Quantifying tree canopy nitrification across European forests

Fluxes and chemical composition of precipitation is substantially changed after passing through tree canopies, particularly in the case of atmospheric nitrogen compounds, with important implications on forest nitrogen cycling. The causes of these changes, however, have mostly focused on the passive role of foliar surfaces to scavenge pollutants from the atmosphere and to ion exchange processes, while biological processes involving microbes hidden in the phyllosphere have been less investigated. We combined triple oxygen isotopes approach and molecular analyses with the aim of quantifying canopy nitrification and identify microbes responsible for it, respectively. Ten sites included in the European ICP Forests monitoring network, chosen along climate and nitrogen deposition gradients, were selected to include the two most dominant tree species in Europe (Fagus sylvatica L. and Pinus sylvestris L.). Specifically, in this study we: 1) estimated the relative contribution of nitrate derived from biological canopy nitrification vs. atmospheric deposition by using δ¹⁸O and Δ¹⁷O of nitrate collected in water samples, i.e., in the open field (bulk deposition) and underneath tree canopies (throughfall); 2) quantified the functional genes related to nitrification for the two dominant tree species in European forests by using next-generation sequence analyses. Based on the isotope approach, we found that up to 80% of the nitrate reaching the soil via throughfall derived from biological transformations in the phyllosphere, equivalent to a flux of gross canopy nitrification of up to 5.76 kg N ha^-1 y^-1. The fraction of microbiologically derived nitrate increased with raising nitrogen deposition, thus suggesting that the process can be substrate limited. Molecular analyses confirmed the presence on foliar surfaces of bacterial and archaeal autotrophic ammonia oxidisers and bacterial autotrophic nitrite oxidisers across the investigate European forests. Our study demonstrates the potential of integrating stable isotopes with molecular analyses to advance our understanding on key processes underpinning forest nitrogen cycling, which should no longer exclude microbial processes occurring in the phyllosphere.