DOI: 10.7280/d19x0d
title: Data from: Pollinator visitation rate and effectiveness vary with flowering phenology

description: Premise of the Study – Flowering time may influence pollination
 success and seed set through a variety of mechanisms, including seasonal
 changes in total pollinator visitation or the composition and
 effectiveness of pollinator visitors. Methods – We investigated
 mechanisms by which changes in flowering phenology influence pollination
 and reproductive success of Mertensia
 ciliata (Boraginaceae). We manipulated flowering onset of potted
 plants and assessed the frequency and composition of pollinator visitors,
 as well as seed set. We tested whether floral visitors differed in their
 effectiveness as pollinators by measuring pollen receipt and seed set
 resulting from single visits to virgin flowers. Key Results –
 Despite a five-fold decrease in pollinator visitation over four weeks, we
 detected no significant difference in seed set among plants blooming at
 different times. On a per-visit basis, each bumblebee transferred more
 conspecific pollen than did a solitary bee or a fly. The proportion of
 visits by bumblebees increased over the season, countering the decrease in
 visitation rate so that flowering time had little net effect on seed set.
 Conclusions – This work illustrates the need to consider
 pollinator effectiveness, along with changes in pollinator visitation and
 species composition to understand the mechanisms by which phenology
 impacts levels of pollination.
description: Methodology: Phenology Manipulation
 Experimental Design To test the hypothesis
 that differences in the timing of flowering alters visitation rate and
 types of potential pollinator visitors, resulting in lower pollination
 success and seed set, we manipulated flowering phenology of potted M.
 ciliata plants. Sixty plants were collected from Rustler’s Gulch in 2012
 and overwintered in the ground at RMBL. In 2015, we induced the plants to
 flower at different times using natural variation in temperature and light
 found along an elevation gradient in Gunnison National Forest. To inhibit
 flowering, potted plants were moved to Schofield Pass in early June and
 placed in a shaded snowbank under a shade-shelter. Each week, 10 randomly
 selected plants were moved back to RMBL, where the higher light and warmer
 temperatures at low elevation induced them to flower.
 Upon flowering, 10 plants per week were moved from RMBL to a
 meadow near the original source population. Plants were arranged 30 cm
 apart into two randomized arrays of five plants, with 2 m between arrays.
 Arrays were located 50 meters away from unmanipulated M.
 ciliata populations. Forty plants flowered and were included in the
 experiment, for a total of four phenology groups spanning four weeks (June
 23-July 20). Pollinator response measurements
 Plants in each phenology group were left open to pollination for
 one week. During that week, we conducted pollinator observations. We
 tracked pollinator identity and the number of flowers visited during
 multiple 30-minute observation periods between the hours of 9:00 and
 16:00. At the beginning of each observation period, open flowers per
 potted plant were counted. Visitors were counted as pollinators if they
 crawled inside the corolla. The order of observations was randomized
 between the two arrays for a given week. We completed 15 hours of
 observations per phenology group, except in week four because of lower
 flower abundance. The floral abundance among arrays
 during the first three weeks ranged from 12 to 86, with a mean of 78.27 ±
 12 (Mean ± SEM) flowers per array (Fig. 1). The floral abundance in week
 four was significantly lower than in previous weeks (F3,
 39=10.15, P&lt;0.0001) and we observed zero pollinators during our
 first 15 hours of observations that week. To compensate for the difference
 in floral display in week four, we increased the number of available
 flowers by adding cut stems in plant picks to each pot and completed an
 additional 15 hours of observations. We combined the data from both rounds
 of observations in our analyses of pollinator visitation rate and
 pollinator type for week four. At the end of each week,
 individual flowers in each phenology group were labeled and bagged with
 fine mesh jewelry bags (Uline, Pleasant Prairie, WI, USA) to prevent
 further pollination and loss of seeds. To standardize conditions after
 pollination exposure, all plants remained in the field until seeds were
 collected. Soil moisture and floral trait
 measurements Because the goal of this experiment was to
 assess the how differences in flowering time alone impact pollination and
 seed set, we also monitored soil moisture and tracked floral traits
 (corolla width, corolla length, and nectar) that were found to vary with
 changes in water availability. We monitored the soil moisture in each pot
 as volumetric water content (VWC) with measurements every week using a
 12-cm probe (Campbell Scientific, Edmonton, Alberta, Canada) inserted in
 the ground at the center of each pot. For all tagged
 flowers, we measured corolla width at the opening of the tube and corolla
 length from the base of the calyx to a randomly chosen
 corolla lobe. Fitness component measurements
 To measure deposition of hetero- and conspecific pollen load,
 stigmas were collected after the corollas fell from the flowers and stigma
 squashes were made with fuchsin gel (Kearns & Inouye 1993). For an
 average of 5.2 ± 0.7 flowers per plant, we counted the number of
 conspecific and heterospecific pollen grains using a compound microscope
 at 200Í. Mertensia ciliata may produce up to four
 one-seeded nutlets per flower. We counted the total number of seeds
 produced per flower for all labeled flowers (measured as described by
 Forrest & Thomson 2010). We calculated the average seeds per
 flower for each potted plant as (number of mature seeds / number of
 flowers). Mature seeds from tagged flowers were collected in coin
 envelopes and transported to the University of California, Irvine to be
 weighed. We calculated mean seed mass for each plant as (mass of collected
 seeds / number of collected seeds). References
 Cited: Forrest, J. & Thomson, J.D. (2010).
