INTRODUCTION
When females mate with more than one male within a single reproductive cycle (i.e., polyandry), post-copulatory sexual selection is hypothesized to favor male reproductive traits that allow them to outcompete rivals in their race to fertilize a females’ ova (i.e., sperm competition; Parker 1970) and female traits that allow them to preferentially bias sperm use in favor of certain males over others (i.e., cryptic female choice; Thornhill 1983; Eberhard 1996) and to prevent polyspermy (Kim et al. 1996; Firman and Simmons 2013; Firman 2018). Consequently, female reproductive traits that enable post-copulatory control and selective fertilization of ova are predicted to occur in polyandrous – but not monogamous – species. This prediction has been poorly tested, however, likely due to lack of attention to female reproductive traits compared to male reproductive traits in post-copulatory sexual selection studies (Orr et al. 2020), the technical challenges of examining covert mechanisms of ‘cryptic female choice’ for internally fertilizing species (reviewed in Firman et al. 2017; Ng et al. 2018), and the difficulty of disentangling these female mechanisms from sperm competition and sexual conflict (Simmons and Wedell 2020).
Despite the challenges with demonstrating cryptic female choice, several studies across diverse taxa have provided empirical support that females are capable of preferentially biasing and controlling sperm use (reviewed in Firman et al. 2017; Firman 2020). For example, in feral fowl (Gallus domesticus ), females use muscular contractions to eject sperm from socially subdominant males to prevent insemination and fertilization of their ova (Pizzari and Birkhead 2000); in Japanese macaques (Macaca fuscata ), females increase orgasm-like muscular contractions after mating with a socially dominant male (Troisi and Carosi 1998), which increases sperm retention within their reproductive tract (Baker and Bellis 1993); and in red flour beetles (Tribolium castaneum ), females appear to be in control of the observed sperm precedence patterns based on male copulatory behavior (Edvardsson and Göran 2000). There is also evidence that physical structures within the female reproductive tract enables female control of sperm use across taxa. For instance, in fruit flies (Drosophila melanogaster ), female sperm storage organs allow them to control the timing and use of sperm stored after copulation with multiple males (Manier et al. 2010). Moreover, many female vertebrates possess a tube-like passageway to their ovaries (i.e., the oviduct), and there is evidence in birds and mammals that features of this structure (Holt and Fazeli 2016), such as its length (Gomendio and Roldan 1993; Anderson et al. 2006), positively correlate with relative testis size, a proxy for sperm competition level (reviewed in Simmons and Fitzpatrick 2012; Vahed and Parker 2012; Lüpold et al. 2020). These findings suggest that the variable structural architecture of the female reproductive tract may have evolved to regulate sperm uptake (Suarez 2008; Tung and Suarez 2021) by selecting for only those sperm cells that are able to bypass its challenging features (Holt and Fazeli 2016; Suarez 2016) while excluding pathogens or microbes (Tung et al. 2015; Holt and Fazeli 2016; Rowe et al. 2020).
The composition of fluids within the female reproductive tract may provide yet another potential mechanism of female control within internally fertilizing species, given that their biochemical properties have been shown to change after insemination, vary throughout the tract, and modulate sperm motility and migration to the ova and, thus, the outcomes of fertilization (reviewed in Holm and Ridderstråale 1998; Hunter et al. 2011; Kirkman-Brown and Smith 2011; Holt and Fazeli 2016; Ng et al. 2018; Gasparini et al. 2020). For example, fluids within the reproductive tract can vary in their viscoelastic properties (Johansson et al. 2000; Rodríguez-Martínez et al. 2005; Suarez 2016), which can influence sperm motility patterns and trajectory (Tung et al. 2015; Holt and Fazeli 2016; Tung and Suarez 2021). In humans, mucus coats the entire female reproductive tract, and sperm must swim through viscoelastic cervical mucous as well as the cumulus mass en route to the oocyte (Kirkman-Brown and Smith 2011); a previous study used artificial insemination to demonstrate that this change in fluidic properties effectively serves as a barrier, allowing only more motile and morphologically normal sperm to pass through to the oviduct (Hanson and Overstreet 1981). Moreover, a pH gradient has been demonstrated throughout the female reproductive tract of different mammals, with the uterine environment being more acidic (i.e., less alkaline) than the oviductal environment (reviewed in Ng et al. 2018). Alkaline environments have been shown to increase sperm velocity and induce sperm hyperactivation in mammals, in part through the activation of essential sperm-specific CatSper protein channels (Kirichok et al. 2006; Lishko et al. 2010) that increases sperm intracellular calcium concentrations (Ho and Suarez 2001; Suarez 2008) and subsequently increases their flagella beat frequency and velocity (Brokaw et al. 1974; Suarez et al. 1993). In boars (Sus scrofa ), high calcium environments lead to greater sperm motility, whereas low calcium environments cause sperm cells to stick to oviductal epithelium and be less motile (Petrunkina et al. 2001). Together these studies suggest that the chemical composition of female reproductive fluids provides a promising mechanism for female sperm control driven by post-copulatory sexual selection, but whether these fluidic properties differ between polyandrous and monogamous species remains unknown.
In this study, we test whether the composition of female reproductive tract fluids diverge between species that have evolved under divergent mating systems in Peromyscus mice. More specifically, we collected fluids from two distinct regions of the reproductive tract – the uterus and the oviduct – for three polyandrous species (P. maniculatus , P. leucopus , and P. gossypinus ) and their closely related monogamous congeners (P. californicus, P. eremicus, and P. polionotus ; Turner et al. 2010; Bedford and Hoekstra 2015). From these fluids, we measured viscosity, pH, and calcium concentration, all of which have been shown to significantly impact sperm movements in other taxa. We compared these physiological properties between species that evolved under polyandry to those that evolved under monogamy to examine associations between mating strategy and potential mechanisms of post-copulatory female control. From these data, we were also able to establish for each species whether a gradient for each of these properties exists within the reproductive tract and indirectly assess how that might impact sperm motility and their unique ability to form collective groups within these mice (Hook et al. 2022).