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.