Pollen Depletion

To compare the rates of pollen depletion among C. virginica populations, we assessed the amount of pollen available in individual flowers (an estimate of the pollen standing crop) during the period of insect activity, from mid-morning until early or late afternoon. Each hour, we arbitrarily chose eight flowers scattered throughout the local plant population and collected the anthers from each flower into a microcentrifuge tube containing 1 mL of 70% ethanol. We chose flowers regardless of the number of dehisced anthers. We began anther collection each morning when the flowers’ anthers began to dehisce, usually between 8:45 and 10:00, but occasionally as late as 13:15 because of cool or wet weather. We concluded anther collection when insect visitation ceased or flowers began to close, between 13:00 and 15:00. We counted the number of pollen grains available in each flower sample using a Coulter Multisizer 3 particle counter (Beckman Coulter Inc., Brea, California). Prior to counting, we suspended pollen in 20 mL 0.9% saline. To obtain a representative count of the number of pollen grains in each flower, we took the average of four 1mL subsamples and multiplied the mean by the total volume of the sample. These counts included the number of pollen grains in undehisced anthers because undehisced anthers opened and emptied during the counting process.