DOI: 10.5061/dryad.8pk0p2nz2
title: Large floral displays increase self-pollination but do not affect visitation rates in Clarkia concinna

description: Optimal floral display (the number of simultaneously open flowers) is
 frequently viewed as a balance between pollinator attraction and avoidance
 of pollinator-mediated interfloral selfing (geitonogamy). However, the
 most beneficial display size may be affected by pollinator abundance,
 pollinator identity, and other environmental variables. We determined the
 effects of individual floral display and patch size on pollination
 outcomes in natural populations of a mixed-mating annual plant, Clarkia
 concinna. We predicted that individual plants with larger floral displays
 would have both greater pollinator visitation and more geitonogamy.
 Additionally, we tested whether the effects of floral display would be
 greater in smaller patches of plants. Larger individual floral displays
 increased total geitonogamy, but the effect of display on geitonogamy
 differed among pollinator taxa. Visitation rates were lower in small
 patches for some pollinators. However, individual display size did not
 affect per flower visitation rate in small or large patches. Therefore, if
 we only consider the expected tradeoff between pollinator attraction and
 geitonogamy, the number of simultaneously open flowers appears excessive.
 In this species, large floral display size requires explanations beyond
 increased pollinator attraction, such as temporal constraints on flowering
 imposed by a limited growing season and the benefits of additional
 low-quality offspring.
description: In the springs of 2014 and 2015, we conducted five studies on the
 floral display size, patch size, and pollinator assemblage of
 <em>Clarkia concinna</em> in northern Napa, southeastern Lake,
 and northwestern Yolo Counties, California. Here, <em>Clarkia
 concinna </em>grows in mixed serpentine chaparral and riparian
 woodland communities. Mixed serpentine chapparal is dominated here by
 evergreen shrubs such as <em>Arctostaphylos viscida</em>,
 <em>Ceanothus jepsonii, </em>and <em>Quercus
 durata</em>, and includes a <em>Pinus sabiniana
 </em>overstory. Riparian woodlands are comprised of a fairly dense
 but generally low canopy of <em>Populus fremontii, Aesculus
 californiaca, Cercis occidentalis, Salix laevigata </em>and
 additional medium-sized trees, with a shrub layer of <em>Rhus
 trilobata, Rosa californica, </em>and others. These habitats grade
 into each other in locations where the rocky serpentine hillsides meet
 ephemeral or permanent streams. In both habitat types, <em>Clarkia
 concinna </em>populations occur patchily on steep slopes and road
 cuts with shallow soil and limited additional surrounding vegetation. In
 both communities, <em>Clarkia concinna </em>plants grow to
 similar sizes and are visited by similar species—all floral visitors with
 more than five total observations in the study were seen in both chaparral
 and riparian woodlands. Studies 1, 4, and 5 were conducted at 6
 populations in both plant communities (Table 1). Studies 2 and 3 occurred
 at Davis Creek, a very large population (&gt; 100,000 plants) growing
 in riparian woodland in the Donald and Sylvia McLaughlin Natural Reserve.
 All studies in both years were restricted to patches in peak flowering, to
 minimize temporal changes in pollinator abundance (Thompson 1981; 1982).
