The tropical Andes are a species-rich and nitrogen-limited system, susceptible to increased nitrogen (N) inputs from the atmosphere. However, our understanding of the impacts of increased N input on belowground systems, in particular on protists and their role in nutrient cycling, remains limited. We explored how increased N affects protists in tropical montane rainforests in Ecuador using high-throughput sequencing (HTS) of environmental DNA from two litter layers. In addition, we manipulated the amount of arbuscular mycorrhizal fungi (AMF) and mesofauna, both playing a significant role in N cycling and interacting in complex ways with protist communities. We found that N strongly affected protist community composition in both layers, while mesofauna reduction had a stronger effect on the lower layer. Changes in concentration of the AMF marker lipid had little effect on protists. In both layers, the addition of N increased phagotrophs and animal parasites and decreased plant parasites, while mixotrophs decreased in the upper layer but increased in the lower layer. In the upper layer with higher AMF concentration, mixotrophs decreased, while in the lower layer, photoautotrophs increased and plant parasites decreased. With reduced mesofauna, phagotrophs increased and animal parasites decreased in both layers, while plant parasites increased only in the upper layer. The findings indicate that to understand the intricate response of protist communities to environmental changes, it is critical to thoroughly analyze these communities across litter and soil layers, and to include HTS.
