Sex and reproductive condition shape thermal acclimation strategy in a plethodontid salamander

Sex and reproductive condition shape thermal acclimation strategy in a plethodontid salamander | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sex and reproductive condition shape thermal acclimation strategy in a plethodontid salamander Kevin J. Moore , Brittany M. Winter , View ORCID Profile Jeanette B. Moss doi: https://doi.org/10.1101/2025.09.28.679080 Kevin J. Moore 1 Department of Biological Sciences, Virginia Tech , Blacksburg, VA 24061, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brittany M. Winter 1 Department of Biological Sciences, Virginia Tech , Blacksburg, VA 24061, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeanette B. Moss 1 Department of Biological Sciences, Virginia Tech , Blacksburg, VA 24061, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jeanette B. Moss For correspondence: jbmoss{at}vt.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The question of whether males and females differ in their responses to temperature is essential to understanding adaptive capacities in a warming world. However, sex has traditionally been neglected in the field of thermal ecology, and our understanding of the factors that promote sex differences in thermal plasticity is underdeveloped. Here, we investigate the independent and interactive effects of sex and reproductive condition on thermal acclimation capacity in a plethodontid salamander ( Plethodon cinereus ). We carried out two stop-flow respirometry experiments, with salamanders acclimated to one of two thermal environments designed to simulate a ‘Cool’ or ‘Warm’ breeding season. In the first experiment, we compared the thermal acclimation responses of males and gravid females across a gradient of three ecologically relevant test temperatures and observed distinct patterns of sexual dimorphism of standardized metabolic rate (SMR) between thermal treatments that were not attributable to differences in body size. Specifically, gravid females in the warm treatment showed reduced SMR relative to gravid females in the cool treatment, particularly at higher test temperatures. In the second experiment, we expanded on our initial findings by directly testing the contribution of reproductive condition to observed sex differences in thermal acclimation capacity. By repeating the experiment of the early breeding season but including a third group of non-gravid females, we: (1) recapitulated our original finding – that gravid females exhibit a pronounced response to thermal acclimation relative to males; and (2) showed that female thermal acclimation responses are absent in nongravid females, and therefore we concluded that responses are contingent on reproductive condition. Taken together, our results provide a first glimpse into how sex and reproductive condition contribute to intraspecific variation in thermal acclimation capacity in an amphibian and underscore the need for more hypothesis-driven studies to directly test when, where, and how such patterns arise. Introduction The capacity of an organism to acclimate its biological rates in response to changing thermal conditions (i.e., thermal acclimation capacity) is a widespread, often beneficial form of phenotypic plasticity that increases individual tolerance and performance at a given temperature and may contribute to population resilience under rapidly changing climates ( Angilletta 2009 ; Rohr et al. 2018 ; Havird et al. 2020 ). Strategies such as the active up-or down-regulation of metabolism relative to basal rates can be an effective mechanism for balancing energy losses and gains at temperatures that are suboptimal for activity ( Angilletta 2009 ; Riddell, Odom, et al. 2018 ; Havird et al. 2020 ). For example, heat-induced reductions in metabolic rate (i.e., metabolic depression ) are common in ectotherms living in warm environments and presumably magnify energy savings during extended bouts of inactivity ( Feder et al. 1982 ; Christian et al. 1999 ; Guppy and Withers 1999 ; Liao et al. 2021 ; Muñoz et al. 2022 ), whereas mitigating prohibitively cold temperatures often involves inflating metabolic rates above expected levels to extend the range of physiological function (i.e., metabolic compensation ; ( Rogers et al. 2007 ; Gaston et al. 2009 ; Bozinovic et al. 2011 ; Catenazzi 2016 ; Watanabe and Payne 2023 )). Accounting for this plasticity can dramatically alter ecological predictions under climate change, and is therefore imperative to accurately modeling population responses ( Riddell, Odom, et al. 2018 ; Briscoe et al. 2023 ). However, species- and population-level measures of thermal acclimation capacity are often derived from few reference individuals, which may not accurately reflect the population as a whole ( Bennett et al. 2019 ). Indeed, recent work has documented tremendous inter-individual variation in thermal acclimation strategies ( Seebacher et al. 2015 ; Riddell, McPhail, et al. 2018 ; Hui et al. 2020 ; Liao et al. 2021 ), although knowledge of the factors that account for this variation remains fragmentary ( Bennett et al. 2019 ; Bodensteiner et al. 2021 ; Cocciardi and Ohmer 2024 ). One poorly understood axis of inter-individual variation in thermal acclimation capacity is sex ( Pottier et al. 2021 ; Hangartner et al. 2022 ). Male and female animals differ in a broad range of morphological, physiological, and behavioral traits. These adaptations are often exaggerated during reproductive periods, when the sexes tend to conform to stereotyped patterns of energy storage and expenditure associated with their distinct reproductive strategies. Where females typically adopt conservative strategies, accumulating and provisioning energy gradually towards costly egg production, males tend to take more risks, accumulating energy in short bursts and expending reserves in active pursuit of reproductive opportunities ( Beck et al. 2003 ; Czenze et al. 2017 ; Tarka et al. 2018 ). Such distinct approaches to energy use may favor different strategies for coping with thermal variation. For example, a study of the striped marsh frog uncovered male-biased metabolic compensation responses to cold temperatures during the early spring breeding season ( Rogers et al. 2007 ), suggesting that thermal acclimation may be enhanced in males relative to females when there is a benefit to mating performance. Conversely, several studies have revealed female bias in capacity to