DOI: 10.5061/dryad.s4mw6m9bg
title: Data from: pollinators limit seed production in an early blooming rare plant: evidence of a mismatch between plant phenology and pollinator emergence

description: The reproductive ecology of rare plants is seldom studied, yet the
 persistence of plant populations depends on successful mutualisms with
 pollinators.  As atmospheric temperatures rise, phenology of
 plants and pollinators may become mismatched.  We investigated
 the reproduction of Trifolium barnebyi (Barneby’s Clover), a mat-forming
 perennial endemic to central Wyoming, USA that grows in the crevices of
 sandstone bedrock.  Our objectives were to evaluate a method for
 monitoring changes in cover as well as assess the pollination and seed-set
 of T. barnebyi.  We established five monitoring transects using a
 divided frame to estimate percent cover.  We conducted seed-set
 experiments at three locations to measure self-pollination and the degree
 to which pollinating insects limited seed production.  We used
 vane traps and bee bowls to capture pollinators, and examined pollen
 carried on bees.  Percent cover along transects declined over the
 4 year period and was associated with spring precipitation. Trifolium
 barnebyi did not self-pollinate and relied on pollinators to produce
 seeds.  The number and mass of viable seeds per flower, and the
 number of bees captured increased as the season progressed, indicating
 that more and larger seeds were made when more pollinators were present.
  Blooming of T. barnebyi ranged between April and June depending
 on the microhabitat the plant lived in and we observed much higher seed
 production in later-blooming plants.  Pollen from T. barnebyi was
 primarily carried by Andrena bees, although we found smaller amounts of
 pollen on seven other bee genera.  A mismatch in timing between
 blooming and pollinating insect emergence could limit seed production in
 T. barnebyi if plants bloom earlier over time and bee emergence does not
 follow the same phenology.  Rare plants can be pollinated by rare
 pollinators showing that conserving these pollinators is crucial for rare
 plants and early blooming species may be higher at risk.
description: Monitoring To assess the cover and asexual
 reproduction of <em>T. barnebyi</em>, we monitored five
 transects annually in July between 2018 and 2021. We established two
 transects at the Weiser Knoll site (Weiser Slope and Weiser Seedlings) and
 the Hall Creek site (Hall 1 and Hall 2), and one transect at South Red
 Canyon (Fig. 1). Each transect consisted of a belt between 6.9 and 18 m in
 length where a plot frame (30 x 30 cm) with sixteen 7.5 x 7.5 cm cells was
 placed on the ground along the belt to assess cover (Fig. 2).  We used a
 plot frame to monitor <em>T. barnebyi </em>because the compact
 and extensive mats of this species make distinguishing individuals
 difficult without destructive sampling.  We placed the plot frame on one
 side of the belt at all sites expect South Red Canyon and Weiser Knoll
 Seedling, where we placed the frame above and below the belt.  We counted
 the number of cells with T. barnebyi present within each frame at 30 cm
 intervals.  Cells were scored as present (1) or absent (0) to calculate
 the percent of cells in which live <em>T. barnebyi </em>were
 present. Seed-Set Experiments We
 measured the seed production of <em>T. barnebyi</em> to
 estimate the degree to which pollinators may limit sexual reproduction. 
 We selected three areas along the rim of Red Canyon where <em>T.
 barnebyi</em> is most abundant (North Red Canyon, South Red Canyon
 and Weiser Knoll) to measure seed-set. We measured seed-set of bagged,
 open, and hand-pollinated flowers to estimate seed production from self-
 and cross-pollination.  We selected 20 <em>T. barnebyi</em>
 plants from 15 April through 16 May 2019. Each plant cluster received one
 of each treatment.  Bagged treatments restricted pollinator access and
 measured the degree to which flowers can self-pollinate by placing 1
 mm<sup>2</sup> mesh bags over flowers before blooming. Open
 treatments left blooms accessible to local pollinators and measured
 ambient levels of seed-set.  The hand-pollinated treatment added excess
 pollen in addition to local pollinators to measure seed production when
 pollen was not limiting.  Pollen came from plants at least 50 m away and
 we delicately brushed collected anthers on the stigma of the treatment
 bloom.  Blooms were bagged with mesh bags before (bagged treatment) or
 after (open and hand-pollinated treatments) flowers bloomed to contain the
 developing seeds and were held in place with fishing line weighted with
 color-coded eye bolts.  For the hand-pollinated treatment, we recorded the
 number of flowers pollinated, and marked and recorded the flowers not
 ready for pollination in each flowerhead.  We recorded the date that each
 flower in the hand-pollinated treatment bloomed and was hand-pollinated. 
 We monitored treatments and collected fruits when flowerheads were ripe,
 from 4 June through 17 July 2019.  Flowerheads were placed in paper bags,
 returned to the laboratory and dried at room temperature.
