Biological nitrogen transformations within the tree canopy: seasonal variations and microbial contributions to nitrogen fluxes in a Mediterranean Holm oak forest

Human activities have increased atmospheric reactive nitrogen (N) deposition, with important consequences for ecosystem biogeochemical cycles. In forest ecosystems, tree canopies act as active filters that intercept, transform and redistribute atmospheric N before it reaches to the soil. These canopy-level processes determine the chemical forms of N that become available for biological uptake or are transferred to the soil. Despite their importance, the temporal variability of these processes remains poorly understood, particularly in Mediterranean ecosystems with pronounced seasonal contrasts.

In this study, we investigated the seasonal dynamics of atmospheric N transformations within the canopy of a Mediterranean Holm oak forest (Quercus ilex / Q. rotundifolia) in Spain. We focused on canopy-scale nitrification processes and the abundance of N-fixing and nitrifying microorganisms associated with the phyllosphere and precipitation. Canopy N fluxes were quantified, nitrate sources were identified using Δ¹⁷O isotopic signatures, and microbial abundances were quantified by qPCR, to explore seasonal dynamics and their environmental drivers.

Our results show that the canopy acted as a net sink for atmospheric N throughout the year, indicating that N inputs did not exceed ecosystem demand and suggesting potential N limitation. The nitrate measured in throughfall samples indicated a predominantly atmospheric origin during most of the year (76–92%). In contrast, during summer up to 76% of the nitrate was derived from in situ biological processes at canopy level. These enhanced biological transformations were correlated with weather conditions, particularly the higher temperatures and dry conditions typical of summer, which may favour nitrification and promote the accumulation of N compounds on leaf surfaces. Reduced plant activity and lower N uptake during summer further prolong N residence time within the canopy, increasing the likelihood of microbial transformations. Both archaeal and bacterial nitrifiers, as well as N-fixing microorganisms, were detected year-round in the phyllosphere and precipitation. Archaeal nitrifiers consistently outnumbered bacterial ones, and showed a marked increase during summer, driven by higher radiation, temperature and lower humidity. This pattern suggests that archaea may play a significant important role in nitrification, coinciding with the highest nitrification rates observed in summer. These findings highlight the crucial function of canopy processes in regulating N fluxes in Mediterranean forests, particularly during summer. The seasonal dynamics of biological transformations and microbial communities emphasise the influence of environmental conditions on N cycling.