acclimate to high temperatures, which could reflect their slow pace-of-life and high reproductive investment ( Edmands 2021 ; Pottier et al. 2021 ; Vermandele et al. 2024 ). Because population growth is often more sensitive to the survival and fecundity of one sex (e.g., ‘female demographic dominance’), the potential for sex-biased plasticity presents a critical, albeit relatively unexplored factor influencing population persistence ( Fox et al. 2019 ; Hangartner et al. 2022 ; Gissi et al. 2023 ). Despite growing interest of recent years (e.g., ( Huey and Pianka 2007 ; Lailvaux 2007 ; Bodensteiner et al. 2021 ; Edmands 2021 ; Gunderson 2021 ; Pottier et al. 2021 ; Hangartner et al. 2022 )), the number of empirical studies explicitly testing for sex differences in thermal response continues to lag behind theory. In their 2021 meta-analysis, Pottier et al. found that over three quarters of eligible studies on thermal acclimation capacity (446 of 586) failed to report or did not consider the sex of experimental subjects. Of the remaining eligible studies, overall effects were heterogeneous and supported few systematic patterns, suggesting that where it arises, sexual dimorphism in thermal response may be context dependent. For instance, countless field studies have documented variation in thermal traits associated with reproductive condition or breeding stage (e.g., ( Charland and Gregory 1990 ; Grinevitch et al. 1995 ; Finkler et al. 2003 ; Galliard et al. 2003 ; Hammill et al. 2004 ; Isaac and Gregory 2004 ; Finkler 2006 ; Rogers et al. 2007 ; Wilson et al. 2007 ; Darnell et al. 2013 ; Allen and Levinton 2014 ; Beal et al. 2014 ; Vaughn et al. 2014 ; Webber et al. 2015 ; Woolrich-Piña et al. 2015 ; Parker et al. 2019 ; Levine et al. 2024 ), which could confound detection of sex differences. Given practical limitations of acquiring detailed physiological and behavioral data from multiple demographic classes when forecasting climate change responses, there is a need to identify when, where, and how sex-specific responses to temperature are likely to emerge in nature ( Pottier et al. 2021 ; Hangartner et al. 2022 ). Lungless salamanders (Family: Plethodontidae) are well suited for investigating the role of sex and reproductive condition in driving thermal acclimation patterns. As small-bodied amphibians with small home ranges and narrow climatic tolerances, plethodontids rely on physiological acclimation mechanisms to function successfully across their natural geographic range ( Gifford and Kozak 2012 ; Markle and Kozak 2018 ; Riddell et al. 2024 ), as well as in the face of climatic extremes ( Novarro et al. 2018 ; Riddell, Odom, et al. 2018 ; Muñoz et al. 2022 ). Yet the potential for sex-specific selection pressures to shape patterns of thermal acclimation has received almost no attention in climate projections for this group. Addressing this limitation is critical, as reproduction profoundly impacts energy balance ( Fitzpatrick 1973 ; Crespi 2001 ; Finkler and Cullum 2002 ; Takahashi 2002 ; Gillette 2003 ) and behavior ( Gergits and Jaeger 1990 ; Dyal 2006 ; Leclair et al. 2008 ; Jaeger et al. 2016 ) and in many species spans the entire year ( Crespi 2001 ; Fisher-Reid et al. 2024 ). Moreover, reproductive life history strategies tend to be highly sexually dimorphic in plethodontids. For example, whereas male gamete production is a relatively rapid process that relies minimally on stored energy and occurs annually ( Sayler 1966 ; Werner 1969 ; Nagel 1977 ; Takahashi and Pauley 2010 ; Fisher-Reid et al. 2024 ), females spend up to nine months yolking a single clutch of eggs, and may reproduce as infrequently as every four years ( Gillette 2003 ; Leclair et al. 2008 ; Takahashi and Pauley 2010 ). Here, we enlist the eastern red-backed salamander ( Plethodon cinereus ), a species widespread across Eastern U.S. deciduous forests, to compare physiological thermal acclimation strategies between the sexes across their nearly eight-month breeding season. Due to sex differences in energy allocation to reproduction, we predicted that metabolic compensation in response to cool temperature acclimation would be male-biased, whereas metabolic depression in response to warm temperature acclimation would be female-biased. Our results provide a first glimpse into how sex and reproductive condition contribute to intraspecific variation in thermal acclimation capacity in an amphibian. Methods Study System Mountain Lake Biological Station (MLBS) sits atop Salt Pond Mountain, a 1,160m summit located in southwest Virginia. This site has long served as the epicenter of field research on the eastern red-backed salamander and details of natural history, behavior, and breeding phenology are particularly well documented ( Fisher-Reid et al. 2024 ). Though many populations of red-backed salamanders show color polymorphism, the “leadback” phase is virtually absent at MLBS, allowing us to focus our analyses to the level of sex. Most females at MLBS follow a biennial cycle of reproduction, such that sexually mature females fall into two equally occurring reproductive classes: gravid and non-gravid ( Gillette 2003 , Moss et al., unpublished). This pattern of biennial female reproduction appears to hold generally across the range of P. cinereus, and reflects the steep energetic requirements of producing direct-developing eggs given limited growing seasons to support fat accumulation ( Leclair et al. 2008 ; Petranka 2010 ; Takahashi and Pauley 2010 ; Fisher-Reid et al. 2024 ). As far as we know, sexually mature male P. cinereus breed every year ( Fisher-Reid et al. 2024 ). The breeding season at MLBS lasts from September–April, with the majority of mating (i.e., as evidenced from tracking occurrences of spermatophores in female vents) appearing to fall within two seasonal peaks: October and March ( Gillette 2003 ). Temperatures during the prolonged breeding season are highly variable but generally cool ( Fig 1A ). For our thermal acclimation treatments, we chose two ranges of temperatures to simulate ‘Cool,’ or current (8 ° C nights, 14.5 ° C days) and ‘Warm,’ or projected future (13 ° C nights, 19.5 ° C days) breeding season conditions. Temperatures were programmed to ramp on a 12:12 D:L photoperiod, with light and temperature cycles reversed to ensure physiological measurements aligned with the active period for nocturnal salamanders. This design would allow us to test whether salamanders acclimated to cool versus warm conditions