The abundance of key N-cycling genes involved in anammox, ammonification, assimilatory and dissimilatory N reduction, denitrification, nitrification and N₂-fixation detected by GeoChip increased as soil aggregate size decreased; and the particular key genes abundance (e.g., ureC, amoA, narG, nirS/K) and their corresponding activity were uncoupled. Aggregate fraction exerted significant impacts on N-cycling microbial taxonomic composition, which was significantly shaped by soil nutrition. Taken together, these findings indicate the important roles of soil aggregates in differentiating N-cycling metabolic potential and taxonomic composition, and provide empirical evidence that nitrogen metabolism potential and community are uncoupled due to aggregate heterogeneity.     

Temperature effects on carbon and nitrogen metabolism in some Maritime Antarctic freshwater phototrophic communities

Biofilms growing on ice and benthic mats are among the most conspicuous biological communities in Antarctic landscapes and harbour a high diversity of organisms. These communities are consortia that make important contributions to carbon and nitrogen input in non-marine Antarctic ecosystems. Here, we study the effect of increasing temperatures on the carbon and nitrogen metabolism of two benthic communities on Byers Peninsula (Livingston Island, Maritime Antarctica): a biofilm dominated by green algae growing on seasonal ice, and a land-based microbial mat composed mainly of cyanobacteria. Inorganic carbon photoassimilation, urea and nitrate uptake and N₂-fixation (acetylene reduction activity) rates were determined in situ in parallel at five different temperatures (0, 5, 10, 15, 25°C) using thermostatic baths. The results for the cyanobacterial mat showed that photosynthesis and N₂-fixation responded positively to increased temperatures, but urea and NO₃⁻ uptake rates did not show a significant variation related to temperature. This microbial mat exhibits relatively low activity at 0°C whereas at higher temperatures (up to 15°C), N₂-fixation rate increased significantly. Similarly, the maximum photosynthetic activity increased in parallel with temperature and showed no saturation up to 25°C. In contrast, the ice biofilm displayed higher photosynthetic activity at 0°C than at the other temperatures assayed, and it showed elevated photoinhibition at warmer temperatures.     

N2 Fixation in Feather Mosses is a Sensitive Indicator of N Deposition in Boreal Forests

Nitrogen (N) fixation in the feather moss–cyanobacteria association represents a major N source in boreal forests which experience low levels of N deposition; however, little is known about the effects of anthropogenic N inputs on the rate of fixation of atmospheric N₂ in mosses and the succeeding effects on soil nutrient concentrations and microbial community composition. We collected soil samples and moss shoots of Pleurozium schreberi at six distances along busy and remote roads in northern Sweden to assess the influence of road-derived N inputs on N₂ fixation in moss, soil nutrient concentrations and microbial communities. Soil nutrients were similar between busy and remote roads; N₂ fixation was higher in mosses along the remote roads than along the busy roads and increased with increasing distance from busy roads up to rates of N₂ fixation similar to remote roads. Throughfall N was higher in sites adjacent to the busy roads but showed no distance effect. Soil microbial phospholipid fatty acid (PLFA) composition exhibited a weak pattern regarding road type. Concentrations of bacterial and total PLFAs decreased with increasing distance from busy roads, whereas fungal PLFAs showed no distance effect. Our results show that N₂ fixation in feather mosses is highly affected by N deposition, here derived from roads in northern Sweden. Moreover, as other measured factors showed only weak differences between the road types, atmospheric N₂ fixation in feather mosses represents a highly sensitive indicator for increased N loads to natural systems.     

Bryophyte-Cyanobacteria Associations during Primary Succession in Recently Deglaciated Areas of Tierra del Fuego (Chile)

