Testosterone and estradiol predict male calling performance, but not performance-related tradeoffs, in competitive signaling environments in Cope’s gray treefrogs (Hyla chrysoscelis)

Testosterone and estradiol predict male calling performance, but not performance-related tradeoffs, in competitive signaling environments in Cope’s gray treefrogs (Hyla chrysoscelis) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Testosterone and estradiol predict male calling performance, but not performance-related tradeoffs, in competitive signaling environments in Cope’s gray treefrogs (Hyla chrysoscelis) Alexander Baugh, Megan Freiler, Liam Halstead, Alexandra Kozak, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8695050/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Acoustic advertisement signals in anurans are classic models for understanding how endocrine mechanisms regulate courtship behavior. However, our understanding of how steroids influence male calling performance remains limited because most studies examine single hormones or isolated call traits. Here we quantified three plasma steroids—testosterone (T), estradiol (E2), and corticosterone (CORT)—from male Cope's gray treefrogs ( Hyla chrysoscelis ) recorded in natural male–male calling pairs, relating these concentrations to multiple calling components including call rate, duration, and effort. We used principal components analysis to describe a male's overall calling performance and his calling strategy (how he navigated a performance tradeoff between longer calls and faster rates). We analyzed these relationships at the population level and within male pairs using multiple regressions including all three hormones as predictors. At the population level, T and E 2 positively correlated with calling performance, whereas calling strategy was unrelated to hormones. Within male pairs, differences in T also positively correlated with differences in calling performance. Despite their collinearity, T and E 2 independently and together explained variation in calling performance. CORT showed no association with calling performance or strategy at either level, and temperature and body condition did not explain variation in calling or hormones. These results show that gonadal activation translates into asymmetries in calling performance between competitors while leaving performance-related tradeoffs unaffected. By revealing strong positive correlations between gonadal hormones and energetic investment in calling, this study demonstrates how female preferences for high-performing callers can impose sexual selection on endocrine mechanisms regulating courtship behavior. androgen corticosterone courtship call estradiol sexual selection testosterone vocalization Figures Figure 1 Figure 2 Introduction Sexually selected signals rank among the most physiologically demanding behaviors animals perform, requiring sustained energetic investment and exposing signalers to tradeoffs between reproduction, maintenance, and survival (Andersson 1994 ). This may be particularly true for species in which signaling occurs in highly competitive social environments, such as a lek or chorus, where males signal in close proximity to attract females (e.g., Vehrencamp et al. 1989 ). Research on acoustic communication in anurans has been especially informative in this regard given the conspicuous nature of frog advertisement calls, the ability to precisely measure and experimentally reproduce calls, the competitiveness of frog breeding choruses, and the numerous demonstrations that fine-scale variation in call structure, timing, and energetic output governs both male–male interactions and female mate choice (reviewed in Gerhardt and Huber 2002 ; Wells 2007 ; Narins et al. 2007 ). Calling is exceptionally costly in anurans: sustained call production elevates metabolic rate several-fold, accelerates depletion of energetic reserves, and is tightly coupled to mating success (Ryan et al. 1983 ; Taigen and Wells 1985 ; Emerson and Hess 2001 ). Across vertebrates, the expression and performance of sexually selected communication signals is tied to endocrine regulation, with steroid hormones rapidly tracking and modulating signaling effort and with signal production imposing measurable metabolic costs (Amorim et al. 2002 ; Remage-Healey and Bass 2005 ). The conservation of neuroendocrine systems thus makes the study of hormonal influences on signaling in anurans applicable across vertebrates (Arch and Narins 2009 ). In Cope’s gray treefrog ( Hyla chrysoscelis ), and its tetraploid sister species ( Hyla versicolor ), male advertisement calling represents an energetically demanding performance phenotype under sexual selection. Males attract females in dense breeding choruses by producing short calls composed of discrete pulses generated by repeated contractions of the laryngeal and trunk musculature (McLister et al. 1995 ; Girgenrath and Marsh 1997, 1999). Pulse rate is species specific, and females prefer calls with pulse rates near the population mean (Gerhardt 1991, Ward et al. 2013 ). The spectral frequencies of pulses are not strongly correlated with body size, and females exhibit weak and inconsistent preferences based on frequency differences (Schrode et al. 2012 ). The number of pulses per call (hereafter, ‘call duration’), together with call rate, defines a male’s overall calling effort, determined as the product of call duration and rate (Ward et al. 2013 ; Krueger et al. in review). A male’s calling behavior is a strong predictor of the metabolic costs of signaling, with longer calls, faster call rates, and higher overall effort reflecting both greater physiological investment and performance capacity (Taigen and Wells 1985 ; Wells and Taigen 1986; Grafe 1997 ; McLister 2001 ; Schwartz and Rahmeyer 2006 ). Males producing calls with more pulses tend to do so at slower rates and there is also negative genetic covariance between these two components of calling effort (Sullivan and Hinshaw 1992 ; Gerhardt et al. 1996 ; Schwartz et al. 2002; Welch et al. 2014 ; Reichert et al. 2024 ). As the competitiveness of local signaling environments increases, males add pulses to lengthen their calls and reduce their call rate while maintaining or slightly increasing their overall calling effort (Wells and Taigen 1986; Reichert and Gerhardt 2012 ). Female mate choice is biased toward these costly signals: experimental studies show that females have strong directional preferences for males that produce longer calls at faster rates—and thus signal with higher calling effort—and that these preferences persist across realistic acoustic environments and competitive contexts (Gerhardt 1991; Gerhardt et al. 1996 , 2000; Bee 2008 ; Ward et al. 2013 ; Velez et al. 2013; Tanner et al. 2017 , 2025 ; Krueger et al. in review). Importantly, female evaluation of calling effort reflects the integration of information across multiple calls rather than isolated acoustic traits, placing sustained calling effort—rather than fine-scale spectral or temporal variation—at the center of sexual selection in this system (Ward et al. 2013 ; Tanner et al. 2017 ; Krueger et al. in review). A longstanding hypothesis in behavioral endocrinology is that such energetically demanding sexual signals are regulated by interacting endocrine axes. Gonadal steroids such as testosterone produced by hypothalamic–pituitary–gonadal (HPG) axis have long been implicated in facilitating male sexual displays by enhancing vocal motor output, neuromuscular performance, and motivational state (Houck et al. 1996 ; Leary and Baugh 2020 ). Estradiol (E 2 ), although historically understudied in males, is increasingly recognized as a biologically relevant component of the male HPG axis (e.g. Freiler et al. 2026 ). Beyond developmental effects, E 2 can act acutely on neural and muscular systems, modulating excitability, endurance, and metabolic efficiency—traits directly relevant to sustained calling behavior (Campbell and Febbraio 2001 ; Balthazart and Ball 2006 ; Remage-Healy and Joshi 2012). Thus far, however, E 2 has rarely been examined explicitly in studies of male frog advertisement calling (but see: Schmidt 1983 ; Hoffmann and Kloas 2012a , b ). Furthermore, glucocorticoids released by the hypothalamic-pituitary-adrenal/inter-renal axis (HPA/I axis), such as corticosterone (CORT), regulate energy mobilization during periods of high metabolic demand and have been proposed to either facilitate sustained signaling or suppress reproductive effort when energetic reserves are limited. Emerson’s Energetics–Hormone Vocalization (EHV) model integrates these ideas, predicting that prolonged calling elevates glucocorticoids, which may in turn inhibit gonadal steroid production and down-regulate sexual signaling (Emerson and Hess 2001 ). Although influential, empirical support for this framework in anurans has been mixed, with studies reporting weak, absent, or context-dependent hormone–behavior relationships, particularly for glucocorticoids (reviewed in Leary and Baugh 2020 ). Progress on such questions has been slowed by two major limitations of previous studies. First, most studies rely on analyzing single acoustic traits despite evidence that both female preferences and male performance are inherently multivariate. Second, few studies address the additional challenge that males do not signal in isolation but instead compete against other nearby males to attract females. Here, we combine paired acoustic recordings of neighboring males in breeding choruses with endocrine sampling in H. chrysoscelis to investigate how gonadal and inter-renal hormones relate to male calling behavior in a competitive context. We measured male calling behavior and circulating T, E 2 , and CORT to address two complementary questions. First, do gonadal and inter-renal hormones explain variation in calling behavior across males at the population level? Second, do asymmetries in endocrine state between nearby males competing in a chorus predict asymmetries in calling behavior? By including contrasts of two competing males, we aimed to isolate endocrine effects while controlling for shared environmental, temporal, and acoustic conditions. Materials and methods Acoustic recordings, blood collection, and biometrics We conducted paired-male recordings of the western genetic lineage of Cope’s gray treefrog (Booker et al., 2022 ) from wetlands located in the Carver Park Reserve (Carver County, MN, USA, 44°52'33.5" N, 93°41'03.4" W ) during peak calling periods (June 10–20, 2024; 2200–2330). Each recording involved two neighboring males calling in close proximity in a breeding chorus, allowing us to quantify variation in calling behavior at the within-pair level in a competitive signaling environment ( n = 14 pairs). Field procedures followed a standardized protocol performed by a three-person team (audio manager, microphone operator, and one blood-sampling technician). Pairs of males were located visually using red headlamps in shallow water and selected only if (a) both were actively calling, (b) each male within a pair was the other’s nearest calling neighbor (within-pair separation: median = 1.83 m, mean = 3.69 m, SD = 3.29 m), and (c) they were positioned sufficiently apart from neighboring males to isolate each dyad to obtain recordings suitable for analysis (Fig. 1 ). Directional microphones (Sennheiser ME66; Sennheiser Electronic Corporation, Old Lyme, CT, USA) were placed in front of each male (∼1 m distance), and a 10-min simultaneous recording (Zoom H8, Zoom Corporation, Tokyo, Japan) was initiated (SM1 & SM2). Immediately after the 10-min recording window, we sampled blood via cardiac puncture following established methods validated in Hyla (e.g., Bastien et al. 2018 ; Baugh 2024 ). Briefly, we rapidly (ca. 3 min; range = 1–7 min measured to nearest minute) collected blood (ca. 50 µL) using a 30-gauge insulin syringe (BD Micro-fine U-100, 0.3 mL) pre-rinsed with heparin. Whole blood was stored at 4° C on wet ice for 2–4 h and then centrifuged (7500 RPM for 10 min; Eppendorf 5418 at 8° C). The plasma fraction was stored at -20° C for three weeks and then shipped on dry ice to Swarthmore College where samples were stored at -80° C for 1 month until assayed. Immediately after blood collection, we measured water temperature (Fluke 62 Max + IR thermometer, Everett, WA, USA; accuracy: ±1.0 C), body mass (to the nearest 0.01 g) and body length (to the nearest 0.01 mm) as snout-vent length (SVL). Body condition estimates can be used as an indicator for energy reserves (Leary and Harris 2013 ). We calculated a body condition index (hereafter ‘BCI’) by obtaining standardized residual values from a linear regression of SVL on body mass. