ENAGenomicsUniversity of Munich (LMU)https://www.ebi.ac.uk/ena/browser/view/PRJNA1137278The transfer of pollen from one plant individual to the next by an animal agent, i.e. animal pollination, represents one of the most important ecosystem functions and facilitates plant cross-pollination. Different tools, such as microscopy and more recently DNA metabarcoding, have been used to investigate pollen transport and by that the interactions between plants and pollinators underlying this pollination service. In particular DNA metabarcoding has proven to be a fast and reliable method to determine the origin of mixed pollen carried by animals. Hitherto this method was primarily focused on the pollinators' side of the interaction through analysing pollen samples from foraging pollinators or nest environments. While this approach provides detailed information on pollinator dietary intake patterns or preferences, it does not allow for inferences on the plant's perspective, e.g., the type or amount of viable con- and heterospecific pollen received. We aimed to explore the potential of DNA metabarcoding as a tool to analyse heterospecific pollen transfer to plants in semi-natural and agricultural landscapes along a land use intensity gradient. For this, plant stigmas of three closely related Ranunculus species (R. acris, R. bulbosus, and R. repens) were collected on 20 grassland plots differing in land-use intensity, i.e. grazing, mowing and fertilization, and thus flowering plant diversity, in Germany and subjected to internal transcribed spacer 2 (ITS2) metabarcoding.We found the species richness of flowering plants on plots to significantly affect the richness of heterospecific pollen found on Ranunculus stigmas. Interestingly, lowest heterospecific pollen diversity were found in intermediately intense landscapes, while low and high land use intensities showed each higher heterospecific pollen diversity. Our findings demonstrate that DNA pollen metabarcoding can be applied to make inferences on pollen transfer between flowers by assessing heterospecific pollen diversity and that pollen transfer is strongly affected by the diversity of the surrounding plant community. This novel approach consequently allows for taking the plants' perspective and assess the flower fidelity of pollinators and potentially even pollination outcomes in different environments.ENAfalseUnveiling heterospecific pollen deposition in Ranunculus plants along a land-use gradient through DNA metabarcoding2025-09-082025-09-08PRJNA1137278