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Neuroendocrinology of sex-role reversal

Journal: Integrative and Comparative Biology

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Manuscript Type: Symposium article

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Complete List of Authors: Lipshutz, Sara; Indiana University Bloomington, Biology Rosvall, Kimberly; Indiana University Bloomington, Biology

sex-role reversal, female competition, neural sensitivity, testosterone, Keywords: sex steroid

http://mc.manuscriptcentral.com/icbiol Page 1 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 1 Neuroendocrinology of sex-role reversal 4 2 Sara E. Lipshutz1,*, Kimberly A. Rosvall1,2 5 3 6 4 1. Department of Biology, Indiana University, Bloomington, IN, 47405 7 5 2. Center for the Integrated Study of Animal Behavior, Indiana University, Bloomington, IN, 8 47405 9 6 10 7 11 8 *Corresponding author 12 9 Full address: Department of Biology, Jordan Hall Rm. 142, 1001 E. 3rd St., Indiana University, 13 10 Bloomington, IN 47405-7005, USA 14 11 Email address: [email protected] 15 12 Phone number: (812) 855-4555 16 13 Fax: (812) 855-6705 17 14 18 15 Running title: Neuroendocrinology of sex-role reversal 19 16 For Peer Review 20 17 Word count: 4,349 21 18 22 23 19 Abstract 24 25 20 Females of some species are considered sex-role reversed, meaning that they face stronger 26 27 competition for mates compared to males. While much attention has been paid to behavioral 28 21 29 30 22 and morphological patterns associated with sex-role reversal, less is known about its 31 32 23 physiological regulation. Here, we evaluate activational and organizational hypotheses relating 33 34 24 to the neuroendocrine basis of sex-role reversal. We refute the most widely tested activational 35 36 25 hypothesis for sex differences in androgen secretion; sex-role reversed females do not have 37 38 26 higher levels of androgens in circulation than males. However, we find some evidence that the 39 40 27 activational effects of androgens may be sex-specific; circulating androgen levels correlate with 41 42 28 some competitive phenotypes in sex-role reversed females. Organizational effects may explain 43 44 29 these relationships, considering that early exposure to sex steroids can shape later sensitivity to 45 46 30 hormones, often in sex-specific ways. We review evidence that sex-role reversed females have 47 48 49 31 higher tissue-specific sensitivity to androgens than males, at least in some species and tissues. 50 51 32 Moving forward, experimental and correlative studies on the ontogeny and expression of sex- 52 53 33 role reversal will help reveal the mechanisms that generate sex-specific behaviors and sex 54 55 34 roles. 56 57 58 1 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 2 of 27

1 2 3 35 “Why are males masculine, females feminine and occasionally vice-versa?” 4 5 36 – GC Williams, 1975 6 7 37 8 9 10 38 An Introduction to Sex-Role Reversal 11 12 39 Why are females and males different? Biologists have been trying to answer this question 13 14 40 since Darwin first postulated that ‘instinct’ (behavior) may be shaped by natural selection (1859). 15 16 41 In his Victorian era, male animals were considered dominant and promiscuous, whereas female 17 18 42 animals were thought to be coy and subdued. In the intervening years, we have learned a lot 19 For Peer Review 20 43 about ‘sex roles’ across the animal kingdom. For many species, as Darwin and his 21 22 44 contemporaries observed, males face stronger competition for mating opportunities than females, 23 24 45 and females tend to conduct the majority of parental care (Darwin 1871; Clutton-Brock 1991; 25 26 46 Andersson 1994). Whereas territorial aggression and promiscuity were historically considered 27 28 male traits, however, behavioral ecologists now recognize that intrasexual competition and 29 47 30 31 48 multiple-mating are adaptive and widespread behaviors in females of many species (Clutton- 32 33 49 Brock 2009; Rosvall 2011; Hare and Simmons 2018). Sex-role reversal (SRR) occurs when 34 35 50 sexual selection among females is stronger than sexual selection acting among males (Vincent 36 37 51 et al. 1992; Kvarnemo and Ahnesjo 1996). SRR females are characterized by phenotypes 38 39 52 typically associated with males, including morphological traits like heavier body mass, larger 40 41 53 weaponry, and more ornamentation, as well as behavioral traits like higher territorial aggression 42 43 54 or more intense courtship rituals. These traits are thought to facilitate female-female competition 44 45 55 for mates and breeding territories (Emlen and Oring 1977; Gwynne 1991). 46 47 Classic work by Bateman (1948) and Trivers (1972) attributed the evolution of sex roles 48 56 49 50 57 to anisogamy, although this has proven challenging to reconcile with sex-role reversal. In 51 52 58 general, male gametes (sperm) are smaller and more numerous than nutrient-rich female 53 54 59 gametes (ova), and so males may be more available to mate than females, who may be 55 56 60 predisposed to caring for their offspring based on this initial asymmetry in parental investment. 57 58 2 59 60 http://mc.manuscriptcentral.com/icbiol Page 3 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 61 For decades, this anisogamy argument dominated theory on the evolution of sex roles: sex 4 5 62 differences in initial parental investment drive the degree of mating competition and direction of 6 7 63 sexual selection (reviwed in Hoquet, 2020). However, anisogamy cannot explain the evolution of 8 9 10 64 SRR because SRR-females are nonetheless female; they always produce the larger gamete, 11 12 65 even when they compete more than males. Recent theoretical work indicates that additional 13 14 66 factors including multiple paternity, adult mortality, and sex ratios may generate co-evolutionary 15 16 67 feedbacks that influence the strength and sex-specificity of sexual selection (Kokko and 17 18 68 Jennions 2008; Fromhage and Jennions 2016). Despite these advances to our understanding of 19 For Peer Review 20 69 the ultimate drivers of SRR, much less is known about the proximate mechanisms that give rise 21 22 70 to SRR. 23 24 71 Sex steroids are logical candidates for the physiological regulation of SRR, because 25 26 72 these hormones are associated with many sexually dimorphic traits, especially those related to 27 28 mating and mating competition (Adkins-Regan 2005). Sex steroids act in two main ways, the 29 73 30 31 74 first of which operates early in life during a critical period when exposure to a hormone (or lack 32 33 75 thereof) can permanently organize tissue structure and function i.e. organizational effects 34 35 76 (Phoenix et al. 1959; Arnold and Breedlove 1985), which determine whether later exposure to a 36 37 77 hormone can bring about a phenotypic effect. One of the primary modes of action for 38 39 78 organizational effects is to change the anatomical distribution and/or abundance of sex steroid 40 41 79 receptors, early in the life and often lasting into adulthood (Moore et al. 1998). Activational 42 43 80 effects typically occur during adulthood, when animals change aspects of their phenotype in 44 45 81 response to changing hormone levels in circulation. For sex steroids, hormone secretion is 46 47 regulated by the hypothalamic-gonadal-pituitary (HPG) axis, when external stimuli prompt the 48 82 49 50 83 hypothalamus to secrete gonadotropin-releasing hormone (GnRH). GnRH then stimulates the 51 52 84 pituitary to release gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone 53 54 85 (FSH) into the bloodstream. These gonadotropins signal to the gonads to initiate gametogenesis 55 56 86 as well as produce sex steroids including estrogen, progesterone, and testosterone, as well as 57 58 3 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 4 of 27

