Plant identity in pollen pellets
To identify the plants bees collected pollen from, each individual
pollen pellet was crushed with a small pestle in a 1.5mL microcentrifuge
tube, followed by the addition of a Safarin-O pollen staining solution.
Ten µL of homogenate was then placed onto a microscope slide, topped
with a coverslip and sealed along the edges using clear nail polish
(Jones, 2012). Using light microscopy, we counted and identified 500
pollen grains in each sample at 400x magnification. We verified that
this is an appropriate sample size by rarefaction using the ‘rarecurve’
function in the ‘vegan’ package (Oksanen et al., 2019) in R (R Core Team
2013) (Appendix: Fig. S1). We made a library of visually distinct pollen
morphotypes, and each pollen grain was identified at the lowest possible
plant taxonomic level using reference samples collected from the study
site, and online atlases (flickr.com/photos/161453633@N02/collections;
paldat.org; blogs.cornell.edu/pollengrains; globalpollenproject.org).
Following identification, the proportional abundance of each visually
distinct pollen morphotype within a pollen pellet was calculated at both
the plant family and pollen morphotype levels. We included a pollen
morphotype if it occurred in at least 3% of all pollen identified
within a pellet (≥ 15 out of 500 pollen grains).