Pollen dispersal is a key evolutionary and ecological process, but the degree to which variation in the density of concurrently flowering conspecific plants (i.e., co-flowering density) shapes pollination patterns remains understudied. We monitored co-flowering density and corresponding pollination patterns of the insect-pollinated palm Oenocarpus bataua in northwestern Ecuador and found that the influence of co-flowering density on these patterns was scale-dependent: high neighborhood densities were associated with reductions in pollen dispersal distance and gametic diversity of progeny arrays, whereas we observed the opposite pattern at the landscape scale. In addition, neighborhood co-flowering density also impacted forward pollen dispersal kernel parameters, suggesting that low neighborhood densities encourage pollen movement and may promote gene flow and genetic diversity. Our work reveals how co-flowering density at different spatial scales influences pollen movement, which in turn informs our broader understanding of the mechanisms underlying patterns of genetic diversity and gene flow within populations of plants.

         We recorded monthly phenological state for all adults (n=181) within a core 130-ha plot by visiting each tree within the study plot and recording the number of reproductive structures. In 2015, we surveyed a 250 m buffer zone around the 130-ha plot and recorded 60 additional adults, all mapped and genotyped, but for which phenology was not recorded. This yielded 241 geo-located and genotyped candidate fathers, of which 181 also had monthly phenology data for analysis. To collect progeny for genetic analysis, we collected ripe fruits directly from the infructescences of O. bataua individuals; we refer to these trees as ‘maternal’ trees and pollen sources as ‘paternal’ trees, noting that the same individual could be both. We randomly sampled maternal trees with complete phenology records. We germinated the seeds in a nursery and collected and stored a tissue sample from the first leaf of each seedling. For each maternal tree, we used monthly phenology data to calculate the co-flowering density in the time frame that the maternal tree was flowering. We calculated density at two spatial scales: the ‘landscape’ scale, defined as the entire 130-ha study plot, and ‘neighborhood’ scale, defined as the average effective pollination neighborhood (A_ep) of all progeny arrays included in this study, which was a circular area with a radius of ~ 320 m and an area of 33.40 ha. If the boundary of the neighborhood area extended beyond that of the study plot, neighborhood co-flowering density was estimated using the subset of individuals within the neighborhood area for which phenology data were available.

       We extracted genomic DNA from 962 offspring leaf samples representing 43 progeny arrays collected from 35 maternal trees. All samples were genotyped using 11 microsatellite loci through Polymerase Chain Reaction, following established protocols. We genotyped the 181 adults (pollen sources and maternal trees) in the study plot and the 60 additional adults in the 250 m buffer around the study plot using equivalent methods, totaling 241 genotyped parental trees. We used offspring and adult genotypes in the program CERVUS v. 3.0.3 (Marshall et al., 1998) to assign paternity with the aim of (1) calculating the average A_ep, (2) quantifying the ‘observed’ distance pollen dispersed from paternal sources to maternal trees, and (3) estimating the paternal contribution to the genetic diversity of each progeny array. We used critical trio (Δ) values with at least 80% confidence and the following simulation parameters used previously for parentage analysis in our study area.

Folders and Files: Folders are .zip files

 

1. Folder name - Distance calculation: the files in this folder were used to calculate the distance that pollen dispersed from CERVUS assigned paternal trees to maternal trees.

1a. File name - data.csv. This is the data to be read into R. 

1ai. Variables: MotherID is the genetic sample code for the maternal tree. 

UTM1 is the first coordinate for the mother tree. 

UTM2 is the second coordinate for the maternal tree. Candidate father is the assigned paternal tree in CERVUS. 

UTM1 is the first coordinate for the paternal tree. 

UTM2 is the second coordinate for the paternal tree. 

1b. File name - AssignedFathers.csv. This file is the list of CERVUS assigned fathers (column 3) for each offspring (column 1), corresponding to each maternal tree (column 2). 

1c. File name - code.R. This is the annotated R script. 

1d. File name - output.csv. This is the output from the R script. You should get the maternal tree id/progeny array (column 2), the mean pollen dispersal distance (column 3), standard deviation (column 4), and variance (column 5).

 

2. Folder name - Modeling_Figures1-3: This is the files in this folder were used to complete the main modeling results and generate figures 1-3 in the manuscript. 

2a. File name - data.csv. File with the summary data for each progeny array. 

2ai. Variables: Tag is the unique maternal ID tag name.

ID is the non-unique maternal ID.

