DOI: 10.5061/dryad.fttdz091s
title: Data from: Mismatch between pollen and pistil size causes asymmetric mechanical reproductive isolation across Phlox species

description: Characterizing the mechanisms of reproductive isolation (RI) between
 lineages is key to determining how new species are formed and maintained.
 In flowering plants, interactions between the reproductive organs of the
 flower –the pollen and the pistil– serve as the last barrier to
 reproduction before fertilization. As such, these pollen-pistil
 interactions are both complex and important for determining a suitable
 mate. Here, we test whether differences in style length (a part of the
 pistil) generate a postmating prezygotic mechanical barrier between five
 species of perennial Phlox wildflowers with geographically overlapping
 distributions. We perform controlled pairwise reciprocal crosses between
 three species with long styles and two species with short styles to assess
 crossing success (seed set). We find that heterospecific seed set is
 broadly reduced compared to conspecific cross success and reveal a
 striking asymmetry in heterospecific crosses between species with
 different style lengths. To determine the mechanism underlying this
 asymmetric reproductive isolating barrier we assess pollen tube growth in
 vivo and in vitro. We demonstrate that pollen tubes of short-styled
 species do not grow long enough to reach the ovaries of long-styled
 species. We find that short-styled species also have smaller pollen and
 that both within and between species pollen diameter is highly correlated
 with pollen tube length. Our results support the hypothesis that the small
 pollen of short-styled species lacks resources to grow pollen tubes long
 enough to access the ovaries of the long-styled species, resulting in an
 asymmetrical, mechanical barrier to reproduction. Such reproductive
 isolating mechanisms, combined with additional pollen-pistil
 incompatibilities, may be particularly important for closely related
 species in geographic proximity that share pollinators.
description: ## Paper: Mismatch between pollen and pistil size causes asymmetric
 mechanical reproductive isolation across *Phlox* species Authors: Anna F.
 Feller, Grace Burgin, Nia Lewis, Rohan Prabhu, Robin Hopkins
 [https://doi.org/10.5061/dryad.fttdz091s](https://doi.org/10.5061/dryad.fttdz091s) This dataset contains the raw data files from experiments (described in detail in the paper) to assess seed set, floral morphology, pollen size, and pollen tube growth in and between six closely related species of *Phlox* flowers, as well as the R code files used to visualize and statistically analyze these data.  ## Description of the data and file structure **Data files:** * ids.csv - contains information on all individuals used in the experiments to assess seed set, floral morphology, pollen size, and pollen tube growth * *ID:* individual ID, composed of 'species abbreviation-population number-individual number' * *species*: scientific species name (without the preceding genus name '*Phlox*') * *population ID*: population ID, composed of 'species abbreviation-population number' * *species abbreviation* - abbreviation of the species name (DIV: *Phlox divaricata*, GLA: *Phlox glaberrima* (ssp. interior), MAC: *Phlox maculata*, PAN: *Phlox paniculata*, PIL: *Phlox pilosa* (ssp. pilosa, North) * *lat*: latitude * *lon*: longitude * Seed_Set_20231116.csv - contains the results of the crossing experiment to assess seed set * *female individual ID*: the individual used as the pollen receiver in a cross (the ID is composed of 'species abbreviation-population number-individual number') * *female species abbr*.: abbreviation of the species name of the individual used as the pollen receiver (DIV: *Phlox divaricata*, GLA: *Phlox glaberrima* (ssp. interior), MAC: *Phlox maculata*, PAN: *Phlox paniculata*, PIL: *Phlox pilosa* (ssp. pilosa, North) * *male individual ID*: the individual used as the pollen donor in a cross (the ID is composed of 'species abbreviation-population number-individual number').  * *male species abbr*.: abbreviation of the species name of the individual used as the pollen donor (DIV: *Phlox divaricata*, GLA: *Phlox glaberrima* (ssp. interior), MAC: *Phlox maculata*, PAN: *Phlox paniculata*, PIL: *Phlox pilosa* (ssp. pilosa, North) * *cross name*: 'ID of individual used as the pollen receiver x ID of individual used as the pollen donor' * *cross*: 'abbreviation of the species name of the individual used as the pollen receiver x abbreviation of the species name of the individual used as the pollen donor' * *cross type*: specifies if a cross was a cross within (heterospecific; 'het') or between (conspecific ; con') species * *crossed flowers*: the number of crossed flowers per cross * *number of seeds*: the number of seeds obtained in a cross * Sides.csv, Styles.csv, Corollas.csv - contain the floral morphological measurements measured with ImageJ from 2D-scans of flowers (see Fig. 2 in paper) * *Individual*: individual ID, composed of 'species abbreviation-population number-individual number' * *Calyx height (mm)*: height of the calyx in mm * *Tube length (mm)*: length of the corolla tube in mm * *Petal limb length (mm)*: length of a petal limb in mm * *Style length (mm)*: length of the style in mm * *Corolla Area (mm^2)*: area of the corolla in mm2 (square millimeters) * *Corolla radius (mm)*: radius if the corolla in mm * Pollen_Size_Measurements.csv - contains the pollen diameter measurements measured in ImageJ from microscope images of pollen (the first column can be ignored) * *Area*: pollen area in um2 (square micrometers) * *Perim*.: pollen perimeter in um (micrometers) * *Filename*: name of the input image file in tif format  * IDs_batch1.csv, IDs_batch2.csv, IDs_batch3.csv, IDs_batch4.csv - contain info on which sample is associated with which microscope images in the files containing in vitro pollen tube length measurements (the 'Results...' files below) *   *Individual*: individual ID, composed of 'species abbreviation-population number-individual number' * *SNAP#*: number of the image file * Results_batch1.csv, Results_batch2.csv, Results_batch3.csv, Results_batch4.csv - contain the pollen diameter and pollen tube length measurements of the in vitro experiment; each measurement comes on two lines (pollen diameter and then pollen tube length) * *Batch*: number of batch (images were collected in four batches) * *Label*: image file name * *Angle*: angle of drawn line to measure pollen diameter (irrelevant, to be ignored) * *Length*: the first of two lines of a measurement that always go together contains the pollen diameter measurement in um (micrometer), the second the pollen tube length measurement in um (micrometer) ## Code/Software **Analysis scripts:** * Occurrence_Data.R - R script to plot occurrence data obtained from GBIF and locations of experimental samples; input files are 'ids.csv' (see Data files) and the GBIF occurrence data below * Seed_Set.R - R script to visualize and analyze the data from the crossing experiments; input file is 'Seed_Set_20231116.csv' * Floral_Measurements_and_Pollen_Size.R - R script to visualize and analyze the morphological data; input files are 'Sides.csv', 'Styles.csv', 'Corollas.csv', 'Pollen_Size_Measurements.csv'. * Pollen_Tubes.R - R script to visualize and analyze the data from the in vitro pollen tube growth experiments; input files are 'IDs_batch1.csv', 'IDs_batch2.csv', 'IDs_batch3.csv', 'IDs_batch4.csv', 'Results_batch1.csv', 'Results_batch2.csv',' Results_batch3.csv', 'Results_batch4.csv' These R scripts assume that all R scripts are in a folder called R/ANALYSIS, that all data files are in a folder called R/DATA (with the exception of the GBIF data, see 'Occurrence_Data.R' and Table S2 in the Supplementary Materials), and that there is a folder called R/OUTPUT to store plots and other output files. ## Sharing/Access information Occurrence data from GBIF (used for plotting Fig. 1 in paper with R-script 'Occurrence_Data.R'): * [https://doi.org/10.15468/dl.3x8u9c](https://doi.org/10.15468/dl.3x8u9c) * [https://doi.org/10.15468/dl.2v5xt6](https://doi.org/10.15468/dl.2v5xt6) * [https://doi.org/10.15468/dl.d7ubmp](https://doi.org/10.15468/dl.d7ubmp) * [https://doi.org/10.15468/dl.33crrv](https://doi.org/10.15468/dl.33crrv) * [https://doi.org/10.15468/dl.sf37vs](https://doi.org/10.15468/dl.sf37vs) * [https://doi.org/10.15468/dl.yg3nh9](https://doi.org/10.15468/dl.yg3nh9)