1 2 3 87 11-ketotestosterone in fishes (Schulz et al. 2010). Many researchers have found important 4 5 88 connections between sex steroids, their cellular mechanisms of action, and the evolution of 6 7 89 sexually selected traits, linking the physiological origins of diversity in mating phenotypes across 8 9 10 90 animals (Wingfield et al. 1990; Soma 2006; Fuxjager and Schuppe 2018; Lipshutz et al. 2019; 11 12 91 Cox 2020). Critically, these mechanisms operate in both sexes (Staub and De Beer 1997), 13 14 92 providing the opportunity to investigate how variation in sex steroid signaling may contribute to 15 16 93 the origin and expression of SRR. 17 18 94 Here, we evaluate two key hypotheses on the role of sex steroids in the evolution of 19 For Peer Review 20 95 SRR. First, we examine the evidence that SRR is explained by activational effects of sex steroid 21 22 96 secretion, focusing on sex differences in levels of androgens in circulation, as well as co- 23 24 97 variation between androgens and competitive phenotypes in SRR species. Next, we assess 25 26 98 hypotheses relating to the organizational basis of SRR, asking whether androgen exposure 27 28 early in development may explain SRR behavior and morphology, and/or sex differences in 29 99 30 31 100 tissue-level sensitivity to androgens (e.g. androgen receptor abundance). We focus primarily on 32 33 101 SRR birds and fishes, which have received the most attention to date, and we draw inferences 34 35 102 from species with conventional sex roles. 36 37 103 38 39 104 H1: SRR stems from activational effects of sex steroids 40 41 105 Do females and males differ in androgen levels in circulation? 42 43 106 Two decades ago, Eens and Pinxten (2000) reviewed studies from field endocrinology to 44 45 107 evaluate the hypothesis that SRR females have male-typical physiological mechanisms, 46 47 specifically that testosterone secretion may be higher in SRR females, a reversal from the 48 108 49 50 109 conventional pattern. Evidence from three SRR avian species did not support this hypothesis: 51 52 110 during mating competition and courtship, males have higher testosterone in circulation 53 54 111 compared with females (spotted sandpiper (Actitis macularius), Rissman and Wingfield 1984; 55 56 112 Wilson's phalarope (Phalaropus tricolor), Fivizzani et al. 1986; red-necked phalarope 57 58 4 59 60 http://mc.manuscriptcentral.com/icbiol Page 5 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 113 (Phalaropus lobatus), Gratto-Trevor et al. 1990), reflecting patterns seen in species with 4 5 114 conventional sex roles. 6 7 115 To re-evaluate this hypothesis 20 years later, we conducted a meta-analysis on sex 8 9 10 116 differences in circulating androgens in SRR species, including both testosterone and its more 11 12 117 potent metabolite 5-alpha dihydrotestosterone (DHT), both of which bind to the androgen 13 14 118 receptor. For the six SRR avian species with relevant data, we compiled mean androgen levels 15 16 119 from HormoneBase (Vitousek et al. 2018) or the primary literature, using data reported in the 17 18 120 text or measured from figures using WebPlotDigitizer (Rohatgi 2019) (Supplementary File 1). 19 For Peer Review 20 121 Our analysis therefore updates Eens and Pinxten’s findings with more recent studies on 21 22 122 androgen secretion in SRR avian species including black coucals (Centropus grillii), barred 23 24 123 buttonquails (Turnix suscitator), and northern jacanas (Jacana spinosa) (Goymann and 25 26 27 124 Wingfield 2004; Voigt and Goymann 2007; Voigt 2016; Lipshutz and Rosvall 2020). To estimate 28 29 125 the standardized effect size of sex differences in androgens across SRR species, we used 30 31 126 random effects models in the package metafor in R (Viechtbauer 2010). We ran separate 32 33 127 models comparing females to males in different breeding stages (i.e. courting vs. caring), as 34 35 128 male androgen levels typically decline with parental care (Wingfield et al. 1990). We also ran a 36 37 129 mixed effects model with breeding stage as a modulator, to compare the influence of breeding 38 39 130 stage on sex differences in levels of androgens in circulation. 40 41 First, focused on data from females and males, sampled when both sexes were 42 131 43 44 132 competing and courting, our analysis showed an average negative and significant effect size (μ 45 46 133 = -1.14, z = -5.24, p < 0.0001), indicating that SRR males have higher androgens than females 47 48 134 (Figure 1). Thus, during the period of time when both sexes are seeking mates, SRR females 49 50 135 secrete androgens in ways that are similar to females of species with conventional sex roles, 51 52 136 and SRR males follow patterns similar to males with conventional roles. However, this 53 54 137 courtship-stage model had significant heterogeneity in sex differences (I2 = 0.56, Q = 22.23, df = 55 56 57 58 5 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 6 of 27

