Tree species identity, mycorrhizal associations, and land-use legacy shape soil protist diversity and community composition through litter quality and soil properties

Soil protists are phylogenetically and functionally diverse microscopic eukaryotes that play key roles in nutrient cycling, microbial filtering, and soil food-web stability. However, the drivers shaping their diversity and community composition remain poorly understood. Using a 50-year-old common-garden experiment in Denmark with six European tree species representing arbuscular mycorrhizal (AM)(e.g. ash and maple) and ectomycorrhizal (ECM)(e.g. lime, beech, oak, and Norway spruce) associations, we combined high-throughput 18S rRNA gene amplicon sequencing of DNA with detailed analyses of soil physicochemical properties and litter quality to assess how tree species, mycorrhizal association, and land-use legacy shape soil protist communities. Protist alpha diversity varied significantly among tree species, driven mainly by litter quality rather than soil properties. AM-associated ash supported higher richness than ECM-associated spruce and former forest soils hosted greater diversity than former croplands. Beta diversity was influenced by tree species and soil chemistry; AM species were linked to higher pH and low C:N ratios, whereas ECM species were associated with greater organic carbon and higher C:N ratio. Apicomplexa dominated across all soils, likely reflecting their high dispersal potential and ecological resilience in terrestrial environments. Different protist groups responded to litter and soil properties: Stramenopiles thrived in alkaline, nutrient-rich soils; Cercozoa and Ciliophora in nutrient-poor, high C:N soils; whereas Amoebozoa showed no clear correlations. This long-term experiment demonstrated that tree species identity and traits; mediated through litter quality, soil management, and soil properties; are key determinants of soil protist diversity and community structure.