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (#2301-40692A). Acoustic analyses We analyzed recordings of advertisement calls using Adobe Audition 2025 (Adobe Systems Inc., San Jose, CA, USA). Because our primary hypothesis concerned endocrine predictors of sustained calling performance, we focused on the three call traits known to be determinants of the energetic costs of calling and under strong sexual selection due to female mate choice: call rate (calls min⁻¹), call duration (pulses call⁻¹), and calling effort (pulses min⁻¹). For each recorded call, we measured its period (defined as the interval between the onsets of two successive calls) and call duration (defined as the number of pulses in the call). From these values, we computed an instantaneous call rate (calls min⁻¹) as the reciprocal of each call period, and an instantaneous calling effort (pulses min⁻¹) as the product of the instantaneous call rate and call duration for each call. Then for each male, we averaged values over all the calls he produced during the 10-min recording to determine his mean call rate, call duration, and calling effort (see Fig. 1 ). Hormone analyses Our hormone analyses followed procedures described in more detail elsewhere (Baugh 2024 ). Briefly, we used a liquid diethyl-ether double extraction protocol. Following extraction, we froze samples at -80◦C for 1–2 days or we resuspended immediately (assay buffer, provided by kit) and allowed samples to reconstitute for 12 h at 4°C. In males, optimal reconstitutions are at 1:10 for CORT and E 2 and 1:80 for T (Baugh 2024 ). We estimated steroid concentrations using EIA kits (DetectX® kits, Arbor Assays, Ann Arbor, MI) for 17β-estradiol (E 2 ; Cat. No. KB30, Donkey anti-Sheep IgG), testosterone (T; Cat. No. K032, Goat anti-Rabbit IgG), and corticosterone (CORT; Cat. No. K014, Donkey anti-Sheep IgG). We analyzed samples in duplicate following manufacturer’s instructions. We used two duplicate internal standards (a single pooled sample of H. chrysoscelis plasma) per plate for calculation of intra-assay coefficients of variation (CV). Detection limits and sensitivities, respectively, were 2.05 pg mL − 1 and 2.21 pg mL − 1 for E 2 ; 9.92 pg mL − 1 and 30.6 pg mL − 1 for T; and 16.9 pg mL − 1 and 18.6 pg mL − 1 for CORT. Cross-reactivity was 100% for E 2 , 3.2% for estrone sulfate, and 2.5% for estrone; 100% for T, 3.53% for 5a-dihydrotestosterone, and 0.27% for androstenedione; 100% for CORT, 12.3% for desoxycorticosterone, 0.62% for aldosterone, 0.38% for cortisol. Average intra-assay variations were 4.2% for E 2 , 9.3% for T, and 2.6% for CORT. Statistical analyses We assessed the ability of circulating hormones to predict male calling behavior using linear regression to address endocrine effects at both the population level and at the level of adjacent competing males. Hormone concentrations were log₁₀-transformed to improve normality of residuals and then standardized ( z -scored). For each of our three acoustic measures of calling behavior (call rate, call duration, and calling effort), we standardized ( z ) the individual means for each variable across males and then used principal component analyses (PCA) to reduce the acoustic data to a smaller set of orthogonal variables. Across all three PCA implementations, PC1 consistently loaded positively on all three call-effort traits, whereas PC2 captured a rate–complexity trade-off, confirming stable biological interpretation despite minor sample size differences. To provide descriptive context for the multiple regressions, we calculated Pearson correlations between hormones, among our three measures of calling behavior, and between hormones and calling behavior using the population-level dataset with complete data ( n = 24 males). To assess whether BCI or water temperature explained additional variation in calling beyond endocrine predictors, we conducted a series of nested model comparisons within the population-level framework. We compared a base model including standardized log 10 T, log 10 E 2 , and log 10 CORT to models additionally including BCI, water temperature, or both covariates. Model comparisons were evaluated using partial F-tests and Akaike’s Information Criterion (AIC). All analyses were conducted in R (v. 4.4.2). Significance was assessed at α = 0.05, and effect sizes are reported alongside test statistics where appropriate. Results Calling behavior On average, males produced advertisement calls consisting of about 28 pulses at a rate of about 12 calls per minute, yielding an average calling effort of about 337 pulses min -1 (Fig. 1 ; Table 1 ). These values are close to population averages reported in an earlier study of the same population (Ward et al. 2013 ). Across individuals, mean calling effort varied by a factor of more than 4.0 (range: 120 to 507 pulses min -1 ). Males within pairs also varied in calling (see Fig. 1 ). As expected, there were significant positive correlations between calling effort and both call rate and call duration, which themselves were negatively correlated (Table 1 ). Thus, we observed the phenotypic tradeoff between call rate and call duration reported in previous studies. Table 1 Summary statistics from sound analysis and principal component analysis Variable Calling effort Call rate Call duration PC1 PC2 Calling effort 336.9 ± 81.2 pulses min − 1 1.00 0.06 Call rate r = 0.53 p = 0.005 12.2 ± 2.7 calls min − 1 0.48 0.87 Call duration r = 0.56 p = 0.003 r = -0.39 p = 0.044 28.1 ± 6.1 pulses call − 1 0.61 -0.79 Eigenvalue 1.60 1.40 Variance explained 53.2% 46.4% The mean ± SD values ( n = 27) for the three acoustic variables are shown along the diagonal along with the Pearson product moment correlations ( r ) between variables and their associated p values (two-tailed). Also shown are the factor loadings for the first and second principal components (PC1 and PC2) along with their associated eigenvalues and variance explained. A PCA of standardized call traits ( n = 27 males with complete calling data) yielded two informative axes that together explained 99.6% of the variance (Table 1 ). PC1 explained 53.2% of the variance and loaded positively on all three acoustic variables, with the heaviest loading on calling effort. Thus, PC1 captured variation in well-established metrics of calling performance in gray treefrogs (i.e., higher call rates, longer calls, and higher calling effort). For use in regression analyses, values for PC1 were multiplied by − 1 so that higher scores represent greater overall calling performance. PC2 explained 46.4% of the variance and primarily contrasted call rate (0.89) against call duration (− 0.81), with minimal contribution of calling effort (0.06). We interpret this result as PC2 capturing orthogonal variation in calling strategy consistent with a performance trade-off between call rate and call duration: some males produce many short calls and others produce fewer longer calls, and this is a trade-off because both strategies can occur at similar overall calling efforts. Hormones Average concentrations for each hormone (Table 2 ) were typical of patterns observed in vocally active males in these populations (Baugh 2024 ; Freiler et al. 2026 ). Circulating T showed the greatest variation among males (coefficient of variation, CV = 1.6), whereas E 2 varied over a much narrower range (CV = 0.17), and CORT showed intermediate variability (CV = 0.69). Table 2 Summary statistics from hormones Variable Testosterone Estradiol CORT Testosterone (CV = 1.6) 10.0 ± 16.6 ng mL − 1 Estradiol (CV = 0.17) r = 0.39 p = 0.057 206.9 ± 36.3 pg mL − 1 CORT (CV = 0.69) r = 0.17 p = 0.420 r = 0.28 p = 0.189 1.37 ± 0.98 ng mL − 1 The mean ± SD values ( n = 24) for the three hormones are shown along the diagonal along with the Pearson product moment correlations ( r ) between variables and their associated p values (two-tailed). Hormone-behavior relationships in the population Pearson correlations between each hormone and each call parameter indicated that gonadal hormones were positively correlated with metrics of calling behavior, especially calling effort (SM3). We used multiple linear regression to test whether circulating hormones predicted variation in calling behavior across males at the population level. We included all males complete with all three metrics of calling behavior and all three hormones ( n = 24 males). We calculated PC scores from this subset of males which yielded a component structure consistent (SM4) with the PCA used earlier (see Calling behavior). We then fit linear multiple regression models predicting PC1 and PC2 from standardized (z scores) values of log 10 T, log 10 E 2 , and log 10 CORT: PC = β₀ + β₁(T) + β₂(E 2 ) + β₃(CORT) + ε This model was significant for PC1 and revealed that circulating hormones explained substantial variation in overall calling performance at the population level ( R ² = 0.50, adjusted R ² = 0.42; F 3,20 = 6.65, p = 0.003; Fig. 2 a; SM5). Both gonadal hormones showed significant, positive partial effects on calling performance (PC1). T was a positive predictor of PC1 when controlling for E 2 and CORT (β = 0.55 ± 0.21 SE, t = 2.58, p = 0.018, 95% CI [0.11, 1.00]). Interpreted in biologically meaningful units, a one–standard deviation increase in T (not log-transformed) was associated with a 0.55 SD increase in overall calling effort (PC1). E 2 also independently predicted PC1 (β = 0.52 ± 0.22 SE, t = 2.37, p = 0.028, 95% CI [0.06, 0.98]). A one SD increase in T predicted a 0.55 SD increase in PC1 (~ 45 pulses min − 1 ), whereas a comparable increase in estradiol predicted a 0.52 SD increase (~ 42 pulses min − 1 ), indicating that both gonadal steroids contribute similarly sized effects on calling effort at the population level. In contrast, CORT did not predict PC1 (β = −0.09 ± 0.21 SE, t = − 0.42, p = 0.680). None of the hormones predicted orthogonal variation in calling strategy (PC2). The PC2 regression model was not significant overall ( R ² = 0.10, adjusted R ² = −0.03; F₃,₂₀ = 0.78, p = 0.522), and no individual predictor approached significance (all p > 0.173). Water temperature and BCI did not improve model fit and were thus not included (SM6). Hormone-behavior relationships between competing males To test whether these population‑level associations also explain competitive asymmetries, we next examined hormone and calling differences within male pairs. We used multiple regression to test whether hormonal differences between competing males predicted differences in calling behavior. We included all pairs complete with all three calling metrics and all three hormones ( n = 10 pairs). We calculated PC scores from this set of 10 pairs (20 males) which yielded a component structure consistent with both PCAs used previously (SM4). For each pair, we calculated differences in PC scores and hormone levels between the two paired males (designated L and R): ΔPCl = PC L – PC R ΔHormone = Hormone L – Hormone R This difference approach controls for shared environmental, temporal, and acoustic conditions within each dyad and thus helps isolate endocrine predictors of relative calling performance. We then fit multiple regression models predicting ΔPC1 and ΔPC2 from ΔT, ΔE 2 , and ΔCORT: ΔPC = β₀ + β₁(ΔT) + β₂(ΔE 2 ) + β₃(ΔCORT) + ε This model was significant for PC1 ( R ² = 0.79, adjusted R ² = 0.68; F 3,6 = 7.43, p = 0.019; Fig. 2 b; SM5), indicating differences in hormone levels between competitors strongly predicted differences in calling performance. Differences in T were a strong predictor of performance asymmetries: males with higher T than their opponent had substantially higher calling efforts (β = 1.24 ± 0.41 SE, t = 3.06, p = 0.022, 95% CI [0.25, 2.24]). Differences in E 2 showed a positive but non-significant association with ΔPC1 (β = 0.50 ± 0.27 SE, t = 1.84, p = 0.12), whereas CORT differences did not predict performance asymmetries (β = 0.24 ± 0.36 SE, t = 0.65, p = 0.54). As in our population level approach, hormonal asymmetries did not predict differences in calling strategy (ΔPC2). The ΔPC2 regression model was not significant ( R ² = 0.16, adjusted R ² = −0.25; F₃,₆ = 0.39, p = 0.76), and no hormone predictor approached significance (all p > 0.40). Discussion Female preference and selection on endocrine traits A key insight from decades of anuran bioacoustics is that female frogs attend closely to a male’s calling performance, including call duration and call rate, calling effort, and overall acoustic output, as indicators of competitive ability (reviewed in Gerhardt and Huber 2002 ; Wells 2007 ; Narins et al. 2007 ). Thus, selection should favor endocrine mechanisms that enable males to sustain high-performance calling in the context of ecological and social constraints. Our results provide a physiological complement to this classic sensory and behavioral framework by showing that coordinated gonadal activation strongly predicts variation in calling performance, but not calling strategy, both across males and between direct competitors. If T or E 2 facilitates sustained motor output or buffers stress-related suppression of signaling, then female preferences for higher calling performance may indirectly select for endocrine phenotypes that balance energetic investment against stress responsiveness (Leary and Knapp 2014 ; Leary and Baugh 2020 ). In this way, female choice for acoustic performance would shape not only signal structure and signaling behavior but also the hormonal architecture underlying male signaling effort. Gonadal hormones and calling performance Regression analyses at the levels of both the population and