1 2 3 138 10, p = 0.014), indicating that not every species demonstrated the same pattern. For example, 4 5 139 female and male barred buttonquails did not differ in levels of DHT in circulation (Voigt 2016). 6 7 140 When we analyzed androgen levels in SRR birds during the period of time when males 8 9 10 141 are parenting, however, the effect size was not significant (μ = -0.019, z = -0.14, p = 0.89), 11 12 142 indicating no overall sex difference in circulating androgen levels (Figure 1). This parental-stage 13 14 143 model did not have significant heterogeneity in sex differences (I2 = 0, Q = 4.81, df = 7, p = 15 16 144 0.68), indicating similar patterns across species. During the parental care phase of breeding, 17 18 145 these SRR males and females did not differ significantly in levels of testosterone and DHT in 19 For Peer Review 20 146 circulation, meaning the sex differences in androgen levels seen during courtship are ablated 21 22 147 during periods of male parental care. Indeed, direct comparison of the courtship-stage and 23 24 148 parental-stage data into a single model shows that breeding stage explained 79% of the 25 26 149 variation in effect size (QM = 18.52, df = 1, p < 0.0001). In males with conventional sex roles, 27 28 androgen levels decline during parental care, though not typically to female-like levels 29 150 30 31 151 (Wingfield et al. 1990; Hirschenhauser and Oliveira 2006). Our finding – that androgens in SRR 32 33 152 males fall to levels as low as females during parental care – may represent an extreme 34 35 153 reduction in androgen sexual dimorphism, potentially suggesting that SRR drives lower T levels 36 37 154 in males, higher T levels in females, or some combination of the two (sensu Goymann and 38 39 155 Wingfield 2014). We find it interesting that in SRR species, circulating androgen levels are most 40 41 156 similar between the sexes during the breeding stage when the sexes are most behaviorally 42 43 157 divergent, i.e. female competition and male parental care. Clearly, sex differences in levels of 44 45 158 androgens in circulation alone do not explain SRR. 46 47 Beyond T and DHT, fewer studies have measured other sex steroids or prohormones in 48 159 49 50 160 SRR species, and evidence to date is mixed as to whether secretion of these hormones is 51 52 161 sexually dimorphic. For example, a study of black coucals suggests that secretion of the 53 54 162 androgenic precursors androstenedione and DHEA is similar between the sexes, regardless of 55 56 163 breeding stage (Goymann and Wingfield 2004). Estradiol levels are typically higher in females 57 58 6 59 60 http://mc.manuscriptcentral.com/icbiol Page 7 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 164 than males in conventional species, a pattern that is reflected in some SRR species (Fivizzani et 4 5 165 al. 1986), but not others, for which estradiol levels are similar between the sexes, or higher than 6 7 166 expected in SRR males (Rissman and Wingfield 1984; Goymann and Wingfield 2004; Voigt 8 9 10 167 2016). Progesterone levels are also typically higher in females than males, which is reflected in 11 12 168 some SRR species (Fivizzani et al. 1986; Gratto-Trevor et al. 1990), but not others (Voigt 2016). 13 14 169 In SRR broadnosed pipefish (Syngnathus typhle) and greater pipefish (Syngnathus acus), 15 16 170 breeding males have higher 11-ketotestosterone than brooding males (Mayer et al. 1993), a 17 18 171 pattern reflected in non-SRR teleost fish (Knapp et al. 1999; Mayer et al. 2004), but circulating 19 For Peer Review 20 172 hormones have been challenging to measure in sygnathids (Scobell and Mackenzie 2011). 21 22 173 Together, these studies suggest that the sex steroid profiles of SRR females and males are 23 24 174 similar to their counterparts with conventional sex roles, largely confirming the conclusions of 25 26 175 Eens and Pinxten (2000). 27 28 29 176 30 31 177 Are activational effects of androgens sexually dimorphic? 32 33 178 Androgen levels themselves are only part of the regulation of androgen-mediated 34 35 179 phenotypes, and there is good evidence that the sexes may differ in their gene regulatory 36 37 180 responses to androgens (Van Nas et al. 2009; Peterson et al. 2014). Thus, androgens may 38 39 181 differentially affect females and males in SRR species, even when hormone levels themselves 40 41 182 largely follow patterns of sexual dimorphism seen in species with conventional sex roles. Sex- 42 43 183 specific activational effects of androgens can be assessed with (a) correlations that directly link 44 45 184 SRR behaviors and morphological traits with sex steroids, or (b) experimental treatments 46 47 between sex steroids and traits of interest. 48 185 49 50 186 One approach to understand the physiological regulation of SRR is to link individual 51 52 187 variation in endocrine phenotypes directly with variation in competitive traits, including 53 54 188 ornamentation, weaponry, and body size. Correlational support for this idea does exist in SRR 55 56 189 species, although it is quite limited. For instance, levels of testosterone in circulation positively 57 58 7 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 8 of 27

1 2 3 190 correlate with melanin throat patch and body condition in female barred buttonquails (Muck and 4 5 191 Goymann 2011). In female northern jacanas, testosterone secretion positively correlates with 6 7 192 the size of weaponry, wing spurs, but this relationship was not found in males (Lipshutz and 8 9 10 193 Rosvall 2020). Thus, despite low levels of testosterone in circulation in SRR females, there is 11 12 194 some evidence that testosterone is related to the regulation of competitive traits in SRR females 13 14 195 in some way. Other studies find that non-steroid hormones may regulate competitive traits in 15 16 196 relation to SRR. In the two-spotted goby (Gobiusculus flavescens), a species with dynamic sex 17 18 197 roles that change from conventional to reversed (Forsgren et al. 2004), pigmentation of the 19 For Peer Review 20 198 female belly ornament is regulated by the pituitary hormone prolactin as well as alpha- 21 22 199 melanocyte stimulating hormone, but this ornament does not appear to be regulated by the sex 23 24 200 steroids testosterone, 11-keto-testosterone, or estradiol in this example (Sköld et al. 2008). 25 26 201 Experimental data to directly address sex-specific activational effects of androgens in 27 28 SRR species is also limited, but indirect evidence suggests that both morphological and 29 202 30 31 203 behavioral traits involved in mating competition respond to experimental manipulation of sex 32 33 204 steroids. Early work in the SRR Wilson’s and Red-necked phalaropes, for which females have 34 35 205 brighter nuptial plumage, found that exogenous testosterone induces nuptial plumage in both 36 37 206 females and males, suggesting that nuptial feather growth is androgen-dependent (Johns 38 39 207 1964). In male gulf pipefish (Syngnathus scovelli), exposure to estrogen impacts the 40 41 208 development of the iridescent transverse band (Partridge et al. 2010), a sexually selected 42 43 209 ornament in females (Flanagan et al. 2014). These findings parallel work in species with 44 45 210 conventional roles, for which testosterone implants in females increase male-typical traits 46 47 including courtship displays (Day et al. 2007), vocalizations (Nottebohm 1980; Chiver and 48 211 49 50 212 Schlinger 2019), and nuptial plumage (Lindsay et al. 2016). In other words, the activational 51 52 213 effects of sex steroids can reverse sex-specific phenotypes in SRR males in a manner similar to 53 54 214 non-SRR females. For competitive behaviors like aggression, the link with testosterone in SRR 55 56 215 species has mixed support. In female barred buttonquails, implantation with testosterone did not 57 58 8 59 60 http://mc.manuscriptcentral.com/icbiol Page 9 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 216 increase aggression, and territorial challenge decreased levels of testosterone in circulation 4 5 217 (Muck and Goymann 2019). In female black coucals, testosterone did not differ between 6 7 218 challenged and unchallenged females, but territorial challenge decreased levels of progesterone 8 9 10 219 in circulation, and progesterone implants reduced female aggression (Goymann et al. 2008). 11 12 220 Similarly, the lack of a relationship between testosterone and aggressive behavior is reflected in 13 14 221 many species with conventional sex roles (Wingfield et al. 2019). In sum, variation in androgens 15 16 222 in circulation may explain some competitive phenotypes but not others, and these relationships 17 18 223 can vary by sex, suggesting we must also look beyond activational hypotheses on the origin and 19 For Peer Review 20 224 expression of SRR. 21 22 225 23 24 226 H2: SRR stems from organizational effects of sex steroids 25 26 227 Organizational effects of sex steroids deliver some promise as proximate regulators of 27 28 SRR (Adkins-Regan 2012). An ontogenetic hypothesis for SRR was proposed by Fivizzani et al. 29 228 30 31 229 (1986), drawing from observations in many species that exposure to sex steroid hormones early 32 33 230 in development can generate sex differences in later responsiveness to these hormones in 34 35 231 adulthood (Arnold 2009). For instance, exposure to high testosterone early in life, in utero, or in 36 37 232 ovo can influence suites of sexual characteristics (vom Saal and Bronson 1980; Hotchkiss et al. 38 39 233 2007), a phenotypic effect that is at least partly mediated by tissue level changes in sensitivity to 40 41 234 sex steroids (Mori et al. 2010; Pfannkuche et al. 2011). Sensitivity is comprised of a number of 42 43 235 factors, including sex steroid receptors, as well as enzymes that produce steroid hormones and 44 45 236 convert them into more or less active forms (Ball and Balthazart 2008). In particular, 46 47 testosterone can be locally converted by the enzymes aromatase and 5-alpha-reductase to the 48 237 49 50 238 metabolites estradiol and DHT (Schmidt et al. 2008). These sex steroids bind to estrogen (ER) 51 52 239 and androgen receptors (AR), respectively, initiating downstream transcriptional effects on 53 54 240 peripheral and neural tissues that influence the expression of diverse mating phenotypes 55 56 241 (Fuxjager and Schuppe 2018). Sex steroid sensitivity can be evaluated by measuring the 57 58 9 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 10 of 27

