Airborne Plant e DNA Reflects Local Biodiversity: A Multi‐Method Assessment in a Botanical Garden

ABSTRACT

Airborne environmental DNA (eDNA) is emerging as a powerful tool for noninvasive assessment of terrestrial biodiversity. We evaluated the effectiveness of three analytical pipelines for characterizing plant diversity in a botanical greenhouse, with a particular focus on how particle size and sampling height influence detection. Air was sampled for up to 4 days using active Total Suspended Particulate samplers, a 14‐stage impactor, and a vertical filter stack, all equipped with quartz filters. To assess methodological variability and identify optimal approach, three analytical pipelines were applied: (A) Illumina amplicon sequencing with a QIIME‐based workflow, and (B) Illumina and (C) Nanopore shotgun sequencing processed with Kraken2. Of ~2100 plant genera recorded in the greenhouse and adjacent garden, ~86% were considered detectable based on rbcL, trnL, ITS1, and ITS2 barcode coverage. Pipeline A (32 samples) detected 149 genera, 88% of which were known from the garden; 55% of species‐level assignments matched cataloged species, with ~11% attributable to misannotations or contamination. Pipelines B (6 samples) and C (2 samples) detected 76 and 88 genera, respectively, with 81%–82% corresponding to garden taxa and 50%–51% confirmed at species level. The two shotgun pipelines produced highly similar taxonomic profiles despite samples being collected 7 weeks apart, and both were strongly complementary to the amplicon results. Size‐dependent sampling yielded elevated detections in the 2.5 μm impactor fraction, indicate substantial contributions from sub‐pollen particles, while larger fractions (> 40 μm) also contained plant eDNA, suggesting multiple particle carriers or aggregates. Overall, airborne eDNA provided robust detection of local plant biodiversity and yielded new insights into particle‐size distributions relevant to eDNA transport and deposition.