Soil diazotrophs sustain nitrogen fixation under high nitrogen enrichment via adjustment of community composition.

Biological nitrogen (N) fixation, an important pathway of N, inputs from the atmosphere to Earth's ecosystems, is well demonstrated to decline under N input. However, it remains unclear why N fixers sustain N fixation in many forests under high atmospheric N deposition. To address this knowledge gap, we analyzed the response of the diazotroph community to low N loads (short-term and low N addition; 3-year N addition at the rates of 25-50 kg N ha-1 year-1) vs high loads (chronic and high N addition; 9-year N addition at the rate of 150 kg N ha-1 year-1) in forest soils using high-throughput sequencing. Rates of N fixation decreased under low and high N loads (by 13%-27% and 10%-12%, respectively). Richness and alpha diversity (ACE and Chao1) of the soil diazotroph community decreased under low but not high N loads. Approximately 67.1%-74.4% of the nifH gene sequences at the OTU level overlapped between the control and low N loads, but only 52.0%-53.6% of those overlapped between the control and high N loads, indicating a larger shift of diazotroph community composition under high N loads. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ concentrations under high N loads did not have negative impacts on the structure and function of the diazotroph community. These findings indicate that diazotrophs sustain N fixation under high N deposition via adjustment of their community composition in forest soils. IMPORTANCE: This study examined the changes in soil diazotroph community under different loads of simulated N deposition and analyzed its relationship with N fixation rates in in five forests using high-throughput sequencing. The magnitudes of N fixation rates reduced by low N loads were higher than those by high N loads. Low N loads decreased richness and diversity of diazotroph community, whereas diazotroph community structure remained stable under high N loads. Compared with low N loads, high N loads resulted in a less similarity and overlap of nifH gene sequences among the treatments and a larger adjustment of diazotroph community. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ under high N loads did not have negative impacts on diazotroph community structure and N fixation. Based on these findings, it is urgently needed to incorporate the loads of N deposition and the composition of diazotroph community into terrestrial N-cycling models for accurate understanding of N inputs in forest ecosystems.

Long-term nitrogen and phosphorus addition have stronger negative effects on microbial residual carbon in subsoils than topsoils in subtropical forests.

Highly weathered lowland (sub)tropical forests are widely recognized as nitrogen (N)-rich and phosphorus (P)-poor, and the input of N and P affects soil carbon (C) cycling and storage in these ecosystems. Microbial residual C (MRC) plays a crucial role in regulating soil organic C (SOC) stability in forest soils. However, the effects of long-term N and P addition on soil MRC across different soil layers remain unclear. This study conducted a 12-year N and P addition experiment in two typical subtropical plantation forests dominated by Acacia auriculiformis and Eucalyptus urophylla trees, respectively. We measured plant C input (fine root biomass, fine root C, and litter C), microbial community structure, enzyme activity (C/N/P-cycling enzymes), mineral properties, and MRC. Our results showed that continuous P addition reduced MRC in the subsoil (20-40 cm) of both plantations (A. auriculiformis: 28.44% and E. urophylla: 28.29%), whereas no significant changes occurred in the topsoil (0-20 cm). N addition decreased MRC in the subsoil of E. urophylla (25.44%), but had no significant effects on A. auriculiformis. Combined N and P addition reduced MRC (34.63%) in the subsoil of A. auriculiformis but not in that of E. urophylla. The factors regulating MRC varied across soil layers. In the topsoil (0-10 cm), plant C input (the relative contributions to the total variance was 20%, hereafter) and mineral protection (47.2%) were dominant factors. In the soil layer of 10-20 cm, both microbial characteristics (41.3%) and mineral protection (32.3%) had substantial effects, whereas the deeper layer (20-40 cm) was predominantly regulated by microbial characteristics (37.9%) and mineral protection (18.8%). Understanding differential drivers of MRC across soil depth, particularly in deeper soil layers, is crucial for accurately predicting the stability and storage of SOC and its responses to chronic N enrichment and/or increased P limitation in (sub)tropical forests.

Nitrogen addition accelerates litter decomposition and arsenic release of Pteris vittata in arsenic-contaminated soil from mine.