1 2 3 242 protein or mRNA abundance of sex steroid receptors and metabolic enzymes, and several 4 5 243 aspects of sex steroid sensitivity and metabolism have been measured in SRR species, at least 6 7 244 in some peripheral and neural tissues. 8 9 10 245 11 12 246 Do females and males differ in sensitivity to sex steroids? 13 14 247 The first study to address this question in a SRR system focused on Wilson’s 15 16 248 phalaropes, in which females showed higher 5-alpha and 5-beta reductase activity in the skin 17 18 249 (Schlinger et al. 1989). These differences may explain why females have brighter nuptial 19 For Peer Review 20 250 plumage than males, but sex differences in neural androgen metabolism did not explain SRR 21 22 251 behavior; the sexes did not differ in 5-alpha or 5-beta reductase in the neural tissues sampled. 23 24 252 Furthermore, courting male Wilson’s phalaropes had higher aromatase activity in the 25 26 253 hypothalamus than females, a pattern found in non-SRR species (Balthazart 1991). We are 27 28 aware of two additional studies that have examined neural sensitivity in SRR species, including 29 254 30 31 255 black coucals (Voigt and Goymann 2007) and barred buttonquails (Voigt 2016). These studies, 32 33 256 like the earlier study in Wilson’s phalaropes, focus on the vertebrate social behavior network, an 34 35 257 assemblage of steroid-sensitive brain regions that regulate mating, sexual, and social behaviors 36 37 258 (Goodson 2005; Maney and Goodson 2011). In black coucals and barred buttonquails, AR 38 39 259 mRNA abundance in the nucleus taeniae was higher in females compared to males, suggesting 40 41 260 that SRR females may be able to ‘do more with less’ testosterone in circulation. In species with 42 43 261 conventional sex roles, variation in sex steroid sensitivity in the nucleus taeniae may explain 44 45 262 variation in aggression, even when hormone levels in the blood do not (Rosvall et al. 2012; 46 47 Horton et al. 2014). Thus, higher androgen sensitivity in the SRR female nucleus taeniae is an 48 263 49 50 264 encouraging explanation for SRR. However, not all studies find such patterns. For instance, 51 52 265 aromatase gene expression was higher in hypothalamic regions in male barred buttonquails 53 54 266 compared to females, a pattern that is comparable to non-SRR Japanese quail (Coturnix 55 56 267 japonica) (Voigt et al. 2009) and also found in Wilson’s phalaropes (Schlinger et al. 1989). 57 58 10 59 60 http://mc.manuscriptcentral.com/icbiol Page 11 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 268 Together, these results suggest modularity of the social behavior network: some aspects of sex 4 5 269 steroid sensitivity can be heightened in some neural tissues, and this may vary between 6 7 270 females and males. 8 9 10 271 Global transcriptomic analyses similarly point to sexual dimorphism in sex steroid 11 12 272 sensitivity as potentially relevant for SRR species. A microarray study comparing conventional 13 14 273 and SRR cichlid species (Julidodchromis spp) examined sex differences in gene expression in 15 16 274 the whole brain (Schumer et al. 2011). This study found that SRR females had globally similar 17 18 275 neural gene expression to males in the conventional species, indicating some masculinization of 19 For Peer Review 20 276 the SRR female brain. Notably, differentially expressed genes between sexes included 21 22 277 aromatase and isotocin, a paralog of arginine vasotocin (AVT), which can co-localize in AR+ 23 24 278 neurons and influence behavior via steroid sensitive circuits (Kabelik et al. 2010). A recent RNA- 25 26 279 seq study of skin and muscle tissue in Gulf pipefish also reported that genes differentially 27 28 expressed between the sexes have an excess of estrogen response elements, suggesting a 29 280 30 31 281 role for sex steroids in the genomic regulation of female ornamentation and body depth 32 33 282 (Anderson et al. 2020). Thus, similar to non-SRR species (Tomaszycki et al. 2009; Wade 2016), 34 35 283 there is potential for sex-biased gene expression to influence sex differences in behavior. 36 37 284 Moving forward, these global analyses have the potential to reveal other important mechanisms 38 39 285 regulating SRR, particularly if they explicitly link specific nuclei with competitive traits in SRR 40 41 286 species and their non-SRR relatives. 42 43 287 44 45 288 What are the ontogenetic origins of sex differences in steroid sensitivity? 46 47 Despite good evidence for sex differences in steroid sensitivity in adults, more research 48 289 49 50 290 is needed to directly link adult data with early life processes in SRR species. To our knowledge, 51 52 291 no studies have experimentally manipulated sex steroid exposure early in life to change SRR 53 54 292 trait development, and only two studies have investigated the hormonal ontogeny of SRR 55 56 293 species. In barred buttonquails, for example, AR mRNA expression levels were higher in female 57 58 11 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 12 of 27

