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The phyllosphere is a challenging microbial habitat in which microorganisms can flourish on organic carbon released by plant leaves but are also exposed to harsh environmental conditions. Here, we assessed the relative importance of canopy position-top, mid, and bottom at a height between 31 and 20 m-and tree species identity for shaping the phyllosphere microbiome in a floodplain hardwood forest. Leaf material was sampled from three tree species-maple (Acer pseudoplatanus L.), oak (Quercus robur L.), and linden (Tilia cordata MILL.)-at the Leipzig canopy crane facility (Germany). Estimated bacterial species richness (Chao1) and bacterial abundances approximated by quantitative PCR of 16S rRNA genes exhibited clear vertical trends with a strong increase from the top to the mid and bottom position of the canopy. Thirty operational taxonomic units (OTUs) formed the core microbiome, which accounted for 77% of all sequence reads. These core OTUs showed contrasting trends in their vertical distribution within the canopy, pointing to different ecological preferences and tolerance to presumably more extreme conditions at the top position of the canopy. Co-occurrence analysis revealed distinct tree species-specific OTU networks, and 55-57% of the OTUs were unique to each tree species. Overall, the phyllosphere microbiome harbored surprisingly high fractions of Actinobacteria of up to 66%. Our results clearly demonstrate strong effects of the position in the canopy on phyllosphere bacterial communities in a floodplain hardwood forest and-in contrast to other temperate or tropical forests-a strong predominance of Actinobacteria.

Canopy Position Has a Stronger Effect than Tree Species Identity on Phyllosphere Bacterial Diversity in a Floodplain Hardwood Forest