competing individuals converged on a robust correlation between gonadal activation and male calling performance. At the population level, hormones strongly (adjusted R ² = 0.42) predicted a multivariate axis of calling performance, demonstrating that integrated HPG-axis activation accounts for substantial among-male variation in the performance of sexual displays. A similar relationship was found comparing nearby competing males, where differences in hormones predicted differences in calling performance with an even larger effect size (adjusted R ² = 0.68). This convergence demonstrates that the loss of statistical power associated with fewer observations ( n = 10 pairs compared to n = 24 males) was more than offset by the paired design’s ability to control for shared environmental, temporal, and other factors. Hence, contrasts between nearby competing individuals provide a particularly powerful and biologically meaningful approach for linking endocrine state to signaling performance. The partial effects of both T and E 2 contributed explanatory power to calling performance, with each hormone remaining a significant predictor when controlling for the other. Testosterone’s positive association with calling performance is consistent with its well-established role in male sexual signaling across vertebrates (Kelley 1986 ; Emerson 2001 ; Wilczynski et al. 2005 ; Adkins-Regan 2005 ; Leary and Harris 2013 ). Testosterone can enhance vocal motor output, increase calling motivation, and support the neuromuscular performance required for sustained calling (Wetzel and Kelley 1983 ; Houck et al. 1996 ; Leary and Baugh 2020 ). Androgens may also act centrally to bias motivational state toward reproductive investment (Wingfield et al. 1990 ) and peripherally to facilitate muscle performance and respiratory capacity (Husak and Irschick 2009 ), thereby scaling overall signaling performance. In this sense, the strong positive association between T and calling performance observed here is consistent with a large body of work linking androgenic state to elevated reproductive effort. The lack of a relationship between hormones and calling strategy (how males distribute their calling effort within and among calls) might further indicate that the circulating endocrine state constrains the overall level of metabolic investment whereas strategic deployment of those efforts is controlled by decision making circuits unmodulated by steroids. In contrast, the finding that E 2 explained additional, non-redundant variation is especially notable given that research on endocrine regulation of male sexual signaling in anurans has historically emphasized androgens and, to a lesser extent, glucocorticoids, with estrogens receiving comparatively little attention despite their activity in male frogs (Schmidt 1983 ; Hoffmann and Kloas 2012a , b ). This may reflect the fact that circulating E 2 is derived primarily from the conversion of androgens in the testes and thus considered incidental rather than a hormone with independent behavioral influence. This pattern mirrors a broader vertebrate literature in which estrogenic effects on male behavior are sometimes subsumed under an androgen-focused framework (Ball and Balthazart 2008 ; but see Balthazart and Ball 2006 ). Yet E 2 receptors and aromatase are abundantly expressed in anuran brain regions involved in vocal production and auditory processing, including the preoptic area and torus semicircularis (e.g., Kelley 1988 ; Wilczynski and Ryan 2010 ), suggesting substantial scope for estrogenic modulation of male signaling, from systemic or local (conversion) sources. Indeed, estradiol is known across vertebrates to influence neuromuscular performance, mitochondrial efficiency, and fatigue resistance, in part through modulation of oxidative metabolism and calcium handling in muscle (e.g., Wideman et al. 2013 ; Ventura-Clapier et al. 2017 ). These effects are especially relevant for prolonged anuran calling, which requires sustained contraction of trunk and laryngeal musculature and imposes substantial metabolic demands (Taigen and Wells 1985 ; Emerson 2001 ). Estradiol can also exert rapid neuromodulatory effects on vocal-motor circuits and motivational centers (Remage-Healey and Bass 2006 ; Leary and Baugh 2020 ), potentially enhancing both the capacity and persistence of high-effort calling. Against this background, our finding that E 2 explains non-redundant variation in male calling performance indicates that estrogenic pathways may play a more direct role in shaping energetically costly advertisement displays than is typically appreciated. Interestingly, experimentally elevated E 2 in Xenopus laevis has been shown to inhibit male courtship calling (Hoffmann and Kloas 2012a ), which occurs through estrogen receptor signaling (Hoffmann and Kloas 2012b ). A future pharmacological experiment is needed to disentangle the stimulatory and inhibitory roles of T and E 2 on aspects of male calling. Future studies will be needed to investigate causal relationships between gonadal hormones and calling performance in gray treefrogs. Experimental manipulations (e.g. elevating, depleting and/or inhibiting T and E 2 ) in calling males, ideally combined with measures of metabolic performance, muscle fatigue, and neural activity during sustained calling bouts, would be particularly informative. Integrating such manipulations with paired recordings, as used here, or in experimental contests (e.g., Reichert and Gerhardt 2012 ) would allow researchers to disentangle hormone-driven changes in signaling capacity from context-dependent motivational effects. Finally, coupling endocrine manipulations of calling males with female phonotaxis assays would provide a powerful test of whether T and E 2 -mediated variation in male calling performance translates into differential mating success, connecting endocrine state, signal production, perception, and selection. HPG and HPI interactions Because T and E 2 are jointly produced components of HPG-axis activation, linked through aromatization, E 2 could help determine whether testosterone-driven high-performance signaling can be maintained by buffering energetic and stress-related constraints on sustained performance or reducing the immune suppressive effects of T alone (Wingfield et al. 1990 ; Remage-Healey and Bass 2006 ; Handa et al. 2014 ). Elevated E 2 may therefore help maintain calling performance under conditions in which activation of the HPI axis would otherwise constrain signaling. This interpretation is consistent with our broader finding that CORT showed little explanatory power for calling performance in this study and suggests that gonadal activation—rather than antagonistic HPG–HPI dynamics—may be the dominant endocrine basis determining calling performance in competitive signaling environments. The lack of evidence for an influence of CORT on male signaling and the positive correlation observed between circulating gonadal hormones and sexually selected calling performance might help clarify another recent discovery related to energetically demanding sexual behavior in this frog. Specifically, male H. chrysoscelis that successfully attract a mate have substantially elevated and correlated plasma hormone concentrations (CORT, androgens and estradiol; Baugh, 2024 ). There are at least two plausible, non-mutually exclusive hypotheses for this pattern: (1) males found in amplexus were chosen by females on the basis of their calling effort, which are positively correlated with gonadal hormone levels (i.e., sexual selection by female choice); or (2) the act of clasping itself rapidly increases steroid hormone secretion (i.e., bidirectionality in hormone-behavior relationships). The former hypothesis is supported by the current study, and the latter hypothesis is also supported by a recent empirical demonstration that induced amplexus in male H. chrysoscelis activates the HPG axis, rapidly elevating both T and E 2 but not CORT (Freiler et al. 2026 ). These findings further support the pleiotropic nature of steroids—gonadal steroids are likely facilitating elevated calling performance and thereby potentially increasing mating success (but see Sullivan and Hinshaw 1992 ) and subsequently becoming further elevated upon clasping to facilitate muscular tone (Kampe and Peters 2013 ) and sperm release (Silla et al. 2019 ). The exact role of androgens versus estrogens in these processes remains to be discovered, for example via aromatase inhibition experiments. Lastly, further study of the role of CORT is also needed given its elevated concentrations in naturally but not artificially amplexed males and lack of correlation with calling behavior. Our conclusion that higher concentrations of gonadal steroids, but not CORT, are associated with higher calling performance requires some qualification. The current study used a 10-min sample of calling activity and a single acute blood sample, with the assumption that these two levels of the phenotype are temporally synchronized and representative more generally. However, a 10-min sample of calling activity during a 10-day period of the breeding season represents a narrow slice of time. The potential influence of CORT on calling performance might only be revealed at extreme concentrations or be relevant only at the performance limits of the males (Marler and Ryan, 1996; Leary and Knapp, 2014 .). In addition, like other lek-breeding frogs (Ryan 1985 ), male gray treefrogs can spend several hours per night, across many nights, calling (Sullivan and Hinshaw 1992 ; Runkle et al. 1994 ; Wells et al. 1995 ; Gerhardt et al. 1996 ; Reichert et al. 2024 ). How males trade-off calling performance in a given bout against performance distributed across an entire night, and across an entire breeding season is not yet well understood. Further, how hormones might provide a mechanistic basis for resolving these temporal-investment tradeoffs is unknown. However, this tradeoff should be important in principle; for example, Sullivan and Hinshaw ( 1992 ) demonstrated that the single best predictor of male mating success in the tetraploid gray treefrog, H. versicolor , is the number of nights a male participates in the chorus, which was also observed in túngara frogs (Ryan 1985 ). Therefore, understanding how males optimize the distribution of vocal effort across different time scales, how interactions between the HPG and HPI axes influence these decisions, and how behavioral and hormonal phenotypes are linked with ecological variables (e.g., foraging; Green 1990 ; Marler and Ryan 1996; Wilhite and Ryan 2024 ; predation risk: Baugh and Ryan 2010 ) and social factors (e.g., female cues; Akre and Ryan 2011 ) represent fruitful directions for further study. Conclusion By linking variation in calling performance to underlying endocrine state in both population-level and paired competitive contexts, the present study provides evidence of physiological mechanisms that subserve male performance in competitive signaling environments. In this regard, this study builds on the enormous body of work by the honoree of this special issue, Peter Narins. A fundamental theme running through Narins’ research has been to understand anuran signaling behaviors in competitive contexts, and this research has profoundly shaped our understanding of acoustic competition among male frogs and its consequences for sexual selection. Most directly related to the present study, Lopez and Narins ( 1991 ) demonstrated that females of the Puerto Rican Coqui, Eleutherodactylus coqui , strongly prefer males calling at higher rates, an earlier finding that helped establish the fundamental importance of female preferences for male calling performance. To better understand how males achieve such performance under competitive conditions, Narins and colleagues have examined the interactions among calling males, often using the evoked vocal response as a powerful assay to reveal dynamic signaling strategies. For example, Narins and Capranica ( 1978 ) identified the functional role of the “Co” note in competitive signaling among Coqui males, Lopez et al. (1988) documented striking plasticity in call intensity and frequency, and Benedix and Narins ( 1999 ) found robust call-rate escalation during simulated intrusions. Narins’ work has also investigated how males cope with the cacophony that results when competing males call in noisy chorus environments. For example, Zelick and Narins ( 1983 ) and Brush and Narins ( 1989 ) showed that males precisely time calls to exploit silent windows and avoid call overlap with rival males, while Lewis et al. ( 2001 ) revealed the use of seismic signals when acoustic channels were masked. Finally, Narins has also shown that vocal interactions extend beyond considerations of the form and timing of acoustic signals by showing that physical aggression between competing males depends on multimodal cues and cross-modal integration during territorial defense (Narins et al. 2003 ; Narins et al. 2005 ). Together, these and other pioneering studies by Peter Narins and his students, postdocs, and collaborators help frame our endocrine findings within a rich tradition of neuroethological research on the behavioral strategies that enable male success in competitive signaling environments. Declarations Funding This work was supported by the Swarthmore College Research Fund to ATB and NSF grant IOS-2154204 to MAB. Author contributions ATB: Conceptualization, Methodology, Resources, Formal analysis, Investigation, Data curation, Writ­ing - original draft, Funding acqui­sition, Supervision, Project administration. MKF: Formal analysis, Investigation, Editing - original draft. LNH: Investigation. AK: Formal analysis, Investigation. MAB: Conceptualization, Methodology, Resources, Formal analysis, Investigation, Writ­ing - original draft, Funding acqui­sition, Supervision, Project administration. Declaration of competing interests The last author (MAB) is a guest editor for this special issue but did not handle this manuscript in any capacity. We have no competing interests to declare. Data availability Complete dataset available in SM7. Acknowledgements We thank the Minnesota Department of Natural Resources for permission to conduct this research, John Moriarty and Steven Hogg with the Three Rivers Park District for after-hours access to frog ponds, and Chris Leary for productive discussions on this research topic. References Adkins-Regan E (2005) Hormones and animal social behavior. Princeton University Press, Princeton. Akre KL, Ryan MJ (2011) Female túngara frogs elicit more complex mating signals from males. Behav Ecol 22:846–853. 