would exhibit relatively higher metabolic rates at low temperatures or lower metabolic rates at high temperatures (metabolic depression), respectively ( Fig. 1B ). determine whether thermal responses vary predictably on the basis of sex and/or reproductive condition following our predictions ( Fig. 1C ), we assayed subjects of both sexes and reproductive classes (gravid and non-gravid). Download figure Open in new tab Figure 1. Schematic overview of thermal acclimation experiment and predictions. (A) Trends in mean ambient temperature at Mountain Lake Biological Station from 2019 to 2022. Shaded bars represent the periods of peak breeding activity in the fall (early breeding season) and spring (late breeding season). Dashed lines represent the temperature ranges chosen for the Cool thermal regime (in blue) and Warm thermal regime (in red). (B) Two predicted patterns of physiological response (depicted as standard metabolic rate, SMR) to thermal acclimation. For a given temperature-SMR relationship (black line), SMR should increase at low temperatures in response to cool acclimation (i.e., metabolic compensation, blue line) and decrease at high temperatures in response to warm acclimation (i.e., metabolic depression, red lines). (C) Predicted patterns of physiological rate and thermal response by sex and reproductive class, with black and white graphs depicting the expected variation in SMR intercepts among groups and arrows depicting the magnitude (depicted as increasing arrow length) and direction of change in SMR in response to either warm (females) or cool (males) thermal acclimation. Experiment 1: Early Breeding Season The goal of our first experiment was to compare thermal responses of male and female P. cinereus in reproductive condition to determine whether thermal acclimation capacity represents a dimension of sex-specific plasticity in this species. We predicted that salamanders acclimated to warm thermal regimes would show depressed metabolic rates at higher test temperatures, as has been demonstrated elsewhere (e.g., ( Feder 1985 ; Bernardo and Spotila 2006 ; Markle and Kozak 2018 ; Muñoz et al. 2022 ); Fig. 1B ). However, if depression of metabolic rate incurs opportunity costs during the breeding season (i.e., due to insufficient delivery of oxygen for energy-demanding activities, such as mate searching), we predicted that males would opt to maintain temperature-specific metabolic rates or even increase metabolic rates when acclimated to warm breeding season conditions. Methods i. Collection and Husbandry A total of 40 salamanders (20 females and 20 males) were collected from the vicinity of MLBS between 30 August 2024 and 20 September 2024. All females were gravid, as confirmed by candling of egg follicles ( Gillette and Peterson 2001 ), and all salamanders had complete tails. Upon collection, we recorded the body mass (within the nearest 0.001 g), snout-vent length (SVL; distance in mm from the tip of the snout to the top of their vent), and total length (TL; distance in mm the tip of the snout to the tips of the tail) of each experimental subject. Prior to weighing, salamanders were gently patted dry with a kimwipe and induced to urinate via gently pressure on the pelvis to remove excess water weight and ensure accurate measurement. All experimental subjects were considered sexually mature adults on the basis of body size (SVL > 34mm). Following initial processing, salamanders were distributed evenly across the two thermal acclimation treatments with respect to size and sex. Thermal regimes and photoperiods were maintained by environmental chambers (Caron Scientific 7000 series) programmed to ramp between the low temperature setpoint between the hours of 0700–1900 (reversed ‘night’) and the high temperature setpoint between the hours of 1900–0700 (reversed ‘day’). Salamanders were housed individually in 120mm petri dishes set on incubator shelves and rotated daily. A moistened paper towel, hand-misted daily with spring water, provided a saturated local humidity environment. Salamanders were fed three times per week, alternating between flightless fruit flies ( Drosophila melanogaster ) dusted with vitamins and white worms ( Enchytraeus albidus ). Paper towels were replaced once weekly. Following recommended best practices for studying thermal acclimation in plethodontids ( Homyack et al. 2010 ; Markle and Kozak 2018 ), salamanders were allowed to acclimate to their respective thermal treatments for a minimum of three weeks (range=24–38 days) before starting physiological trials. In the 10 days leading up to individual trials, food was withheld to ensure subjects were in a post-absorptive state for measuring metabolic rates ( Homyack et al. 2010 ). ii. Stop-Flow Respirometry Protocol Standard metabolic rates (SMR) were assayed across a gradient of three ecologically relevant test temperatures (8, 12, and 16 ° C) by way of automated stop-flow respirometry at a flow rate of 80 ml/min (Appendix S1). All trials were conducted between 0800 and 1600 during the day. To initiate physiological trials, salamanders (N=7 per trial) were weighed within 0.001 g, as above, before being loaded into test chambers. One chamber in the 8-channel multiplexer was left empty to collect baseline data. Three measures of volumetric O2 consumption (VO2) were recorded for each individual per trial using Expedata software, and data transformations were carried out following established manual bolus integration calculations ( Lighton 2019 ). Repeat testing of individual subjects occurred over five days, consisting of three days of trials alternating with one day of rest. The order of test temperatures was randomized for each set of N=7 salamanders. At the conclusion of trials, normal feeding resumed. Two repeat trials could not be completed due to tail loss (N=1 warm-acclimated male at 12 ° C and N=1 cool-acclimated female at 8 ° C). At the end of the experiment, all test subjects were euthanized and dissected to confirm sex and reproductive condition. iii. Statistical Analyses All statistical analyses were carried out using R v 4.2 ( R Core Development Team 2020 ). To analyze variation in SMR across sexes, treatments and test temperatures, we ran a series of linear mixed effects models using the package lme4 ( Bates et al. 2015 ). For each model, log (SMR) was specified as the response variable and subject ID and trial date were specified as random effects. We first investigated whether the scaling relationship