DOI: 10.5061/dryad.ffbg79d6q
title: Influence of sex-organ positions on pollen transfer and self-interference in plants with stylar polymorphisms: An experimental approach using three-dimensional printed flowers

description: Heterostylous plants are characterized by the reciprocal placement of
 stigmas and anthers in the floral morphs. Darwin proposed that this
 arrangement increases the proficiency of disassortative (intermorph)
 pollen transfer. The floral polymorphism may also function to avoid gamete
 wastage by reducing interference between sex organs. Distylous populations
 consist of two morphs: the long-styled morph with stigmas above anthers
 and the short-styled morph with stigmas below anthers. A related floral
 polymorphism – stigma-height dimorphism – involves floral morphs that
 differ in stigma-height but not anther position. We used three-dimensional
 printed artificial flowers, with attached styles and anthers from
 glasshouse-grown Petunia grandiflora, to investigate whether sex-organ
 positioning simulating distyly and two forms of stigma-height dimorphism
 influence patterns of pollen transfer. Artificial flowers were arranged in
 flight cages and pollen transfer within and among flowers was mediated by
 captive bumble bees (Bombus impatiens) from commercial colonies. At the
 end of each trial, we quantified pollen grain number on stigmas and styles
 and the amount remaining in anthers of pollen donors.  We found
 partial support for Darwin’s hypothesis on the probability of pollen
 reaching stigmas of the opposite morph, especially in the S-morph of
 distylous arrays. The number of pollen grains deposited on stigmas was
 influenced by the amount of time bumble bees spent foraging on flowers,
 which was significantly longer in the long-styled morph resulting in
 higher pollen loads in comparison with the short-styled morph.
 Self-interference in the form of self-pollen deposition on stigmas of
 donor flowers depended on the spatial separation of sexual organs. As
 predicted, we found higher self-pollen deposition in stigma-height
 dimorphic than distylous arrays.  Although our results do not
 provide unequivocal support for Darwin’s pollen-transfer hypothesis, they
 confirmed that the morphological arrangement of sexual organs within
 flowers influenced pollen transfer patterns and levels of
 self-interference. They also indicated that variation in sex-organ
 deployment affects bumble bee foraging times with consequences for amounts
 of pollen deposited on stigmas. Although preliminary, our results
 highlight that three-dimensional printed flowers provide future
 opportunities to investigate the functional significance of variation in
 floral design in the context of plant-pollinator interactions.
description: # Influence of sex-organ positions on pollen transfer and
 self-interference in plants with stylar polymorphisms: An experimental
 approach using three-dimensional printed flowers Dataset DOI:
 [10.5061/dryad.ffbg79d6q](10.5061/dryad.ffbg79d6q) ## Description of the
 data and file structure ### Files and variables This dataset and
 accompanying R scripts support an analysis of pollination efficiency in
 experimental arrays simulating different types of stylar polymorphisms
 (e.g., heterostyly, stylar dimorphism). In the experiments, some flowers
 were used as pollen donors and others as pollen recipients. Several
 variables were compared across different "treatments" (types of
 polymorphism), including: * The time bees spent per flower (handling time)
 * The amount of pollen deposited on stigmas and styles of recipient
 flowers * The amount of pollen remaining in the anthers of donor flowers
 The study examines how pollinator behavior affects reproductive metrics
 using generalized linear models (GLMs) and negative binomial models.
 Post-hoc comparisons and visualizations are included. --- **File:
 `Data_3D_2.txt`**\ **Description:**\ Tab-delimited dataset containing the
 experimental data. **Variables (units):** * **Tratamiento**: Type of array
 (simulation of heterostyly, stylar dimorphism, etc.); factor variable
 representing the treatment group. A, donor flower; B-C, receptor flowers
 in distylous arrays when S-morph was donor. D, donor flower; E-F, receptor
 flowers in distylous arrays when L-morph was donor. G, donor flower; H-I,
 receptor flowers in stylar dimorphic arrays. J, donor flower; K-L,
 receptor flowers in relaxed stilya dimorphic arrays. * **Flor**:
 Identifier used to label individual artificial flowers in the experiment;
 factor not used in the analysis. * **Morph**: Flower morph based on the
 stylar dimorphism system: long-style or short-style; factor. *
 **PolenEstg**: Number of pollen grains deposited on stigmas (grains). *
 **PolenEstilo**: Number of pollen grains deposited on styles (grains). *
 **numvisitas**: Number of visits each flower received (number of bee
 entries). * **ttltimeflor**: Total time bees spent on each flower
 (seconds). * **Polentotal**: Total pollen remaining in the anthers of
 donor flowers after trials (number of grains). ## Code/software All
 statistical analyses were performed in R version 4.5.0. (R Core
 Development Team 2025) using the packages: glmmADMB and MuMIn to select
 the random-effect structure lme4 MASS and nlme for the linear and
 generalized effect models car for the overall significance of the effects
 emmeans for multiple comparisons after Type-III analysis of variance
 ggplot2 and gridExtra for graphs representation. **Scripts:** **File:
 `Receptor_analyses.R`**\ **Description:**\ R script used to analyze data
 from recipient flowers and to generate the figures presented in the
 manuscript. GLMs were used to compare pollen loads on stigmas and styles,
 as well as the total time bees spent on flowers across treatments (arrays
 with different polymorphisms). Time spent by bees was also compared
 between flower morphs (short-style vs. long-style). --- **File:
 `Donor_analyses.R`**\ **Description:**\ R script used to analyze data from
 donor flowers and generate corresponding figures for the manuscript. It
 includes comparisons of pollen loads on stigmas and styles, as well as the
 amount of pollen remaining in anthers after the trials, across treatments.
 It is recommended to run the analyses of donor and recipient flowers
 separately.