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Supplementary Files SM1videopair7.mov SM2stillimagepair8.jpg SM3tablepearsonscallhorm.docx SM4supplPCA23tables.docx SM5commonalityanalyses.docx SM6bodyandtempanalysis.docx SM7DATAFILEBaughetal.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviews received at journal 07 Mar, 2026 Reviewers agreed at journal 07 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 02 Feb, 2026 Submission checks completed at journal 29 Jan, 2026 First submitted to journal 25 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8695050","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585274102,"identity":"c41fb46d-4f02-4588-b9df-daa828059517","order_by":0,"name":"Alexander Baugh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACCSBmbJCQA3MYG6CiPIS1WBiDOQdJ0FKR2EC0Fv7Zzc8kv+6QSJ8/I/nZ5487tsmZtzcwPnjbhseSO8fMpGXPSORuuJFmPOPgmdvGMmcOMBvOxaOF4UaCmbRkG1CLdIIxw8G224kzJBLYpHnxaJG/kf4NpCVdfnb6Z4gW+Qfsv/FpMbiRYyb5sU0igeF2DswWBjZmfFoM75wptmZskzDccP9NMcPZttvGEjyJzZJzzuHWIne7fePNn2118vI9xzczVLbdlpNgP3zww5syPN4HAma0WICnAdyA8QdBJaNgFIyCUTCiAQAibFci3AN2zwAAAABJRU5ErkJggg==","orcid":"","institution":"Swarthmore College","correspondingAuthor":true,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Baugh","suffix":""},{"id":585274104,"identity":"d120530a-aa60-4182-a96f-5661ccd97df8","order_by":1,"name":"Megan Freiler","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Megan","middleName":"","lastName":"Freiler","suffix":""},{"id":585274109,"identity":"2158c898-77b4-4c66-97b5-482d158c9710","order_by":2,"name":"Liam Halstead","email":"","orcid":"","institution":"Swarthmore College","correspondingAuthor":false,"prefix":"","firstName":"Liam","middleName":"","lastName":"Halstead","suffix":""},{"id":585274112,"identity":"67bec176-a130-48cd-aace-00f8be966c61","order_by":3,"name":"Alexandra Kozak","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Kozak","suffix":""},{"id":585274117,"identity":"a22de564-8361-4fad-8a79-536f5a1a5092","order_by":4,"name":"Mark Bee","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Bee","suffix":""}],"badges":[],"createdAt":"2026-01-25 22:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8695050/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8695050/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101847469,"identity":"6284b3b1-27d7-47c1-b456-46cdc04cb095","added_by":"auto","created_at":"2026-02-04 09:30:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":205797,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-channel recording depicting waveforms of two adjacent and vocally competing \u003cem\u003eH. chrysoscelis \u003c/em\u003emales (pair #12) from the night of June 19th, 2024. The left channel (green) is Frog 12L and the right channel (blue) is Frog 12R. (a) 10 min full recording. Despite the gaps in calling by 12L, he produced more calls (114) compared to 12R (101) due to shorter inter-call intervals, and he exhibited substantially higher calling effort (pulses/min = 446) compared to 12R (pulses/min = 247). (b) A shorter segment of the 10 min recording that better depicts call rate differences between males 12L and 12R. (c) Sequential calls from 12L call with 28 pulses and 12R with 23 pulses.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/7226ae8401757e6ab3844efa.png"},{"id":102298556,"identity":"1c5790c8-7bcf-4d65-851c-466e4ecccc83","added_by":"auto","created_at":"2026-02-10 10:46:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108383,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Observed values of overall calling performance (PC1) plotted against values predicted by the multiple regression model including standardized log-testosterone, log-estradiol, and log-corticosterone (n = 24 males). PC1 represents a composite axis of calling performance derived from call rate, call duration, and calling effort, with higher values indicating better performance. Points represent individual males and the fitted values reflect the combined effects of all three hormones estimated simultaneously. (b) Observed within-pair differences in calling performance (ΔPC1) plotted against values predicted by the multiple regression model including differences in standardized log-testosterone, log-estradiol, and log-corticosterone (n = 10 pairs). Positive values indicate that the male with higher hormone concentrations exhibited higher calling performance. Predicted values represent the combined effects of endocrine asymmetries between competitors estimated simultaneously.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/76bc01f876b233adec7822fd.png"},{"id":102301166,"identity":"cf8b6705-9f5b-4607-bea6-d6313b82216d","added_by":"auto","created_at":"2026-02-10 11:19:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1046139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/c0675e3c-bcdd-4f6b-9803-93f6acedec29.pdf"},{"id":101847475,"identity":"232a55c4-41f3-45a9-9fbb-d24a46a37edd","added_by":"auto","created_at":"2026-02-04 09:30:41","extension":"mov","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15584957,"visible":true,"origin":"","legend":"","description":"","filename":"SM1videopair7.mov","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/f899eac860e5998c3cae662d.mov"},{"id":101881668,"identity":"62933955-997f-4ca2-b8ad-beb83ca64293","added_by":"auto","created_at":"2026-02-04 15:14:38","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102295,"visible":true,"origin":"","legend":"","description":"","filename":"SM2stillimagepair8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/7c51a56db045c06220f9f2ba.jpg"},{"id":101847471,"identity":"0db3deb0-7ca6-482c-84a7-2c5e7848b1b0","added_by":"auto","created_at":"2026-02-04 09:30:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13252,"visible":true,"origin":"","legend":"","description":"","filename":"SM3tablepearsonscallhorm.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/1643e2f9d55f0b5035f6e0be.docx"},{"id":101847467,"identity":"b3efe7e1-91b4-4629-8ee3-3e3c53bd322b","added_by":"auto","created_at":"2026-02-04 09:30:41","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14036,"visible":true,"origin":"","legend":"","description":"","filename":"SM4supplPCA23tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/4990a8f82065cac913148df0.docx"},{"id":101847474,"identity":"160ad811-feab-4998-b9cd-0111a27363d5","added_by":"auto","created_at":"2026-02-04 09:30:41","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14684,"visible":true,"origin":"","legend":"","description":"","filename":"SM5commonalityanalyses.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/dae6efe25ddbac84af7e4aba.docx"},{"id":101881644,"identity":"3785fe1a-ea94-4b35-a3fc-bd56acc18170","added_by":"auto","created_at":"2026-02-04 15:14:25","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13871,"visible":true,"origin":"","legend":"","description":"","filename":"SM6bodyandtempanalysis.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/2f3ba4b71a3e11f473a74e97.docx"},{"id":101847465,"identity":"d0d6ccd6-9c7d-416e-8bfe-46e690ccc660","added_by":"auto","created_at":"2026-02-04 09:30:41","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":18609,"visible":true,"origin":"","legend":"","description":"","filename":"SM7DATAFILEBaughetal.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8695050/v1/ed9bb946d5b75705954d7159.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Testosterone and estradiol predict male calling performance, but not performance-related tradeoffs, in competitive signaling environments in Cope’s gray treefrogs (Hyla chrysoscelis)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSexually selected signals rank among the most physiologically demanding behaviors animals perform, requiring sustained energetic investment and exposing signalers to tradeoffs between reproduction, maintenance, and survival (Andersson \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). This may be particularly true for species in which signaling occurs in highly competitive social environments, such as a lek or chorus, where males signal in close proximity to attract females (e.g., Vehrencamp et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Research on acoustic communication in anurans has been especially informative in this regard given the conspicuous nature of frog advertisement calls, the ability to precisely measure and experimentally reproduce calls, the competitiveness of frog breeding choruses, and the numerous demonstrations that fine-scale variation in call structure, timing, and energetic output governs both male\u0026ndash;male interactions and female mate choice (reviewed in Gerhardt and Huber \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wells \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Narins et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Calling is exceptionally costly in anurans: sustained call production elevates metabolic rate several-fold, accelerates depletion of energetic reserves, and is tightly coupled to mating success (Ryan et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Taigen and Wells \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Emerson and Hess \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Across vertebrates, the expression and performance of sexually selected communication signals is tied to endocrine regulation, with steroid hormones rapidly tracking and modulating signaling effort and with signal production imposing measurable metabolic costs (Amorim et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Remage-Healey and Bass \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The conservation of neuroendocrine systems thus makes the study of hormonal influences on signaling in anurans applicable across vertebrates (Arch and Narins \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Cope\u0026rsquo;s gray treefrog (\u003cem\u003eHyla chrysoscelis\u003c/em\u003e), and its tetraploid sister species (\u003cem\u003eHyla versicolor\u003c/em\u003e), male advertisement calling represents an energetically demanding performance phenotype under sexual selection. Males attract females in dense breeding choruses by producing short calls composed of discrete pulses generated by repeated contractions of the laryngeal and trunk musculature (McLister et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Girgenrath and Marsh 1997, 1999). Pulse rate is species specific, and females prefer calls with pulse rates near the population mean (Gerhardt 1991, Ward et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The spectral frequencies of pulses are not strongly correlated with body size, and females exhibit weak and inconsistent preferences based on frequency differences (Schrode et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The number of pulses per call (hereafter, \u0026lsquo;call duration\u0026rsquo;), together with call rate, defines a male\u0026rsquo;s overall calling effort, determined as the product of call duration and rate (Ward et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Krueger et al. in review). A male\u0026rsquo;s calling behavior is a strong predictor of the metabolic costs of signaling, with longer calls, faster call rates, and higher overall effort reflecting both greater physiological investment and performance capacity (Taigen and Wells \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Wells and Taigen 1986; Grafe \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; McLister \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Schwartz and Rahmeyer \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Males producing calls with more pulses tend to do so at slower rates and there is also negative genetic covariance between these two components of calling effort (Sullivan and Hinshaw \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Gerhardt et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Schwartz et al. 2002; Welch et al. \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Reichert et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As the competitiveness of local signaling environments increases, males add pulses to lengthen their calls and reduce their call rate while maintaining or slightly increasing their overall calling effort (Wells and Taigen 1986; Reichert and Gerhardt \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Female mate choice is biased toward these costly signals: experimental studies show that females have strong directional preferences for males that produce longer calls at faster rates\u0026mdash;and thus signal with higher calling effort\u0026mdash;and that these preferences persist across realistic acoustic environments and competitive contexts (Gerhardt 1991; Gerhardt et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, 2000; Bee \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ward et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Velez et al. 2013; Tanner et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Krueger et al. in review). Importantly, female evaluation of calling effort reflects the integration of information across multiple calls rather than isolated acoustic traits, placing sustained calling effort\u0026mdash;rather than fine-scale spectral or temporal variation\u0026mdash;at the center of sexual selection in this system (Ward et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tanner et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Krueger et al. in review).