Evaluation of sampling effort required to assess pollen species richness on pollinators using rarefaction

Premise Understanding the flower visitation history of individual pollinators is key in the study of pollination networks, but direct tracking is labor intensive and, more important, does not capture information about the previous interactions of an individual. Therefore, a protocol to detect most of the pollen species on the body surfaces of an individual pollinator could elucidate its flower visitation history. Methods and Results Under a microscope, we observed 6.0-mu L droplets from a sample solution (1.0 or 3.0 mL) containing pollen grains collected from individuals of six major pollinator functional groups. To clarify how many droplets need to be observed to detect all pollen species within the solution, we examined up to 10 droplets collected from each individual insect. Sample-based rarefaction curve analyses of the data showed that we could detect ~90% of the pollen species and the plant-pollinator links in the networks by observing six droplets. Conclusions The rarefaction curve analysis for pollen-on-pollinator studies is a useful preliminary step for minimizing the time and labor required while maximizing the data on the flower visitation history of each individual pollinator and revealing any hidden flower-pollinator interactions.