DOI: 10.5061/dryad.z08kprrrf
title: Data and code for: Relative testis size is associated with vagina length but not sperm storage traits in Galliformes

description: This dataset contains the data and code to replicate the analyses in
 “Relative testis size is associated with vagina length but not sperm
 storage traits in Galliformes.” The study examined relationships among
 sperm storage capacity, sperm storage tubule (SST) morphology, relative
 testis mass (a proxy for post-copulatory sexual selection), and sperm
 traits across 26 Galliforme species. The dataset includes four files:
 RepeatDF.csv (data for repeatability calculations); SummaryBothFolds.csv
 (raw species and fold-level data); SummarySpeciesAvgs.csv (species-level
 averages across both folds); and Analysis.R (code to replicate
 analyses). Data files contain measurements from female
 reproductive tracts, male testis mass, and sperm samples.
description: For each species, one female was dissected; from each, two
 utero-vaginal junction (UVJ) folds (the SST-bearing region of the vagina)
 were analysed. Measurements from each individual include SST area and
 average tubule length measurements (from each UVJ fold), body mass, testis
 mass, vagina length, and sperm length (from 1–3 males per species; see
 dataset for details). A phylogenetic tree from Stein et al. (2015) is also
 required and available via Dryad (https://doi.org/10.5061/dryad.p2pn8).
description: # Data and code for: Relative testis size is associated with vagina length
 but not sperm storage traits in Galliformes Dataset DOI:
 [10.5061/dryad.z08kprrrf](10.5061/dryad.z08kprrrf) ## Description of the
 data and file structure We dissected and measured the reproductive tract
 of 26 species of Galliformes, extracting and imaging the utero-vaginal
 junction (UVJ) and sperm storage tubules (using fluorescence microscopy).
 We also collected and measured testes and sperm of 20 species, which –
 combined with data from Liao et al (2019) – resulted in testes and sperm
 length measurements from 22 species of Galliformes (see methods in paper
 for further details). The following three datasets and R script contain
 the data and code required to replicate analyses investigating the
 relationships between female reproductive tract morphology, sperm
 competition intensity, and sperm traits, whilst accounting for
 phylogenetic and allometric relationships. Phylogeny is provided by Stein
 et al., (2015).  ## Files and variables ### File: RepeatDF.csv
 **Description:**  * This dataset contains measurement data including Utero
 Vaginal Junction tissue area and tubule tissue area, sperm storage tubule
 length measurements and morphological categorisations for one randomly
 chosen image (from the pool of images containing SSTs within each sample),
 for the purpose of calculating measurement and categorisation
 repeatability between 2 observers. - Repeatability was calculated for the
 measurement of tubule tissue area within each image, and for individual
 tubule length measurements chosen haphazardly by observer 1. * To test
 whether haphazard choice of SSTs was a robust indication of true average
 SST length within the sample, observer 2 haphazardly chose 5 additional
 tubules to compare against the original subset. - Repeatability was also
 calculated for the morphological categorisation of tubules, using the
 categorisation criteria provided in Assersohn et al., 2024.
 **Abbreviations:**  SST - Sperm Storage Tubule UVJ - Utero-Vaginal
 Junction **NA values:** NA values in this file represent missing data
 values. * NA values in the SST length measurement columns for Observer 1
 represent cases where either the was no SST tissue in the sample, there
 was SST tissue but it was not measurable (e.g., SSTs overlapped, or were
 not clearly defined), or there were fewer than 5 measurable SSTs. * NA
 values in the SST length measurements columns for Observer 2 represent the
 cases above, but additionally, NA values are present because only 1
 individual per species was measured by Observer 2.  ##### Variables *
 tip.label: Latin name corresponding to the tip labels in the phylogeny *
 SpeciesCommon: Species common name * Sample: Unique sample ID given in the
 format SpeciesCodeSampleNumber_FoldNumber e.g. BF02_F1 is BlackFrancolin2,
 fold 1. * Measurerer: Either observer 1 (primary observer) or observer 2
 (second observer measurements for repeatability purposes). *
 ImageNumber: Which image in the fold the data pertains to. *
 EmptySpace_um2: The amount of space in the image not taken up by tissue.
 Calculated using particle analysis in ImageJ. * ImageSize_um2: The total
 size of the image.  * TotalUVJTissue_um2: Image size minus empty space, to
 give the total amount of UVJ tissue within the image. *
 TotalSSTTissue_um2: Total amount of tubule tissue within the image.