of SMR with body mass differed between the sexes or across treatments by modeling SMR for each group separately and deriving mass-scaling coefficients from the slope of the fixed effect of body mass at the start of the trial. We then ran a fully parameterized model including two-way and three-way interactions between body mass, treatment, and sex. Based on a lack of significant interaction terms, we retained body mass as a covariate but excluded statistical interactions in all subsequent models. Differences in SMR between sexes and treatments were visualized using mass-adjusted estimates, which were derived by dividing VO2 in ul/hr by starting body mass. To test for significant thermal acclimation responses to treatment and determine whether these differed between the sexes, we expanded our base model to include sex, treatment, and their interaction as fixed effects. To investigate more specifically how males and females responded to thermal acclimation across the gradient of test temperatures, separate models were constructed for each sex including treatment, test temperature, and their interaction as fixed effects. To identify significant pairwise differences in SMR across temperatures and treatments, post-hoc tests were carried out using the package emmeans ( Lenth et al. 2022 ). Results Subjects showed marginally significant sex differences in mass upon collection (F = 3.293, P = 0.077), with gravid females being larger on average (1.288 ± 0.235) than males (1.148 ± 0.258). However, the initial mass of subjects did not differ between the acclimation treatments (Males: t 0.582, P = 0.568; Females: t = 0.666; P = 0.513). Mass-scaling coefficients of SMR did not differ significantly between sexes or across treatments (X 2 = 0.008, P = 0.928; Fig. S1). Rather, we observed distinct patterns of sexual dimorphism of SMR between thermal treatments not attributed to differences in body size ( Fig. 2 ). Whereas sex had no overall effect on SMR across the three test temperatures (X 2 = 0.064, P = 0.801), both thermal treatment (X 2 = 7.373, P = 0.007) and the interaction between sex and thermal treatment (X 2 = 4.448, P = 0.035) were significant predictors of SMR. SMR generally increased with test temperature, and thermal acclimation treatment had no effect on this relationship in males (X 2 = 1.497, P = 0.473). Yet a distinct pattern emerged in gravid females, who showed a strong interactive effect of thermal treatment and test temperature on SMR (X 2 = 14.214, P = 0.0008). Specifically, gravid females reduced SMR in the warm treatment relative to the cool treatment (X 2 = 10.11, P = 0.014), a trend primarily attributed to metabolic depression at the highest test temperature (16 ° C: Tukey ratio = 3.81, P = 0.006). Download figure Open in new tab Figure 2. Standard metabolic rate (mass-adjusted VO2, in ul/hr) measured at three test temperatures for two classes of salamanders following 3–5 weeks thermal acclimation in one of two treatments (Cool or Warm) in the fall: gravid females (N=10 cool, N=10 warm, in gold) and males (N=10 cool, N=10 warm, in blue). Asterisks denote significant within-sex treatment differences at a given test temperature in a linear mixed effects model (* < 0.05, ** < 0.001, * * * < 0.0001). Experiment 2: Late Breeding Season Building on the results of Experiment 1, the goal of our second experiment was to determine whether sex-specific patterns of thermal response in P. cinereus are attributable to intrinsic sex differences or to reproductive condition. A secondary goal was to track potential variation in thermal response of reproductive males and females across the reproductive season. We predicted that if sex differences in thermal plasticity are due to differences in reproductive strategy, then gravid females collected late in the breeding season should show thermal responses similar to or more pronounced than gravid females collected early in the breeding season, whereas non-gravid females would show thermal responses more similar to males ( Fig. 1C ). Methods i. Collection and Husbandry A total of 84 salamanders (28 gravid females, 28 nongravid females, and 28 males) were collected from the vicinity of MLBS between 12 March 2025 and 28 March 2025. Females were candled to determine reproductive stage. Late stage eggs characteristic of gravid females in the spring are highly prominent due to their size (>1.5mm; gravid) and easily distinguished from absent or very early stage follicles (34mm. However, we show that our main effects hold whether these three individuals (range = 32.53–33.50mm) are retained or eliminated from the dataset (see Appendix S2). Subjects were distributed evenly across the two thermal acclimation treatments with respect to size, sex, and reproductive class and acclimated for 19–29 days before starting physiological trials. iv. Stop-Flow Respirometry Protocol Standard metabolic rates (SMR) were assayed as before except trials were only conducted at a single test temperature (16 ° C). This decision was made based on results of Experiment 1 suggesting that differences between groups are most pronounced at high test temperatures. Thus, each group of N=7 test subjects underwent a single day of trials and resumed normal feeding immediately afterwards. One subject (a non-gravid female in the warm thermal treatment) did not complete its trial due to mortality. All subjects were released at their point of capture at the conclusion of their trial. v. Statistical Analyses To analyze variation in SMR across reproductive classes and treatments, we ran a series of linear mixed effects models as in Experiment 1, specifying log (SMR) as the response variable and trial date as a random effect. Mass-scaling coefficients were derived as before, and we screened for interaction effects before deciding to retain body mass as a covariate in all subsequent models. Differences in SMR between reproductive classes and treatments were visualized using mass-adjusted estimates, which were derived by dividing VO2 in ul/hr by starting body mass. To test for significant thermal acclimation responses to treatment and determine whether these differed between reproductive classes, we expanded our base model to include reproductive class, treatment, and their interaction as fixed effects. To identify significant pairwise differences in SMR across reproductive classes and treatments, post-hoc tests were carried out using the package emmeans (Lenth et al. 2022). Finally, to test for significant