The arsenic (As) release from litter decomposition of As-hyperaccumulator (Pteris vittata L.) in mine areas poses an ecological risk for metal dispersion into the soil. However, the effect of atmospheric nitrogen (N) deposition on the litter decomposition of As-hyperaccumulator in the tailing mine area remains poorly understood. In this study, we conducted a microcosm experiment to investigate the As release during the decomposition of P. vittata litter under four gradients of N addition (0, 5, 10, and 20 mg N g-1). The N10 treatment (10 mg N g-1) enhanced As release from P. vittata litter by 1.2-2.6 folds compared to control. Furthermore, Streptomyces, Pantoea, and Curtobacterium were found to primarily affect the As release during the litter decomposition process. Additionally, N addition decreased the soil pH, subsequently increased the microbial biomass, as well as hydrolase activities (NAG) which regulated N release. Thereby, N addition increased the As release from P. vittata litter and then transferred to the soil. Moreover, this process caused a transformation of non-labile As fractions into labile forms, resulting in an increase of available As concentration by 13.02-20.16% within the soil after a 90-day incubation period. Our findings provide valuable insights into assessing the ecological risk associated with As release from the decomposition of P. vittata litter towards the soil, particularly under elevated atmospheric N deposition.

Negative responses of terrestrial nitrogen fixation to nitrogen addition weaken across increased soil organic carbon levels.

The traditional view holds that biological nitrogen (N) fixation is energetically expensive and thus, facultative N fixers reduce N fixation rates while obligate N fixers are excluded by non-N fixers as soil N becomes rich. This view, however, contradicts the phenomenon that N fixation does not decline in many terrestrial ecosystems under N enrichment. To address this paradoxical phenomenon, we conducted a meta-analysis of N fixation and diazotroph (N-fixing microorganism) community structure in response to N addition across terrestrial ecosystems. N addition inhibited N fixation, but the inhibitory effect weakened across increased soil organic carbon (SOC) concentrations. The response ratios of N fixation (including free-living, plant-associated, and symbiotic types) to N addition were lower in the ecosystems with low SOC concentrations (<10 mg/g) than in those with medium or high SOC concentrations (10-20 and > 20 mg/g, respectively). The negative N-addition effects on diazotroph abundance and diversity also weakened across increased SOC levels. Among the climatic and soil factors, SOC was the most important predictor regarding the responses of N fixation and diazotroph community structure to N addition. Overall, our study reveals the role of SOC in affecting the responses of N fixation to N addition, which helps understand the relationships of biological N fixation and N enrichment as well as the mechanisms of terrestrial C and N coupling.

Unexpected high retention of 15 N-labeled nitrogen in a tropical legume forest under long-term nitrogen enrichment.

The responses of forests to nitrogen (N) deposition largely depend on the fates of deposited N within the ecosystem. Nitrogen-fixing legume trees widely occur in terrestrial forests, but the fates of deposited N in legume-dominated forests remain unclear, which limit a global evaluation of N deposition impacts and feedbacks on carbon sequestration. Here, we performed the first ecosystem-scale 15 N labeling experiment in a typical legume-dominated forest as well as in a nearby non-legume forest to determine the fates of N deposition between two different forest types and to explore their underlying mechanisms. The 15 N was sprayed bimonthly for 1 year to the forest floor in control and N addition (50 kg N ha-1  year-1 for 10 years) plots in both forests. We unexpectedly found a strong capacity of the legume forest to retain deposited N, with 75 ± 5% labeled N recovered in plants and soils, which was higher than that in the non-legume forest (56 ± 4%). The higher 15 N recovery in legume forest was mainly driven by uptake by the legume trees, in which 15 N recovery was approximately 15% more than that in the nearby non-legume trees. This indicates higher N-demand by the legume than non-legume trees. Mineral soil was the major sink for deposited N, with 39 ± 4% and 34 ± 3% labeled N retained in the legume and non-legume forests, respectively. Moreover, N addition did not significantly change the 15 N recovery patterns of both forests. Overall, these findings indicate that legume-dominated forests act as a strong sink for deposited N regardless of high soil N availability under long-term atmospheric N deposition, which suggest a necessity to incorporate legume-dominated forests into N-cycling models of Earth systems to improve the understanding and prediction of terrestrial N budgets and the global N deposition effects.

Nitrogen addition promotes soil microbial beta diversity and the stochastic assembly.