 Calculated using particle analysis. * Observer1SST1Length: The first SST
 chosen by observer 1, and measured by both observer 1 and observer 2 (to
 test measurement repeatability) * Observer1SST2Length: The second SST
 chosen by observer 1, and measured by both observer 1 and observer 2 (to
 test measurement repeatability) * Observer1SST3Length: The third SST
 chosen by observer 1, and measured by both observer 1 and observer 2 (to
 test measurement repeatability) * Observer1SST4Length: The fourth SST
 chosen by observer 1, and measured by both observer 1 and observer 2 (to
 test measurement repeatability) * Observer1SST5Length: The fifth SST
 chosen by observer 1, and measured by both observer 1 and observer 2 (to
 test measurement repeatability) * Observer1.AverageSSTLength: The average
 length of observer 1 SSTs 1:5 (to test SST length measurement
 repeatability) * Observer2SST1Length: The first SST chosen by observer 2
 haphazardly (to test whether haphazard choice of SSTs influences
 repeatability of average SST length). * Observer2SST2Length: The second
 SST chosen by observer 2 haphazardly (to test whether haphazard choice of
 SSTs influences repeatability of average SST length). *
 Observer2SST3Length: The third SST chosen by observer 2 haphazardly (to
 test whether haphazard choice of SSTs influences repeatability of average
 SST length). * Observer2SST4Length: The fourth SST chosen by observer 2
 haphazardly (to test whether haphazard choice of SSTs influences
 repeatability of average SST length). * Observer2SST5Length: The fifth SST
 chosen by observer 2 haphazardly (to test whether haphazard choice of SSTs
 influences repeatability of average SST length). *
 Observer2.AverageSSTLength: The average length of SSTs chosen by observer
 1 or observer 2 (to test whether haphazard choice of SSTs influences
 repeatability of average SST length). * Complex: Whether the sample
 contains SSTs categorised as 'complex' (either branched, coiled
 or agglomerate), by both observer 1 and 2. * StraightUnbranched: Whether
 the sample contains SSTs categorised as 'straight unbranched'. *
 StraightBranched: Whether the sample contains SSTs categorised as
 'straight branched', by both observer 1 and 2. * Coiled: Whether
 the sample contains SSTs categorised as 'coiled', by both
 observer 1 and 2. * Agglomerate: Whether the sample contains SSTs
 categorised as 'agglomerate', by both observer 1 and 2. ###
 File: SummaryBothFolds.csv **Description:**  * This dataset contains
 species information and measurement data for sperm storage tubule length
 and density, from the image of highest tubule density within each sample.
 Each individual (1 individual per species) is associated with 2 fold
 measurements.  - Within species, repeatability was calculated for SST
 tissue and length measurements. **Abbreviations:**  SST - Sperm Storage
 Tubule **NA values:** NA values in this file represent missing data
 values. * For species that we lack male testis mass and sperm length
 measurements, NA values appear in 'TotalTestisMassMean_g' and
 'MeanTotalSpermLength_um' representing missing data. For this
 reason, data is not applicable for columns; 'n_sperm_sampled';
 'n_males_sampled'; 'SpermLengthDataSource'; and
 'TestisMassDataFrom'.  * For several species, testis mass and
 sperm length measurements were obtained from a separate study (Liao et
 al., 2019), and in these cases NA values in the
 'n_sperm_sampled' and 'n_males_sampled' columns
 represent information missing/not imported from this study.  #####
 Variables * Species: Species common name * tip.label: Latin name
 corresponding to the tip labels in the phylogeny. * Sample: Unique sample
 ID given in the format SpeciesCodeSampleNumber_FoldNumber e.g. BF02_F1 is
 BlackFrancolin2, fold 1. * SSTTissue.DensestImageOnly: Total amount of
 tubule tissue within the image of highest tubule density. *
 SSTLength.DensestImageOnly: Average length of tubules within the image of
 highest tubule density. ### File: SummarySpeciesAvgs.csv **Description:** 
 * This dataset contains species average measurement data from Galliformes
 reproductive tract, testis mass and sperm samples.  * Mean values for each
 individual (across both folds) are given for SST tissue area and SST
 length, for both the entire sample and for the image of highest tubule
 density. Female body mass and vagina length measurements are also given
 per individual. * Mean total sperm length is provided, including
 information about the numbers of individuals (and number of sperm)
 sampled. For 2 individuals, some data has been taken from Wen Bo Liao et
 al., 2019 (as indicated in the datasheet). * Information is also given
 about the morphological categorisation of tubules within the samples (as
 determined by Assersohn et al., 2024. See methods therein).
