N₂-fixation is linked to the ability to encroach in African savanna trees

Encroachment is a globally ubiquitous phenomenon, characterised by increasing indigenous tree densities in savanna and grassland. Encroachment has been attributed to rising atmospheric CO2 concentrations fertilising tree growth and shifting the competitive balance between trees and grasses. However, only a subset of savanna tree species are currently described as encroachers, raising the hypothesis that CO2 responsiveness differs among species. Within southern African savannas, encroachment is driven primarily by nitrogen (N2)-fixing species, implying the CO2 response may be mediated via traits that enhance plant-available N. Using an open-topped chamber system, we experimentally manipulated atmospheric CO2 concentrations and soil moisture for 12 savanna tree species (six encroachers and six non-encroachers) under ambient (a)CO2 (~397.9 ppm) or elevated (e)CO2 (~545.1 ppm) treatments and water limited or well-watered soil moisture treatments. We measured N-dynamics traits including nodule mass fraction (NMF), leaf δ15N, stem δ15N, and percentage of N derived from fixation (%Ndfa). We found that encroachers and non-encroachers differ in short-term N-dynamics but share similar long-term N allocation strategies. Encroachers exhibited lower leaf δ15N, indicating greater utilisation of N2 fixation products to meet immediate short-term protein synthesis. Long-term N allocation strategies (NMF, stem δ15N, and %Ndfa) were similar between encroachers and non-encroachers, with plants fixing more N2 under the eCO2 and well-watered treatment. In encroachers, leaf and stem δ¹⁵N were unrelated, in contrast to the positive relationship in non-encroachers, pointing to distinct tissue-level N allocation, possibly reflecting differential N utilisation and retention. We demonstrate the significance of N2 fixation in mediating the CO2 responsiveness of encroaching savanna trees. N2 fixation increases plant-available N, likely enabling encroachers to meet immediate N demands even under water limitation and increasing CO2. The potential feedback loop, where eCO2 enhances photosynthesis, facilitating greater C allocation for N2 fixation, helps to explain the ecological success of the subset of species driving encroachment under increased atmospheric CO2.