Biological nitrogen fixation (BNF) is a critical source of new N for terrestrial ecosystems, yet how BNF is regulated and organized over time remains unclear. Here, we investigated four taxonomically distinct N-fixers in longleaf pine savannas and determined their contribution to ecosystem BNF and their response to fire and N availability over stand development. We examined BNF in 59 1-ha plots across gradients of age (2 – 227 years) and fire frequency at two sites (Fort Benning and Eglin Air Force Base) in the southeastern US. We hypothesized that the four groups of fixers would fix at different rates, but that total BNF would decline with increasing N availability and stand age. We quantified BNF by herbaceous legumes, biological soil crusts, soil heterotrophic bacteria, and foliar endophytes of longleaf pine. We estimated the balance of available N (i.e. surplus or deficiency) in each plot as the difference between N supply (mineralization and deposition) and N demand from biomass growth. We fit linear regressions to evaluate the effects of stand age, site, time since fire and N availability on BNF for all four groups over ecosystem development, and performed separate analyses on mature stands to determine how fire frequency affects BNF.
Longleaf pine ecosystems house a diversity of N-fixers, and their niches are maintained even when stands reach maturity. However, the four groups of N-fixers differed in their contribution to BNF over time and how they responded to fire. Legumes fixed two orders of magnitude more N than the other groups, and rates remained constant over stand age. This finding challenges the expectation that BNF peaks in early stages of development in temperate terrestrial ecosystems. Soil crusts fixed the most N in younger stands, but foliar endophytes fixed the most N in older stands, which had the most foliar biomass. Fire frequency reduced BNF by heterotrophic bacteria, which was driven by organic matter abundance. In contrast, legume BNF increased with fire frequency and decreased with months since fire, suggesting that fire promotes legume N-fixation over annual and decadal time scales. Soil N supply did not affect BNF in any group, suggesting that obligate fixation strategies prevail in longleaf pine savannas. In total, site explained the most variation in BNF rates, where Fort Benning supported more BNF than Eglin AFB, which raises questions about landscape-level drivers (i.e., climate, parent material) of BNF in these ecosystems.