1 2 3 294 hatchlings in every brain area investigated, and this sex difference persisted into adulthood for 4 5 295 the mediobasal hypothalamus, lateral septum, and nucleus taeniae (Voigt 2016). Notably, these 6 7 296 patterns differ from the closely related Japanese quail, a non-SRR species in which AR mRNA 8 9 10 297 abundance is equal in hatchling females and males (Voigt et al. 2009). Another study focused 11 12 298 more on testosterone levels in circulation in black coucals, a species that displays the typical 13 14 299 SRR pattern in which adult females are larger than males (Andersson 1995). Specifically, 15 16 300 Goymann and colleagues (2005) found that female nestlings grow faster and fledge with a 17 18 301 larger body mass, compared to males. Although female nestlings do not have higher levels of 19 For Peer Review 20 302 testosterone in circulation compared to males, structural growth rates are related to testosterone 21 22 303 in females but not males (Goymann et al. 2005). Considering connections between androgen 23 24 304 exposure and growth, which set the stage for sexual dimorphism in competitive traits in many 25 26 305 species (Hews and Moore 1995; Cox et al. 2015), these patterns indirectly suggest 27 28 organizational effects as plausible drivers of SRR. In non-SRR species, gonadally derived 29 306 30 31 307 hormones early in development shape neural substrate for activation in adulthood in a sex- 32 33 308 specific manner, wherein sexual differentiation results from organizational alignment between 34 35 309 gonadal and neural phenotypes (McCarthy 2016). To what extent are gonadal and neural 36 37 310 phenotypes mismatched, or more modular, in SRR species? Future work treating embryos 38 39 311 and/or juveniles with testosterone or aromatase inhibitors are needed to explicitly test the role of 40 41 312 sex steroids in the development of SRR behavior and morphology. 42 43 313 44 45 314 Conclusions and future directions 46 47 We evaluated activational and organizational hypotheses linking sex steroids with the 48 315 49 50 316 development and expression of SRR. Our meta-analysis of sex differences in androgen 51 52 317 secretion found that SRR species follow the pattern of conventional species: males have higher 53 54 318 levels of androgens in circulation during courtship. Despite stronger selection to compete for 55 56 319 mates, SRR females are still females -- they produce ova, solicit copulation, and typically prefer 57 58 12 59 60 http://mc.manuscriptcentral.com/icbiol Page 13 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 320 to mate with males. Even at low levels, however, androgens correlate with some competitive 4 5 321 phenotypes in SRR females, suggesting that activational effects of androgens may be important 6 7 322 in the expression of SRR. These relationships could be reconciled via sex-specific changes in 8 9 10 323 sensitivity to androgens in the neural and peripheral tissues that influence ‘reversed’ traits in 11 12 324 SRR species, such as territorial aggression, plumage coloration, or growth. However, it is still 13 14 325 unclear whether and how developmental androgen exposure drives SRR in adults. 15 16 326 With these findings in mind, we see three research initiatives that can move the field 17 18 327 towards greater understanding of the role of hormones in the evolution of SRR. First, evidence 19 For Peer Review 20 328 thus far suggests that tissue-specific sensitivity and organizational effects of androgens may 21 22 329 generate SRR, but we need more experiments. Effectively testing these hypotheses will require 23 24 330 manipulation of sex steroid levels, metabolism, and/or sensitivity, as well as account for 25 26 331 phylogenetic history, for instance using paired designs that directly compare SRR species with 27 28 non-SRR close relatives. Finding that some SRR phenotypes can be un-reversed, whereas 29 332 30 31 333 others are fixed, could point to the influence of activational, organizational, or direct genetic 32 33 334 effects (Adkins-Regan 2005). 34 35 335 Second, we focused on the regulation of competitive traits, but similar hypotheses can 36 37 336 apply to the regulation of parental care in SRR males. Although parental care is outside the 38 39 337 scope of this review, and historically not part of the definition of SRR (Ah-King and Ahnesjö 40 41 338 2013), sex steroids and other hormones like oxytocin, vasopressin, and prolactin are important 42 43 339 in the regulation of parental care (Smiley 2019; Storey et al. 2020). In many SRR species, 44 45 340 males conduct the majority of parental care and have higher levels of prolactin in circulation 46 47 than females (Oring et al. 1986, 1988; Gratto-Trevor et al. 1990). In other words, SRR males 48 341 49 50 342 may have female-typical physiological mechanisms related to parenting. Whether these parental 51 52 343 mechanisms are the same that regulate competitive traits (i.e. pleiotropy) or whether these traits 53 54 344 are independently regulated in relation to SRR is less clear. 55 56 57 58 13 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 14 of 27

