DOI: 10.5061/dryad.6t1g1jx7p
title: Maternal quality, paternal effects, and sibling interactions influence seed size in the eelgrass, Zostera marina

description: Seed size is an essential determinant of germination and survival in
 angiosperms. Zostera marina, one of few marine angiosperms, is a key
 foundation species present in coastal marine ecosystems, and edge-of-range
 population persistence is increasingly reliant on seed production. While
 environmental conditions have been invoked to explain regional patterns of
 seed size variation, far less is known about seed size differences within
 a plant. In this study, we genotype and measure individual seeds across
 parent plants to investigate the relative contributions of maternal,
 paternal and offspring traits on seed weight. Specifically, we investigate
 how parent heterozygosity, paternal siring success, outcrossing, a
 size-number tradeoff, and sibling interactions influence both seed weight
 (mg) and cross-sectional area (mm2). Here, we provide all
 necessary files to conduct the entirety of analyses used in the
 manuscript. 
description: <em>1. Study Sites and Sample
 Collection  </em> Three
 seagrass meadows in the intracoastal waters of Topsail Sound, North
 Carolina were sampled to characterize <em>Zostera marina</em>
 seed size variation. Meadows were on a narrow shelf classified as shallow
 subtidal (depth &lt; 2m MLLW) between the Intracoastal Waterway and
 the adjacent shoreline (34.22 N, 77.37 W; Fig. S1). On 4 May 2021, near
 the end of the eelgrass reproductive season, flowering shoots were
 haphazardly collected at least 5m apart, yielding 35 shoots total. Samples
 were transported on ice to UNCW’s Center for Marine Science, and the
 morphological characteristics of each shoot were recorded. Specifically,
 each rhipidium (branching reproductive structure), spathe (seed-containing
 branch within a rhipidium), and seed position was labeled in order of
 decreasing proximity to the rhizome (i.e., basal positions were given a
 value of 1; Fig. S2).  
   <em>2. Seed
 Measurements and Genotyping </em> 
 Seeds were removed from spathes, blotted dry, and tested for
 viability using the “squeeze test” by gently compressing individual seeds
 with a pair of tweezers (Marion and Orth 2010). Those with a seed coat
 that compressed were considered nonviable. Viable seeds were then weighed
 (mg) with a microbalance and photographed at ×10 magnification on a stereo
 microscope (Fisher Scientific, MA, USA). Length (mm), width (mm), and
 cross-sectional area (mm<sup>2</sup>) were measured for each
 seed using ImageJ software (Schneider et al. 2012). Cross-sectional area
 of seeds was then calculated from the following equation: <em>v
 </em>=<em> πab</em>, where <em>a</em> =
 radius of length and <em>b</em> = radius of width
 (Wyllie-Echeverria et al. 2003). DNA was extracted from viable seed
 samples using a PowerPlant® Pro DNA Isolation Kit (QIAGEN, Hilden, GER).
 Ten microsatellite loci previously described for Z. marina (Reusch et al.
 1999, Reusch 2000, Oetjen and Reusch 2007, Oetjen et al. 2010; Table S1)
 were amplified in two multiplex Polymerase Chain Reactions (PCR).
 Individual primer working stocks contained 1 μL of 10 μM fluorescently
 labeled forward primer and 10 μL each of 50 μM unlabeled forward and
 reverse primers diluted in 80 μL of ddH2O. Primers were then combined into
 two primer mixes – each containing five different primers (Table A1). PCR
 conditions for all multiplex conditions were as follows: 95.0°C for 15
 min; 2 cycles of 94.0°C for 15 s, 60.0°C for 30 s, 72.0°C for 45 s; 2
 cycles of 94.0°C for 15 s, 59.0°C for 30 s, 72.0°C for 45 s; 2 cycles of
 94.0°C for 15 s, 58.0°C for 30 s, 72.0°C for 45 s; 2 cycles of 84.0°C for
 15 s, 57.0°C for 30 s, 72.0°C for 45 s; 28 cycles of 94.0°C for 15 s,
 56.0°C for 30 s, 72.0°C for 45 s; and a final 2 min extension at 72.0°C.
