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.