Vertical stratification drives additive prokaryotic diversity in beech forest floors, while site conditions shape boundary layers

The forest floor is a key interface regulating carbon and nutrient processing and transfer to the mineral soil, yet it is threatened by climate-change-driven reductions in organic layer mass. How its prokaryotic microbiome is structured across the fine-scale vertical gradient from fresh litter to mineral topsoil remains poorly resolved. We characterised prokaryotic communities across eight sequential layers spanning fresh litter, organic layers at different stages of decomposition, and mineral topsoil in three temperate beech forests using 16S rRNA gene amplicon sequencing. The decomposition stage was the dominant driver of community assembly, resulting in a strongly vertically stratified prokaryotic microbiome. While alpha diversity peaked within the fragmented litter layers, the overall high diversity of the forest floor was primarily an additive effect of vertical stratification. Site-specific effects were pronounced in the litter layer and the mineral topsoil, but diminished in the intermediate organic layers, where high local heterogeneity masked between-site differences. Redundancy analysis further showed that the environmental drivers of community structure shifted with depth, from litter quality in the upper horizons to mineral-associated properties in the mineral topsoil. At the same time, predicted 16S rRNA gene copy numbers indicate that the humified layers harbour the highest abundance of oligotrophic life strategists in the profile. By resolving the fine-scale vertical structure of the forest floor prokaryotic microbiome, our results provide a baseline for predicting how climate-change induced loss of organic layer mass threatens layer-specific communities, particularly the oligotrophic taxa in the humified layers, with consequences for the carbon turnover, tree nutrition, and nutrient cycling functions they mediate.