 Studies 1, 2, and 3 looked for an overall effect of floral display size on
 pollinator attraction and geitonogamy by comparing of pollinator movement,
 pollen analogue (pigment) deposition, and seed traits. Studies 4 and 5
 tested whether the effect of floral display size depended on patch size by
 comparing visitation and pollen deposition between small and large
 patches. <em>Clarkia concinna </em>is a
 self-compatible annual plant native to a variety of habitats throughout
 California’s Northern Coast Ranges with disjunct occurrences in the
 Northern Sierra Foothills. Individuals produce 1 to more than 100
 four-merous, fuchsia-colored, lobed and clawed flowers (average of 6 in
 natural populations) on leafy compound spikes. Flowers are protandrous and
 self-compatible, and fruits are elongate capsules containing an average of
 about 50 ovules (range 1-124; Groom 1998). Seeds<em>
 </em>germinate at the beginning of the winter rains and plants bloom
 for a few weeks in May or June. Across the species range, mean
 precipitation during its growing season (October-June) varies from
 approximately 500 mm in the southeast to 1800 mm in the
 northwest. Study 1) How does floral display size affect
 pollinator movement? In order to compare pollinator
 movement among plants with different floral display sizes, we observed
 floral visitors to <em>C. concinna </em>during peak flowering
 in 2014 (May 21<sup>st</sup> through June
 8<sup>th</sup>). Observations took place throughout the day
 7:40-19:20. Each observation began with a visitor arriving at a new plant
 and ended when it left the patch or stopped foraging for approximately 15
 seconds. A visit included any contact with the interior surface of the
 corolla or reproductive parts. Most observed visitor taxa were found to be
 effective pollinators in a previous study (Miller <em>et
 al.</em> 2014). We counted the number of flowers visited per plant
 after the initial flower was visited, giving the total visits for which
 geitonogamy could occur (consecutive visits). Plants with one open flower
 could not have consecutive visits and were not included. We used numbered
 flags to temporarily mark visited plants and counted the number open
 flowers per plant (floral display size) after the completion of the
 foraging bout. Rare instances when visitors returned to the same flower or
 plant after visiting elsewhere were counted as separate visits. We
 identified all visitors to genus. Visitor preference for either male or
 female phase flowers could reduce the effectiveness of consecutive visits
 as a measure of geitonogamy. In initial observations, we generally did not
 observe such bias in flower choice except for two small solitary bee
 genera, <em>Cerotina</em> and
 <em>Lasioglossum</em>. These bees were uncommon visitors that
 exclusively pollen-collected on male-phase flowers. Emasculation did not
 reduce visitation in a study of the related <em>Clarkia
 xantiana</em>, supporting the idea that visitors do not cue in on
 flower phase (Moeller <em>et al.</em> 2012). Floral display
 size and consecutive visit data were approximately lognormally
 distributed, and were log transformed. We tested for a positive linear
 relationship between the log of floral display size and the log of
 consecutive visits. To test for variation among pollinator species in this
 relationship, we selected the eight pollinator genera with more than 50
 observations. We compared the log of consecutive visits made by visitors
 among these genera using an ANCOVA with log of display size as the
 covariate. Study 2) How does floral display size affect
 pollen deposition? Although high numbers of
 within-plant transitions strongly indicate the potential for geitonogamy,
 high pollen carryover or erratic pollinator behavior may reduce the
 relative amount of within-plant pollen transfer (Geber 1985). We compared
 the amounts of self and outcross pollen movement across floral display
 sizes by manipulating display and comparing the deposition of fluorescent
 pollen pigment. From May 18<sup>th</sup> through June
 7<sup>th</sup>, 2014, we selected 140 focal plants in large
 patches (greater than 250 open flowers) that had at least 6 male phase and
 1 female phase flowers. We marked the most recently female phase flower on
 the main stem (highest) with thread and removed any other female phase
 flowers on the plant by pinching off the perianth just above the ovary. We
 chose plants with no prior pollen deposition on the focal flower stigma.