\u003c/p\u003e \u003cp\u003eA longstanding hypothesis in behavioral endocrinology is that such energetically demanding sexual signals are regulated by interacting endocrine axes. Gonadal steroids such as testosterone produced by hypothalamic\u0026ndash;pituitary\u0026ndash;gonadal (HPG) axis have long been implicated in facilitating male sexual displays by enhancing vocal motor output, neuromuscular performance, and motivational state (Houck et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Leary and Baugh \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Estradiol (E\u003csub\u003e2\u003c/sub\u003e), although historically understudied in males, is increasingly recognized as a biologically relevant component of the male HPG axis (e.g. Freiler et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Beyond developmental effects, E\u003csub\u003e2\u003c/sub\u003e can act acutely on neural and muscular systems, modulating excitability, endurance, and metabolic efficiency\u0026mdash;traits directly relevant to sustained calling behavior (Campbell and Febbraio \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Balthazart and Ball \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Remage-Healy and Joshi 2012). Thus far, however, E\u003csub\u003e2\u003c/sub\u003e has rarely been examined explicitly in studies of male frog advertisement calling (but see: Schmidt \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Hoffmann and Kloas \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Furthermore, glucocorticoids released by the hypothalamic-pituitary-adrenal/inter-renal axis (HPA/I axis), such as corticosterone (CORT), regulate energy mobilization during periods of high metabolic demand and have been proposed to either facilitate sustained signaling or suppress reproductive effort when energetic reserves are limited. Emerson\u0026rsquo;s Energetics\u0026ndash;Hormone Vocalization (EHV) model integrates these ideas, predicting that prolonged calling elevates glucocorticoids, which may in turn inhibit gonadal steroid production and down-regulate sexual signaling (Emerson and Hess \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Although influential, empirical support for this framework in anurans has been mixed, with studies reporting weak, absent, or context-dependent hormone\u0026ndash;behavior relationships, particularly for glucocorticoids (reviewed in Leary and Baugh \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Progress on such questions has been slowed by two major limitations of previous studies. First, most studies rely on analyzing single acoustic traits despite evidence that both female preferences and male performance are inherently multivariate. Second, few studies address the additional challenge that males do not signal in isolation but instead compete against other nearby males to attract females.\u003c/p\u003e \u003cp\u003eHere, we combine paired acoustic recordings of neighboring males in breeding choruses with endocrine sampling in \u003cem\u003eH. chrysoscelis\u003c/em\u003e to investigate how gonadal and inter-renal hormones relate to male calling behavior in a competitive context. We measured male calling behavior and circulating T, E\u003csub\u003e2\u003c/sub\u003e, and CORT to address two complementary questions. First, do gonadal and inter-renal hormones explain variation in calling behavior across males at the population level? Second, do asymmetries in endocrine state between nearby males competing in a chorus predict asymmetries in calling behavior? By including contrasts of two competing males, we aimed to isolate endocrine effects while controlling for shared environmental, temporal, and acoustic conditions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAcoustic recordings, blood collection, and biometrics\u003c/h2\u003e \u003cp\u003eWe conducted paired-male recordings of the western genetic lineage of Cope\u0026rsquo;s gray treefrog (Booker et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) from wetlands located in the Carver Park Reserve (Carver County, MN, USA, 44\u0026deg;52'33.5\" N, 93\u0026deg;41'03.4\" W ) during peak calling periods (June 10\u0026ndash;20, 2024; 2200\u0026ndash;2330). Each recording involved two neighboring males calling in close proximity in a breeding chorus, allowing us to quantify variation in calling behavior at the within-pair level in a competitive signaling environment (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14 pairs). Field procedures followed a standardized protocol performed by a three-person team (audio manager, microphone operator, and one blood-sampling technician). Pairs of males were located visually using red headlamps in shallow water and selected only if (a) both were actively calling, (b) each male within a pair was the other\u0026rsquo;s nearest calling neighbor (within-pair separation: median\u0026thinsp;=\u0026thinsp;1.83 m, mean\u0026thinsp;=\u0026thinsp;3.69 m, SD\u0026thinsp;=\u0026thinsp;3.29 m), and (c) they were positioned sufficiently apart from neighboring males to isolate each dyad to obtain recordings suitable for analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Directional microphones (Sennheiser ME66; Sennheiser Electronic Corporation, Old Lyme, CT, USA) were placed in front of each male (\u0026sim;1 m distance), and a 10-min simultaneous recording (Zoom H8, Zoom Corporation, Tokyo, Japan) was initiated (SM1 \u0026amp; SM2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmediately after the 10-min recording window, we sampled blood via cardiac puncture following established methods validated in \u003cem\u003eHyla\u003c/em\u003e (e.g., Bastien et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Baugh \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, we rapidly (ca. 3 min; range\u0026thinsp;=\u0026thinsp;1\u0026ndash;7 min measured to nearest minute) collected blood (ca. 50 \u0026micro;L) using a 30-gauge insulin syringe (BD Micro-fine U-100, 0.3 mL) pre-rinsed with heparin. Whole blood was stored at 4\u0026deg; C on wet ice for 2\u0026ndash;4 h and then centrifuged (7500 RPM for 10 min; Eppendorf 5418 at 8\u0026deg; C). The plasma fraction was stored at -20\u0026deg; C for three weeks and then shipped on dry ice to Swarthmore College where samples were stored at -80\u0026deg; C for 1 month until assayed. Immediately after blood collection, we measured water temperature (Fluke 62 Max\u0026thinsp;+\u0026thinsp;IR thermometer, Everett, WA, USA; accuracy: \u0026plusmn;1.0 C), body mass (to the nearest 0.01 g) and body length (to the nearest 0.01 mm) as snout-vent length (SVL). Body condition estimates can be used as an indicator for energy reserves (Leary and Harris \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). We calculated a body condition index (hereafter \u0026lsquo;BCI\u0026rsquo;) by obtaining standardized residual values from a linear regression of SVL on body mass. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (#2301-40692A).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAcoustic analyses\u003c/h3\u003e\n\u003cp\u003eWe analyzed recordings of advertisement calls using Adobe Audition 2025 (Adobe Systems Inc., San Jose, CA, USA). Because our primary hypothesis concerned endocrine predictors of sustained calling performance, we focused on the three call traits known to be determinants of the energetic costs of calling and under strong sexual selection due to female mate choice: call rate (calls min⁻\u0026sup1;), call duration (pulses call⁻\u0026sup1;), and calling effort (pulses min⁻\u0026sup1;). For each recorded call, we measured its period (defined as the interval between the onsets of two successive calls) and call duration (defined as the number of pulses in the call). From these values, we computed an instantaneous call rate (calls min⁻\u0026sup1;) as the reciprocal of each call period, and an instantaneous calling effort (pulses min⁻\u0026sup1;) as the product of the instantaneous call rate and call duration for each call. Then for each male, we averaged values over all the calls he produced during the 10-min recording to determine his mean call rate, call duration, and calling effort (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eHormone analyses\u003c/h3\u003e\n\u003cp\u003eOur hormone analyses followed procedures described in more detail elsewhere (Baugh \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, we used a liquid diethyl-ether double extraction protocol. Following extraction, we froze samples at -80◦C for 1\u0026ndash;2 days or we resuspended immediately (assay buffer, provided by kit) and allowed samples to reconstitute for 12 h at 4\u0026deg;C. In males, optimal reconstitutions are at 1:10 for CORT and E\u003csub\u003e2\u003c/sub\u003e and 1:80 for T (Baugh \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We estimated steroid concentrations using EIA kits (DetectX\u0026reg; kits, Arbor Assays, Ann Arbor, MI) for 17β-estradiol (E\u003csub\u003e2\u003c/sub\u003e; Cat. No. KB30, Donkey anti-Sheep IgG), testosterone (T; Cat. No. K032, Goat anti-Rabbit IgG), and corticosterone (CORT; Cat. No. K014, Donkey anti-Sheep IgG). We analyzed samples in duplicate following manufacturer\u0026rsquo;s instructions. We used two duplicate internal standards (a single pooled sample of \u003cem\u003eH. chrysoscelis\u003c/em\u003e plasma) per plate for calculation of intra-assay coefficients of variation (CV). Detection limits and sensitivities, respectively, were 2.05 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2.21 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for E\u003csub\u003e2\u003c/sub\u003e; 9.92 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 30.6 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for T; and 16.9 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 18.6 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CORT. Cross-reactivity was 100% for E\u003csub\u003e2\u003c/sub\u003e, 3.2% for estrone sulfate, and 2.5% for estrone; 100% for T, 3.53% for 5a-dihydrotestosterone, and 0.27% for androstenedione; 100% for CORT, 12.3% for desoxycorticosterone, 0.62% for aldosterone, 0.38% for cortisol. Average intra-assay variations were 4.2% for E\u003csub\u003e2\u003c/sub\u003e, 9.3% for T, and 2.6% for CORT.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eWe assessed the ability of circulating hormones to predict male calling behavior using linear regression to address endocrine effects at both the population level and at the level of adjacent competing males. Hormone concentrations were log₁₀-transformed to improve normality of residuals and then standardized (\u003cem\u003ez\u003c/em\u003e-scored). For each of our three acoustic measures of calling behavior (call rate, call duration, and calling effort), we standardized (\u003cem\u003ez\u003c/em\u003e) the individual means for each variable across males and then used principal component analyses (PCA) to reduce the acoustic data to a smaller set of orthogonal variables. Across all three PCA implementations, PC1 consistently loaded positively on all three call-effort traits, whereas PC2 captured a rate\u0026ndash;complexity trade-off, confirming stable biological interpretation despite minor sample size differences.