1 2 3 345 Finally, like many areas of animal behavior, the study of SRR will surely be enhanced by 4 5 346 integrated research that explicitly connects ultimate eco-evolutionary processes driving SRR 6 7 347 with the proximate neuroendocrine factors that generate trait variation. Ecological feedbacks 8 9 10 348 between the social environment and maternal physiology are a natural area of focus due to 11 12 349 potential links between adult trait variation and early life processes. Maternal effects have yet to 13 14 350 be tested in SRR species, but evidence from non-SRR species suggests that high-competition 15 16 351 environments can influence maternal testosterone allocation to yolk (Bentz et al. 2016). The 17 18 352 association of sex-role reversal with male-biased adult sex ratios (Liker et al. 2013) suggests 19 For Peer Review 20 353 the potential for maternal effects and ecological feedback, as hormones can also influence sex 21 22 354 ratios (Navara 2013). This raises the possibility that some physiological regulation of SRR may 23 24 355 be environmentally plastic, an exciting arena for future study. 25 26 356 27 28 Funding 29 357 30 31 358 This work was supported by a National Science Foundation Postdoctoral Fellowship (grant 32 33 359 number 1907134); and an Indiana University Provost’s Travel Award for Women in Science to 34 35 360 SEL. Any opinion, findings, and conclusions or recommendations expressed in this material are 36 37 361 those of the authors and do not necessarily reflect the views of the National Science 38 39 362 Foundation. 40 41 363 42 43 364 Acknowledgements 44 45 365 We thank the organizers of the SICB symposium on female perspectives of reproduction. SEL 46 47 thanks Christie Riehl and Elizabeth Derryberry for encouraging this topic. Illustrations by Mae 48 366 49 50 367 Berlow. 51 52 368 53 54 369 Figure 1. Mean effect sizes with corresponding 95% confidence intervals for sex differences in 55 56 370 levels of androgens in circulation between females and males in courtship or parental stages. 57 58 14 59 60 http://mc.manuscriptcentral.com/icbiol Page 15 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 371 Black square sizes represent corresponding sampling variances. Grey diamonds represent the 4 5 372 estimated true effect from the male courtship model (μ = -1.14) and male parenting random- 6 7 373 effects model (μ = -0.019). 8 9 10 374 11 12 375 References 13 14 376 Adkins-Regan E. 2005. Hormones and Animal Social Behavior Princeton, NJ: Princeton 15 16 377 University Press. 17 18 378 Adkins-Regan E. 2012. Hormonal organization and activation: Evolutionary implications and 19 For Peer Review 20 379 questions. Gen Comp Endocrinol 176:279–85. 21 22 380 Ah-King M, Ahnesjö I. 2013. The “sex role” concept: An overview and e valuation. Evol Biol 23 24 381 40:461–70. 25 26 382 Anderson AP, Flanagan SP, Rose E, Jones AG. 2020. The estrogen-responsive transcriptome 27 28 of female secondary sexual traits in the Gulf pipefish. J Hered esaa008. 29 383 30 31 384 Andersson M. 1994. Sexual selection, Monographs in behavior and ecology Princeton: 32 33 385 Princeton Univ. Press. 34 35 386 Andersson M. 1995. Evolution of reversed sex roles, sexual size dimorphism, and mating 36 37 387 system in coucals (Centropodidae, Aves). Biol J Linn Soc 54:173–81. 38 39 388 Arnold AP. 2009. The organizational-activational hypothesis as the foundation for a unified 40 41 389 theory of sexual differentiation of all mammalian tissues. Horm Behav 55:570–78. 42 43 390 Arnold AP, Breedlove SM. 1985. Organizational and activational effects of sex steroids on brain 44 45 391 and behavior: A reanalysis. Horm Behav 19:469–98. 46 47 Ball GF, Balthazart J. 2008. Individual variation and the endocrine regulation of behaviour and 48 392 49 50 393 physiology in birds: a cellular/molecular perspective. Philos Trans R Soc B Biol Sci 51 52 394 363:1699–1710. 53 54 395 Balthazart J. 1991. Testosterone metabolism in the avian hypothalamus. J Steroid Biochem Mol 55 56 396 Biol 40:557–70. 57 58 15 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 16 of 27

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1 2 3 449 Hews DK, Moore MC. 1995. Influence of androgens on differentiation of secondary sex 4 5 450 characters in tree lizards, Urosaurus ornatus. Gen Comp Endocrinol. 6 7 451 Hirschenhauser K, Oliveira RF. 2006. Social modulation of androgens in male vertebrates: 8 9 10 452 Meta-analyses of the challenge hypothesis. Anim Behav 71:265–77. 11 12 453 Hoquet T. 2020. Bateman (1948): rise and fall of a paradigm? Anim Behav In press. 13 14 454 Horton BM, Hudson WH, Ortlund EA, Shirk S, Thomas JW, Young ER, Zinzow-Kramer WM, 15 16 455 Maney DL. 2014. Estrogen receptor α polymorphism in a species with alternative 17 18 456 behavioral phenotypes. Proc Natl Acad Sci 111:1443–48. 19 For Peer Review 20 457 Hotchkiss AK, Lambright CS, Ostby JS, Parks-Saldutti L, Vandenbergh JG, Gray Jr LE. 2007. 21 22 458 Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple 23 24 459 development, and reproductive tract morphology in female Sprague-Dawley rats. Toxicol 25 26 460 Sci 96:335–45. 27 28 Johns JE. 1964. Testosterone-induced nuptial feathers in phalaropes. Condor 66:449–55. 29 461 30 31 462 Kabelik D, Morrison JA, Goodson JL. 2010. Cryptic regulation of vasotocin neuronal activity but 32 33 463 not anatomy by sex steroids and social stimuli in opportunistic desert finches. Brain Behav 34 35 464 Evol 75:71–84. 36 37 465 Knapp R, Wingfield JC, Bass AH. 1999. Steroid hormones and paternal care in the plainfin 38 39 466 midshipman fish (Porichthys notatus). Horm Behav 35:81–89. 40 41 467 Kokko H, Jennions MD. 2008. Parental investment, sexual selection and sex ratios. J Evol Biol 42 43 468 21:919–48. 44 45 469 Kvarnemo C, Ahnesjo I. 1996. The dynamics of operational sex ratios and competition for 46 47 mates. Trends Ecol Evol 11:404–8. 48 470 49 50 471 Liker A, Freckleton RP, Székely T. 2013. The evolution of sex roles in birds is related to adult 51 52 472 sex ratio. Nat Commun 4:1–6. 53 54 473 Lindsay WR, Barron DG, Webster MS, Schwabl H. 2016. Testosterone activates sexual 55 56 474 dimorphism including male-typical carotenoid but not melanin plumage pigmentation in a 57 58 18 59 60 http://mc.manuscriptcentral.com/icbiol Page 19 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 475 female bird. J Exp Biol 219:3091–99. 4 5 476 Lipshutz SE, George EM, Bentz AB, Rosvall KA. 2019. Evaluating testosterone as a phenotypic 6 7 477 integrator: From tissues to individuals to species. Mol Cell Endocrinol 496:110531. 8 9 10 478 Lipshutz SE, Rosvall KA. 2020. Testosterone secretion varies in a sex- and stage-specific 11 12 479 manner: Insights on the regulation of competitive traits from a sex-role reversed species. 13 14 480 Gen Comp Endocrinol 292:113444. 15 16 481 Maney DL, Goodson JL. 2011. Neurogenomic mechanisms of aggression in songbirds, 17 18 482 Advances in Genetics. 19 For Peer Review 20 483 Mayer I, Borg B, Pall M. 2004. Hormonal control of male reproductive behaviour in rishes: A 21 22 484 stickleback perspective. Behaviour 141:1499–1510. 23 24 485 Mayer I, Rosenqvist G, Borg B, Ahnesjö I, Berglund A, Schulz RW. 1993. Plasma levels of sex 25 26 486 steroids in three species of pipefish (Syngnathidae). Can J Zool 71:1903–7. 27 28 McCarthy MM. 2016. Multifaceted origins of sex differences in the brain. Philos Trans R Soc B 29 487 30 31 488 Biol Sci 371. 32 33 489 Moore MC, Hewsf DK, Knapp R. 1998. Hormonal control and evolution of alternative male 34 35 490 phenotypes: Generalizations of models for sexual differentiation. Am Zool 38:133–51. 36 37 491 Mori H, Matsuda KI, Tsukahara S, Kawata M. 2010. Intrauterine position affects estrogen 38 39 492 receptor α expression in the ventromedial nucleus of the hypothalamus via promoter DNA 40 41 493 methylation. Endocrinology 151:5775–81. 42 43 494 Muck C, Goymann W. 2011. Throat patch size and darkness covaries with testosterone in 44 45 495 females of a sex-role reversed species. Behav Ecol 20:1312–19. 46 47 Muck C, Goymann W. 2019. Exogenous testosterone does not modulate aggression in sex-role- 48 496 49 50 497 reversed female Barred Buttonquails, Turnix suscitator. J Ornithol 160:399–407. 51 52 498 Navara KJ. 2013. Hormone-mediated adjustment of sex ratio in vertebrates. Integr Comp Biol 53 54 499 53:877–87. 55 56 500 Nottebohm F. 1980. Testosterone triggers growth of brain vocal control nuclei in adult female 57 58 19 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 20 of 27