 Pollen in this species is large and colorful enough in this species that
 we could observe deposition with a hand lens. We varied floral display
 size by leaving 1, 2, 4, or 6 of the male phase flowers on the focal plant
 and removing additional flowers. Although large plants can have greater
 than six simultaneously open male-phase flowers, the range of treatment
 display sizes was comparable with most natural display sizes. We used a
 pipe cleaner to brush all anthers of the remaining male-phase flowers with
 blue, orange, green, yellow, or pink fine-grained florescent powdered
 pigment (self pigment; <em>Series JST-300</em><em>,
 </em><em>Radiant Color</em>, Richmond, CA,<em>
 USA</em>). We coated the anthers of the 50 nearest male phase
 flowers with a different color of pigment (outcross pigment). The
 surrounding treated flowers were within a one-meter radius of the focal
 plant. Replicates were at least 10 meters apart and pigment colors were
 changed between adjacent replicates. The presence of pigment did not
 appear to affect visitation—we observed all common visitors foraging
 freely on treated flowers. Treatments were initiated between 7:00 and
 12:00. After 6-10 hours, we collected the focal stigma
 and quantified the amount of self and outcross pigment deposition the same
 evening using a dissecting microscope. There was no effect of time on
 pollen deposition (r<sup>2 </sup>&lt;0.01, N = 136, P =
 0.81) Three stigmas were lost or eaten, resulting in sample sizes per
 treatment of 34 or 35. We separately ranked focal and surrounding pigment
 deposition as: no pigment (0), 1 fluorescent grain to approximately 5%
 stigma surface covered (1), 5% to 20% coverage (2), 20% to 40% coverage
 (3), and &gt;40% coverage (4). Pigment as a pollen surrogate may
 overestimate pollen movement—its smaller size may result in higher amounts
 of wind transfer (Thompson <em>et al.</em> 1986) However most
 pigment deposition was associated with pollen deposition, indicating the
 pigment is an adequate pollen surrogate. We tested whether the proportion
 of stigmas receiving any pigment depended on display size with
 c<sup>2 </sup>contingency test. For stigmas that received
 pigment (N = 105), we compared self and outcross pigment scores among
 display sizes using Kruskal-Wallis and post-hoc Steel-Dwass All Pairs
 tests (the nonparametric equivalent of a Tukey test).
 Study 3) How does floral display size affect seed size and
 number? If large floral displays increase the
 deposition of self-pollen, then floral display size may be negatively
 correlated with offspring quantity (fewer seeds matured) or quality
 (smaller seeds) due to early-acting inbreeding depression. Alternatively,
 plants may be able to discriminate against self pollen, and display size
 will not affect seed set (Jones 1994). We tested whether floral display
 size affects seed weight or number, by selecting 130 total plants on May
 19<sup>th</sup> and May 30<sup>th</sup>, 2014.
 Focal plants were separated by at least 5 meters and had one unpollinated
 freshly-female phase flower (confirmed with a hand lens) and 6 or more
 male phase flowers. On each plant, we removed all other female phase
 flowers and left 0, 1, 2, 4, or 6 male phase flowers. We also removed buds
 that were likely to bloom while the focal flower was still open. Because
 plant size and flower position affect seed traits in this species, we
 measured the basal stem diameter with calipers (a proxy for size), and the
 position of each focal flower along the main stem (with position 1 being
 the lowest and first to bloom). After corollas senesced, we placed a small
 piece of transparent adhesive tape around the top of the capsule to
 prevent seed loss. Mature fruits were collected on June
 25<sup>th</sup>, 2014, and ripe seeds were counted and weighed
 collectively after air-drying. Twenty-four fruits were missing or eaten
 prior to collection, resulting in per-treatment sample sizes between 19
 and 23 plants. We compared weight per seed and seed number among floral
 display size classes with an ANCOVA, using stem diameter and stem position
 as covariates. Study 4) How does patch size affect the
 pollinator environment? The previous three studies were
 conducted in relatively large patches of plants (hundreds to thousands of
 individuals). However, the relationship between display size, visitation,
 and geitonogamy may depend on patch size. Therefore, we tested for an
 effect of patch size on visitor frequency and identity with an
 observational study. During peak flowering in 2015 (May