\u003c/p\u003e \u003cp\u003eTo provide descriptive context for the multiple regressions, we calculated Pearson correlations between hormones, among our three measures of calling behavior, and between hormones and calling behavior using the population-level dataset with complete data (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24 males). To assess whether BCI or water temperature explained additional variation in calling beyond endocrine predictors, we conducted a series of nested model comparisons within the population-level framework. We compared a base model including standardized log\u003csub\u003e10\u003c/sub\u003eT, log\u003csub\u003e10\u003c/sub\u003eE\u003csub\u003e2\u003c/sub\u003e, and log\u003csub\u003e10\u003c/sub\u003eCORT to models additionally including BCI, water temperature, or both covariates. Model comparisons were evaluated using partial F-tests and Akaike\u0026rsquo;s Information Criterion (AIC). All analyses were conducted in R (v. 4.4.2). Significance was assessed at α\u0026thinsp;=\u0026thinsp;0.05, and effect sizes are reported alongside test statistics where appropriate.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCalling behavior\u003c/h2\u003e \u003cp\u003eOn average, males produced advertisement calls consisting of about 28 pulses at a rate of about 12 calls per minute, yielding an average calling effort of about 337 pulses min\u003csup\u003e-1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These values are close to population averages reported in an earlier study of the same population (Ward et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Across individuals, mean calling effort varied by a factor of more than 4.0 (range: 120 to 507 pulses min\u003csup\u003e-1\u003c/sup\u003e). Males within pairs also varied in calling (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As expected, there were significant positive correlations between calling effort and both call rate and call duration, which themselves were negatively correlated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, we observed the phenotypic tradeoff between call rate and call duration reported in previous studies.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary statistics from sound analysis and principal component analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalling effort\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCall rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCall duration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePC1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePC2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalling effort\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e336.9\u0026thinsp;\u0026plusmn;\u0026thinsp;81.2 pulses min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCall rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.53\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 calls min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCall duration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.56\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e =\u0026nbsp;-0.39\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 pulses call\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEigenvalue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariance explained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eThe mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) for the three acoustic variables are shown along the diagonal along with the Pearson product moment correlations (\u003cem\u003er\u003c/em\u003e) between variables and their associated \u003cem\u003ep\u003c/em\u003e values (two-tailed). Also shown are the factor loadings for the first and second principal components (PC1 and PC2) along with their associated eigenvalues and variance explained.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA PCA of standardized call traits (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27 males with complete calling data) yielded two informative axes that together explained 99.6% of the variance (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PC1 explained 53.2% of the variance and loaded positively on all three acoustic variables, with the heaviest loading on calling effort. Thus, PC1 captured variation in well-established metrics of calling \u003cem\u003eperformance\u003c/em\u003e in gray treefrogs (i.e., higher call rates, longer calls, and higher calling effort). For use in regression analyses, values for PC1 were multiplied by \u0026minus;\u0026thinsp;1 so that higher scores represent greater overall calling performance. PC2 explained 46.4% of the variance and primarily contrasted call rate (0.89) against call duration (\u0026minus;\u0026thinsp;0.81), with minimal contribution of calling effort (0.06). We interpret this result as PC2 capturing orthogonal variation in calling \u003cem\u003estrategy\u003c/em\u003e consistent with a performance trade-off between call rate and call duration: some males produce many short calls and others produce fewer longer calls, and this is a trade-off because both strategies can occur at similar overall calling efforts.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHormones\u003c/h3\u003e\n\u003cp\u003eAverage concentrations for each hormone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were typical of patterns observed in vocally active males in these populations (Baugh \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Freiler et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Circulating T showed the greatest variation among males (coefficient of variation, CV\u0026thinsp;=\u0026thinsp;1.6), whereas E\u003csub\u003e2\u003c/sub\u003e varied over a much narrower range (CV\u0026thinsp;=\u0026thinsp;0.17), and CORT showed intermediate variability (CV\u0026thinsp;=\u0026thinsp;0.69).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary statistics from hormones\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTestosterone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstradiol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCORT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTestosterone\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(CV\u0026thinsp;=\u0026thinsp;1.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;16.6 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstradiol\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(CV\u0026thinsp;=\u0026thinsp;0.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.39\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e206.9\u0026thinsp;\u0026plusmn;\u0026thinsp;36.3 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCORT\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(CV\u0026thinsp;=\u0026thinsp;0.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.17\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e =\u0026nbsp;0.28\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eThe mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24) for the three hormones are shown along the diagonal along with the Pearson product moment correlations (\u003cem\u003er\u003c/em\u003e) between variables and their associated \u003cem\u003ep\u003c/em\u003e values (two-tailed).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eHormone-behavior relationships in the population\u003c/h3\u003e\n\u003cp\u003ePearson correlations between each hormone and each call parameter indicated that gonadal hormones were positively correlated with metrics of calling behavior, especially calling effort (SM3). We used multiple linear regression to test whether circulating hormones predicted variation in calling behavior across males at the population level. We included all males complete with all three metrics of calling behavior and all three hormones (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24 males). We calculated PC scores from this subset of males which yielded a component structure consistent (SM4) with the PCA used earlier (see Calling behavior). We then fit linear multiple regression models predicting PC1 and PC2 from standardized (z scores) values of log\u003csub\u003e10\u003c/sub\u003eT, log\u003csub\u003e10\u003c/sub\u003eE\u003csub\u003e2\u003c/sub\u003e, and log\u003csub\u003e10\u003c/sub\u003eCORT:\u003c/p\u003e \u003cp\u003ePC\u0026thinsp;=\u0026thinsp;β₀ + β₁(T) + β₂(E\u003csub\u003e2\u003c/sub\u003e) + β₃(CORT) + ε\u003c/p\u003e \u003cp\u003eThis model was significant for PC1 and revealed that circulating hormones explained substantial variation in overall calling performance at the population level (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.50, adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.42; F\u003csub\u003e3,20\u003c/sub\u003e = 6.65, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; SM5). Both gonadal hormones showed significant, positive partial effects on calling performance (PC1). T was a positive predictor of PC1 when controlling for E\u003csub\u003e2\u003c/sub\u003e and CORT (β\u0026thinsp;=\u0026thinsp;0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 SE, \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.58, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018, 95% CI [0.11, 1.00]). Interpreted in biologically meaningful units, a one\u0026ndash;standard deviation increase in T (not log-transformed) was associated with a 0.55 SD increase in overall calling effort (PC1). E\u003csub\u003e2\u003c/sub\u003e also independently predicted PC1 (β\u0026thinsp;=\u0026thinsp;0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 SE, \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.37, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028, 95% CI [0.06, 0.98]). A one SD increase in T predicted a 0.55 SD increase in PC1 (~\u0026thinsp;45 pulses min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), whereas a comparable increase in estradiol predicted a 0.52 SD increase (~\u0026thinsp;42 pulses min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating that both gonadal steroids contribute similarly sized effects on calling effort at the population level. In contrast, CORT did not predict PC1 (β = \u0026minus;0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 SE, \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.42, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.680). None of the hormones predicted orthogonal variation in calling strategy (PC2). The PC2 regression model was not significant overall (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.10, adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; = \u0026minus;0.03; F₃,₂₀ = 0.78, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.522), and no individual predictor approached significance (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.173). Water temperature and BCI did not improve model fit and were thus not included (SM6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHormone-behavior relationships between competing males\u003c/h2\u003e \u003cp\u003eTo test whether these population‑level associations also explain competitive asymmetries, we next examined hormone and calling differences within male pairs. We used multiple regression to test whether hormonal differences between competing males predicted differences in calling behavior. We included all pairs complete with all three calling metrics and all three hormones (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 pairs). We calculated PC scores from this set of 10 pairs (20 males) which yielded a component structure consistent with both PCAs used previously (SM4). For each pair, we calculated differences in PC scores and hormone levels between the two paired males (designated L and R):\u003c/p\u003e \u003cp\u003eΔPCl\u0026thinsp;=\u0026thinsp;PC\u003csub\u003eL\u003c/sub\u003e \u0026ndash; PC\u003csub\u003eR\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eΔHormone\u0026thinsp;=\u0026thinsp;Hormone\u003csub\u003eL\u003c/sub\u003e \u0026ndash; Hormone\u003csub\u003eR\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eThis difference approach controls for shared environmental, temporal, and acoustic conditions within each dyad and thus helps isolate endocrine predictors of relative calling performance. We then fit multiple regression models predicting ΔPC1 and ΔPC2 from ΔT, ΔE\u003csub\u003e2\u003c/sub\u003e, and ΔCORT:\u003c/p\u003e \u003cp\u003eΔPC\u0026thinsp;=\u0026thinsp;β₀ + β₁(ΔT) + β₂(ΔE\u003csub\u003e2\u003c/sub\u003e) + β₃(ΔCORT) + ε\u003c/p\u003e \u003cp\u003eThis model was significant for PC1 (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.79, adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.68; F\u003csub\u003e3,6\u003c/sub\u003e = 7.43, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; SM5), indicating differences in hormone levels between competitors strongly predicted differences in calling performance. Differences in T were a strong predictor of performance asymmetries: males with higher T than their opponent had substantially higher calling efforts (β\u0026thinsp;=\u0026thinsp;1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 SE, \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.06, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022, 95% CI [0.25, 2.24]). Differences in E\u003csub\u003e2\u003c/sub\u003e showed a positive but non-significant association with ΔPC1 (β\u0026thinsp;=\u0026thinsp;0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 SE, \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.84, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.12), whereas CORT differences did not predict performance asymmetries (β\u0026thinsp;=\u0026thinsp;0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 SE, t\u0026thinsp;=\u0026thinsp;0.65, p\u0026thinsp;=\u0026thinsp;0.54). As in our population level approach, hormonal asymmetries did not predict differences in calling strategy (ΔPC2). The ΔPC2 regression model was not significant (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.16, adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; = \u0026minus;0.25; F₃,₆ = 0.39, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.76), and no hormone predictor approached significance (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.40).