 Following PCR, two reactions were prepared: one containing 0.5 μL of each
 PCR product from each of the multiplex mixes. PCR products were added to 9
 μL of highly deionized formamide (HiDi) and 0.4 μL of GeneScan-600 (LIZ)
 size standard (Applied Biosystems, Foster City, CA, USA) for capillary
 sequencing on an ABI Prism 3130XL Genetic Analyzer. Fragments were scored
 using Applied Biosystems Microsatellite Analysis Software (ThermoFisher
 Scientific Inc.)  
 <em>3. Paternity
 Analyses </em> 
 Paternity assignment and parent genotype
 reconstruction were performed using all offspring genotypes in COLONY
 v2.0.7.0 (Jones & Wang, 2010; Wang, 2019). COLONY input parameters
 included a polygamous mating system for both sexes, possible inbreeding,
 and a monecious, diploid species. The maternal plant of each seed (i.e.,
 in which seeds are at least maternal half-siblings) was specified. The
 maximum likelihood approach with a long run, medium-likelihood precision,
 and a genotyping error rate of 1% was performed. Both maternal and
 paternal genotypes were reconstructed using the maximum likelihood
 approach and a probability threshold of 0.925 to infer the most likely
 parental genotypes based on observed sibship patterns. Seeds were
 categorized as selfed if the putative father had the same multi-locus
 genotype as the known mother. It was also noted whether a seed's
 basal neighbor (i.e., the seed closer to the base of the plant) was
 fertilized via outcrossing or selfing. Paternity skew was used as a proxy
 for within-spathe seed relatedness and calculated following Neff et al.
 (2008). A value of 0 indicates no skew in which all sires contribute
 equally to seeds within a spathe (seeds would be some combination of half-
 and full-siblings), and a value approaching 0.5 indicates maximum skew in
 which all seeds were sired by a single father (all seeds would be
 full-siblings). Paternal siring success was defined as the extent to which
 a sire monopolized available ovules on a plant and was measured as the
 proportion of viable seeds per shoot sired by a particular father.
 Heterozygosity of the putative sires (as well as the maternal shoots and
 individual seeds) was calculated as the proportion of heterozygous alleles
 across successfully reconstructed loci.   
   
 2.5 Statistical Analyses   
 Statistical analyses were
 conducted in RStudio with R v4.2.1 (R Core Team, 2022; Posit Team, 2023).
 Data were tested for outliers, collinearity, and normal distribution (Zuur
 et al. 2007; see Figure S3 in the supporting information for the
 relationships among fixed effects). All model residuals were visually
 inspected for normal distribution (DHARMa; Hartig & Lohse, 2022),
 and all figures were generated using the package “ggplot2” (Wickham et
 al., 2016).   To test the
 fixed effect of maternal heterozygosity on reproductive shoot
 characteristics, generalized linear models (GLMs) were fit to the total
 number of seeds, viable seeds (offset by the number of seeds), spathes,
 and rhipidia per shoot using a Poisson distribution as well as to the mean
 and CV of seed weight and area per shoot using a gamma distribution and a
 log link function (lme4; Bates<em> </em>et
 al<em>.,</em> 2015). To test the size-number tradeoff, GLMs
 were fit between the proportion of viable seeds, the total number of
 seeds, and mean seed weight per shoot using a gamma distribution and a log
 link function.  To first test
 the fixed effects of rhipidium position, spathe position, and seed
 position on individual seed size, a GLM was fit to seed weight and area
 using a gamma distribution and a log link function (lme4; Bates<em>
 </em>et al<em>.</em><em>,</em> 2015). This
 model was also fit to reproductive mode (selfed vs. outcrossed), as
 previous work has shown that outcrossing decreases in more distal spathes
 (Sgambelluri et al. 2024). An ANOVA and Tukey's Honest Significant
 Difference test was performed on the model outputs to assess the
 significance of each fixed effect (car; Fox & Weisberg,
 2019).  To then test the fixed
 effects of maternal heterozygosity, paternal heterozygosity, offspring
 heterozygosity, outcrossing, paternal siring success, within-spathe seed
 relatedness, basal neighbor effects, and the proportion of viable seeds
 per shoot on individual seed size, generalized linear mixed models (GLMMs)
 were fit to weight and area using a gamma distribution and a log link
 function (lme4; Bates<em> </em>et
 al<em>.</em><em>,</em> 2015). Here, the random
 effects of rhipidium and spathe position were included to account for
 observed positional differences (see Results) and a random effect of
 maternal identity was added to account for possible differences among
 mothers. Models were run with different combinations of fixed and random
 effects, and those garnering the highest Akaike weight were considered top
 models for inference (AICcmodavg; Mazerolle, 2023). To then evaluate the
 relative impact of maternity, paternity, and developmental timing on seed
 size, an REML variance components analysis (VCA) was performed with spathe
 position, maternal identity, and paternal identity as random effects and
 with individual seed weight as the response variable.  