 2<sup>nd</sup> to May 19<sup>th</sup>), we
 observed groups of 20 flowers centrally located in varying patch sizes for
 121 20-minute observation periods between 10:00 and 16:30. Patches ranged
 from 20 to approximately 10,000 open flowers, and we did not reuse the
 same patch on the same day. Patches were groups of <em>Clarkia
 concinna</em> separated from others by at least ten meters. We
 identified all visitors to genus and counted the total flower visits and
 visits per taxa. We used a generalized linear model (GLM) to test for a
 relationship between the log of patch size and total visits. We also
 included observation date as a predictive variable. We modelled visits
 with a Poisson distribution and a log link function. In addition to total
 visits, we ran separate GLMs for <em>Apis </em>and
 <em>Bombylius</em>, the two taxa with more than 100 visits in
 the data set. Finally, for observation periods with at least one visitor
 (N = 87) we conducted a regression analysis of the Shannon-Weiner species
 diversity index against the log of patch size, including date as a random
 variable. Study 5) Do the effects of floral display
 size depend on patch size? If pollinators are less
 abundant in small patches, plants with small displays may be pollen
 limited in small, but not large patches. We manipulated both patch size
 and floral display size to test for a significant interaction between
 these variables. From May 2<sup>nd</sup> to May
 17<sup>th</sup>, 2015, we selected single unpollinated
 (confirmed with a handlens) freshly-female flowers on a total 204 focal
 plants with at least 6 male-phase flowers. For half the plants, we created
 very small patches (&lt; 50 additional open flowers within 2 meters of
 the focal plant, mean 14.8, s.d. = 8.9). In order to avoid excessive
 destruction of plants, we used natural patchiness in plant distribution,
 supplemented by thinning excess plants within a 10-meter radius of a focal
 plant. Other focal plants were chosen in large patches (&gt; 250
 flowers).  Focal plants in small and large patches received one of 3
 display treatments: 1) all additional open flowers and large buds removed,
 2) six unmanipulated male-phase flowers left on the plant, or 3) six
 male-phase flowers left on the plant, but emasculated by removing the
 anthers with forceps. For the treatments with six unmanipulated and six
 emasculated flowers, we maintained the display size until the focal flower
 senesced by removing flowers as they aged and controlling the number of
 newly opening flowers daily, emasculating them in bud in the emasculated
 treatment. Unlike studies 2 and 3, we emasculated the focal flower,
 ensuring any pollen deposition was pollinator mediated. Differences
 between the emasculated large display treatment (3) and the small display
 treatment (1) are attributable to differences in display attraction.
 Differences between the emasculated large display treatment (3) and the
 unmanipulated large display treatment (2) are due to within plant
 pollinator movement. An equal number of replicates per
 treatment (2 or 3) were initiated on each of 13 days between May
 2<sup>nd</sup> to May 17<sup>th</sup>. Replicates
 were set up between 7:00 and 10:00, before the bulk of pollinator
 activity, and ran until the corolla senesced (1-5 days). Concurrently
 running replicates were spaced more than 10 meters apart. Four focal
 flowers were eaten, but they were replaced the next day, resulting in even
 sample sizes of 34 per treatment. We checked focal flowers for pollen
 deposition with a hand lens and corolla senescence 3 times per day,
 between 7:00 and 9:00, between 12:00 and 14:00, and between 17:00 and
 19:00. We calculated the time to initial pollen deposition and time to
 corolla senescence to the nearest third of a day. Once the corolla wilted,
 we collected the stigma. The same evening, we applied basic fuchsin jelly
 (Kearns and Inouye 1993) to a collected stigma, heated it, squashed it
 between two microsope slides, and counted the stained pollen grains under
 a dissecting microscope. We used Cox Proportional Hazards to test for an
 effect of patch size, display size, and their interaction on time to
 pollen deposition and time to senescence. We also included date as an
 independent variable, as among-day differences in weather and location
 could affect the response variables. We conducted an ANOVA to test for an
 effect of patch size, display size, their interaction, and the random
 effect of date on the number pollen grains deposited on focal
 stigmas.