 We cleaned, counted, and weighed seed pods and seeds to estimate
 the degree to which <em>T. barnebyi </em>self-pollinated or
 depended on pollinators. Each<em> T. barnebyi </em>flower
 produced one legume seed pod, and there were multiple flowers per
 flowerhead.  We counted the number of flowers per flowerhead and the
 number of ovules per flower using a dissecting microscope.  We weighed the
 pods and seeds together, because the unfertilized ovules were too small to
 remove from the pods without damaging. We calculated mean seed mass by
 dividing total mass by the number of seeds.  We noted seeds that appeared
 viable by size, and counted and weighed seeds for each plant.  We tested
 viability of the seeds using Tetrazolium staining, which measures the
 germinative potential of seeds (Lindenbain 1965).  We placed the seeds
 between moistened paper towels for 24 hours, cut them to expose the
 endosperm, and immersed them in tetrazolium solution for 24 hours.  The
 endosperm of viable seeds turned pink or red indicating respiration, while
 the endosperm of non-viable seeds remained white.
 Pollinators We collected pollinators at the
 same sites as the seed-set experiments to estimate which insects pollinate
 this rare plant.  We deployed seven pollinator stations across sites for
 24-48 hours 10 times between 15 April and 21 June 2019.  Pollinator
 stations consisted of one blue vane trap (vane trap hereafter; colored
 plastic; 37 cm height by 14 cm diameter; left dry; SpringStar) and three
 bee bowls (148 mL polystyrene vials; 10 cm  height by 6 cm diameter;
 Thornton Plastic Co.) painted fluorescent yellow, white or fluorescent
 blue (Royal Exterior Latex Flat House Paint, Ace Hardware Corp., Oak
 Brook, Illinois) filled with soapy water.  We recorded the location, date
 and time we deployed and retrieved pollinator stations, weather, and other
 notes for each sampling event.  Pollinator stations were used to estimate
 the abundance and diversity of pollinators within the <em>T.
 barnebyi </em>population, and to assess which pollinators
 collect<em> T. barnebyi </em>pollen.  We washed (bee bowls
 only) and pinned bees and identified them to genus using Michener et al.
 (1994).  Pollen We identified pollen
 carried on bees to estimate who pollinated <em>T.
 barnebyi</em>.  We collected and identified (Dorn 2001) flowers from
 plant species that were blooming at the same time as <em>T. barnebyi
 </em>and we made a library by mounting pollen grains on slides.  We
 prepared pollen from the scopa or corbicula of individual bees (one hind
 leg for all bees except those with scopa on abdomen, mainly Megachilidae).
 We assumed that the pollen on the scopa or corbicula of bees represented
 the flowers they had visited based on evidence of pollen identified from 6
 body regions of Megachilidae for a rare <em>Penstemon</em>
 (Tepedino et al. 2006).  We performed acetolysis and stained pollen with
 Safranin O from flowers and bees to make features on pollen grains clearer
 before slide mounting (Jones 2014).  We scanned the entire slide to count
 and identify pollen grains from female bees with scopa
 (non-cleptoparasites) or corbicula under a compound microscope at
 200x.  Analyses We used generalized
 linear models (glm), mixed-effects models (lmer) and linear regression
 (lm) to estimate differences among variables.  We estimated differences in
 precipitation, air temperature and cover of <em>T. barnebyi
 </em>using glm analyzed with the Gamma distribution after viewing
 histograms of the data.  Differences in the number, mass and viability of
 seeds were assessed using mixed effects models where plant cluster was the
 random effect, and treatment and site were fixed effects using lme4
 package (Bates et al. 2015).  When a categorical fixed effect had α ≤
 0.05, we calculated estimated marginal means using the emmeans package
 (Lenth 2021) to estimate which treatments or sites differed.  We used
 linear regression to estimate how the number of viable seeds changed with
 the number of pollinators captured and day of year.  We transformed the
 number and mass of seeds using ln(x+1) because they were not normally
 distributed or had non-constant variance.  All analyses were done in
 Program R (R Core Team 2017).
description: All analyses were done in Program R (R Core Team 2017).