 Finally, to test the fixed effects of
 maternal heterozygosity, the number of seeds per spathe, the proportion of
 selfed seeds per spathe, and within-spathe seed relatedness on variance in
 seed size, GLMMs were fit to the mean and the coefficient of variation (CV
 =<em> </em><em>σ/µ</em>, where
 <em>σ</em> = standard deviation and <em>µ</em> =
 sample mean) in seed weight and area within spathes.  
  
  
description: # Seed size variation in the eelgrass, *Zostera marina*
 [https://doi.org/10.5061/dryad.6t1g1jx7p](https://doi.org/10.5061/dryad.6t1g1jx7p) # Data Collection & Description ## 1. Study Sites and Sample Collection  *Zostera marina* shoots were sampled in the intracoastal waters of Topsail Sound, North Carolina. The morphological characteristics of each shoot were recorded. Specifically, each rhipidium (branching reproductive structure), spathe (seed-containing branch within a rhipidium), and seed position was labeled in order of decreasing proximity to the rhizome (i.e., basal positions were given a value of 1; Fig. S2). ## 2. Seed Measurements and Genotyping  Viable seeds were removed from spathes, weighed (mg) with a microbalance, and photographed at ×10 magnification on a stereo microscope. Length (mm), width (mm), and cross-sectional area (mm^2) were measured for each seed using ImageJ software. Cross-sectional area of seeds was then calculated from the following equation: *v* = π*ab*, where *a* = radius of length and *b* = radius of width. DNA was extracted from viable seed samples using a PowerPlant® Pro DNA Isolation Kit (QIAGEN, Hilden, GER). Ten microsatellite loci previously described for *Z. marina* (Table S1) were amplified and scored via the methods found in the full data description.  ## 3. Paternity Analyses  Paternity assignment and parent genotype reconstruction were performed using known, within-shoot half-siblings and all offspring genotypes in COLONY v2.0.7.0 (Jones and Wang, 2010; Wang, 2019). COLONY input parameters included a polygamous mating system for both sexes, possible inbreeding, and a monecious, diploid species. The maximum likelihood approach with a long run, medium-likelihood precision, and a genotyping error rate of 1% was performed. An allele probability threshold of 0.925 was used for parent genotype reconstruction. Seeds were categorized as selfed if the detected father had the same genotype as the known mother. Paternity skew was calculated (Neff *et al.*, 2008) and used as a proxy for within-spathe seed relatedness; spathes with only one detected sire were assigned a paternity skew value of 0.5. As such, a value of 0 indicates no skew in which all sires contribute equally to seeds within a spathe, and a value approaching 0.5 indicates maximum skew in which all seeds were sired by a single father. Paternal siring success was measured as the proportion of sampled seeds per shoot sired by a particular father. Heterozygosity of the putative sires (as well as the maternal shoots and individual seeds) was calculated as the proportion of heterozygous alleles across successfully reconstructed loci. ## 4. Description of the data Offspring genotypes were associated to their respective seed on each parent plant; and data exploration was performed in which outliers in both outliers in both dependent and independent variables were identified, and the appropriate distribution was assigned to each dependent variable. Data was then quality controlled to remove any outliers and control for uneven sample size. Quality controlled data was then organized by variable measured - mass or cross-sectional area - and on spatial scales corresponding to varying degrees of seagrass morphological structures - on a 'per seed,' 'per spathe,' and 'per shoot' basis. A subset of data was formed by rerunning COLONY using only offspring with 9-10 successfully amplified and scored loci. This dataset is referred to as the "reduced" dataset throughout. Offspring genotypes were again associated to their respective seed on each parent plant. Quality controlled data was then organized on spatial scales corresponding to varying degrees of seagrass morphological structures - on a 'per seed,' 'per spathe,' and 'per shoot' basis for only the measure of seed mass. ## 5. Supplementary material Supplementary material including Figure S1, Figure S2, and Table S1 have been