variation in SMR and thermal responses across the reproductive season, we combined all SMR estimates obtained from tests at 16 ° C and fitted separate models for each sex, which included season, treatment, and their interaction as fixed effects. Results Subjects in the three reproductive classes differed significantly in mass (F = 26.468, P < 0.0001), with gravid females (1.195 ± 0.215 g) weighing significantly more than either non-gravid females (0.896 ± 0.210) or males (0.895 ± 0.107). Effects of body mass on SMR did not differ significantly among treatments or sexes (X 2 =2.544, P = 0.280; Fig. S2). We detected no significant overall effect of reproductive class on SMR (X 2 = 1.758, P = 0.415) but there was a significant overall treatment effect (X 2 = 23.927, P < 0.0001) as well as a significant interaction between class and treatment (X 2 = 11.628, P = 0.003; Fig. 3 ). Specifically, gravid females in the warm thermal treatment showed significant reductions in SMR compared to counterparts in the cool thermal treatment (gravid females: Tukey ratio = 4.887, P = 0.0001; non-gravid females: Tukey ratio = 3.819, P = 0.004; males: Tukey ratio = 3.270; P = 0.020) and non-gravid females in the warm thermal treatment (Tukey ratio = 3.070, P = 0.035). Download figure Open in new tab Figure 3. Standard metabolic rate (mass-adjusted VO2, in ul/hr) measured at 16 ° C in three classes of salamanders following three weeks of thermal acclimation at one of two treatments (Cool or Warm) in the spring: gravid females (N=14 cool, N=14 warm), non-gravid females (N=14 cool, N=13 warm), and males (N=14 cool, N=14 warm). Asterisks denote significant within-class treatment differences in a linear mixed effects model (* < 0.05, ** < 0.001, * * * < 0.0001). Seasonal Comparisons To determine whether physiological rates differ across the breeding season, we compared SMR estimates at 16 ° C from gravid females and males assayed early (Fall) and late (Spring) in the breeding season ( Fig. 4 ). Significant reductions in SMR were observed between the Fall and Spring in both gravid females (X 2 = 5.486, P = 0.019) and males (X 2 = 5.595, P = 0.018). We observed no significant interactions between season and treatment, suggesting that the pattern of response to thermal acclimation was consistent across seasons despite differences in base physiology. Download figure Open in new tab Figure 4. Standard metabolic rate (mass-adjusted VO2, in ul/hr) measured at 16 ° C at two points in the breeding season: Fall (September–November, early breeding season) and Spring (March–April, late breeding season). Three classes of salamanders are represented: Gravid females (in gold), non-gravid females (in coral), and males (in blue). Asterisks denote significant within-class season differences in a linear mixed effects model (* < 0.05, ** < 0.001, * * * < 0.0001). Discussion The question of whether males and females differ in their responses to temperature change is essential to understanding adaptive capacities in a warming world, yet our basic understanding of when and where sex differences in thermal plasticity are likely to arise remains limited. Here, we tested for sex differences in thermal acclimation capacity in a plethodontid salamander, P. cinereus , across its protracted reproductive cycle. Our prediction was that reproductively active males and females would adopt distinct thermal acclimation strategies expected to optimize fitness relative to their sex-specific reproductive roles. We found patterns consistent with significant metabolic depression in gravid female, but not male salamanders, in response to warm temperature acclimation across the breeding season. Sex differences were not detected when sampling was restricted to non-gravid females at the same time of year, suggesting that patterns are driven by reproductive condition. Below, we discuss these results in the context of salamander thermal ecology and thermal ecology more broadly. No evidence for male-biased metabolic cold compensation during the breeding season In cold-adapted ectotherms, enhanced physiological and/or behavioral plasticity of breeding males in response to low temperature acclimation has been suggested to convey sex-specific reproductive benefits, i.e., by enabling mating and courtship behaviors at temperatures that would otherwise be too cold to support activity ( Rogers et al. 2007 ; Kiss et al. 2009 ; Moss et al. 2023 ). Given that the breeding season of P. cinereus at MLBS coincides with the fall and spring, when surface temperatures during nocturnal activity periods frequently drop below 5°C ( Fig. 1A ), we predicted that adult male salamanders acclimated to a cool thermal regime would show metabolic compensation at low temperatures compared to males acclimated to warmer temperatures. On the contrary, we found no evidence for metabolic compensation in males during the peak breeding season, with SMR at the coldest test temperature (8°C) being indistinguishable between thermal acclimation treatments. It is possible that sex differences in respiratory capacity at low temperatures manifest in more subtle ways than we examined here; for example, Rogers et al. (2007) observed no sex variation in whole-organism metabolism of frogs acclimated to autumn-like conditions but documented significant male bias in mitochondrial and enzyme activity in muscles used for calling and amplexus. Additional investigations may be warranted to investigate the sensitivity of specific, behaviorally relevant tissues to cold acclimation in salamanders. Female-biased metabolic depression at warm temperatures is associated with reproduction Metabolic depression in response to sustained high temperature exposure is widespread among ectotherms, including lungless salamanders ( Feder 1985 ; Bernardo and Spotila 2006 ; Markle and Kozak 2018 ; Muñoz et al. 2022 ), for which such plasticity is expected to enhance resilience by reducing maintenance energy costs and mitigating evaporative water loss ( Riddell, Odom, et al. 2018 ; Riddell et al. 2019 ). In the current study, we observed metabolic depression in female, but not male P. cinereus, in response to warm temperature acclimation. Significant differences in SMR were observed between acclimation treatments at 16°C, the highest test temperature, but not at 8°C or 12°C, consistent with metabolic depression ( Fig. 2 ). This result held for animals tested in both the fall and spring and appears to be driven by female reproductive