description: # Trifolium barnebyi pollination and monitoring ## Description of the data
 and file structure 2021TrifoliumBarnebyiClimateAndMonitoringData.csv
 Monthly temperature in degrees Fahrenheit and total precipitation in
 inches in Lander Wyoming (nearest weather station;
 https://wrcc.dri.edu/cgi-bin/cliMAIN.pl?wy5390); Spring precipitation
 totals in inches; Percent occupied cells in monitoring frame by location;
 All by year 2018-2021 no NA values 2021TrifoliumBarnebyiSeedSet.csv has
 the seed set treatment, number of flowers, number of viable seeds, day of
 year harvested, location of plant. Variables: * Treatment (Bagged =
 pollinators excluded from flowerhead throughout blooming, HP = hand
 pollinated with extra pollen with pollinator access throughout blooming,
 Open = pollinator access throughout blooming); * PlantNo (unique plant
 identifier); * NoFlower (number of flowers on flowerhead); * NoOvules
 (number of ovules in the flowerhead); * OvulesPerFlw (mean ovules per
 flower in the flowerhead); * NoPodsGreaterThan2mm (number of pods wider
 than 2mm in the flowerhead, wide enough to support viable seeds); *
 NoWidePodsPerFlw (mean pods per flower that were wide enough to support
 viable seeds in the flowerhead); * TotalWtOfPods (total weight of pods in
 the flowerhead); * MeanWtPerOvuleInPod (mean weight - in grams - per ovule
 per pod); * NoViableSeeds (number of viable seeds in flowerhead - from
 Tetrazolium test); * TotalWtOfViableSeeds (total weight of viable seeds -
 in grams - in flowerhead); * TotalWtOfViableSeedsUG (total weight of
 viable seeds - in micrograms - in flowerhead); * HPJulianDay (Julian
 Calendar Day of hand pollination of that plant); * Area (location of plant
 in study area); * Trip (time period plant started treatments); *
 NoViableSeedsPerFlw (mean viable seeds pre flower in flowerhead). NA
 values in this table indicate a loss of material for data evaluation (e.g.
 flowerheads blew or washed away). 2021TrifoliumBarnebyiBees.csv has the
 slide/insect number, whether or not pollen was mounted onto a slide from
 that insect, insect taxonomic identification, trapping dates, location of
 traps. Variables: * Slide_ID (unique bee identifier); * MountLegPollen
 (whether pollen from bee was mounted or not); * InsectOrder InsectGenus
 InsectSubgenus InsectSpecies (classification of bee); * Sex (sex of bee);
 * InsectCount (number of identification labels to be made); * Project
 (project bee was collected for); * DeployDate (date - month/day/year -
 blue vane trap was deployed); * DeployTime (24 hour time of day trap
 deployed); * Deploy (combination of DeployDate and DeployTime); *
 RetrievalDate (date - month/day/year - blue vane trap was retrieved); *
 RetreiveTime (24 hour time of day trap retrieved); * Retrieve (combination
 of RetrievalDate and RetreiveTime); * Julian (Julian Calendar Day of trap
 retrieval); * Trip (time period of trap depolyment); * Week (week during
 study of trap deployment); * TimeOut_hr (length of time - hours - of trap
 deployment); * InsectHr (InsectCount divided by TimeOut_hr or mean per
 hour); * Trap (portion of trap bee was in, vane or cups); * Site (site of
 trap deployment); * Location (site and repetition name); * Rep (repetition
 number at that site); * InsectNotes (any notes associated with bee) NA
 values in this table indicate no relevant data
 2021TrifoliumBarnebyiPollen.csv has the slide/bee number, magnification at
 which the slide was examined, Pollen taxonomic identification, and number
 of pollen grains counted. no NA values ## Methods Data was derived from
 the following sources: Monitoring To assess the cover and asexual
 reproduction of T. barnebyi, we monitored five transects annually in July
 between 2018 and 2021. We established two transects at the Weiser Knoll
 site (Weiser Slope and Weiser Seedlings) and the Hall Creek site (Hall 1
 and Hall 2), and one transect at South Red Canyon (Fig. 1). Each transect
 consisted of a belt between 6.9 and 18 m in length where a plot frame (30
 x 30 cm) with sixteen 7.5 x 7.5 cm cells was placed on the ground along
 the belt to assess cover (Fig. 2). We used a plot frame to monitor T.