1 2 3 501 canaries. Brain Res 189:429–36. 4 5 502 Oring LW, Fivizzani AJ, Colwell MA, El Halawani ME. 1988. Hormonal changes associated with 6 7 503 natural and manipulated incubation in the sex-role reversed Wilson’s phalarope. Gen 8 9 10 504 Comp Endocrinol 72:247–56. 11 12 505 Oring LW, Fivizzani AJ, El Halawani ME, Goldsmith A. 1986. Seasonal changes in prolactin and 13 14 506 luteinizing hormone in the polyandrous spotted sandpiper, Actitis macularia. Gen Comp 15 16 507 Endocrinol 62:394–403. 17 18 508 Partridge C, Boettcher A, Jones AG. 2010. Short-term exposure to a synthetic estrogen disrupts 19 For Peer Review 20 509 mating dynamics in a pipefish. Horm Behav 58:800–807. 21 22 510 Peterson MP, Rosvall KA, Taylor CA, Lopez JA, Choi J-H, Ziegenfus C, Tang H, Colbourne JK, 23 24 511 Ketterson ED. 2014. Potential for sexual conflict assessed via testosterone-mediated 25 26 512 transcriptional changes in liver and muscle of a songbird. J Exp Biol 217:507–17. 27 28 Pfannkuche KA, Gahr M, Weites IM, Riedstra B, Wolf C, Groothuis TGG. 2011. Examining a 29 513 30 31 514 pathway for hormone mediated maternal effects--yolk testosterone affects androgen 32 33 515 receptor expression and endogenous testosterone production in young chicks (Gallus 34 35 516 gallus domesticus). Gen Comp Endocrinol 172:487–93. 36 37 517 Phoenix C, Goy R, Gerall A, Young W. 1959. Organizing action of prenatally administered 38 39 518 testosterone propionate on the tissues mediating mating behavior in the female guinea pig. 40 41 519 Endocrinology 65:369–382. 42 43 520 Rissman EF, Wingfield JC. 1984. Hormonal correlates of polyandry in the spotted sandpiper, 44 45 521 Actitis macularia. Gen Comp Endocrinol 56:401–5. 46 47 Rohatgi A. 2019. WebPlotDigitizer. . 48 522 49 50 523 Rosvall KA. 2011. Intrasexual competition in females: Evidence for sexual selection? Behav 51 52 524 Ecol. 22:1131-1140. 53 54 525 Rosvall KA, Bergeon Burns CM, Barske J, Goodson JL, Schlinger BA, Sengelaub DR, 55 56 526 Ketterson ED. 2012. Neural sensitivity to sex steroids predicts individual differences in 57 58 20 59 60 http://mc.manuscriptcentral.com/icbiol Page 21 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 527 aggression: implications for behavioural evolution. Proc R Soc B Biol Sci 279:3547–55. 4 5 528 Schlinger BA, Fivizzani AJ, Callard G V. 1989. Aromatase, 5 alpha- and 5 beta-reductase in 6 7 529 brain, pituitary and skin of the sex-role reversed Wilson’s phalarope. J Endocrinol. 8 9 10 530 Schmidt KL, Pradhan DS, Shah AH, Charlier TD, Chin EH, Soma KK. 2008. Neurosteroids, 11 12 531 immunosteroids, and the Balkanization of endocrinology. Gen Comp Endocrinol 157:266– 13 14 532 74. 15 16 533 Schulz RW, de França LR, Lareyre J-J, LeGac F, Chiarini-Garcia H, Nobrega RH, Miura T. 17 18 534 2010. Spermatogenesis in fish. Gen Comp Endocrinol 165:390–411. 19 For Peer Review 20 535 Schumer M, Krishnakant K, Renn SCP. 2011. Comparative gene expression profiles for highly 21 22 536 similar aggressive phenotypes in male and female cichlid fishes (Julidochromis). J Exp Biol 23 24 537 214:3269–78. 25 26 538 Scobell SK, Mackenzie DS. 2011. Reproductive endocrinology of Syngnathidae. J Fish Biol 27 28 78:1662–80. 29 539 30 31 540 Sköld HN, Amundsen T, Svensson PA, Mayer I, Bjelvenmark J, Forsgren E. 2008. Hormonal 32 33 541 regulation of female nuptial coloration in a fish. Horm Behav 54:549–56. 34 35 542 Smiley KO. 2019. Prolactin and avian parental care: New insights and unanswered questions. 36 37 543 Horm Behav 111:114–30. 38 39 544 Soma KK. 2006. Testosterone and aggression: Berthold, birds and beyond. J Neuroendocrinol 40 41 545 18:543–51. 42 43 546 Staub NL, De Beer M. 1997. The role of androgens in female vertebrates. Gen Comp 44 45 547 Endocrinol 108:1–24. 46 47 Storey AE, Alloway H, Walsh CJ. 2020. Dads: Progress in understanding the neuroendocrine 48 548 49 50 549 basis of human fathering behavior. Horm Behav 119:104660. 51 52 550 Tomaszycki ML, Peabody C, Replogle K, Clayton DF, Tempelman RJ, Wade J. 2009. Sexual 53 54 551 differentiation of the zebra finch song system: potential roles for sex chromosome genes. 55 56 552 BMC Neurosci 10:24. 57 58 21 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 22 of 27