description: # Large floral displays increase self-pollination but do not affect
 visitation rates in Clarkia concinna
 [https://doi.org/10.5061/dryad.8pk0p2nz2](https://doi.org/10.5061/dryad.8pk0p2nz2) ## Description of the data and file structure We determined the effects of individual floral display and patch size on pollination outcomes in natural populations of a mixed-mating annual plant, *Clarkia concinna*. We conducted a series of 5 studies. We tested for a tradeoff between pollinator attraction and geitonogamy avoidance by comparing total visitation rates and geitonogamy across floral display sizes. We predicted that larger floral displays would have both higher per-flower visitation rates and increased geitonogamy. We then investigated how patch size may affect this tradeoff. We predicted that flowers in small patches would have lower visitation rates from fewer pollinator taxa. Additionally, we tested whether patch size changes the effects of floral display size. We factorially manipulated patch size and floral display size and tested for differences in pollination success. We predicted that large floral displays would increase visitation more in small than large patches. ### Files and variables #### File: Floral\_display\_and\_geitonogamy\_data\_study\_1.csv **Description:** Data for study 1 ##### Variables obs: row number genus: name of the insect or bird genus date: date observation occurred in yearmonthday format. All observations took place in May and June of 2014 time: time of day the observation occurred using a 24 hour clock hour:minute format ind: an identification number of individual floral visitor. Numbers repeat for each genus. Each individual may have visited multiple plants on one foraging bout and is therefore represented on multiple rows fl visits: the number of flowers the floral visitor visited consecutively on one plant fl total: the total number of open flowers (both male and female phase) on one plant notes: comments on species identity or visitor behavior #### File: Floral\_display\_and\_geitonogamy\_data\_study\_2.csv **Description:** Data for study 2 ##### Variables replicate: unique identifying number associated with each plant date: date the plant was treated with fluorescent pigment in yearmonthday format. All observations took place in May and June of 2014 \# flrs on focal plant: the number of male phase flowers left on the plant after the treatment (removal of all other flowers) was applied. All focal plants had one open female phase flower with no pollen visible on the stigma target dye color: color of fluorescent pigment applied to the anthers of all the remaining male phase flowers of the focal plant surr dye color: color of fluorescent pigment applied to the anthers of the fifty male-phase flowers nearest the focal plant. All treated flowers were within 1 meter of the focal plant time out: time of day the treatment was initiated in a 24 hour clock using a 24 hour clock hour:minute format time coll: time of day the stigma from the focal flower was collected using 24 hour clock hour:minute format dye receipt?:  categorization of whether or not the focal stigma received any fluorscent pigment target dye: rank categorization of the amount of pigment coverage that matched the color of the pigment applied to the male phase flowers on the focal plant. 0 = no pigment, 1 = 1 fluorscent grain to approximately 5 % of the stigma surface, 2 = 5-20% coverage, 3 = 20-40% coverage, 4 = &gt; 40% coverage surr dye: rank categorization of the amount of pigment coverage that matched the color of the pigment applied to the male phase flowers on the 50 nearest male phase flowers. 0 = no pigment, 1 = 1 fluorscent grain to approximately 5 % of the stigma surface, 2 = 5-20% coverage, 3 = 20-40% coverage, 4 = &gt; 40% coverage location: brief discription of the location of the flower patch. Contact author for more specific site descriptions. notes: any additional notes on location and observations #### File: Floral\_display\_and\_geitonogamy\_data\_study\_3.csv **Description:** Data for study 3 ##### Variables replicate: unique identifying number associated with each plant \# flrs: number of male phase flowers that were left on the plant after removing all other open or nearly open flowers. All plants had one focal flower that was in female phase. st dia: diameter of the plant main stem in millimeters, measured with calipers at the base of the main stem of the plant fl l: the length of the focal flower corolla in millimeters. Petals were stretched flat and measured with a ruler from the tip of the top petal to the tip of the bottom petal fl sta: the length of the focal (female phase) flower stamen (filiment plus anther) in millimeters, measured with a ruler from the top of the hypanthium to the tip of the longest anther fl sty: the length of the focal (female phase) flower style in millimeters, measured with a ruler from the top of the hypanthium to the base of the stigma fl 50cm: count of the number of male phase flowers on plants rooted within 50 centimeters of the focal plant, not including flowers on the focal plant stem pos: the position along the main stem of the focal (female phase) flower, with 1 representing the most basal position of a flower (inclusive of previously senesced flowers). Explanation for NA can be found in the notes column. \# fruits (fertilized): total number of fruits on the entire focal plant that contained fertilized seeds. Explanation for NA can be found in the notes column. seed #: total number of fertilized