DOI: 10.5061/dryad.1c59zw3zj
title: Characterizing each step of pollination in the wildflower, Phlox drummondii, reveals a single butterfly species predominates in the pollinator assemblage

description: Premise: A central goal of pollination biology is to connect plants with
 the identity of their pollinator(s). While predictions based on floral
 syndrome traits are extremely useful, direct observation can reveal
 further details of a species’ pollination biology. The wildflower, Phlox
 drummondii, has a floral syndrome consistent with pollination by a variety
 of lepidoptera. We describe pollination in P. drummondii and use empirical
 data to test this prediction. Methods: We directly observe each step of
 the pollination process in P. drummondii. First, we observe 55.5 hours of
 floral visitation throughout the day/evening (7:00-20:30) at sites across
 the species range. We use a temporal pollinator exclusion experiment to
 determine the contribution of diurnal and nocturnal pollination to
 reproductive output. We then quantify P. drummondii pollen pickup and
 deposition by the dominant floral visitor, Battus philenor. Finally, we
 test the effect of B. philenor visitation on P. drummondii reproductive
 output by quantifying fruit set following visitation to greenhouse-grown
 flowers. Results: B. philenor is the primary pollinator of P. drummondii.
 Pollination is largely diurnal, and we observe a variety of lepidopteran
 visitors during this period. However, B. philenor is by far the most
 frequent visitor, representing 88.5% of all observed floral visits. We
 show that B. philenor is not only an extremely common visitor but also an
 effective pollinator by demonstrating that individuals transfer pollen
 between flowers and that a single visit can elicit fruit set. Conclusion:
 Our data are consistent with the syndrome prediction of lepidopteran
 pollination and further reveal a single butterfly species, B. philenor, as
 the primary pollinator. Collectively, our study demonstrates the
 importance of empirical pollinator observations, adds to our understanding
 of pollination mechanics, and offers a specific case study of butterfly
 pollination.
description: Study species: <em>P. drummondii</em> (Polemoniaceae)
 is an annual herb native to central Texas that occurs along roadsides,
 fields, and pastures. Seeds germinate in early spring and individuals
 flower and fruit from April through June. Flowers are radially symmetric
 with a diameter of 18 to 24mm (Figure 1A,B) (Wherry 1955). Floral buds
 open in the morning (07:00-9:00) in both field and greenhouse conditions.
 If unpollinated, open flowers last approximately 2-4 days before wilting.
 Fruits begin developing 1-2 days after pollination and each flower can
 produce a single fruit. <em>P. drummondii</em> has an active
 self-incompatibility system and displays low rates of autonomous selfing
 (~1%) (Roda and Hopkins 2019). Previous studies within
 the <em>Phlox</em> genus demonstrate visitation by a broad
 group of lepidopteran pollinators (Strakosh and Ferguson 2005; Wiggam and
 Ferguson 2005). Like other members of its genus, <em>P.
 drummondii</em> exhibits floral traits consistent with a
 lepidopteran pollination syndrome (Faegri and Van Der Pijl 1979; Grant and
 Grant 1965; Stebbins 1970). Flowers produce nectar at the base of a
 narrow, fused, tubular corolla ranging in length from 13 to 17mm (Wherry
 1955). Sexual organs (stigma and anthers) are fully inserted within the
 corolla tube. Brightly colored petals are light-blue/purple throughout
 most of the species range and emit a faint fragrance (Hopkins and Rausher
 2012).  Observing floral visitation: To characterize
 the diversity and frequency of floral visitors, we conducted 55.5 hours of
 direct pollinator observations. We observed visitation throughout the
 day/evening (07:00 to 20:30). Coinciding with peak bloom, observations
 were conducted from April 23, 2022 to May 4, 2022. We identified three
 focal populations for pollinator observations throughout Central Texas
 (Appendix S1). These focal study sites were located on rural roadsides or
 in a protected meadow at the Brackenridge Field Station. At each site, we
 delimited a patch area ranging from 30 to 140 square meters. Observers
 stood just outside the patch boundary for 1 hour observation periods and
 noted every animal interacting with flowers inside the study patch.