 barnebyi because the compact and extensive mats of this species make
 distinguishing individuals difficult without destructive sampling. We
 placed the plot frame on one side of the belt at all sites expect South
 Red Canyon and Weiser Knoll Seedling, where we placed the frame above and
 below the belt. We counted the number of cells with T. barnebyi present
 within each frame at 30 cm intervals. Cells were scored as present (1) or
 absent (0) to calculate the percent of cells in which live T. barnebyi
 were present (Supplementary Table 1). Seed-Set Experiments We measured the
 seed production of T. barnebyi to estimate the degree to which pollinators
 may limit sexual reproduction. We selected three areas along the rim of
 Red Canyon where T. barnebyi is most abundant (North Red Canyon, South Red
 Canyon and Weiser Knoll) to measure seed-set. We measured seed-set of
 bagged, open, and hand-pollinated flowers to estimate seed production from
 self- and cross-pollination. We selected 20 T. barnebyi plants from 15
 April through 16 May 2019. Each plant cluster received one of each
 treatment. Bagged treatments restricted pollinator access and measured the
 degree to which flowers can self-pollinate by placing 1 mm2 mesh bags over
 flowers before blooming. Open treatments left blooms accessible to local
 pollinators and measured ambient levels of seed-set. The hand-pollinated
 treatment added excess pollen in addition to local pollinators to measure
 seed production when pollen was not limiting. Pollen came from plants at
 least 50 m away and we delicately brushed collected anthers on the stigma
 of the treatment bloom. Blooms were bagged with mesh bags before (bagged
 treatment) or after (open and hand-pollinated treatments) flowers bloomed
 to contain the developing seeds and were held in place with fishing line
 weighted with color-coded eye bolts. For the hand-pollinated treatment, we
 recorded the number of flowers pollinated, and marked and recorded the
 flowers not ready for pollination in each flowerhead. We recorded the date
 that each flower in the hand-pollinated treatment bloomed and was
 hand-pollinated. We monitored treatments and collected fruits when
 flowerheads were ripe, from 4 June through 17 July 2019. Flowerheads were
 placed in paper bags, returned to the laboratory and dried at room
 temperature. We cleaned, counted, and weighed seed pods and seeds to
 estimate the degree to which T. barnebyi self-pollinated or depended on
 pollinators. Each T. barnebyi flower produced one legume seed pod, and
 there were multiple flowers per flowerhead. We counted the number of
 flowers per flowerhead and the number of ovules per flower using a
 dissecting microscope. We weighed the pods and seeds together, because the
 unfertilized ovules were too small to remove from the pods without
 damaging. We calculated mean seed mass by dividing total mass by the
 number of seeds. We noted seeds that appeared viable by size, and counted
 and weighed seeds for each plant. We tested viability of the seeds using
 Tetrazolium staining, which measures the germinative potential of seeds
 (Lindenbain 1965). We placed the seeds between moistened paper towels for
 24 hours, cut them to expose the endosperm, and immersed them in
 tetrazolium solution for 24 hours. The endosperm of viable seeds turned
 pink or red indicating respiration, while the endosperm of non-viable
 seeds remained white. Pollinators We collected pollinators at the same
 sites as the seed-set experiments to estimate which insects pollinate this
 rare plant. We deployed seven pollinator stations across sites for 24-48
 hours 10 times between 15 April and 21 June 2019. Pollinator stations
 consisted of one blue vane trap (vane trap hereafter; colored plastic; 37
 cm height by 14 cm diameter; left dry; SpringStar) and three bee bowls
 (148 mL polystyrene vials; 10 cm height by 6 cm diameter; Thornton Plastic
 Co.) painted fluorescent yellow, white or fluorescent blue (Royal Exterior
 Latex Flat House Paint, Ace Hardware Corp., Oak Brook, Illinois) filled
 with soapy water. We recorded the location, date and time we deployed and
 retrieved pollinator stations, weather, and other notes for each sampling
 event. Pollinator stations were used to estimate the abundance and
 diversity of pollinators within the T. barnebyi population, and to assess
 which pollinators collect T. barnebyi pollen. We washed (bee bowls only)
 and pinned bees and identified them to genus using Michener et al. (1994).
 Pollen We identified pollen carried on bees to estimate who pollinated T.
 barnebyi. We collected and identified (Dorn 2001) flowers from plant
 species that were blooming at the same time as T. barnebyi and we made a
 library by mounting pollen grains on slides. We prepared pollen from the
 scopa or corbicula of individual bees (one hind leg for all bees except
 those with scopa on abdomen, mainly Megachilidae). We assumed that the
 pollen on the scopa or corbicula of bees represented the flowers they had
 visited based on evidence of pollen identified from 6 body regions of
 Megachilidae for a rare Penstemon (Tepedino et al. 2006). We performed
 acetolysis and stained pollen with Safranin O from flowers and bees to
 make features on pollen grains clearer before slide mounting (Jones 2014).
 We scanned the entire slide to count and identify pollen grains from
 female bees with scopa (non-cleptoparasites) or corbicula under a compound
 microscope at 200x. ## Code/Software We used generalized linear models
 (glm), mixed-effects models (lmer) and linear regression (lm) to estimate
 differences among variables. We estimated differences in precipitation,
 air temperature and cover of T. barnebyi using glm analyzed with the Gamma
 distribution after viewing histograms of the data. Differences in the
 number, mass and viability of seeds were assessed using mixed effects
 models where plant cluster was the random effect, and treatment and site
 were fixed effects using lme4 package (Bates et al. 2015). When a
 categorical fixed effect had 0.05, we calculated estimated marginal means
 using the emmeans package (Lenth 2021) to estimate which treatments or
 sites differed. We used linear regression to estimate how the number of
 viable seeds changed with the number of pollinators captured and day of
 year. We transformed the number and mass of seeds using ln(x+1) because
 they were not normally distributed or had non-constant variance. All
 analyses were done in Program R (R Core Team 2017).