DOI: 10.5061/dryad.9cnp5hqkp
title: Extensive pollinator sharing does not promote character displacement in two orchid congeners

description: Pollinator sharing between close relatives can be costly, which can
 promote pollination niche partitioning and floral divergence. This should
 be reflected by a higher species divergence in sympatry than in allopatry.
 We tested this hypothesis in two orchid congeners with overlapping
 distributions and flowering times. We characterized floral traits and
 pollination niches and quantified pollen limitation in 15 pure and mixed
 populations, and we measured phenotypic selection on floral traits and
 performed controlled crosses in one mixed site. Most floral traits
 differed between species, yet pollinator sharing was extensive. Only the
 timing of scent emission diverged more in mixed than in pure sites, and
 this was not mirrored by the timing of pollinator visitation. We did not
 detect divergent selection on floral traits. Seed production was pollen
 limited in most populations, but not more severely in mixed than in pure
 sites. Interspecific crosses produced the same or a higher proportion of
 viable seeds than intraspecific ones. The two orchid species attract the
 same pollinator species despite showing divergent floral traits. Yet, this
 does not promote character displacement, implying a low cost of pollinator
 sharing. Our results highlight the importance of characterizing both
 traits and ecological niches in character displacement studies.
description: To assess whether divergence in floral traits and pollination
 niches between <em>Gymnadenia conopsea</em> and <em>G.
 densiflora</em> was higher in mixed than in pure sites, in 2019 we
 measured floral traits (phenology, morphology and floral scent) in 15
 populations, located in six pure <i>G. conopsea</i> sites,
 three pure <i>G. densiflora</i> sites, and three mixed sites,
 where populations of both species were sampled. In 2020, we characterized
 pollination niches and quantified pollen limitation in 10 of those 15
 populations (located in three pure <i>G. conopsea</i> sites,
 two pure <i>G. densiflora</i> sites, and three mixed sites,
 where populations of both species were sampled except in Hörninge, where
 too few <i>G. densiflora</i> plants were detected this year).
 Finally, in one population (Ismantorp), we performed controlled crosses to
 evaluate the strength of post-pollination barriers between the two
 species. <strong>Phenology
 data:</strong> We visited each population every two days to record
 flowering start (<i>i.e.</i> day on which the first flower had
 opened) and end (<i>i.e.</i> day on which the last flower
 wilted; not recorded in <i>G. densiflora</i>). Note that no
 phenology data could be collected in Ryd.
 <strong>Morpholoy data:</strong> On each plant, we
 measured one of the lowermost flowers for corolla length, corolla width
 and spur length (distance from corolla to spur tip) to the nearest 0.1 mm
 using a digital caliper. At the end of the reproductive season, we
 measured plant height and we counted the number of flowers.
 <strong>Floral scent data:</strong> We extracted
 floral scents emitted by each plant twice - once during the day and once
 during the following night - using dynamic headspace adsorption. Scent
 samples were analyzed by Gas Chromatography-Mass Spectrometry. We
 estimated emission rates of individual compounds using the peak area of
 the internal standard and the total emission rate by summing emission
 rates of individual compounds. The total emission rate was then divided by
 the number of open flowers to estimate the flower emission rate. Scent
 composition was studied using relative amounts of individual compounds
 (expressed in percentages of the blend).
 <strong>Pollinator data:</strong> We recorded diurnal
 and nocturnal pollinator visits in each of the ten populations for four to
 five days and nights during the flowering peak. For each pollinator visit
 scored on the videos, we identified the pollinator species and we recorded
 the duration of the visit and the number of flowers probed by the
 pollinator. We then calculated the pollinator visitation rate by dividing
 the number of visits that a plant received on a given day or night by the
 recording time. In addition, we patrolled for three days and nights in
 each population to catch flower visitors. Insects that were observed
 feeding from <i>Gymnadenia</i> flowers were caught with a hand
 net, killed by freezing and identified to the species level and the number
 of pollinia they carried was recorded.
 <strong>Pollen supplementation
 experiment:</strong> In ten populations, we randomly assigned 30
 plants to an open- or a supplementally hand-pollinated treatment (N=15 per
 treatment). We visited each population every two days and performed hand
 pollinations continuously as flowers opened using pollinia from unmarked
 plants. At the end of the reproductive season, we counted the number of
 fruits produced by each plant and we weighed three fruits per plant to
 estimate mean fruit mass. We then estimated female reproductive success
 as: number of fruits × mean fruit mass (<i>i.e.</i> total
 fruit mass). <strong>Crossing
 experiment:</strong> In one popualtion, we selected 30 plants (15 of
 each species) and randomly assigned three flowers per plant to each of the
 following treatments: pollination using either (i) self-pollen, (ii)
 outcross, conspecific pollen, or (iii) heterospecific pollen. Plants were
 bagged before and after the crosses to exclude pollinators. After fruit
 collection, seeds were observed under a dissecting microscope as described
 in Söderquist <i>et al.</i> (2020) to estimate seed viability
 as the proportion of seeds bearing an embryo.
 Statistical analyses applied to these datasets are detailed in
 the related article.
description: Sites: ALB: Alböke (pure G. conopsea
 site) GRA: Gråborg (mixed site) HOR:
 Hörninge (mixed site) ISM: Ismantorp (mixed
 site) OST: Österskog (pure G. densiflora
 site) RYD: Ryd (pure G. conopsea site)
 SAN: Sandby borg (pure G. conopsea site) SEG:
 Segerstad (pure G. conopsea site) STO: Störlinge (pure
 G. conopsea site) TOR: Torpmossen (pure G. densiflora
 site) TRI: Triberga (pure G. conopsea site)
 VAN: Vanserum (pure G. densiflora site)