 Consequences of variation in flowering time within and among individuals
 of Mertensia fusiformis (Boraginaceae), an early spring
 wildflower. American Journal of Botany, 97:38-48.
   Methodology: Pollinator
 Effectiveness To estimate the single-visit pollinator
 effectiveness of different insect visitors, in 2016 we measured pollen
 receipt and seed set resulting from single visits to virgin M.
 ciliata flowers in wild populations at Rustler’s Gulch (June 27-July 12)
 and Schofield Pass (July 12-29). Experimental
 Design Unopened flowering cymes on individual ramets
 were bagged with fine mesh jewelry bags to provide a supply of virgin
 flowers. For each single-visit ramet, two additional ramets in the same
 clone were bagged to serve as controls. The first control group remained
 bagged throughout the experiment to serve as a control for
 self-pollination. Non-production of seeds by plants in the bagged-control
 group would indicate that despite being self-compatible, M.
 ciliata flowers are not self-pollinating and therefore require insect
 pollination. Cymes in the second control group (hereafter open control)
 were made available to pollinators during observation periods but not
 observed to be visited, thus serving as a control for missed visits by
 observers. Each group of three ramets, including the single-visit ramet
 and two control ramets, belonged to the same M. ciliata clone, and no
 clone was used for more than one group of three.
 Pollinator visit data Bags were removed from
 flowering cymes on single-visit and open control ramets during observation
 periods. We recorded the number of flowers available on single-visit and
 open control ramets during each observation period. Once a single visit to
 a single flower was received on the single-visit ramet, the visited flower
 was marked on the calyx with permanent marker and both the open control
 and single-visit ramet were re-bagged to prevent further pollination and
 loss of seeds. For each visitor, we recorded the pollinator identity and
 number of flowers visited. Only insects that crawled inside of a corolla
 were counted as visitors. When a single visit was observed to an open
 control ramet, we re-designated that ramet as a single-visit ramet and
 marked the visited flower(s). We monitored 235 marked
 flowers from 95 ramets that received single visits, as well as 65 open
 control and 74 bagged control ramets. The most common pollinators,
 bumblebees (Bombus spp.) and flies (Muscoidea, hereafter flies), accounted
 for 97.5% of visits, with solitary bees (Osmia spp. 2.1%) and a syrphid
 fly (Syrphidae 0.4%) making up the rest. We excluded the syrphid fly from
 our analyses. Floral trait measurements
 For single-visit ramets, we measured corolla width and length of
 marked flowers, or if marked flowers were withered or had fallen off, we
 calculated mean corolla width and length from up to five randomly selected
 flowers on the same ramet. In a few cases, more than one flower was
 visited on the single-visit ramet. When this happened, we marked the calyx
 of each visited flower, and used mean trait values (e.g., pollen receipt,
 seed set, etc.) for these ramets in our analyses.
 Fitness component measurements To measure
 conspecific and heterospecific pollen receipt, we collected stigmas from
 marked flowers on single-visit ramets, as well as one randomly selected
 flower from each open control ramet. Stigmas were collected after the
 corollas fell from the flowers and stigma squashes were made with fuchsin
 gel (Kearns & Inouye 1993). For an average of 15.5 ± 3.4 flowers
 per visitor/control type we counted the number of conspecific and
 heterospecific pollen grains using a compound microscope at
 200x. For each single-visit ramet, we counted the
 number of seeds produced per marked flower and, when a ramet had more than
 one marked flower, calculated mean seed set of marked flowers as (number
 of mature seeds / number of marked flowers). For each open and bagged
 control ramet, we calculated mean seed set per flower as (number of mature
 seeds / number of bagged flowers). Mature seeds were collected in coin
 envelopes and transported to the University of California, Irvine to be
 weighed. We calculated mean seed mass per flower as (mass of collected
 seeds / number of collected seeds). Ramets that failed to set seed because
 of herbivory or accidental damage were excluded from analyses.
description: Location of study: Plants used in this experiment were wild M.
 ciliata populations along Rustler’s Gulch (38° 59.6'N, 107° 0.5’W;
 3,009 m. a.s.l.) and Schofield Pass (39°00'54.98'' N, 107°
 2'49.40'' W; 3,263 m.a.s.l.). For all
 details see:
 gallagher&campbell_nonSpatialData_phenologyExperiment.pdf gallagher&campbell_nonSpatialData_pollinatorEffectiveness.pdf