 Visitors were recorded if any part of the animal’s body inserted into the
 corolla tube opening. Species identity was assessed visually. We conducted
 a handful of additional observation hours near range edges to assess
 potential variation in pollinator assemblage across space (Appendix
 S1). Temporal pollinator exclusion: We used temporal
 pollinator exclusion to assess the contribution of diurnal pollination,
 nocturnal pollination and autonomous selfing towards overall reproductive
 output. In April 2022, a population of <em>P. drummondii</em>
 was identified at the Brackenridge Field Laboratory in Austin, TX (30.2832
 N, -97.7801 W) near the geographic center of the species range (Appendix
 S1). Throughout an approximately 100m<sup>2</sup> area, 86
 experimental plants were randomly chosen and assigned to one of four
 pollinator treatments: total exclusion (n=15), diurnal exclusion (n=23),
 nocturnal exclusion (n=23), and no exclusion (n=25). All open flowers and
 fruits were removed before covering plants in fine-mesh cages to exclude
 pollinators of all sizes. At 07:00 each morning, fine-mesh cages were
 removed from the nocturnal exclusion group. At 19:00 each evening,
 fine-mesh cages were removed from the diurnal exclusion group. The total
 exclusion group remained covered while the no exclusion group remained
 uncovered. After five days, new but unopened buds were removed and all
 flowers that opened during the experiment were counted (as measured by
 remaining calyx number). All plants were then covered with exclusion cages
 and monitored for fruit set. We counted the number of fruits on days five
 and eight after beginning treatment. On day eight, we observed minor plant
 death (n=84 live plants remaining) and caterpillar herbivory resulting in
 the removal of entire inflorescences leading to some uncertainty in final
 reproductive success. We implemented a binomial mixed model using the lme4
 R package (v1.1-30) to model fruit set in our exclusion conditions (Bates
 et al. 2015). Each individual flower that opened during the experiment was
 represented in the data set as either fruit present or fruit absent. 124
 flowers were recorded in the total exclusion treatment, 309 in the diurnal
 exclusion treatment, 235 in the diurnal treatment and 250 in the no
 exclusion treatment. Because our dataset includes multiple flowers from
 the same individual, we included plant individual as a random effect in
 the model. To compare fruit set across treatment groups, we conducted
 pairwise post-hoc Tukey-Kramer tests using the glht() function in the
 multcomp R package (v1.4-20) (Hothorn, Bretz, and Westfall
 2008).  Quantifying pollen transfer: To determine if
 visitors transfer pollen between flowers, we assessed both pollen pickup
 and deposition by insect visitors. We characterized pollen pickup by
 capturing insects foraging on roadside populations of <em>P.
 drummondii</em> and quantifying pollen grains on proboscises. These
 insects were captured outside of patches used for observation and
 represented a small fraction of all insects observed in the area. Because
 <em>Battus philenor</em> was by far the most frequent visitor,
 most insects captured and included in our analysis were <em>B.
 phileno</em>r (n=24). We collected at least one representative
 individual of each pollinator type observed visiting <em>P.
 drummondii </em>(n=1 <em>Hyles lineata</em>; n=4 Skipper
 spp.; n=2 ) except for the Common Buckeye (Junonia coenia) which was only
 seen making a single floral visit throughout all observation
 periods. After capture, insects were immediately
 immobilized in a glassine envelope and placed on ice. Specimens were
 stored at -20°C. Following twenty-four hours in a humidifying chamber to
 increase pliability, we pinned each specimen through the thorax onto
 glassine covered Styrofoam. The proboscis was carefully unrolled with a
 clean pin and length was measured. To remove pollen from the proboscis for
 counting, the dorsal, ventral and lateral sides of the unrolled proboscis
 were rubbed with a gelatin-glycerin cube (Kearns and Inouye 1993). We
 melted the gelatin-glycerin cube onto a microscope slide and imaged the
 entirety of the melted gelatin in brightfield at 2.5x magnification using
 the Zeiss Axioimager (Jena, Germany). Pollen grain number per image was
 quantified automatically using ImageJ and confirmed by manual count
 (Schindelin et al. 2012). Based on the literature and
 extensive experience observing <em>Phlox</em> pollen using a
 light microscope, we identified pollen grains matching
 <em>Phlox</em> pollen’s distinctive morphology in size and
 shape (Wherry 1955; Roda and Hopkins 2019; Suni and Hopkins 2018; Buthod
 and Skvarla 2014). Pollen grains consistent with
 <em>Phlox</em> pollen morphology were included in our
 quantifications. Another <em>Phlox</em>, <em>P.