condition, as no sex bias was detected when considering only non-gravid females. Hence, it is the process of vitellogenesis specifically, not other life history stages supporting reproduction, that shape the observed female-biased thermal acclimation strategy. This result is interesting in the light of what we know about female plethodontid reproductive cycles. At higher latitudes and elevations, where short growing seasons limit food intake and much of vitellogenesis occurs overwinter, females must attain a critical minimum body size before they may reproduce ( Fitzpatrick 1973 ; Feder 1983 ; Crespi 2001 ; Leclair et al. 2008 ), and frequently skip years of reproduction to recoup depleted fat reserves ( Gillette 2003 ; Leclair et al. 2008 ; Takahashi and Pauley 2010 ). The paradoxical outcome of this gradual accumulation strategy is that by the start of the breeding season, additional food intake is no longer necessary for females to proceed with vitellogenesis and yolk uptake; stored fat is simply shunted from growth to reproduction ( Crespi 2001 ). We propose that the thermal environment could interact with female reproductive strategies to reinforce an extreme energy conservation approach during vitellogenesis, stimulating metabolic depression to mitigate higher rates of energy turnover and preserve energy allocation to reproduction independent of food intake. Our data stand in contrast to the limited number of studies to date on metabolism in gravid female salamanders, which report significantly elevated mass-adjusted SMR when compared to males and non-gravid females ( Fitzpatrick 1973 ; Finkler and Cullum 2002 ). Clearly, more studies are needed to determine the precise contribution of thermal environment, as well as other ecological factors, to female energetics across the protracted reproductive cycle. Both sexes exhibit seasonal metabolic rhythms While we did not observe any changes in the general pattern of thermal acclimation of either males or gravid females across the reproductive cycle, comparison of measurements taken between fall and spring did reveal seasonal metabolic rhythms. Specifically, salamanders of both sexes showed significantly lower temperature-specific SMR when measured late in the reproductive cycle (spring) compared to early in the reproductive cycle (fall). To our knowledge, this pattern has not been previously documented in P. cinereus and could offer intriguing insights into seasonal patterns of energy allocation and activity. While we originally predicted that males at our study location would show higher metabolic rates in the fall compared to the spring to align energetic expenditure with seasonal peaks in breeding condition (i.e., circulating testosterone peaks in October at our study location ( Church and Okazaki 2002 )), the same pattern was not anticipated for females, who have been reported to emerge from brumation early in spring to maximize foraging time ( Crespi 2001 ). A recent study investigating seasonal acclimatization of metabolic rates in spring-breeding spotted salamanders ( Ambystoma maculatum ) revealed a similar pattern of reduced SMR in the spring relative to the fall ( Giacometti and Tattersall 2025 ), suggesting that seasonal rhythms may be attributed more to replenishing energy stores ahead of winter brumation than to breeding activities specifically. More research is needed to understand the adaptive significance of seasonal variation in resting metabolism. Conclusions and Future Directions Our study provides new insights into the drivers of intraspecific variation in thermal traits in amphibians, a poorly understood topic ( Cocciardi and Ohmer 2024 ). In the last decade, lungless salamanders have become models for the study of thermal ecology owing to their heightened physiological sensitivity ( Sears et al. 2019 ; Riddell et al. 2024 ); however, a majority of this work has focused on characterizing macrogeographic variation within and between species ( Riddell and Sears 2015 ; Markle and Kozak 2018 ; Muñoz et al. 2022 ). The contributions of sex or reproductive condition to seasonal patterns of thermal acclimation is virtually unstudied in this group. Given that the plethodontid reproductive cycle spans the majority of the year and is generally synonymous with the active season, forecasting the potential impacts of increasing thermal variability on wild salamander populations requires accounting for reproductive condition, especially given female demographic dominance in most populations. Whereas a previous study considering only adult males and juveniles reported an absence of physiological acclimation to warming conditions in two cold-adapted populations of P. cinereus ( Muñoz et al. 2022 ), here we report pronounced physiological acclimation responses in gravid females from a montane population, suggesting that different demographic classes are likely to vary in their adaptive capacities. More studies are needed to evaluate the generalizability of this result across the extensive geographic range of P. cinereus , and to examine how patterns of thermal acclimation map onto other performance traits of interest, such as surface activity, reproductive success, and survival. More broadly, our findings speak to the importance of accounting for sex in studies of thermal ecology. Not unlike the majority of scientific disciplines ( Tannenbaum et al. 2019 ), the field of thermal ecology has a long-held tradition of ignoring sex as an explanatory variable ( Huey and Pianka 2007 ; Lailvaux 2007 ; Bodensteiner et al. 2021 ; Edmands 2021 ; Gunderson 2021 ; Pottier et al. 2021 ; Hangartner et al. 2022 ). Despite a recent surge of interest in sex-specific thermal traits yielding several meta-analyses ( Huey and Pianka 2007 ; Edmands 2021 ; Pottier et al. 2021 ; Hangartner et al. 2022 ), systematic patterns remain elusive, offering few insights into when and how sex may be important for designing studies. By taking a hypothesis-driven approach in an amphibian with sexually dimorphic reproductive life histories, we show that sex does account for differences in thermal acclimation strategies, but that reproductive stage matters. Future empirical studies must take both variables into account when testing for sex variation in thermal ecology, especially if population growth is limited by fecundity selection. differences in thermal acclimation capacity. Data Accessibility Statement For the purposes of submission, all data transformation macros, extracted raw data, and analysis scripts have been uploaded along with the manuscript files for review. Upon acceptance, all the aforementioned data will be made publicly available in the Data Dryad repository. Ethical Approval All procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee (Protocol #23-250). Acknowledgements We would like to thank Dr. Vincent Farallo, Dr. Eric Riddell, and the technical support staff at Sable Systems International, in particular Jaco Klok and Barbara Joos, for their generous guidance and advice on the methodologies used for this study. This study was supported by Virginia Tech start-up funds to J.B.M. References ↵ Angilletta MJ . 