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFemale preference and selection on endocrine traits\u003c/h2\u003e \u003cp\u003eA key insight from decades of anuran bioacoustics is that female frogs attend closely to a male\u0026rsquo;s calling performance, including call duration and call rate, calling effort, and overall acoustic output, as indicators of competitive ability (reviewed in Gerhardt and Huber \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wells \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Narins et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thus, selection should favor endocrine mechanisms that enable males to sustain high-performance calling in the context of ecological and social constraints. Our results provide a physiological complement to this classic sensory and behavioral framework by showing that coordinated gonadal activation strongly predicts variation in calling performance, but not calling strategy, both across males and between direct competitors. If T or E\u003csub\u003e2\u003c/sub\u003e facilitates sustained motor output or buffers stress-related suppression of signaling, then female preferences for higher calling performance may indirectly select for endocrine phenotypes that balance energetic investment against stress responsiveness (Leary and Knapp \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Leary and Baugh \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this way, female choice for acoustic performance would shape not only signal structure and signaling behavior but also the hormonal architecture underlying male signaling effort.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGonadal hormones and calling performance\u003c/h2\u003e \u003cp\u003eRegression analyses at the levels of both the population and competing individuals converged on a robust correlation between gonadal activation and male calling performance. At the population level, hormones strongly (adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.42) predicted a multivariate axis of calling performance, demonstrating that integrated HPG-axis activation accounts for substantial among-male variation in the performance of sexual displays. A similar relationship was found comparing nearby competing males, where differences in hormones predicted differences in calling performance with an even larger effect size (adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.68). This convergence demonstrates that the loss of statistical power associated with fewer observations (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 pairs compared to \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24 males) was more than offset by the paired design\u0026rsquo;s ability to control for shared environmental, temporal, and other factors. Hence, contrasts between nearby competing individuals provide a particularly powerful and biologically meaningful approach for linking endocrine state to signaling performance.\u003c/p\u003e \u003cp\u003eThe partial effects of both T and E\u003csub\u003e2\u003c/sub\u003e contributed explanatory power to calling performance, with each hormone remaining a significant predictor when controlling for the other. Testosterone\u0026rsquo;s positive association with calling performance is consistent with its well-established role in male sexual signaling across vertebrates (Kelley \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Emerson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Wilczynski et al. \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Adkins-Regan \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Leary and Harris \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Testosterone can enhance vocal motor output, increase calling motivation, and support the neuromuscular performance required for sustained calling (Wetzel and Kelley \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Houck et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Leary and Baugh \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Androgens may also act centrally to bias motivational state toward reproductive investment (Wingfield et al. \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and peripherally to facilitate muscle performance and respiratory capacity (Husak and Irschick \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), thereby scaling overall signaling performance. In this sense, the strong positive association between T and calling performance observed here is consistent with a large body of work linking androgenic state to elevated reproductive effort. The lack of a relationship between hormones and calling strategy (how males distribute their calling effort within and among calls) might further indicate that the circulating endocrine state constrains the overall level of metabolic investment whereas strategic deployment of those efforts is controlled by decision making circuits unmodulated by steroids.\u003c/p\u003e \u003cp\u003eIn contrast, the finding that E\u003csub\u003e2\u003c/sub\u003e explained additional, non-redundant variation is especially notable given that research on endocrine regulation of male sexual signaling in anurans has historically emphasized androgens and, to a lesser extent, glucocorticoids, with estrogens receiving comparatively little attention despite their activity in male frogs (Schmidt \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Hoffmann and Kloas \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003eb\u003c/span\u003e). This may reflect the fact that circulating E\u003csub\u003e2\u003c/sub\u003e is derived primarily from the conversion of androgens in the testes and thus considered incidental rather than a hormone with independent behavioral influence. This pattern mirrors a broader vertebrate literature in which estrogenic effects on male behavior are sometimes subsumed under an androgen-focused framework (Ball and Balthazart \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; but see Balthazart and Ball \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Yet E\u003csub\u003e2\u003c/sub\u003e receptors and aromatase are abundantly expressed in anuran brain regions involved in vocal production and auditory processing, including the preoptic area and torus semicircularis (e.g., Kelley \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Wilczynski and Ryan \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), suggesting substantial scope for estrogenic modulation of male signaling, from systemic or local (conversion) sources. Indeed, estradiol is known across vertebrates to influence neuromuscular performance, mitochondrial efficiency, and fatigue resistance, in part through modulation of oxidative metabolism and calcium handling in muscle (e.g., Wideman et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ventura-Clapier et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These effects are especially relevant for prolonged anuran calling, which requires sustained contraction of trunk and laryngeal musculature and imposes substantial metabolic demands (Taigen and Wells \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Emerson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Estradiol can also exert rapid neuromodulatory effects on vocal-motor circuits and motivational centers (Remage-Healey and Bass \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Leary and Baugh \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), potentially enhancing both the capacity and persistence of high-effort calling. Against this background, our finding that E\u003csub\u003e2\u003c/sub\u003e explains non-redundant variation in male calling performance indicates that estrogenic pathways may play a more direct role in shaping energetically costly advertisement displays than is typically appreciated. Interestingly, experimentally elevated E\u003csub\u003e2\u003c/sub\u003e in \u003cem\u003eXenopus laevis\u003c/em\u003e has been shown to inhibit male courtship calling (Hoffmann and Kloas \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e), which occurs through estrogen receptor signaling (Hoffmann and Kloas \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e). A future pharmacological experiment is needed to disentangle the stimulatory and inhibitory roles of T and E\u003csub\u003e2\u003c/sub\u003e on aspects of male calling.\u003c/p\u003e \u003cp\u003eFuture studies will be needed to investigate causal relationships between gonadal hormones and calling performance in gray treefrogs. Experimental manipulations (e.g. elevating, depleting and/or inhibiting T and E\u003csub\u003e2\u003c/sub\u003e) in calling males, ideally combined with measures of metabolic performance, muscle fatigue, and neural activity during sustained calling bouts, would be particularly informative. Integrating such manipulations with paired recordings, as used here, or in experimental contests (e.g., Reichert and Gerhardt \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) would allow researchers to disentangle hormone-driven changes in signaling capacity from context-dependent motivational effects. Finally, coupling endocrine manipulations of calling males with female phonotaxis assays would provide a powerful test of whether T and E\u003csub\u003e2\u003c/sub\u003e-mediated variation in male calling performance translates into differential mating success, connecting endocrine state, signal production, perception, and selection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHPG and HPI interactions\u003c/h2\u003e \u003cp\u003eBecause T and E\u003csub\u003e2\u003c/sub\u003e are jointly produced components of HPG-axis activation, linked through aromatization, E\u003csub\u003e2\u003c/sub\u003e could help determine whether testosterone-driven high-performance signaling can be maintained by buffering energetic and stress-related constraints on sustained performance or reducing the immune suppressive effects of T alone (Wingfield et al. \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Remage-Healey and Bass \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Handa et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Elevated E\u003csub\u003e2\u003c/sub\u003e may therefore help maintain calling performance under conditions in which activation of the HPI axis would otherwise constrain signaling. This interpretation is consistent with our broader finding that CORT showed little explanatory power for calling performance in this study and suggests that gonadal activation\u0026mdash;rather than antagonistic HPG\u0026ndash;HPI dynamics\u0026mdash;may be the dominant endocrine basis determining calling performance in competitive signaling environments.\u003c/p\u003e \u003cp\u003eThe lack of evidence for an influence of CORT on male signaling and the positive correlation observed between circulating gonadal hormones and sexually selected calling performance might help clarify another recent discovery related to energetically demanding sexual behavior in this frog. Specifically, male \u003cem\u003eH. chrysoscelis\u003c/em\u003e that successfully attract a mate have substantially elevated and correlated plasma hormone concentrations (CORT, androgens and estradiol; Baugh, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). There are at least two plausible, non-mutually exclusive hypotheses for this pattern: (1) males found in amplexus were chosen by females on the basis of their calling effort, which are positively correlated with gonadal hormone levels (i.e., sexual selection by female choice); or (2) the act of clasping itself rapidly increases steroid hormone secretion (i.e., bidirectionality in hormone-behavior relationships). The former hypothesis is supported by the current study, and the latter hypothesis is also supported by a recent empirical demonstration that induced amplexus in male \u003cem\u003eH. chrysoscelis\u003c/em\u003e activates the HPG axis, rapidly elevating both T and E\u003csub\u003e2\u003c/sub\u003e but not CORT (Freiler et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). These findings further support the pleiotropic nature of steroids\u0026mdash;gonadal steroids are likely facilitating elevated calling performance and thereby potentially increasing mating success (but see Sullivan and Hinshaw \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and subsequently becoming further elevated upon clasping to facilitate muscular tone (Kampe and Peters \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and sperm release (Silla et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The exact role of androgens versus estrogens in these processes remains to be discovered, for example via aromatase inhibition experiments. Lastly, further study of the role of CORT is also needed given its elevated concentrations in naturally but not artificially amplexed males and lack of correlation with calling behavior.