Aligning terrestrial eDNA sampling and analytical choices to effectively capture both rare species and compositional variation in grassland plant communities

Abstract Vascular plants are a major component of terrestrial diversity, yet they are overrepresented among the world’s threatened species. To effectively manage this biodiversity crisis, data with high spatiotemporal resolution are crucial, yet often lacking for plants. Environmental DNA analysis (eDNA) is capable of rapid detection of biodiversity by metabarcoding the collection of DNA molecules retrieved from environmental samples such as soil cores. The technology may soon support the generation of broad-scale longitudinal plant community data, yet much methodological work on sampling strategies and analytical choices remains if soil-based eDNA is to become a reliable tool for monitoring terrestrial plant communities. Therefore, this dual purpose study in seven Swedish semi-natural grasslands investigated if and when eDNA-generated community data can be used as a stand-alone information source 1) to inform on the presence of a rare, small-statured grassland specialist (Gentianella campestris) and 2) to simultaneously infer community compositional change. We demonstrate eDNA to be an effective means of finding a rare species in a highly taxonomically diverse habitat, uncovering G. campestris DNA in 31% of the core samples. Evidence suggests the eDNA signal reflects recent spatio-temporal population dynamics at fine spatial scales. Although the entire plant community was not uncovered, molecular community data proved a representative subset, effectively capturing changes in community diversity and composition at plot sizes commonly used for plant surveys. Choices surrounding typical RRA-filtering had significant bearing on eDNA’s discriminating power: filtering may overly conservatively remove true observations of a rare species, while filtering highly localized plot noise led to more robust patterns emerging in species richness and plant composition turn-over. Given careful alignment of study goals and sampling strategies, soil-based eDNA may already provide a stand-alone tool for generating reliable, scalable and observer-independent longitudinal data for unveiling and monitoring changes in plant diversity in terrestrial habitats. Competing Interest Statement The authors have declared no competing interest. Footnotes Funding information MT and JP received funding from the Swedish Institute for Water and Air Pollution Research (SIVL) for this work. JP also held grants by FORMAS (nr 2018-00961, 2022-01803), and MT was part of the Mistra Finbio research programme (Dnr 2020/10). Data availability statement Upon acceptance, all raw data will be archived in an online Dryad digital repository (Plue & Töpel, 2026).