Bryophyte establishment represents a positive feedback process that enhances soil development in newly exposed terrain. Further, biological nitrogen (N) fixation by cyanobacteria in association with mosses can be an important supply of N to terrestrial ecosystems, however the role of these associations during post-glacial primary succession is not yet fully understood. Here, we analyzed chronosequences in front of two receding glaciers with contrasting climatic conditions (wetter vs drier) at Cordillera Darwin (Tierra del Fuego) and found that most mosses had the capacity to support an epiphytic flora of cyanobacteria and exhibited high rates of N₂ fixation. Pioneer moss-cyanobacteria associations showed the highest N₂ fixation rates (4.60 and 4.96 µg N g⁻¹ bryo. d⁻¹) very early after glacier retreat (4 and 7 years) which may help accelerate soil development under wetter conditions. In drier climate, N₂ fixation on bryophyte-cyanobacteria associations was also high (0.94 and 1.42 µg N g⁻¹ bryo. d⁻¹) but peaked at intermediate-aged sites (26 and 66 years). N₂ fixation capacity on bryophytes was primarily driven by epiphytic cyanobacteria abundance rather than community composition. Most liverworts showed low colonization and N₂ fixation rates, and mosses did not exhibit consistent differences across life forms and habitat (saxicolous vs terricolous). We also found a clear relationship between cyanobacteria genera and the stages of ecological succession, but no relationship was found with host species identity. Glacier forelands in Tierra del Fuego show fast rates of soil transformation which imply large quantities of N inputs. Our results highlight the potential contribution of bryophyte-cyanobacteria associations to N accumulation during post-glacial primary succession and further describe the factors that drive N₂-fixation rates in post-glacial areas with very low N deposition.     

Increases of Soil C, N, and P Pools Along an Acacia Tree Density Gradient and Their Effects on Trees and Grasses

Nitrogen (N) fixing trees including many species of Acacia are an important though variable component of savanna ecosystems. It is known that these trees enrich the soil with carbon (C) and N, but their effect on the combined C:N:P stoichiometry in soil is less well understood. Theory suggests that they might reduce available phosphorus (P), creating a shift from more N-limited conditions in grass-dominated to more P-limited conditions in tree-dominated sites, which in turn could feed back negatively on the trees’ capacity to fix N. We studied the effects of Acacia zanzibarica tree density upon soil and foliar N:P stoichiometry, and the N₂-fixation rates of trees and leguminous herbs in a humid Tanzanian savanna. Foliar N:P ratios and N₂-fixation rates of trees remained constant across the density gradient, whereas soil C, N and organic P pools increased. In contrast, the N:P ratio of grasses increased and N₂-fixation rates of leguminous herbs decreased with increasing tree density, indicating a shift towards more P-limited conditions for the understory vegetation. These contrasting responses suggest that trees and grasses have access to different sources of N and P, with trees being able to access P from deeper soil layers and perhaps also utilizing organic forms more efficiently.     

Short-term Nitrogen Fixation by Legume Seedlings and Resprouts After Fire in Mediterranean Old-fields

Fires may greatly alter the N budget of a plant community. During fire nitrogen is lost to the atmosphere. Although high light availability after fire promotes N₂-fixation, the presumably high soil N availability could limit N₂-fixation activity. The latter limitation might be particularly acute in legume seedlings compared with resprouts, which have immediate access to belowground stored carbon. We wished to learn whether early post-fire conditions were conducive to N₂-fixation in leguminous seedlings and resprouts in two types of grassland and in a shrubland and whether seedlings and resprouts incurred different amounts of N₂-fixation after fire. We set 18 experimental fires in early autumn on 6 plots, subsequently labelling 6 subplots (2 × 2 m²) in each community with ¹⁵NH₄⁺-N (99 atom % excess). For 9 post-fire months we measured net N mineralisation in the top 5 cm of soil and we calculated the fraction of legume N derived from the atmosphere (%Ndfa) in seedlings and resprouts. We used two independent estimates of the amounts of N derived from non-atmospheric sources in potentially N₂-fixing plants: mean soil pool abundance and the ¹⁵N-enrichment of non-legumes. Despite substantial soil net N mineralisation in all burned community types (about 2.6 g Nm⁻² during the first nine months after fire), the %Ndfa of various legume species was 52–99%. Legumes from both grasslands showed slightly higher N₂-fixation values than shrubland legumes. As grassland legumes grew in more belowground dense communities than shrubland legumes, we suggest that higher competition for soil resources in well established grass-resprouting communities may enhance the rate of N₂-fixation after fire. In contrast to our hypothesis, legume seedlings and resprouts from the three plant communities studied, had similar %Ndfa and apparently acquired most of their N from the atmosphere rather than from the soil.     

