Effects of a 12-Year Nitrogen Addition Experiment on Protist Communities in a Boreal Forest, Heilongjiang Province, China

Escalating nitrogen (N) deposition has profound effects on terrestrial ecosystems; however, the mechanisms underlying soil protist community responses in cold-temperate regions remain poorly understood. This study investigated the effects of long-term N addition on soil protist communities at Nanwenghe Reserve in the Greater Khingan Mountains, where a 12-year continuous N addition experiment was initiated in 2011 and has been maintained annually thereafter. Four N addition levels were applied: control (CK, 0), low N (LN, 25), medium N (MN, 50), and high N (HN, 75 kg N ha−1 yr−1). During the 2023 growing season, soil protist communities were characterized using 18S rRNA gene sequencing. Our results demonstrated that N addition significantly altered the protist community structure, with significant interactive effects between N addition and season. Phagotrophic and phototrophic protists exhibited significantly stronger responses to N addition than parasitic protists. Phototrophic communities showed the strongest compositional differentiation (NMDS, p = 0.002), whereas parasitic protists exhibited no significant response to N addition. PLS-PM analysis indicated that soil nutrient factors primarily exerted indirect effects on protist communities by altering microbial diversity and plant characteristics, whereas biotic factors, particularly fungal and plant diversity, showed stronger direct regulatory effects on protist community dynamics. These findings reveal how N addition influences soil protist communities through the soil–plant–microbial pathway, providing a scientific basis for assessing soil microbial food web stability in high-latitude forests.

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