Nitrogen (N) deposition is one of major environmental concerns and alters the microbial communities in the pedosphere. A central debate in governing microbial community is on the relative importance of deterministic (ecological selection) vs. stochastic processes (dispersal, drift, diversification or speciation), which consequently limited our understanding of microbial assembly in response to N addition. Here, we conducted a global analysis of high-throughput sequencing data to reveal the mechanisms of N-addition effects on soil microbial communities. The results show that N addition significantly shifted the microbial community structure and promoted microbial beta diversity, particularly in the N-limited ecosystems. Changes in microbial structure and beta diversity increased significantly as the N addition rate, study duration, and the degree of soil acidification increased. The stochastic processes are more important than the deterministic processes for microbial community assembly, while N addition significantly increase the importance of stochastic processes whether the phylogenetic relationship is considered or not. Overall, the current study highlights the important of ecological stochasticity in regulating microbial assembly under N deposition scenarios.

Long-term effects of 7-year warming experiment in the field on leaf hydraulic and economic traits of subtropical tree species.

Rising temperature associated with climate change may have substantial impacts on forest tree functions. We conducted a 7-year warming experiment in subtropical China by translocating important native forest tree species (Machilas breviflora, Syzygium rehderianum, Schima superba and Itea chinensis) from cooler high-elevation sites (600 m) to 1-2°C warmer low-elevation sites (300 and 30 m) to investigate warming effects on leaf hydraulic and economic traits. Here, we report data from the last 3 years (Years 5-7) of the experiment. Warming increased leaf hydraulic conductance of S. superba to meet the higher evaporative demand. M. breviflora (300 m), S. rehderianum, S. superba and I. chinensis (300 and 30 m) exhibited higher area-based and mass-based maximum photosynthetic rates (Aa and Am , respectively) related to increasing stomatal conductance (gs ) and stomatal density in the wet season, which led to rapid growth; however, we observed decreased growth of M. breviflora at 30 m due to lower stomatal density and decreased Aa in the wet season. Warming increased photosynthetic nitrogen-use efficiency and photosynthetic phosphorus-use efficiency, but reduced leaf dry mass per unit area due to lower leaf thickness, suggesting that these tree species allocated more resources into upregulating photosynthesis rather than into structural investment. Our findings highlight that there was trait variation in the capacity of trees to acclimate to warmer temperatures such that I. chinensis may benefit from warming, but S. superba may be negatively influenced by warming in future climates.

Effects of human disturbance activities and environmental change factors on terrestrial nitrogen fixation.

Biological nitrogen (N) fixation plays an important role in terrestrial N cycling and represents a key driver of terrestrial net primary productivity (NPP). Despite the importance of N fixation in terrestrial ecosystems, our knowledge regarding the controls on terrestrial N fixation remains poor. Here, we conducted a meta-analysis (based on 852 observations from 158 studies) of N fixation across three types of ecosystems with different status of disturbance (no management, restoration [previously disturbed], and disturbance [currently disturbed]) and in response to multiple environmental change factors (warming, elevated carbon dioxide [CO2 ], increased precipitation, increased drought, increased N deposition, and their combinations). We explored the mechanisms underlying the changes in N fixation by examining the variations in soil physicochemical properties (bulk density, texture, moisture, and pH), plant and microbial characteristics (dominant plant species numbers, plant coverage, and soil microbial biomass), and soil resources (total carbon, total N, total phosphorus (P), inorganic N, and inorganic P). Human disturbance inhibited non-symbiotic N fixation but not symbiotic N fixation. Terrestrial N fixation was stimulated by warming (+152.7%), elevated CO2 (+19.6%), and increased precipitation (+73.1%) but inhibited by increased drought (-30.4%), N deposition (-31.0%), and combinations of available multiple environmental change factors (-14.5%), the extents of which varied among biomes and ecosystem compartments. Human disturbance reduced the N fixation responses to environmental change factors, which was associated with the changes in soil physicochemical properties (2%-56%, p < .001) and the declines in plant and microbial characteristics (3%-49%, p ≤ .003) and soil resources (6%-48%, p ≤ .03). Overall, our findings reveal for the first time the effects of multiple environmental change factors on terrestrial N fixation and indicate the role of human disturbance activities in inhibiting N fixation, which can improve our understanding, modeling, and prediction of terrestrial N budgets, NPP, and ecosystem feedbacks under global change scenarios.

Substrate stoichiometry determines nitrogen fixation throughout succession in southern Chinese forests.

