Pollen Removal and Quantification
Pollen was removed from the exterior of insect bodies following the protocol by Lucas et al. (2018). Each specimen was washed in 500 μL of wash solution containing 2% PVP and 1% SDS (buffer solution) using a 1.5 mL Eppendorf tube. For larger specimens (>8 mm) additional wash solution was added until they were submerged. The specimens and controls were agitated by hand for 1 minute and then centrifuged at 15800 g for 20 seconds. Afterwards they were incubated for 5 minutes and shaken for an additional 20 seconds to resuspend pollen. The insects were then removed from the tube and stored in 95% ethanol. The remaining washing solution and suspended pollen were centrifuged at 15800 g for 5 minutes. The supernatant was discarded, and samples stored at -20°C.
The pollen pellet was resuspended in 250 μL of 95% ethanol by vortexing for 4 minutes. Samples that were difficult to homogenize were heated at 56°C for 5 minutes and vortexed for an additional 4 minutes. An aliquot of 50 μL was taken and dried in a sterile incubation oven for quantification and the remainder was used for metabarcoding. Pollen counts were determined for each sample using a Multisizer 3 Coulter Counter (Beckman Instruments, Fullerton, CA, USA). A blank of 10 mL of Isoton II diluent was measured in a 30 mL cuvette and used to calibrate the machine. The pollen sample was suspended in 300 μL of diluent by vortexing for 10-20 seconds. This pollen suspended diluent was added to the measuring cuvette. Additional diluent was added to reach 11 mL of liquid. The cuvette was gently vortexed for 3-5 seconds to homogenize the sample. The Coulter counter was then used to quantify the number of particles in the size range 10-120 μm for three 1 mL samples.