2009 . Thermal Adaptation . Oxford University Press . [accessed 2023 May 30 ]. doi: 10.1093/acprof:oso/9780198570875.001.1 OpenUrl CrossRef ↵ Rohr JR et al. 2018 . The complex drivers of thermal acclimation and breadth in ectotherms . Ecology Letters . 21 ( 9 ): 1425 – 1439 . doi: 10.1111/ele.13107 OpenUrl CrossRef PubMed ↵ Havird JC et al. 2020 . Distinguishing between active plasticity due to thermal acclimation and passive plasticity due to Q 10 effects: Why methodology matters Fox C, editor . Funct Ecol . 34 ( 5 ): 1015 – 1028 . doi: 10.1111/1365-2435.13534 OpenUrl CrossRef ↵ Riddell EA , Odom JP , Damm JD , Sears MW . 2018 . Plasticity reveals hidden resistance to extinction under climate change in the global hotspot of salamander diversity . Sci Adv . 4 ( 7 ): eaar5471 . doi: 10.1126/sciadv.aar5471 OpenUrl FREE Full Text ↵ Feder ME , Lynch JF , Shaffer HB , Wake DB . 1982 . Field body temperatures of tropical and temperature zone salamanders . Smithsonian Herpetological information Service. [published online ahead of print] [accessed 2024 Dec 9 ]. http://repository.si.edu/xmlui/handle/10088/8242 ↵ Christian KA , Bedford GS , Schultz TJ . 1999 . Energetic consequences of metabolic depression in tropical and temperate-zone lizards . Aust J Zool . 47 ( 2 ): 133 – 141 . doi: 10.1071/zo98061 OpenUrl CrossRef ↵ Guppy M , Withers P. 1999 . Metabolic depression in animals: physiological perspectives and biochemical generalizations . Biological Reviews . 74 ( 1 ): 1 – 40 . doi: 10.1017/S0006323198005258 OpenUrl CrossRef PubMed Web of Science ↵ Liao M et al. 2021 . Physiological determinants of biogeography: The importance of metabolic depression to heat tolerance . Global Change Biology . 27 ( 11 ): 2561 – 2579 . doi: 10.1111/gcb.15578 OpenUrl CrossRef PubMed ↵ Muñoz D , Miller D , Schilder R , Campbell Grant EH . 2022 . Geographic variation and thermal plasticity shape salamander metabolic rates under current and future climates . Ecology and Evolution . 12 ( 1 ): e8433 . doi: 10.1002/ece3.8433 OpenUrl CrossRef ↵ Rogers KD , Thompson MB , Seebacher F. 2007 . Beneficial acclimation: sex specific thermal acclimation of metabolic capacity in the striped marsh frog (Limnodynastes peronii ). Journal of Experimental Biology . 210 ( 16 ): 2932 – 2938 . doi: 10.1242/jeb.008391 OpenUrl Abstract / FREE Full Text ↵ Gaston KJ et al. 2009 . Macrophysiology: A Conceptual Reunification . The American Naturalist . 174 ( 5 ): 595 – 612 . doi: 10.1086/605982 OpenUrl CrossRef PubMed Web of Science ↵ Bozinovic F , Calosi P , Spicer JI . 2011 . Physiological correlates of geographic range in animals . Annu Rev Ecol Evol Syst . 42 ( 1 ): 155 – 179 . doi: 10.1146/annurev-ecolsys-102710-145055 OpenUrl CrossRef ↵ Catenazzi A. 2016 . Ecological implications of metabolic compensation at low temperatures in salamanders . PeerJ . 4 : e2072 . doi: 10.7717/peerj.2072 OpenUrl CrossRef PubMed ↵ Watanabe Y , Payne N. 2023 . Thermal sensitivity of metabolic rate mirrors biogeographic differences between teleosts and elasmobranchs . Nature Communications . 14 . doi: 10.1038/s41467-023-37637-z OpenUrl CrossRef ↵ Briscoe NJ et al. 2023 . Mechanistic forecasts of species responses to climate change: The promise of biophysical ecology . Global Change Biology . 29 ( 6 ): 1451 – 1470 . doi: 10.1111/gcb.16557 OpenUrl CrossRef PubMed ↵ Bennett S , Duarte CM , Marbà N , Wernberg T. 2019 . Integrating within-species variation in thermal physiology into climate change ecology . Philosophical Transactions of the Royal Society B: Biological Sciences . 374 ( 1778 ): 20180550 . doi: 10.1098/rstb.2018.0550 OpenUrl CrossRef PubMed ↵ Seebacher F , Ducret V , Little AG , Adriaenssens B. 2015 . Generalist–specialist trade-off during thermal acclimation . Royal Society Open Science . 2 ( 1 ): 140251 . doi: 10.1098/rsos.140251 OpenUrl CrossRef PubMed ↵ Riddell EA , McPhail J , Damm JD , Sears MW . 2018 . Trade-offs between water loss and gas exchange influence habitat suitability of a woodland salamander Sinclair B, editor . Funct Ecol . 32 ( 4 ): 916 – 925 . doi: 10.1111/1365-2435.13030 OpenUrl CrossRef ↵ Hui TY et al. 2020 . Timing metabolic depression: Predicting thermal stress in extreme intertidal environments . The American Naturalist . 196 ( 4 ): 501 – 511 . doi: 10.1086/710339 OpenUrl CrossRef PubMed ↵ Bodensteiner BL et al. 2021 . Thermal adaptation revisited: How conserved are thermal traits of reptiles and amphibians? J Exp Zool . 335 ( 1 ): 173 – 194 . doi: 10.1002/jez.2414 OpenUrl CrossRef ↵ Cocciardi JM , Ohmer MEB . 2024 . Drivers of intraspecific variation in thermal traits and their importance for resilience to global change in amphibians . Integrative and Comparative Biology . 64 ( 3 ): 882 – 899 . doi: 10.1093/icb/icae132 OpenUrl CrossRef PubMed ↵ Pottier P et al. 2021 . Sexual (in)equality? A meta-analysis of sex differences in thermal acclimation capacity across ectotherms . Functional Ecology . 35 ( 12 ): 2663 – 2678 . doi: 10.1111/1365-2435.13899 OpenUrl CrossRef ↵ Hangartner S , Sgrò CM , Connallon T , Booksmythe I. 2022 . Sexual dimorphism in phenotypic plasticity and persistence under environmental change: An extension of theory and meta-analysis of current data Pantel J, editor . Ecology Letters . 25 ( 6 ): 1550 – 1565 . doi: 10.1111/ele.14005 OpenUrl CrossRef PubMed ↵ Beck CA , Bowen WD , Iverson SJ . 2003 . Sex differences in the seasonal patterns of energy storage and expenditure in a phocid seal . Journal of Animal Ecology . 72 ( 2 ): 280 – 291 . doi: 10.1046/j.1365-2656.2003.00704.x OpenUrl CrossRef ↵ Czenze ZJ , Jonasson KA , Willis CKR . 2017 . Thrifty females, frisky males: Winter energetics of hibernating bats from a cold climate . Physiological and Biochemical Zoology . 90 ( 4 ): 502 – 511 . doi: 10.1086/692623 OpenUrl CrossRef ↵ Tarka M et al. 2018 . Sex differences in life history, behavior, and physiology along a slow-fast continuum: a meta-analysis . Behav Ecol Sociobiol . 