1 2 3 553 Trivers RL. 1972. Parental investment and sexual selection. In: Campbell B, editor. Sexual 4 5 554 Selection and the Descent of Man: The Darwinian Pivot Chicago: Aldine. p. 136–79. 6 7 555 Van Nas A, Guhathakurta D, Wang SS, Yehya N, Horvath S, Zhang B, Ingram-Drake L, 8 9 10 556 Chaudhuri G, Schadt EE, Drake TA, Arnold AP, Lusis AJ. 2009. Elucidating the role of 11 12 557 gonadal hormones in sexually dimorphic gene coexpression networks. Endocrinology 13 14 558 150:1235–49. 15 16 559 Viechtbauer W. 2010. Conducting meta-analyses in R with the metafor. J Stat Softw 36:1–48. 17 18 560 Vincent A, Ahnesjo I, Berglund A, Rosenqvist G. 1992. Pipefishes and seahorses: Are they all 19 For Peer Review 20 561 sex role reversed? Trends Ecol Evol 7:237–41. 21 22 562 Vitousek MN, Johnson MA, Donald JW, Francis CD, Fuxjager MJ, Goymann W, Hau M, Husak 23 24 563 JF, Kircher BK, Knapp R, Martin LB, Miller ET, Schoenle LA, Uehling JJ, Williams TD. 25 26 564 2018. Data Descriptor: HormoneBase, a population-level database of steroid hormone 27 28 levels across vertebrates. Sci Data 5:1–7. 29 565 30 31 566 Voigt C. 2016. Neuroendocrine correlates of sex-role reversal in barred buttonquails. Proc R 32 33 567 Soc B Biol Sci 283. 34 35 568 Voigt C, Ball GF, Balthazart J. 2009. Sex differences in the expression of sex steroid receptor 36 37 569 mRNA in the quail brain. J Neuroendocrinol 21:1045–62. 38 39 570 Voigt C, Goymann W. 2007. Sex-role reversal is reflected in the brain of African black coucals 40 41 571 (Centropus grillii). Dev Neurobiol 67:1560–73. 42 43 572 vom Saal FS, Bronson FH. 1980. Sexual characteristics of adult female mice are correlated with 44 45 573 their blood testosterone levels during prenatal development. Science (80) 208:597 – 599. 46 47 Wade J. 2016. Genetic regulation of sex differences in songbirds and lizards. Philos Trans R 48 574 49 50 575 Soc B Biol Sci 371. 51 52 576 Wingfield JC, Hegner RE, Dufty AM, Ball GF. 1990. The “Challenge Hypothesis”: Theoretical 53 54 577 implications for patterns of testosterone secretion, mating systems, and breeding 55 56 578 strategies. Am Nat 136:829–46. 57 58 22 59 60 http://mc.manuscriptcentral.com/icbiol Page 23 of 27 Manuscripts submitted to Integrative and Comparative Biology

1 2 3 579 Wingfield JC, Ramenofsky M, Hegner RE, Ball GF. 2019. Whither the challenge hypothesis? 4 5 580 Horm Behav 104588. 6 7 581 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 23 59 60 http://mc.manuscriptcentral.com/icbiol Manuscripts submitted to Integrative and Comparative Biology Page 24 of 27

1 2 3 4 5 Higher in males Higher in females 6 7 8 9 10 11 12 For Peer Review 13 14

15 Courtship 16 17 18 19 20 21 22 23 24 25

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1 2 3 4 5 Higher in males Higher in females 6 7 8 9 10 11 12 For Peer Review 13 14

15 Courtship 16 17 18 19 20 21 22 23 24 25

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1 2 3 Trait Phenotype Species Breeding F_Mean F_N F_SD 4 5 DHT Circulating HormoneAfrican black coucalCourtship 0.095 21 0.119 6 DHT Circulating HormoneBarred buttonquailCourtship 0.030 14 0.026 7 DHT Circulating HormoneSpotted SandpiperCourtship 0.120 14 0.075 8 9 DHT Circulating HormoneWilson's phalaropeCourtship 0.327 13 0.187 10 testosterone Circulating HormoneAfrican black coucalCourtship 0.567 25 0.295 11 testosterone Circulating HormoneBarred buttonquailCourtship 0.200 14 0.374 12 13 testosterone Circulating HormoneNorthern jacanaCourtship 0.510 12 0.450 14 testosterone Circulating HormoneRed-necked phalaropeCourtship 0.085 8 0.133 15 testosterone Circulating HormoneSpotted SandpiperCourtship 0.630 8 0.651 16 17 testosterone Circulating HormoneSpotted SandpiperCourtship 0.180 14 0.112 18 testosterone Circulating HormoneWilson's phalaropeCourtship 0.505 13 0.343 19 DHT Circulating HormoneSpotted SandpiperParental 0.120 14 0.075 20 21 DHT Circulating HormoneForWilson's PeerphalaropeParental Review0.327 13 0.187 22 testosterone Circulating HormoneAfrican black coucalParental 0.567 25 0.295 23 testosterone Circulating HormoneNorthern jacanaParental 0.510 12 0.450 24 25 testosterone Circulating HormoneRed-necked phalaropeParental 0.085 8 0.133 26 testosterone Circulating HormoneSpotted SandpiperParental 0.630 8 0.651 27 testosterone Circulating HormoneSpotted SandpiperParental 0.180 14 0.112 28 29 testosterone Circulating HormoneWilson's phalaropeParental 0.505 13 0.343 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 M_Mean M_N M_SD Citation Notes 4 5 0.179 10 0.266 Voigt and GoymannValues 2007 came from webplot digitizer 6 0.030 14 0.030 Voigt 2016 7 0.200 9 0.090 Rissman and Wingfield 1984 8 9 0.907 5 0.722 Fivizzani et al. 1986 10 2.157 17 2.095 Goymann and ValuesWingfield came 2004 from hormonebase 11 0.500 14 0.374 Voigt 2016 12 13 5.260 5 4.919 Lipshutz and Rosvall 14 3.979 7 1.926 Gratto-Trevor combinedet al. 1990 data GCE from all 2 years 15 3.710 7 2.513 Oring et al. 1986Values came from hormonebase 16 17 0.950 9 1.230 Rissman and Wingfield 1984 18 3.608 5 2.609 Fivizzani et al. 1986 19 0.090 11 0.066 Rissman and Wingfield 1984 20 21 0.262 24For 0.216PeerFivizzani Review et al. 1986 22 0.590 18 0.573 Goymann and Wingfield 2004 23 0.540 7 0.476 Lipshutz and Rosvall 24 25 0.222 13 0.198 Gratto-Trevor et al. 1990 GCE 26 0.740 12 0.901 Oring et al. 1986 27 0.170 11 0.166 Rissman and Wingfield 1984 28 29 0.625 24 0.740 Fivizzani et al. 1986 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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