included in the FigS1*_*FigS2*_*TableS1.pdf file. Please see Materials and Methods for additional context regarding the supplementary material. ## 6. Sources referenced Jones OR, Wang JL (2010) COLONY: a program for parentage and sibship inference from multilocus genotype data. Molecular Ecology Resources 10:551-222 Neff BD, Pitcher TE, Ramnarine IW (2008) Inter-population variation in multiple paternity and reproductive skew in the guppy. Molecular Ecology 17:2975-2984 Oetjen K, Ferber S, Dankert I, Reusch TBH (2010) New evidence for habitat-specific selection in the Wadden Sea *Zostera marina* populations revealed by genome scanning using SNP and microsatellite markers. Marine Biology 157:81-89 Oetjen K, Reusch TBH (2007) Genome scans detect consistent divergent selection among subtidal vs. intertidal populations of the marine angiosperm *Zostera marina*. Molecular Ecology 16:5156-5167 Wang J (2019) Pedigree reconstruction from poor quality genotype data. Heredity 122:719-728 # Files and Variables ## File: Mass\_area\_comparison.csv **Description:** Comparison of seed mass and seed cross-sectional area ##### Variables * Mass: seed mass (mg) * Area: seed cross-sectional area (mm^2) * NOTE: empty cells denote missing data (i.e., processing error led to uncollected size measurement for a particular sample) ## File: Seed\_area.csv **Description:** Individual measurements of seed cross-sectional area, with corresponding parent plant characteristics, sampling location, position of the seed within the parent plant, and measures of offspring relatedness ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Selfed: whether a seeds was fertilized via outcrossing or selfing * BN: whether a seed's basal (i.e., proximal) neighbor was fertilized via outcrossing or selfing * Paternal ID: number assigned to each sire identified by COLONY * Paternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed paternal loci * Seeds Fertilized: the number of seeds fertilized per sire * Reproductive Skew: the number of fertilized seeds per sire divided by the number of analyzed seeds per shoot fertilized * Sires per Spathe: number of unique identified sires per spathe * Offspring Heterozygosity: proportion of heterozygous alleles across successfully amplified and scored offspring loci * Rhipidium: rhipidium position * Spathe: spathe position * Seed: seed position * Seeds per Shoot: number of seeds per sampled reproductive shoot * Area: seed cross-sectional area (mm^2) * Paternity Skew per Spathe: paternity skew, calculated after Neff et al. (2008) per spathe * Viable Seeds per Shoot: number of viable seeds per sampled reproductive shoot * Prop: proportion of viable seeds per sampled reproductive shoot ## File: Seed\_area\_per\_spathe.csv **Description:** Measurements of seed cross-sectional area averaged within spathes, with corresponding parent plant characteristics, sampling location, position of the seed within the parent plant, and measures of offspring relatedness ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Rhipidium: rhipidium position * Spathe: spathe position * Seeds per Spathe: number of seeds per spathe * Seeds Analyzed per Spathe: number of seeds measured (either mass or cross-sectional area) and genotyped per spathe * % Analyzed per Spathe: percent of seeds measured (either mass or cross-sectional area) and genotyped per spathe * Sires per Spathe: number of unique identified sires per spathe * Selfed Seeds: number of seeds fertilized via selfing per spathe * %Selfed: proportion of seeds fertilized via selfing per spathe * Mean Area per Spathe: seed cross-sectional area (mm^2) averaged within spathes * SD Area per Spathe: seed cross-sectional area (mm^2) standard deviation within spathes * CV Area per Spathe: seed cross-sectional area (mm^2) coefficient of variation within spathes * Paternity Skew per Spathe: paternity skew, calculated after Neff et al. (2008) per spathe ## File: Seed\_mass.csv **Description:** Individual measurements of seed mass, with corresponding parent plant characteristics, sampling location, position of the seed within the parent plant, and measures of offspring relatedness ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Selfed: whether a seeds was fertilized via outcrossing or selfing * BN: whether a seed's basal (i.e., proximal) neighbor was fertilized