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Background Airborne environmental DNA (eDNA) research is an emerging field that focuses on the detection of species from their genetic remnants in the air. The majority of studies into airborne eDNA of plants has until now either focused on single species detection, specifically only pollen, or human health impacts, with no previous studies surveying an entire plant community through metabarcoding. We therefore conducted an airborne eDNA metabarcoding survey and compared the results to a traditional plant community survey. Results Over the course of a year, we conducted two traditional transect-based visual plant surveys alongside an airborne eDNA sampling campaign on a short-grass rangeland. We found that airborne eDNA detected more species than the traditional surveying method, although the types of species detected varied based on the method used. Airborne eDNA detected more grasses and forbs with less showy flowers, while the traditional method detected fewer grasses but also detected rarer forbs with large showy flowers. Additionally, we found the airborne eDNA metabarcoding survey required less sampling effort in terms of the time needed to conduct a survey and was able to detect more invasive species than the traditional method. Conclusions Overall, we have demonstrated that airborne eDNA can act as a sensitive and efficient plant community surveying method. Airborne eDNA surveillance has the potential to revolutionize the way plant communities are monitored in general, track changes in plant communities due to climate change and disturbances, and assist with the monitoring of invasive and endangered species.

Airborne environmental DNA (eDNA) research has typically focused on the detection of pollen from anemophilous terrestrial plant species; however, recent findings have expanded the definition of airborne eDNA to include a variety of eDNA sources, such as leaf and flower fragments. While methods for capturing pollen are well studied, there is less known about how to best analyze and capture more broadly defined bulk airborne eDNA samples. Therefore, this study aimed to identify efficient techniques for the extraction and amplification of airborne eDNA and how best to capture these samples. First, we collected samples using Big Spring Number Eight dust traps and quantified the influence of three different extraction methods (cetrimonium bromide‐chloroform, DNeasy Plant Maxi Kit, and DNeasy PowerPlant Pro DNA Isolation Kit) and two global plant primer sets (targeting the trnL and ITS2 genes) on quantitative polymerase chain reaction (qPCR)‐based terrestrial plant detection. Additionally, we examined how best to capture airborne eDNA by comparing the performance of three difference passive dust collectors (Big Spring Number Eight, Modified Wilson and Cooke, and Marble dust collectors). We found that the detection of airborne eDNA was significantly influenced by both the extraction methods employed and choice of qPCR primer. Additionally, we found that all three traps preformed similarly but the Big Spring Number Eight dust traps trended as consistently one of the most effective, shedding light on the nonhomogeneous nature of airborne eDNA. This study represents the first methodology paper focusing on airborne eDNA as a bulk environmental sample containing more than just pollen. In the future, additional research should examine more about the ecology of airborne eDNA (origin, state, transport, fate), explore active dust collectors compared to the passive traps used here, and develop the application of metabarcoding for airborne eDNA analysis.