The traditional view holds that biological nitrogen (N) fixation often peaks in early- or mid-successional ecosystems and declines throughout succession based on the hypothesis that soil N richness and/or phosphorus (P) depletion become disadvantageous to N fixers. This view, however, fails to support the observation that N fixers can remain active in many old-growth forests despite the presence of N-rich and/or P-limiting soils. Here, we found unexpected increases in N fixation rates in the soil, forest floor, and moss throughout three successional forests and along six age-gradient forests in southern China. We further found that the variation in N fixation was controlled by substrate carbon(C) : N and C : (N : P) stoichiometry rather than by substrate N or P. Our findings highlight the utility of ecological stoichiometry in illuminating the mechanisms that couple forest succession and N cycling.

Effects of urbanization on plant phosphorus availability in broadleaf and needleleaf subtropical forests.

Urbanization, the migration of populations from rural to urban areas, has been causing great stress on natural environments, leading to air pollution and nitrogen (N) deposition, negatively affecting forest health. Although there is evidence that urbanization has changed forest N cycling, little is known about whether urbanization also changes the availability of phosphorus (P), which is important for plant growth and forest productivity. To address this question, we carried out a survey in the Pearl River Delta region, the world's largest urban area in southern China, using two types of representative forests, the evergreen broadleaf forests (BFs) and pine plantations (PPs). The leaf N:P ratios in the two forest types were high (20-50) with a significant increasing pattern along the rural-to-urban gradient. The ratios of leaf P:K and P:Na declined along the rural-to-urban gradient, whereas leaf P content did not change in BF but decreased in PP along the rural-to-urban gradient, suggesting that leaf P became limiting along urbanization. The abundance of actinomycetes and gram-negative bacteria decreased along the rural-to-urban gradient, indicating the negative effects of urbanization on soil microorganisms. Principal component analysis indicated that divergent key factors respond to the urbanization and affect plant P limitation in the two forest types. In BF, broadleaf trees showed a greater response to N deposition from urbanization indicating direct leaf N uptake from N deposition is a key factor for plant P limitation. Alternatively, in PP, our findings suggest soil acidification is an important factor accelerating plant P limitation. Our study revealed that urbanization intensifies plant P limitation in subtropical forests, and the effects vary depending on forest types. Our findings provide empirical information to support the management of forest ecosystems and evaluation of urbanization effects on forest health.

Global pattern and controls of biological nitrogen fixation under nutrient enrichment: A meta-analysis.

Biological nitrogen (N) fixation (BNF), an important source of N in terrestrial ecosystems, plays a critical role in terrestrial nutrient cycling and net primary productivity. Currently, large uncertainty exists regarding how nutrient availability regulates terrestrial BNF and the drivers responsible for this process. We conducted a global meta-analysis of terrestrial BNF in response to N, phosphorus (P), and micronutrient (Micro) addition across different biomes (i.e, tropical/subtropical forest, savanna, temperate forest, grassland, boreal forest, and tundra) and explored whether the BNF responses were affected by fertilization regimes (nutrient-addition rates, duration, and total load) and environmental factors (mean annual temperature [MAT], mean annual precipitation [MAP], and N deposition). The results showed that N addition inhibited terrestrial BNF (by 19.0% (95% confidence interval [CI]: 17.7%-20.3%); hereafter), Micro addition stimulated terrestrial BNF (30.4% [25.7%-35.3%]), and P addition had an inconsistent effect on terrestrial BNF, i.e., inhibiting free-living N fixation (7.5% [4.4%-10.6%]) and stimulating symbiotic N fixation (85.5% [25.8%-158.7%]). Furthermore, the response ratios (i.e., effect sizes) of BNF to nutrient addition were smaller in low-latitude (<30°) biomes (8.5%-36.9%) than in mid-/high-latitude (≥30°) biomes (32.9%-61.3%), and the sensitivity (defined as the absolute value of response ratios) of BNF to nutrients in mid-/high-latitude biomes decreased with decreasing latitude (p ≤ 0.009; linear/logarithmic regression models). Fertilization regimes did not affect this phenomenon (p > 0.05), but environmental factors did affect it (p < 0.001) because MAT, MAP, and N deposition accounted for 5%-14%, 10%-32%, and 7%-18% of the variance in the BNF response ratios in cold (MAT < 15°C), low-rainfall (MAP < 2,500 mm), and low-N-deposition (<7 kg ha-1  year-1 ) biomes, respectively. Overall, our meta-analysis depicts a global pattern of nutrient impacts on terrestrial BNF and indicates that certain types of global change (i.e., warming, elevated precipitation and N deposition) may reduce the sensitivity of BNF in response to nutrient enrichment in mid-/high-latitude biomes.

