DOI: 10.5061/dryad.41ns1rnc0
title: Mechanisms of male-male interference during dispersal of orchid pollen

description: 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.
description: We quantified massula dispersal in a glasshouse at the Ecological
 Field Station of Uppsala University at Skogsby on Öland, Sweden. Bees
 without <em>A. morio</em> pollinaria were captured while
 foraging near the Field Station and maintained in individual vials in a
 refrigerator. We also collected <em>A. morio</em>
 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 &gt; 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
 (<em>r</em> = 0.971, <em>P</em> &lt; 0.001,
 <em>n</em> = 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 <em>A.
 morio</em> 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.
description: 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