via outcrossing or selfing * Paternal ID: number assigned to each sire identified by COLONY * Paternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed paternal loci * Seeds Fertilized: the number of seeds fertilized per sire * Reproductive Skew: the number of fertilized seeds per sire divided by the number of analyzed seeds per shoot fertilized * Sires per Spathe: number of unique identified sires per spathe * Offspring Heterozygosity: proportion of heterozygous alleles across successfully amplified and scored offspring loci * Rhipidium: rhipidium position * Spathe: spathe position * Seed: seed position * Seeds per Shoot: number of seeds per sampled reproductive shoot * Mass: seed mass (mg) * Paternity Skew per Spathe: paternity skew, calculated after Neff et al. (2008) per spathe * Viable Seeds per Shoot: number of viable seeds per sampled reproductive shoot * Prop: proportion of viable seeds per sampled reproductive shoot ## File: Seed\_mass\_per\_spathe.csv **Description:** Measurements of seed mass averaged within spathes, with corresponding parent plant characteristics, sampling location, position of the seed within the parent plant, and measures of offspring relatedness ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Rhipidium: rhipidium position * Spathe: spathe position * Seeds per Spathe: number of seeds per spathe * Seeds Analyzed per Spathe: number of seeds measured (either mass or cross-sectional area) and genotyped per spathe * % Analyzed per Spathe: percent of seeds measured (either mass or cross-sectional area) and genotyped per spathe * Sires per Spathe: number of unique identified sires per spathe * Selfed Seeds: number of seeds fertilized via selfing per spathe * %Selfed: proportion of seeds fertilized via selfing per spathe * Mean Mass per Spathe: seed mass (mg) averaged within spathes * SD Mass per Spathe: seed mass (mg) standard deviation within spathes * CV Mass per Spathe: seed mass (mg) coefficient of variation within spathes * Paternity Skew per Spathe: paternity skew, calculated after Neff et al. (2008) per spathe ## File: Seed\_mass\_-\_reduced\_dataset.csv **Description:** Individual measurements of seed cross-sectional area, with corresponding parent plant characteristics, sampling location, position of the seed within the parent plant, and measures of offspring relatedness formed using the reduced dataset ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Selfed: whether a seeds was fertilized via outcrossing or selfing * BN: whether a seed's basal (i.e., proximal) neighbor was fertilized via outcrossing or selfing * Paternal ID: number assigned to each sire identified by COLONY * Paternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed paternal loci * Seeds Fertilized: the number of seeds fertilized per sire * Reproductive Skew: the number of fertilized seeds per sire divided by the number of analyzed seeds per shoot fertilized * Sires per Spathe: number of unique identified sires per spathe * Offspring Heterozygosity: proportion of heterozygous alleles across successfully amplified and scored offspring loci * Rhipidium: rhipidium position * Spathe: spathe position * Seed: seed position * Seeds per Shoot: number of seeds per sampled reproductive shoot * Mass: seed mass (mg) * Paternity Skew per Spathe: paternity skew, calculated after Neff et al. (2008) per spathe * Viable Seeds per Shoot: number of viable seeds per sampled reproductive shoot * Prop: proportion of viable seeds per sampled reproductive shoot ## File: Seed\_mass\_per\_spathe\_-\_reduced\_dataset.csv **Description:** Measurements of seed mass averaged within spathes, with corresponding parent plant characteristics, sampling location, position of the seed within the parent plant, and measures of offspring relatedness formed using the reduced dataset ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Rhipidium: rhipidium position * Spathe: spathe position * Seeds per Spathe: number of seeds per spathe * Seeds Analyzed per Spathe: number of seeds measured (either mass or cross-sectional area) and genotyped per spathe * % Analyzed per Spathe: percent of seeds measured (either mass or cross-sectional area) and genotyped per spathe * Sires per Spathe: number of unique identified sires per spathe * Selfed seeds: number of seeds fertilized via selfing per spathe * %Selfed: proportion of seeds fertilized via selfing per spathe * Mean Mass per Spathe: seed mass (mg) averaged within spathes * SD Mass per Spathe: seed mass (mg) standard deviation within spathes * CV Mass per Spathe: seed mass (mg) coefficient of variation within spathes * Paternity Skew per Spathe: paternity skew, calculated after Neff et al. (2008) per spathe ## File: Seed\_size\_per\_shoot.csv **Description:** Measurements of seed cross-sectional area and mass averaged within reproductive shoots, with corresponding parent plant characteristics, sampling location, and measures of offspring relatedness ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Rhipidia: number of rhipidia per sampled reproductive shoot * Spathes: number of spathes per sampled reproductive shoot * Seeds: number of total seeds per sampled reproductive shoot * Viable Seeds: number of viable seeds per sampled reproductive shoot * Prop: proportion of viable seeds per sampled reproductive shoot * Analyzed Seeds: number of seeds measured (either mass or cross-sectional area) and genotyped per reproductive shoot * % Analyzed: percent of seeds measured (either mass or cross-sectional area) and genotyped per reproductive shoot * Sires per shoot: number of unique identified sires per shoot * Avg Mass: seed mass (mg) averaged within shoots * Avg Area: seed cross-sectional area (mm^2) averaged within shoots * CV Mass: seed mass (mg) coefficient of variation within shoots * CV Area: seed cross-sectional area (mm^2) coefficient of variation within shoots ## File: Seed\_size\_per\_shoot\_-\_reduced\_dataset.csv **Description:** Measurements of seed mass averaged within reproductive shoots, with corresponding parent plant characteristics, sampling location, and measures of offspring relatedness formed using the reduced dataset ##### Variables * Site: number assigned to each sampled meadow * Maternal ID: number assigned to each sampled reproductive shoot * Maternal Heterozygosity: proportion of heterozygous alleles across successfully reconstructed maternal loci * Rhipidia: number of rhipidia per sampled reproductive shoot * Spathes: number of spathes per sampled reproductive shoot * Seeds: number of seeds per sampled reproductive shoot * Viable Seeds: number of viable seeds per sampled reproductive shoot * Prop: proportion of viable seeds per sampled reproductive shoot * Analyzed Seeds: number of seeds measured (either mass or cross-sectional area) and genotyped per reproductive shoot * % Analyzed: percent of seeds measured (either mass or cross-sectional area) and genotyped per reproductive shoot * Sires per shoot: number of unique identified sires per shoot * Avg Mass: seed mass (mg) averaged within shoots * CV Mass: seed mass (mg) coefficient of variation within shoots ## File: Seed\_coat.csv **Description:** Comparison of seed mass with and without the seed coat, performed on n=50 resampled seeds ##### Variables * Sample: number assigned to each sampled seed * Gram: weight in grams of each sampled seed * Category: whether a seed measurement included the seed coat or did not include the seed coat * Sample: number assigned to each sampled seed * Gram coat: weight in grams of each sampled seed with the seed coat * Gram no coat: weight in grams of each sampled seed without the seed coat * NOTE: this dataset includes replicate measurements formatted in two different ways for statistical analysis # Code/software ## COLONY COLONY v2.0.7.0 (Jones & Wang 2010) was used to assign parent genotypes to each offspring genotype, with which individual heterozygosity, outcrossing, paternity skew (Neff et al. 2008), and reproductive skew were calculated. ## Seed-Area---R-Markdown.html ## Seed-Mass---R-Markdown.html ## Seed-Mass-Reduced-Dataset---R-Markdown.html These files conduct the entirety of analyses used in the manuscript. Source csv files are provided and described as datasets. All utilized R code and outputs are included in these files. ###### Language and Environment R Environment for Statistical Computing ###### Version R v4.2.1 ###### Package Dependencies * dplyr * foreign * rmarkdown * ggsignif * lme4 * AICcmodavg * car * VCA * DHARMa * multcomp * multcompView * ggplot2 * GGally * ggpubr # Assess information There are no other publicly accessible locations of the data, and data was derived from no other sources.