Stoichiometry controls asymbiotic nitrogen fixation and its response to nitrogen inputs in a nitrogen-saturated forest.

Lowland tropical forests with chronic nitrogen (N) deposition and/or abundant N-fixing organisms are commonly rich in N relative to other nutrients. The tropical N richness introduces a paradoxical relationship in which many tropical forests sustain high rates of asymbiotic N fixation despite the soil N richness and the higher energy cost of N fixation than of soil N uptake. However, the mechanism underlying this phenomenon remains unclear. Our study aims to test this phenomenon and examine potential mechanisms of nutrient concentrations vs. substrate stoichiometry in regulating N fixation using multiple linear regression models. We hypothesized that the rates of asymbiotic N fixation would be low in an N-rich forest under N deposition and substrate stoichiometry would explain the variation in N fixation better than nutrient concentrations. We conducted a chronic N-addition experiment in an N-saturated tropical forest in southern China and measured the N fixation rates, carbon (C), N, and phosphorus (P) concentrations, and stoichiometry in different substrates (soil, forest floor, mosses, and canopy leaves). Total N fixation rates were high (10.35-12.43 kg N·ha-1 ·yr-1 ) in this N-saturated forest because of the high substrate C:N and N:P stoichiometry (which explained 13-52% of the variation in N fixation, P < 0.037) rather than substrate nutrient concentrations (P > 0.05). Atmospheric N deposition (34-50 kg N·ha-1 ·yr-1 ) failed to down-regulate asymbiotic N fixation in this forest possibly because the N deposition rate was insufficient to inhibit N fixation or N deposition maintained high N fixation rates by increasing C sequestration in the substrates. Our N-addition experiment showed the insensitivity of N fixation in all the tested substrates to low N addition (50 kg N·ha-1 ·yr-1 ); however, medium and high N addition (100-150 kg N·ha-1 ·yr-1 ) stimulated the moss and foliar N fixation because of the increases in substrate C:N stoichiometry (which explained 30-34% of the variation in N fixation, P < 0.001). Overall, our results emphasize the importance of substrate (particularly mosses and foliage) stoichiometry as a driver of asymbiotic N fixation and sustained N richness in lowland tropical forests.

Different responses of asymbiotic nitrogen fixation to nitrogen addition between disturbed and rehabilitated subtropical forests.

Asymbiotic nitrogen (N) fixation is an important source of new N in ecosystems, and is sensitive to atmospheric N deposition. However, there is limited understanding of asymbiotic N fixation and its response to N deposition in the context of forest rehabilitation. In this study, we measured N fixation rates (acetylene reduction) in different ecosystem compartments (i.e. soil, forest floor, moss Syrrhopodon armatus, and canopy leaves) in a disturbed and a rehabilitated subtropical forest in southern China, under 12years of N treatments: control, low N addition (50kgNha-1yr-1), and medium N addition (100kgNha-1yr-1). The rehabilitated forest had higher nutrient (e.g. N) availability than the disturbed forest. In control plots, N fixation rates in forest floor were higher in the rehabilitated forest than in the disturbed forest, but N fixation rates in other compartments (soil, S. armatus, and canopy leaves) were comparable between the forests. Nitrogen addition significantly suppressed N fixation in soil, forest floor, S. armatus, and canopy leaves in the disturbed forest, but had no significant effect on those compartments in the rehabilitated forest. The main reasons for the negative effects of N addition on N fixation in the disturbed forest were NH4+ inhibition (soil), the P and C limitation (forest floor), and the reduced N dependence on canopy N-fixers (S. armatus and canopy leaves). We conclude that asymbiotic N fixation does not decline with increasing N availability after rehabilitation in the study forests. The inhibitory effects of N addition on asymbiotic N fixation occurred in the disturbed forest but not in the rehabilitated forest, indicating that forest rehabilitation may change the response of ecosystem function (i.e. N fixation) to N deposition, which merits further study in other tropical and subtropical regions.