N2-fixation by methanotrophs sustains carbon and nitrogen accumulation in pristine peatlands

Symbiotic relationships between N₂-fixing prokaryotes and their autotrophic hosts are essential in nitrogen (N)-limited ecosystems, yet the importance of this association in pristine boreal peatlands, which store 25 % of the world’s soil (C), has been overlooked. External inputs of N to bogs are predominantly atmospheric, and given that regions of boreal Canada anchor some of the lowest rates found globally (~1 kg N ha^?1 year^?1), biomass production is thought to be limited primarily by N. Despite historically low N deposition, we show that boreal bogs have accumulated approximately 12–25 times more N than can be explained by atmospheric inputs. Here we demonstrate high rates of biological N₂-fixation in prokaryotes associated with Sphagnum mosses that can fully account for the missing input of N needed to sustain high rates of C sequestration. Additionally, N amendment experiments in the field did not increase Sphagnum production, indicating that mosses are not limited by N. Lastly, by examining the composition and abundance of N₂-fixing prokaryotes by quantifying gene expression of 16S rRNA and nitrogenase-encoding nifH, we show that rates of N₂-fixation are driven by the substantial contribution from methanotrophs, and not from cyanobacteria. We conclude biological N₂-fixation drives high sequestration of C in pristine peatlands, and may play an important role in moderating fluxes of methane, one of the most important greenhouse gases produced in peatlands. Understanding the mechanistic controls on biological N₂-fixation is crucial for assessing the fate of peatland carbon stocks under scenarios of climate change and enhanced anthropogenic N deposition.     

Experimental warming alters the community composition,diversity, and N2 fixation activity of peat moss (Sphagnum fallax) microbiomes

Sphagnum‐dominated peatlands comprise a globally important pool of soil carbon (C) and are vulnerable to climate change. While peat mosses of the genus Sphagnum are known to harbor diverse microbial communities that mediate C and nitrogen (N) cycling in peatlands, the effects of climate change on Sphagnum microbiome composition and functioning are largely unknown. We investigated the impacts of experimental whole‐ecosystem warming on the Sphagnum moss microbiome, focusing on N₂ fixing microorganisms (diazotrophs). To characterize the microbiome response to warming, we performed next‐generation sequencing of small subunit (SSU) rRNA and nitrogenase (nifH) gene amplicons and quantified rates of N₂ fixation activity in Sphagnum fallax individuals sampled from experimental enclosures over 2 years in a northern Minnesota, USA bog. The taxonomic diversity of overall microbial communities and diazotroph communities, as well as N₂ fixation rates, decreased with warming (p < 0.05). Following warming, diazotrophs shifted from a mixed community of Nostocales (Cyanobacteria) and Rhizobiales (Alphaproteobacteria) to predominance of Nostocales. Microbiome community composition differed between years, with some diazotroph populations persisting while others declined in relative abundance in warmed plots in the second year. Our results demonstrate that warming substantially alters the community composition, diversity, and N₂ fixation activity of peat moss microbiomes, which may ultimately impact host fitness, ecosystem productivity, and C storage potential in peatlands.     

The impact of simulated chronic nitrogen deposition on the biomass and N2-fixation activity of two boreal feather moss–cyanobacteria associations

Bryophytes achieve substantial biomass and play several key functional roles in boreal forests that can influence how carbon (C) and nitrogen (N) cycling respond to atmospheric deposition of reactive nitrogen (N_r). They associate with cyanobacteria that fix atmospheric N₂, and downregulation of this process may offset anthropogenic N_r inputs to boreal systems. Bryophytes also promote soil C accumulation by thermally insulating soils, and changes in their biomass influence soil C dynamics. Using a unique large-scale (0.1 ha forested plots), long-term experiment (16 years) in northern Sweden where we simulated anthropogenic N_r deposition, we measured the biomass and N₂-fixation response of two bryophyte species, the feather mosses Hylocomium splendens and Pleurozium schreberi. Our data show that the biomass declined for both species; however, N₂-fixation rates per unit mass and per unit area declined only for H. splendens. The low and high treatments resulted in a 29% and 54% reduction in total feather moss biomass, and a 58% and 97% reduction in total N₂-fixation rate per unit area, respectively. These results help to quantify the sensitivity of feather moss biomass and N₂ fixation to chronic N_r deposition, which is relevant for modelling ecosystem C and N balances in boreal ecosystems.     