 **Abbreviations:**  SST - Sperm Storage Tubule **NA values:** NA values in
 this file represent missing data values. * For species that we lack male
 testis mass and sperm length measurements, NA values appear in
 'TotalTestisMassMean_g' and 'MeanTotalSpermLength_um'
 representing missing data. For this reason, data is not applicable for
 columns; 'n_sperm_sampled'; 'n_males_sampled';
 'SpermLengthDataSource'; and 'TestisMassDataFrom'.  *
 For several species, testis mass and sperm length measurements were
 obtained from a separate study (Liao et al., 2019), and in these cases NA
 values in the 'n_sperm_sampled' and 'n_males_sampled'
 columns represent information missing/not imported from this study.  #####
 Variables * tip.label: Latin name corresponding to the tip labels in the
 phylogeny. * SpeciesCommon: Species common name. *
 SSTTissue.DensestImageOnly: Total amount of tubule tissue within the image
 of highest tubule density (species average). *
 SSTLength.DensestImageOnly: Average length of tubules within the image of
 highest tubule density (species average). *
 SpeciesAverageSSTTissue_um2: Total amount of tubule tissue across the
 entire sample (species average). * SpeciesAverageTubuleLength_um: Average
 length of tubules across the entire sample (species average). *
 FemaleBodyMass_g: Body mass of females. * VaginaLengthMean_mm: Mean vagina
 length.  * TotalTestisMassMean_g: Mean total testis mass (right + left
 testis). * MeanTotalSpermLength_um: Average total sperm length for males.
 * n_sperm_sampled: Number of sperm sampled per species. *
 n_males_sampled: Number of males sampled per species. *
 n_females_sampled: Number of females sampled per species. *
 n_folds_per_female_sampled : Number of folds per female sampled. *
 SpermLengthDataSource: Where sperm length data was obtained from. UoS
 refers to the University of Sheffield (i.e., measurements and samples
 obtained from this study). * TestisMassDataFrom: Where testis mass data
 was obtained from. UoS refers to the University of Sheffield (i.e.,
 measurements and samples obtained from this study). * Complexity: Whether
 the sample contains SSTs categorised as 'complex' (either
 branched, coiled or agglomerate). * StraightUnbranched: Whether the sample
 contains SSTs categorised as 'straight unbranched'. *
 StraightBranched: Whether the sample contains SSTs categorised as
 'straight branched'. ## Code/software ### File: Analysis.R
 **Description:**  * Code to replicate analyses and figure production is
 provided as an R script. *  All analyses were run in R V 4.3.1.  - We
 conduct the majority of these analyses using the measure of SST area and
 length taken from only the image (in each fold) with the highest density
 of tubule tissue (as opposed to taking the absolute values for the entire
 fold). This is because we expect the highest density region of the UVJ to
 be the most functional,and allows us to account for variation in UVJ
 length, the spread of SSTs, and SST functionality along the length of the
 UVJ between species. * Note that We are missing testis mass and sperm
 length data for 4 species. Consequently, any models including these
 variables use separate data with these species removed. **R packages
 required:** rptR; dplyr; ape; caper; geiger; treeplyr; ggtree; ggplot2;
 viridis; phylolm **Script layout:** This script is split into 7 main
 sections: **1) Repeatability** * Measurement repeatability -
 within-individual repeatability (how repeatable are the 2 folds taken per
 individual). * We found them to be highly repeatable. Consequently, the
 average of both folds was taken to give a single value per species. **2)
 Prepare data and tree** * Creating the comparative.data class and objects
 (tree and data combined). Because we are missing testis mass and sperm
 length data for 4 species, we create 2 separate comparative data objects,
 one with all species included (for any model not using testis mass or
 sperm length variables), and one where the species with missing data are
 removed. We create a separate data object for the binary analyses (which
 use phylolm instead of caper) **3) Phylogeny plotting** **4) – 7) Main
 analyses, testing each hypothesis in turn.** **References** Assersohn ,
 K., Richards, J. P., & Hemmings, N. (2024). The surprising complexity
 and diversity of sperm storage structures across Galliformes. *Ecology and
 Evolution*, 14 (6), e11585.
 [https://doi.org/10.1002/ece3.11585](https://doi.org/10.1002/ece3.11585)
 Liao , W. B., Zhong, M. J., & Lüpold, S. (2019). Sperm quality and
 quantity evolve through different selective processes in the Phasianidae.
 *Scientific Reports* , 9 (1), 19278.
 [https://doi.org/10.1038/s41598-019-55822-3](https://doi.org/10.1038/s41598-019-55822-3) Stein , R. W., Brown, J. W., & Mooers, A. Ø. (2015). A molecular genetic time scale demonstrates Cretaceous origins and multiple diversification rate shifts within the order Galliformes (Aves). *Molecular Phylogenetics and Evolution* , 92 , 155–164. [https://doi.org/10.1016/j.ympev.2015.06.005](https://doi.org/10.1016/j.ympev.2015.06.005)