 cuspidata</em>, is found geographically near, although not at, some
 of our study sties. Because of shared pollen morphology, distinguishing
 between pollen of these species is challenging. However, we do not expect
 this to present an issue in our analyses as <em>P.
 cuspidata</em> is highly selfing with low pollinator visitation
 (Levin 1989). We assessed <em>B.
 philenor</em> pollen deposition per visit by allowing wild-caught
 individuals to visit flowers in succession on greenhouse grown plants
 maintained indoors. After each single visit, corollas were removed and
 stigmas were collected, fixed in 80% isopropanol, and stored at 4°C (n=20
 unvisited; n=28 visited by <em>B. philenor</em>). We removed
 corollas and collected stigmas from unvisited flowers as a control.
 Stigmas were then mounted in glycerol media on a slide and imaged in
 brightfield at 10x magnification using the Zeiss Axioscope 7 (Jena,
 Germany). Pollen grain number per image was quantified using ImageJ
 (Schindelin et al. 2012). We used a linear model implemented in the lme4 R
 package (v1.1-30) to test for an effect of <em>B.
 philenor</em> visitation on the number of pollen grains on
 stigmas. Although quantifying pollen transfer by each
 visitor type would be informative, the low visitation rates of
 non-<em>Battus</em> insects made capturing other visitors
 extremely challenging. We were unable to gain adequate sample sizes of
 non-<em>Battus</em> visitors to assess pollen transfer despite
 extensive time in the field. Quantifying pollination
 efficiency: To assess whether <em>B. philenor</em> visitation
 affects reproductive output, we performed a controlled foraging
 experiment. Plants were grown from seed in the greenhouse and maintained
 indoors to exclude pollinators. We captured <em>B.
 philenor</em> foraging on natural populations of <em>P.
 drummondii</em> and allowed them to forage on a ‘pollen donor plant’
 while inside of a mesh cage. Because <em>B. philenor</em> is a
 generalist, wild-foraging insects likely carry some heterospecific pollen
 which may affect overall pollination efficiency. To capture this effect in
 our measurement of efficiency, insects were not cleaned prior to
 controlled foraging. Next, we presented the butterfly
 with a single, new plant and noted the first three flowers visited in
 succession by the butterfly (geitonogamous visitation). Corollas were
 removed following <em>B. philenor</em> visitation and calyxes
 were labeled with tape. On the same plant, we removed corollas from three
 unvisited flowers as a control for autonomous selfing. Additionally, we
 manually applied pollen from the ‘pollen donor plant’ to stigmas of three
 open flowers. Pollen donor and experimentally visited plants were from
 independent genetic lines grown in our lab, to reduce the likelihood of
 sharing incompatibility genotypes. We included 18 plants in our
 experiment. Following pollinator exposure, plants were maintained in the
 greenhouse and monitored for fruit set for one week. We used a binomial
 mixed model using the lme4 R package (v1.1-30) to model fruit set across
 the five pollination treatments (manual outcross, autonomous selfing,
 first, second, and third flower visited by <em>B.
 philenor</em>) (Bates et al. 2015). We included plant individual as
 a random effect in the model. To compare fruit set across treatment
 groups, we conducted pairwise post-hoc Tukey-Kramer tests using the glht()
 function in the multcomp R package (v1.4-20) (Hothorn, Bretz, and Westfall
 2008).