Siring success of flowering plants depends on the fates of male gametophytes, which compete for access to stigmas, stylar resources and ovules. Although rarely considered, pollen may often compete during dispersal, affecting the processes required for export to stigmas; pollen pickup, transport and deposition. We quantified dispersal interference by tracking bee dispersal of stained Anacamptis morio (Orchidaceae) pollen from individual donor flowers and inferred the affected dispersal mechanisms based on the fit of a process-based model. During individual trials, all recipient flowers were either emasculated, precluding interference with donor pollen, or intact, adding potentially interfering pollen to the pollinator. The presence of competing pollinaria on bees reduced pickup of additional pollinaria, doubled the overall proportion of lost donor pollen and reduced total pollen export by 27%. Interference specifically increased loss of donor pollen between successive flower visits and variation in deposition among trials, and likely also reduced pollen contact with stigmas and pollen deposition when contact occurred. Thus, by altering pollen removal, transport and deposition, male-male interference during pollen dispersal can significantly, and perhaps commonly, limit plant siring success.

We quantified massula dispersal in a glasshouse at the Ecological Field Station of Uppsala University at Skogsby on Öland, Sweden. Bees without A. morio pollinaria were captured while foraging near the Field Station and maintained in individual vials in a refrigerator. We also collected A. morio inflorescences with intact pollinaria and clean stigmas from a nearby population and placed them in water-filled vials for use in the experiment.  On each donor inflorescence, we stained both pollinia of one flower by injecting rhodamine pink histochemical stain into the anther sacs with a syringe, which generally dyes all of a pollinium’s massulae. Stain was allowed to dry > 20 min before an inflorescence was used in the experiment. We also collected a neighboring flower from the inflorescence, for which we counted the massulae of one pollinium as an estimate of pollen availability in the stained flower. Massula production correlates very strongly between adjacent flowers within inflorescences (r = 0.971, P < 0.001, n = 18 plants). A bee was allowed to visit a donor flower and, if it removed the stained pollinaria, it was left for a minute to reorient before the experimental trial began. Only A. morio flowers were present in the glasshouse.

The experiment contrasted two types of trials during which a bee with two stained donor pollinia visited a sequence of recipient flowers.  For the 16 emasculated (E) trials, the pollinaria had been removed from all recipient flowers, precluding pollen interference. In contrast, for the 15 intact (I) trials, all recipient flowers had unstained pollinaria that could transfer to the bee and interfere with dispersal of stained donor pollen that the bee already carried. During intact trials, we allowed about 1 min to elapse between visits to successive recipient flowers to permit pollinarium reorientation. To minimize unintended effects of trial sequence, day, time of day, etc., we conducted the two trial types in almost strictly alternating order. Except for one intact trial during which the bee escaped after visiting 10 recipient flowers, bees were caught at the end of each trial and placed in vials in a refrigerator to facilitate removal and counting of both recipient pollinaria and remaining stained massulae on the donor pollinarium (if still present).  Most bees participated in only one trial each. We examined the stigmas of all visited recipient flowers with a dissecting microscope (24x) and counted both the stained (donor) and, if applicable, unstained (recipient) massulae. For intact trials, we also recorded the number of pollinaria removed from recipient flowers to quantify changes in a bee’s maximal (cumulative) pollinarium load. Total loss of donor massulae was estimated as the difference between the initial number (based on an adjacent unvisited donor flower) and the sum of donor massulae deposited on all recipient stigmas and those remaining on the bee at the end of a trial.

Variable	Description
Trial_type	Type of trial (Intact  pollinaria in recipient flowers or Emasculated recipient flowers)
Trial	Trial number
M_init	Initial number of donor massulae
R	Final number of donor pollinaria on bee
Visit	Sequence number of recipient flower in trial
Deposition	Number of donor massulae deposited on stigma of recipient flower