High‐throughput assessment of anemophilous pollen size and variability using imaging cytometry

Pollen grain size relates to plant community structure via pollen dispersal, plant resource allocation into regenerative processes, plant phylogeny and plant genetics (ploidy), or it can be used as a decisive trait for pollen species distinction. However, the availability of pollen size data is limited because of labor- and time-consuming methodological constraints and is classically based on fewer than 50 measured pollen grains per species, thus restricting our knowledge of the temporal and spatial variability of pollen size in response to biotic and abiotic conditions. We addressed this data gap by using imaging flow cytometry (IFC), which allows for high-throughput assessment of pollen size and measured > 500 000 single pollen from 100 anemophilous species that were sampled between 2018 and 2022. We present a workflow for high-throughput data analysis, show the agreement of IFC estimates with literature size estimates and assess pollen size variability in the context of plant phylogeny. Our approach allows us to make statistically robust measurements of pollen size that are not limited by sampling effort and sample throughput to answer broad ecological questions at large temporal and spatial scales.