Making a living while starving in the dark: metagenomic insights into the energy dynamics of a carbonate cave

Carbonate caves represent subterranean ecosystems that are largely devoid of phototrophic primary production. In semiarid and arid regions, allochthonous organic carbon inputs entering caves with vadose-zone drip water are minimal, creating highly oligotrophic conditions; however, past research indicates that carbonate speleothem surfaces in these caves support diverse, predominantly heterotrophic prokaryotic communities. The current study applied a metagenomic approach to elucidate the community structure and potential energy dynamics of microbial communities, colonizing speleothem surfaces in Kartchner Caverns, a carbonate cave in semiarid, southeastern Arizona, USA. Manual inspection of a speleothem metagenome revealed a community genetically adapted to low-nutrient conditions with indications that a nitrogen-based primary production strategy is probable, including contributions from both Archaea and Bacteria. Genes for all six known CO₂-fixation pathways were detected in the metagenome and RuBisCo genes representative of the Calvin–Benson–Bassham cycle were over-represented in Kartchner speleothem metagenomes relative to bulk soil, rhizosphere soil and deep-ocean communities. Intriguingly, quantitative PCR found Archaea to be significantly more abundant in the cave communities than in soils above the cave. MEtaGenome ANalyzer (MEGAN) analysis of speleothem metagenome sequence reads found Thaumarchaeota to be the third most abundant phylum in the community, and identified taxonomic associations to this phylum for indicator genes representative of multiple CO₂-fixation pathways. The results revealed that this oligotrophic subterranean environment supports a unique chemoautotrophic microbial community with potentially novel nutrient cycling strategies. These strategies may provide key insights into other ecosystems dominated by oligotrophy, including aphotic subsurface soils or aquifers and photic systems such as arid deserts.     

N2-fixation can sustain wastewater treatment performance of photogranules under nitrogen-limiting conditions

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen-limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen-replete (N+) and limiting (N−) conditions was assessed in sequencing batch reactors. Photogranules under N− condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and chemical oxygen demand removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N− photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N₂-fixation. These were higher at N− (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N₂-fixation.     

Culturable bacteria community development in postglacial soils of Ecology Glacier, King George Island, Antarctica

Glacier forelands are excellent sites in which to study microbial succession because conditions change rapidly in the emerging soil. Development of the bacterial community was studied along two transects on lateral moraines of Ecology Glacier, King George Island, by culture-dependent and culture-independent approaches (denaturating gradient gel electrophoresis). Environmental conditions such as cryoturbation and soil composition affected both abundance and phylogenetic diversity of bacterial communities. Microbiocenosis structure along transect 1 (severe cryoturbation) differed markedly from that along transect 2 (minor cryoturbation). Soil physical and chemical factors changed along the chronosequence (time since exposure) and influenced the taxonomic diversity of cultivated bacteria, particularly along transect 2. Arthrobacter spp. played a pioneer role and were present in all soil samples, but were most abundant along transect 1. Cultivated bacteria isolated from transect 2 were taxonomically more diverse than those cultivated from transect 1; those from transect 1 tended to express a broader range of enzyme and assimilation activities. Our data suggest that cryoturbation is a major factor in controlling bacterial community development in postglacial soils, shed light on microbial succession in glacier forelands, and add a new parameter to models that describe succession phenomena.     

N and P Cycling in Tanzanian Humid Savanna: Influence of Herbivores, Fire, and N2-Fixation

Availabilities of nitrogen (N) and phosphorus (P) have a strong influence on plant growth and the species composition of savannas, but it is not clear how these availabilities depend on factors such as fire, N₂-fixation, and activities of wild herbivores and cattle. We quantified soil N and P availabilities in various ways (extractable pools, mineralization, resin adsorption) along vegetation gradients within a recently abandoned cattle ranch and a former game reserve in Tanzania (both areas now part of the Saadani National Park). We also assessed annual N and P balances to evaluate how long-term availabilities of N and P are affected by large herbivores, symbiotic N₂-fixation, and fire. The results show that cattle ranching led to a spatial re-distribution of nutrients, with the local accumulation of P being stronger and more persistent than that of N. In the former game reserve, intensively grazed patches of short grass tended to have elevated soil N and P availabilities; however, because quantities of nutrients removed through grazing exceeded returns in dung and urine, the nutrient balances of these patches were negative. In dense Acacia stands, N₂-fixation increased N availability and caused a net annual N input. Fire was the major cause for nutrient losses from tallgrass savanna, and estimated N inputs from the atmosphere and symbiotic N₂-fixation were insufficient to compensate for these losses. Our results call into question the common assumption that N budgets in annually burned savanna are balanced; rather, these ecosystems are a mosaic of patches with both N enrichment and impoverishment, which vary according to the vegetation type.     