\u003c/p\u003e \u003cp\u003eOur conclusion that higher concentrations of gonadal steroids, but not CORT, are associated with higher calling performance requires some qualification. The current study used a 10-min sample of calling activity and a single acute blood sample, with the assumption that these two levels of the phenotype are temporally synchronized and representative more generally. However, a 10-min sample of calling activity during a 10-day period of the breeding season represents a narrow slice of time. The potential influence of CORT on calling performance might only be revealed at extreme concentrations or be relevant only at the performance limits of the males (Marler and Ryan, 1996; Leary and Knapp, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e.). In addition, like other lek-breeding frogs (Ryan \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), male gray treefrogs can spend several hours per night, across many nights, calling (Sullivan and Hinshaw \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Runkle et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Wells et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Gerhardt et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Reichert et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). How males trade-off calling performance in a given bout against performance distributed across an entire night, and across an entire breeding season is not yet well understood. Further, how hormones might provide a mechanistic basis for resolving these temporal-investment tradeoffs is unknown. However, this tradeoff should be important in principle; for example, Sullivan and Hinshaw (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) demonstrated that the single best predictor of male mating success in the tetraploid gray treefrog, \u003cem\u003eH. versicolor\u003c/em\u003e, is the number of nights a male participates in the chorus, which was also observed in t\u0026uacute;ngara frogs (Ryan \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Therefore, understanding how males optimize the distribution of vocal effort across different time scales, how interactions between the HPG and HPI axes influence these decisions, and how behavioral and hormonal phenotypes are linked with ecological variables (e.g., foraging; Green \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Marler and Ryan 1996; Wilhite and Ryan \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; predation risk: Baugh and Ryan \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and social factors (e.g., female cues; Akre and Ryan \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) represent fruitful directions for further study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBy linking variation in calling performance to underlying endocrine state in both population-level and paired competitive contexts, the present study provides evidence of physiological mechanisms that subserve male performance in competitive signaling environments. In this regard, this study builds on the enormous body of work by the honoree of this special issue, Peter Narins. A fundamental theme running through Narins\u0026rsquo; research has been to understand anuran signaling behaviors in competitive contexts, and this research has profoundly shaped our understanding of acoustic competition among male frogs and its consequences for sexual selection. Most directly related to the present study, Lopez and Narins (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) demonstrated that females of the Puerto Rican Coqui, \u003cem\u003eEleutherodactylus coqui\u003c/em\u003e, strongly prefer males calling at higher rates, an earlier finding that helped establish the fundamental importance of female preferences for male calling performance. To better understand how males achieve such performance under competitive conditions, Narins and colleagues have examined the interactions among calling males, often using the evoked vocal response as a powerful assay to reveal dynamic signaling strategies. For example, Narins and Capranica (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1978\u003c/span\u003e) identified the functional role of the \u0026ldquo;Co\u0026rdquo; note in competitive signaling among Coqui males, Lopez et al. (1988) documented striking plasticity in call intensity and frequency, and Benedix and Narins (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) found robust call-rate escalation during simulated intrusions. Narins\u0026rsquo; work has also investigated how males cope with the cacophony that results when competing males call in noisy chorus environments. For example, Zelick and Narins (\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and Brush and Narins (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) showed that males precisely time calls to exploit silent windows and avoid call overlap with rival males, while Lewis et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) revealed the use of seismic signals when acoustic channels were masked. Finally, Narins has also shown that vocal interactions extend beyond considerations of the form and timing of acoustic signals by showing that physical aggression between competing males depends on multimodal cues and cross-modal integration during territorial defense (Narins et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Narins et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Together, these and other pioneering studies by Peter Narins and his students, postdocs, and collaborators help frame our endocrine findings within a rich tradition of neuroethological research on the behavioral strategies that enable male success in competitive signaling environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Swarthmore College Research Fund to ATB and NSF grant IOS-2154204 to MAB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eATB: Conceptualization, Methodology, Resources, Formal analysis, Investigation, Data curation, Writ\u0026shy;ing - original draft, Funding acqui\u0026shy;sition, Supervision, Project administration. \u0026nbsp;MKF: Formal analysis, Investigation, Editing - original draft. LNH: Investigation. AK: Formal analysis, Investigation. MAB: Conceptualization, Methodology, Resources, Formal analysis, Investigation, Writ\u0026shy;ing - original draft, Funding acqui\u0026shy;sition, Supervision, Project administration.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe last author (MAB) is a guest editor for this special issue but did not handle this manuscript in any capacity.\u0026nbsp;We have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplete dataset available in SM7. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Minnesota Department of Natural Resources for permission to conduct this research, John Moriarty and Steven Hogg with the Three Rivers Park District for after-hours access to frog ponds, and Chris Leary for productive discussions on this research topic. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdkins-Regan E (2005) Hormones and animal social behavior. 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Behav Ecol Sociobiol 78:54. https://doi.org/10.1007/s00265-024-03470-7\u003c/li\u003e\n\u003cli\u003eWingfield JC, Hegner RE, Dufty AM, Ball GF (1990) The \u0026ldquo;challenge hypothesis\u0026rdquo;: theoretical implications for patterns of testosterone secretion, mating systems, and breeding strategies. Am Nat 136:829\u0026ndash;846.\u003c/li\u003e\n\u003cli\u003eWingfield JC, Sapolsky RM (2003) Reproduction and resistance to stress: when and how. J Neuroendocrinol 15:711\u0026ndash;724.\u003c/li\u003e\n\u003cli\u003eZelick RD, Narins PM (1983) Intensity discrimination and the precision of call timing in two species of neotropical treefrogs. J Comp Physiol A 153:403\u0026ndash;412. https://doi.org/10.1007/BF00612594\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-comparative-physiology-a","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcpa","sideBox":"Learn more about [Journal of Comparative Physiology A](http://link.springer.com/journal/359)","snPcode":"359","submissionUrl":"https://submission.nature.com/new-submission/359/3","title":"Journal of Comparative Physiology A","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"androgen, corticosterone, courtship call, estradiol, sexual selection, testosterone, vocalization","lastPublishedDoi":"10.21203/rs.3.rs-8695050/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8695050/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcoustic advertisement signals in anurans are classic models for understanding how endocrine mechanisms regulate courtship behavior. However, our understanding of how steroids influence male calling performance remains limited because most studies examine single hormones or isolated call traits. Here we quantified three plasma steroids\u0026mdash;testosterone (T), estradiol (E2), and corticosterone (CORT)\u0026mdash;from male Cope's gray treefrogs (\u003cem\u003eHyla chrysoscelis\u003c/em\u003e) recorded in natural male\u0026ndash;male calling pairs, relating these concentrations to multiple calling components including call rate, duration, and effort. We used principal components analysis to describe a male's overall calling \u003cem\u003eperformance\u003c/em\u003e and his calling \u003cem\u003estrategy\u003c/em\u003e (how he navigated a performance tradeoff between longer calls and faster rates). We analyzed these relationships at the population level and within male pairs using multiple regressions including all three hormones as predictors. At the population level, T and E\u003csub\u003e2\u003c/sub\u003e positively correlated with calling performance, whereas calling strategy was unrelated to hormones. Within male pairs, differences in T also positively correlated with differences in calling performance. Despite their collinearity, T and E\u003csub\u003e2\u003c/sub\u003e independently and together explained variation in calling performance. CORT showed no association with calling performance or strategy at either level, and temperature and body condition did not explain variation in calling or hormones. These results show that gonadal activation translates into asymmetries in calling performance between competitors while leaving performance-related tradeoffs unaffected. By revealing strong positive correlations between gonadal hormones and energetic investment in calling, this study demonstrates how female preferences for high-performing callers can impose sexual selection on endocrine mechanisms regulating courtship behavior.\u003c/p\u003e","manuscriptTitle":"Testosterone and estradiol predict male calling performance, but not performance-related tradeoffs, in competitive signaling environments in Cope’s gray treefrogs (Hyla chrysoscelis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 09:30:33","doi":"10.21203/rs.3.rs-8695050/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-22T07:51:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-21T11:01:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-07T22:03:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205862057133195868808062271131298313145","date":"2026-02-07T16:40:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299569561011182584829937622185172952527","date":"2026-02-03T20:26:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T16:30:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-02T12:13:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-29T05:33:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Comparative Physiology A","date":"2026-01-25T22:07:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-comparative-physiology-a","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcpa","sideBox":"Learn more about [Journal of Comparative Physiology A](http://link.springer.com/journal/359)","snPcode":"359","submissionUrl":"https://submission.nature.com/new-submission/359/3","title":"Journal of Comparative Physiology A","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b74b940-3073-4e81-9394-55e09d6763d2","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-04T14:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 09:30:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8695050","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8695050","identity":"rs-8695050","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}