Date is the month that the maternal tree was in flower with 1 being September 2011 and 48 is August 2015

UTM1 is the first coordinate for each maternal tree

UTM2 is the second coordinate for each maternal tree 

x is the longitude for each maternal tree

y is the longitude for each maternal tree

  Alphak_50 is the alpha diversity measure for male gametes for each progeny array

mean is the mean pollen dispersal distance for each maternal tree

Log_Alpha is log transformed Alphak_50 

Ndensity is estimated neighborhood density

Ldensity is estimated landscape density

Mean_CI is the 95% confidence interval for average pollen dispersal distance

Adiv_CI is the 95% CI for alpha diversity 

Radius is the radius of the neighborhood area for each maternal tree 

AepNDensity is the estimated density of the neighborhood density in the neighborhood area (Aep)

2b. File name - LD_high.csv. Observed pollen dispersal distances for offspring fertilized during high landscape density scenarios. 

2c. File name - LD_low.csv. Observed pollen dispersal distances for offspring fertilized during low landscape density scenarios.

2d. File name - ND.high.csv. Observed pollen dispersal distances for offspring fertilized during high neighborhood density scenarios.

2e. File name - ND.low.csv. Observed pollen dispersal distances for offspring fertilized during low neighborhood density scenarios.

2f. File name - all_dists.csv. Observed pollen dispersal distances for offspring fertilized during all density scenarios. 

2g. File name - Stats_Figure1-3.R. Script for modeling and making figures in R. 

2h. File name - Figure1.tiff. Output for figure 1.

2i. File name - Figure1.tiff. Output for figure 2.

2j. File name - Figure3.tiff. Output for figure 3.

 

3. Folder name - Pollen_kernel_simulations.

3a. File name - Adult_Genotypes_UTMs.csv. This file contains all adult O. bataua genotypes and coordinate information for each adult (UTM1, UTM2). 

3ai. Variables: ID is the genetic sample ID for each individual

Field_Tag is the field ID for each individual

UTM1 is first coordinate

UTM2 is the second coordinate

Mother? Is a yes/no detailing if that individual is a maternal tree

The following columns are microsatellite alleles.

3b. File name - allelePmiDiversity.R. This script is used to calculate allelic diversity of the pollen pool. Modified from the DispersalDiversity R package.

3c. File name - Flowering_IDS.csv. This file contains the Field tag of all flowering maternal trees in the study (Array_Maternal_ID).

3d. File name - Paternal_IDS.csv. This file contains the Field tags of all potential flowering trees (Pat_ID) for each flowering maternal tree (Mat_ID) in the study.

3e. File name - pmiDiversity.R This script is used to calculate allelic diversity of the pollen pool. Modified from the DispersalDiversity R package.

3f. File name - Pollen_Kernel_simulations_2022_01_13_annotated.Rmd. This is an R markdown document that contains the code needed to run the pollen simulation analyses presented in the results.

 

4. Folder name - NmpiAnalysis. 

4a. Folder name - Data. Tab-separated text files containing genotype data in NMpi format for each co-flowering density scenario. Used in the Nmpi analysis.

4b. File name - error_template.txt. Basic template for genotyping errors used in Nmpi analysis.

4c. Folder name - info_files. Contains the info files required in NMpi analysis for each co-flowering density scenario and pollen dispersal kernel. Each combination has 2000 info files with randomly generated initial values.

4d. File name - info_template.txt. Basic template for info.txt file used in Nmpi analysis.

4e. File name - NMPi_analysis 2022_01_13.Rmd. Annotated R markdown file for generating info.txt files and running Nmpi analysis from the command line. Note that the Nmpi analysis will only run on Windows computers.

4f. File name - NMpi.exe. NMpi executable called from NMPi_analysis 2022_01_13.Rmd. See Chybicki 2018 for description: DOI: 10.1111/1755-0998.12710

4g. Folder name - Results. Tab separated files containing the collated results of the Nmpi analysis for each co-flowering density scenario + pollen dispersal kernel combination. These values were summarized and collated using the windowed version of Nmpi.

4h. File name - selection_template.txt. Basic template for selection.txt used in Nmpi analysis

4i. Folder name - temp. Temporary directory to hold the intermediate files of the Nmpi analysis.

4j. File name - wNMpi.exe. Executable to open windowed version of Nmpi. See Chybicki 2018 for description: DOI: 10.1111/1755-0998.12710

 

5. Folder name - Data. 

5a. File name - Adult_Genotypes_UTMs.csv. This file contains all adult O.bataua genotypes and coordinate information for each adult (UTM1, UTM2).

5ai. Variables: ID is the genetic sample ID for each individual

Field_Tag is the field ID for each individual

UTM1 is first coordinate

UTM2 is the second coordinate

Mother? Is a yes/no detailing if that individual is a maternal tree

The following columns are microsatellite alleles. 

5b. File name - Offspring_Genotypes.csv. This is the file that contain all offspring genotypes use in the analyses. 

5bi. Variables: Field_Number is the offspring field ID 

Mother_Tag is the field ID for the maternal tree

Mother_ID is the genetic sample ID of the mother tree

Failed_alleles are the number of alleles for that individual that were not genotyped

The remaining columns are the genotypes for each allele.