2.3.1 Pollen Export
We used pollen export as a proxy for the male component of reproductive success, by quantifying the number of pollen grains removed from an anther by visitors in a 3-hour period. In D. purpurea flowers, two anthers at the front are paired with each other and tend to dehisce simultaneously (Percival & Morgan 1965), as do the two anthers further inside the flower. To find the number of pollen grains removed (= pollen export), we subtracted the number of pollen grains left on a “post-visitation” anther from the number on a “pre-visitation anther” on the same flower. For this, immediately before the beginning of each allotted visitation period (whereupon inflorescences are still bagged), we collected one of the near-dehisced anthers (the “pre-visitation” control anther) in a centrifuge tube. Once dehisced in the tube, we added 70% ethanol until counting. We collected the corresponding paired “post-visitation” anther (which was already dehisced before the visitation period begun) from the same flower immediately after the visitation period of 3 hours and stored it in 70% ethanol. We did not record the number of visits to “post-visitation” anthers, but each flower is likely to have received multiple visits. This was repeated for each plant on separate days to include 2-6 anther comparisons per plant for at least 12 plants per treatment.
We used a haemocytometer counting chamber to estimate the number of pollen grains on the “pre-visitation” and “post-visitation” anthers. Up to 30 minutes prior to counting, tubes containing anthers were sonicated for four lots of 30 seconds to dislodge pollen grains and to homogenise the grains in the ethanol. Immediately before counting, the ethanol-pollen mixture was then vortexed for five seconds and 8µl of the sample was pipetted into the chamber for counting. Pollen grains were counted in grid squares of a known volume (0.1 µL) and multiplied up for an estimate of the total number of pollen grains in the original 200µl sample. We repeated this process four times for each sample and averaged for analysis. We calculated total pollen export by subtracting the number of pollen grains estimated to be left on a post-visitation anther from the number of pollen grains estimated to be on the corresponding paired pre-foraging anther.