Statistical analyses
We performed all statistical analyses using R version 3.4.2 (R Core Team 2016) and created all figures using the ‘ggplot2’ package with R (Wickham 2016). We visually inspected all diagnostic plots (qqplots and plots of the distribution of the residuals against fitted values) to validate model normality. In cases where model assumptions of normality were not, we assessed the normality of the response variable using a Shapiro-Wilk test and transformed variables as needed until normality of models was met. Only the best fitting models are reported here.
To assess differences in the viscosity of female reproductive fluids between polyandrous and monogamous species, we used our dataset of individually tracked particles and pooled these data across species with shared mating systems (categorized as either monogamous or polyandrous). We excluded data that were ±2SD of the mean within each sample because they represented clear outliers. All viscosity values were log-transformed to improve model assumptions of normality. We assessed differences in the viscosity of female reproductive fluids in the uterus and in the oviduct using separate linear models (LM), with log viscosity as the response variable and mating system as the predictor variable. We also analyzed fluidic differences in each tract region within each species using separate linear models, with log viscosity as the response variable and the region of the female reproductive tract as the predictor variable. Last, we used the data sets from three focal samples (P. eremicus , P. gossypinus , and P. polionotus ) to statistically compare viscosity measurements to their dyed mineral oil controls using separate linear models, with log viscosity as the response variable and the sample type as the predictor variable. Post-hoc pairwise comparisons were made using Tukey HSD adjustments for multiple comparisons using the ‘LSmeans’ R package (Lenth 2016).
To assess differences in the pH and calcium levels of female reproductive fluids between polyandrous and monogamous species, we used separate linear models for each region of the tract, with either pH or the log calcium measurements included as the response variable and mating system included as the explanatory variable. Last, we used paired t-tests to conduct pairwise comparisons for pH and calcium measurements from each region of the tract within each species to control for differences among individual females.