Spatial and temporal variation in moss-associated dinitrogen fixation in coniferous- and deciduous-dominated Alaskan boreal forests

Dominant canopy tree species have strong effects on the composition and function of understory species, particularly bryophytes. In boreal forests, bryophytes and their associated microbes are a primary source of ecosystem nitrogen (N) inputs, and an important process regulating ecosystem productivity. We investigated how feather moss-associated N₂-fixation rates and contribution to N budgets vary in time and space among coniferous and broadleaf deciduous forests. We measured N₂-fixation rates using stable isotope (¹⁵N₂) labeling in two moss species (Pleurozium schreberi and Hylocomium splendens) in broadleaf deciduous (Alaska paper birch—Betula neoalaskana) and coniferous (black spruce—Picea mariana) stands near Fairbanks, interior Alaska, from 2013 to 2015. N₂-fixation rates showed substantial inter-annual variation among the 3 years. High N₂-fixation was more strongly associated with high precipitation than air temperature or light availability. Overall, contribution of N₂-fixation to N budgets was greater in spruce than in birch stands. Our results enhance the knowledge of the processes that drive N₂-fixation in boreal forests, which is important for predicting ecosystem consequences of changing forest composition.     

Habitat Specialization Along a Wetland Moisture Gradient Differs Between Ammonia-oxidizing and Denitrifying Microorganisms

Gradients in abiotic parameters, such as soil moisture, can strongly influence microbial community structure and function. Denitrifying and ammonia-oxidizing microorganisms, in particular, have contrasting physiological responses to abiotic factors such as oxygen concentration and soil moisture. Identifying abiotic factors that govern the composition and activity of denitrifying and ammonia-oxidizing communities is critical for understanding the nitrogen cycle. The objectives of this study were to (i) examine denitrifier and archaeal ammonia oxidizer community composition and (ii) assess the taxa occurring within each functional group related to soil conditions along an environmental gradient. Soil was sampled across four transects at four locations along a dry to saturated environmental gradient at a restored wetland. Soil pH and soil organic matter content increased from dry to saturated plots. Composition of soil denitrifier and ammonia oxidizer functional groups was assessed by terminal restriction fragment length polymorphism (T-RFLP) community analysis, and local soil factors were also characterized. Microbial community composition of denitrifiers and ammonia oxidizers differed along the moisture gradient (denitrifier: ANOSIM R?=?0.739, P?<?0.001; ammonia oxidizers: ANOSIM R?=?0.760, P?<?0.001). Individual denitrifier taxa were observed over a larger range of moisture levels than individual archaeal ammonia oxidizer taxa (Wilcoxon rank sum, W?=?2413, P value?=?0.0002). Together, our data suggest that variation in environmental tolerance of microbial taxa have potential to influence nitrogen cycling in terrestrial ecosystems.     

Nitrogen fertilisation reduces grass-induced N2 fixation of tree seedlings from semi-arid savannas

Aims

Coexistence of trees and grasses in nutrient-poor arid savannas may result in competition for soil N. While grasses may be more effective than woody plants in acquiring N from the soil, some leguminous woody species rely on N₂ fixation. We assessed the role of N₂ fixation in the N-budget of Acacia mellifera seedlings by varying N supply and grass competition.

Methods

The contribution of N₂ fixation to the N-budget of Acacia mellifera seedlings with varying N supply and grass competition was determined by measuring growth, nutrient concentrations, and ¹⁵N values.

Results

Tree seedlings were 4-fold taller and had 20-fold more biomass in the absence of grass. Tree foliar δ¹⁵N was lower with (?0.25?±?0.2‰, n?=?9) than without grasses (5.2?±?0.1‰, n?=?64). The contribution of N₂-fixation to the N budget decreased with increasing N supply. Greater reliance on N₂-fixation by trees in the presence of grasses did not result in greater biomass accumulation or tissue [N] relative to tree seedlings grown without grass competition. Tree seedlings competing with grass had significantly more negative δ¹³C (?29.5?±?0.6‰) than seedlings without grass competition (?28.8‰?±?0.5‰).

Conclusions

Induction of N₂-fixation by grass may have resulted from competition for nutrients. N₂-fixation enables tree seedlings to compensate for limited soil N and survive grass competition at a critical and vulnerable developmental stage of germination and establishment.