seeds in the fruit of the focal flower. Seeds were collected and counted once mature, and determined to be fertilized if they were larger than immature seeds and a dark brown in color. Explanation for NA can be found in the notes column. unfert seeds: total number of unfertilized seeds in the fruit of the focal flower. Seeds were collected and counted once mature, and determined to be unfertilized if they were the same size as immature seeds or a light brown in color. Explanation for NA can be found in the notes column. seed weight total (collective): weight of all fertilized seeds, measured in grams on a lab balance. Explanation for NA can be found in the notes column. location: brief discription of the location of the flower patch. Contact author for more specific site descriptions. date established: date the plant received the treatment of flower removal in yearmonthday format. All treatments were started on either May 19th or May 30th, 2014 collected June 25?: filter variable on whether the fruit from the focal flower was collected. Blank rows did not have fruits that were collected notes: additional information on flower position or explaination for why the fruit was not collected and data for fruit and seeds are missing and replaced with NA. NA means no notes because no missing data for that plant. #### File: Floral\_display\_and\_geitonogamy\_data\_study\_4.csv **Description:** Data for study 4 ##### Variables replicate: unique identifying number associated with each observation period. All observations were made on 20 open flowers location: brief discription of the location of the flower patch. Contact author for more specific site descriptions. date: date the plant was treated with fluorescent pigment in yearmonthday format. All observations took place in May and June of 2014 columns D-O: column headers are the name of the insect or bird genus observed. Values are the number of individual visits made by that genus to one of the 20 focal flowers during the 20 minute observation period Total Visits: sum of the individual visits made to one of the 20 focal flowers during the 20 minute observation period patch size: estimate of the total number of open flowers in the entire focal patch, including the 20 focal flowers. Patches were seperated from other patches or individual plants with open flowers by at least 10 meters. Patch sizes of 20-~100 were estimated to the nearest 10 flowers. Patch sizes of ~100-~300 flowers were estimated to the nearest 50 flowers. Patch sizes of ~300-~1000 flowers were estimated to the nearest 100 flowers. Patch sizes of ~&gt;1000 flowers were estimated to the nearest 500 flowers. #### File: Floral\_display\_and\_geitonogamy\_data\_study\_5.csv **Description:** Data for study 5 ##### Variables replicate: unique identifying number associated with each observation period. All observations were made on 20 open flowers location: brief discription of the location of the flower patch. Contact author for more specific site descriptions.  specifics: additional information on the location of the flower patch. Contact author for more specific site descriptions. There was only one site at Research Hill, so we recorded NA to indicate there are no specifics for this site. date: date the plant was treated by removing flowers/anthers in yearmonthday format. All observations took place in May and June of 2014 display treatment: type of treatment given to the focal plant. All focal flowers were emasculated to ensure any pollen deposition was visitor-mediated. 1 = only focal flower was left on focal plant. 6 = the focal flower and 6 unmanipulated male phase flowers were left on the focal plant. 6e = the focal flower and 6 emasculated flowers were left on the focal plant. Treatments were maintained until the focal flower wilted patch size: category of patch of the focal plant. Patches were seperated from other patches or individual plants with open flowers by at least 10 meters. Large = &gt;250 open flowers. Small = &lt; 50 open flowers. \# flrs: total number of flowers open in the patch in addition to the flowers found on the focal plant. The number was recorded only for small patches. NA means missing flower counts for large patches. st dia: diameter of the plant main stem in millimeters, measured with calipers at the base of the main stem of the plant time out: time of day the treatment was initiated in a 24 hour clock using a 24 hour clock hour:minute format t to dep: "length of time until pollen was observed with a hand lens on the stigma of the focal flower. Focal flowers were checked 3 times a day: between 7:00 and 9:00, between 12:00 and 14:00, and between 17:00 and 19:00." t to wilt: "length of time until the focal flower senesced and the stigma was no loger accessable to visitors. Stigmas were collected at this time. Focal flowers were checked 3 times a day: between 7:00 and 9:00, between 12:00 and 14:00, and between 17:00 and 19:00." grains: "total number of pollen grains on the stigma of the focal flower. The evening of collection, we applied basic fuchsin jelly (Kearns and Inouye 1993) to the stigma, heated it, squashed it between two microsope slides, and counted the stained pollen grains under a dissecting microscope" notes: additional information about patterns of pollen deposition or other details. NA means no notes. #### File: Floral\_display\_and\_geitonogamy\_metadata.csv **Description:** Column headers for all the other files in .csv format ## Code/software Data can be viewed in any text editor or spreadsheet program.