DOI: 10.5061/dryad.vdncjsxr0
title: Data from: Temporal variations in the linkages between plants and flower visitors and the pollination success of Primula modesta along the snowmelt gradient

description: Although pollination networks between plants and flower visitors are
 diverse and flexible, seed production of many plant species is restricted
 by pollen limitation. Obligate outcrossers often suffer from low
 pollinator activity or severe interspecific competition for pollinator
 acquisition among co-flowering species. This study focused on seasonal
 changes in plant–flower visitor linkages in an alpine ecosystem and
 examined whether and how this seasonality affected the seed-set of Primula
 modesta, a self-incompatible distylous herb having long-tubed flowers.
 First, we recorded the linkages between plants and flower visitors along
 the snowmelt gradient. Then, pollination experiment was conducted to
 estimate the degree of pollen limitation over the course of flowering
 season of P. modesta. Flower visitors were classified by their tongue
 length based on the morphological matching with P. modesta flowers. As the
 season progressed, plant–visitor linkages became more diverse and
 generalized, and the visitation frequency to P. modesta flowers increased.
 In the later part of the season, however, the seed set of P. modesta was
 significantly reduced due to severe pollen limitation, presumably because
 of increased competition for long-tongued pollinators among co-flowering
 species. The present study revealed that pollinator availability for
 specialist species may be restricted even when plant–visitor linkages are
 diverse and generalized as a whole. In the case of P. modesta,
 morphological matching and competition for pollinators might be the main
 factors explaining this discrepancy.
description: Study site We conducted this study within an
 area of 150 m × 130 m on the north-facing slope of the Happo-One ridge,
 northern Japanese Alps, central Japan (N36°42′0″, E137°50′58″). The local
 topography within the study area gradually changes from fellfield on the
 wind-blown ridge to snowbed at the bottom of valley; the altitude
 difference between the two habitats is about 40 m and the slope angle is
 about 15–20° (see Kameyama et al. 2015a for map). The ridge consists of
 fellfield vegetation dominated by <i>Pinus pumila</i>,
 <i>Juniperus communis</i>, <i>Thymus
 quinquecostatus</i>, <i>Spiraea nipponica</i>,
 <i>Festuca ovina,</i> and lichens. Meanwhile, the slope is
 dominated by dwarf shrubs and herbs such as <i>Rhododendron
 tschonoskii</i>, <i>Rhododendron
 multiflorum</i>,<i> Carex blepharicarpa,</i> and
 <i>Schizocodon soldanelloides</i>. The valley consists of
 herbaceous snowbed vegetation dominated by <em>Carex
 omiana</em>, <em>Eriophorum vaginatum</em> ssp.
 <em>fauriei</em>, <em>Trichophorum
 cespitosum</em>, and <em>Sieversia
 pentapetala</em>. Target species
 <i>Primula modesta</i> is a distylous alpine herb
 which produces one flowering stem with three to five tubular flowers, and
 distributed over a wide range of montane to alpine habitats throughout
 Japan. Because this species possesses a heteromorphic self-incompatibility
 system that almost completely prevents self- and intramorph mating,
 seed-set strongly depends on cross-pollination between two morphs, i.e.,
 long styled pin and short styled thrum flowers. Pollination success of
 <i>P. modesta</i> may be dependent on long-tongued
 pollinators, which probe for nectar at the bottom of the floral tube and
 transfer legitimate pollen grains between reciprocal morphs.
 In the study area, <i>P. modesta</i> is distributed
 widely along the snowmelt gradient, and is one of the earliest bloomers
 within each topographic site (Kameyama, personal observation). Thus, in
 the present study, we defined the early, middle, and late season according
 to the peak flowering time of <i>P. modesta</i> growing on the
 ridge, slope, and valley, i.e., early June, early July, and late July,
 respectively. Community scale linkages between plants
 and flower visitors We observed flower visitors within
 quadrats which were arbitrarily set to include as many flowering patches
 as possible (1 m × 1 m or 2 m × 2 m depending on flower density). The
 number of quadrats set during the early, middle, and late season were 3,
 9, and 9 in 2016, and 2, 7, and 6 in 2017, respectively. Within each
 quadrat, we counted the flowers of all entomophilous plant species;
 apparent anemophilous plant species such as Gyperaceae, Gramineae,
 Juncaceae, and Pinaceae were excluded. We recorded flower visitors when an
 insect other than a spider or ant contacted the reproductive organs of
 flowers. Given that complete identification of flower visitors was
 impossible in the field, we collected specimens (116 in total) and
 identified them to the order, genus, or species level wherever possible.
 Observations were conducted during daylight hours (0830–1450 h) in periods
 of calm weather. The total observation time and number of observation days
 during the early season was 6.2 h over 3 d in 2016 and 7.7 h over 2 d in
 2017. In the middle season, this was 7.8 h over 6 d in 2016 and 11.8 h
 over 5 d in 2017, and in the late season it was 11.7 h over 3 d in 2016
 and 10.0 h over 3 d in 2017. The total number of visits observed during
 the early, middle, and late season was 423, 164, and 1171 in 2016, and 56,
 788, and 943 in 2017, respectively. Plant–flower visitor linkages were
 quantified by the frequency of visits to each plant species
 (flower<sup>−</sup><sup>1</sup>hr<sup>−</sup><sup>1</sup>). Morphological matching between flower visitors and <i>P. modesta</i> To consider the morphological matching between flower visitors and <i>P. modesta</i> flowers, we collected 43 pin and 44 thrum flowers (one flower from each plant). The lateral side of each flower was carefully dissected and a digital photograph was taken using a stereomicroscope (Olympus SZ60) with a digital camera (Olympus E-620). The length of the floral tube and the distance between the floral surface and the short reproductive organ (stigma of thrum flower and anther of pin flower) were measured using Image J (Schneider et al. 2012, Rueden et al. 2017). The body lengths and tongue lengths of flower visitors (116 specimens) were also measured for morphological matching with <i>P. modesta </i>flowers using Image J. Based on the measurements, flower visitors were classified as long-tongued (LT), medium-tongued (MT), short-tongued (ST), or other (data deficient). Seed-set of <i>P. modesta</i> The degree of pollen limitation was evaluated by comparing the actual and potential seed production of <i>P. modesta</i> growing within a 10 m × 10 m quadrat at the ridge, slope, and valley, which correspond to the early, middle, and late season, respectively. To estimate the actual seed production, we collected several fruits (2.9 on average) from arbitrarily selected plants (18.3 on average) growing within a quadrat. The total number of fruits collected was 168 and 148 in 2016 and 2017, respectively. The potential ability to produce seeds was estimated by artificial pollination experiments. Plants growing within a quadrat was arbitrarily selected (17.7 on average) and covered with fine-meshed nylon nets to exclude flower visitors. For each plant, 1–3 flower buds (1.4 on average) were emasculated just before opening, and other flowers were removed. After flower opening, hand pollination was conducted using legitimate pollen grains collected from a single pollen donor growing at least 5 m away from the recipient plant. All fruits derived from this pollination treatment were harvested just before dehiscence. The total number of fruits harvested was 62 and 87 in 2016 and 2017, respectively. The number of seeds were counted using a stereomicroscope (Olympus SZ60).