72 ( 8 ): 132 . doi: 10.1007/s00265-018-2534-2 OpenUrl CrossRef ↵ Edmands S. 2021 . Sex ratios in a warming world: Thermal effects on sex-biased survival, sex determination, and sex reversal . Journal of Heredity . 112 ( 2 ): 155 – 164 . doi: 10.1093/jhered/esab006 OpenUrl CrossRef PubMed ↵ Vermandele F et al. 2024 . When the Going Gets Tough, the Females Get Going: Sex-Specific Physiological Responses to Simultaneous Exposure to Hypoxia and Marine Heatwave Events in a Ubiquitous Copepod . Global Change Biology . 30 ( 10 ): e17553 . doi: 10.1111/gcb.17553 OpenUrl CrossRef PubMed ↵ Fox RJ , Fromhage L , Jennions MD . 2019 . Sexual selection, phenotypic plasticity and female reproductive output . Phil Trans R Soc B . 374 ( 1768 ): 20180184 . doi: 10.1098/rstb.2018.0184 OpenUrl CrossRef PubMed ↵ Gissi E et al. 2023 . Exploring climate-induced sex-based differences in aquatic and terrestrial ecosystems to mitigate biodiversity loss . Nat Commun . 14 ( 1 ): 4787 . doi: 10.1038/s41467-023-40316-8 OpenUrl CrossRef PubMed ↵ Huey RB , Pianka ER . 2007 . Lizard thermal biology: Do genders differ? The American Naturalist . 170 ( 3 ): 473 – 478 . doi: 10.1086/520122 OpenUrl CrossRef PubMed ↵ Lailvaux SP . 2007 . Interactive effects of sex and temperature on locomotion in reptiles . Integrative and Comparative Biology . 47 ( 2 ): 189 – 199 . doi: 10.1093/icb/icm011 OpenUrl CrossRef PubMed ↵ Gunderson A. 2021 . FE Spotlight: Sex, heat and phenotypic plasticity . Functional Ecology . 35 : 2618 – 2620 . doi: 10.1111/1365-2435.13959 OpenUrl CrossRef ↵ Charland MB , Gregory PT . 1990 . The influence of female reproductive status on thermoregulation in a viviparous snake, Crotalus viridis . Copeia . 1990 ( 4 ): 1089 – 1098 . doi: 10.2307/1446493 OpenUrl CrossRef ↵ Grinevitch L , Holroyd SL , Barclay RMR . 1995 . Sex differences in the use of daily torpor and foraging time by big brown bats (Eptesicus fuscus) during the reproductive season . Journal of Zoology . 235 ( 2 ): 301 – 309 . doi: 10.1111/j.1469-7998.1995.tb05146.x OpenUrl CrossRef Web of Science ↵ Finkler MS , Sugalski MT , Claussen DL . 2003 . Sex-related differences in metabolic rate and locomotor performance in breeding spotted salamanders (Ambystoma maculatum) . cope . 2003 ( 4 ): 887 – 893 . doi: 10.1643/h203-110.1 OpenUrl CrossRef ↵ Galliard J-FL , Le Bris M , Clobert J. 2003 . Timing of locomotor impairment and shift in thermal preferences during gravidity in a viviparous lizard . Functional Ecology . 17 ( 6 ): 877 – 885 OpenUrl CrossRef Web of Science ↵ Hammill E , Wilson RS , Johnston IA . 2004 . Sustained swimming performance and muscle structure are altered by thermal acclimation in male mosquitofish . Journal of Thermal Biology . 29 ( 4 ): 251 – 257 . doi: 10.1016/j.jtherbio.2004.04.002 OpenUrl CrossRef ↵ Isaac LA , Gregory PT . 2004 . Thermoregulatory behaviour of gravid and non-gravid female grass snakes (Natrix natrix) in a thermally limiting high-latitude environment . Journal of Zoology . 264 ( 4 ): 403 – 409 . doi: 10.1017/S095283690400593X OpenUrl CrossRef ↵ Finkler MS . 2006 . Effects of temperature, sex, and gravidity on the metabolism of smallmouthed salamanders, Ambystoma texanum, during the reproductive season . Journal of Herpetology . 40 ( 1 ): 103 – 106 OpenUrl ↵ Wilson RS , Hammill E , Johnston IA . 2007 . Competition moderates the benefits of thermal acclimation to reproductive performance in male eastern mosquitofish . Proceedings of the Royal Society B: Biological Sciences . 274 ( 1614 ): 1199 – 1204 . doi: 10.1098/rspb.2006.0401 OpenUrl CrossRef PubMed Web of Science ↵ Darnell MZ , Fowler KK , Munguia P. 2013 . Sex-specific thermal constraints on fiddler crab behavior . Behavioral Ecology . 24 ( 4 ): 997 – 1003 . doi: 10.1093/beheco/art006 OpenUrl CrossRef ↵ Allen BJ , Levinton JS . 2014 . Sexual selection and the physiological consequences of habitat choice by a fiddler crab . Oecologia . 176 ( 1 ): 25 – 34 . doi: 10.1007/s00442-014-3002-y OpenUrl CrossRef PubMed ↵ Beal MS , Lattanzio MS , Miles DB . 2014 . Differences in the thermal physiology of adult Yarrow’s spiny lizards (Sceloporus jarrovii) in relation to sex and body size . Ecol Evol . 4 ( 22 ): 4220 – 4229 . doi: 10.1002/ece3.1297 OpenUrl CrossRef PubMed ↵ Vaughn D , Turnross OR , Carrington E. 2014 . Sex-specific temperature dependence of foraging and growth of intertidal snails . Mar Biol . 161 ( 1 ): 75 – 87 . doi: 10.1007/s00227-013-2316-3 OpenUrl CrossRef ↵ Webber MM , Gibbs AG , Rodríguez-Robles JA. 2015 . Hot and not-so-hot females: Reproductive state and thermal preferences of female Arizona Bark Scorpions (entruroides sculpturatus) . Journal of Evolutionary Biology . 28 ( 2 ): 368 – 375 . doi: 10.1111/jeb.12569 OpenUrl CrossRef PubMed ↵ Woolrich-Piña GA , Smith GR , Lemos-Espinal JA , Ramírez-Silva JP. 2015 . Do gravid female Anolis nebulosus thermoregulate differently than males and non-gravid females? Journal of Thermal Biology . 52 : 84 – 89 . doi: 10.1016/j.jtherbio.2015.06.006 OpenUrl CrossRef PubMed ↵ Parker CA , Geiser F , Stawski C. 2019 . Thermal physiology and activity in relation to reproductive status and sex in a free-ranging semelparous marsupial . Conservation Physiology . 7 ( 1 ): coz073 . doi: 10.1093/conphys/coz073 OpenUrl CrossRef PubMed ↵ Levine RL , Kroger B , Monteith KL . 2024 . Thermal conditions alter the mating behaviour of males in a polygynous system . Functional Ecology . 38 ( 12 ): 2553 – 2563 . doi: 10.1111/1365-2435.14664 OpenUrl CrossRef ↵ Gifford ME , Kozak KH . 2012 . Islands in the sky or squeezed at the top? Ecological causes of elevational range limits in montane salamanders . Ecography . 35 ( 3 ): 193 – 203 . doi: 10.1111/j.1600-0587.2011.06866.x OpenUrl CrossRef Web of Science ↵ Markle TM , Kozak KH . 2018 . Low acclimation capacity of narrow-ranging thermal specialists exposes susceptibility to global climate change . Ecol Evol . 8 ( 9 ): 4644 – 4656 . doi: 10.1002/ece3.4006 OpenUrl CrossRef PubMed ↵ Riddell EA et al. 2024 . Amphibians exhibit extremely high hydric costs of respiration . Integrative and Comparative Biology . 64 ( 2 ): 366 – 376 . doi: 10.1093/icb/icae053 OpenUrl CrossRef ↵ Novarro AJ et al. 2018 . Physiological responses to elevated temperature across the geographic range of a terrestrial salamander . Journal of Experimental Biology . 221 ( 18 ): jeb.178236 . doi: 10.1242/jeb.178236 OpenUrl Abstract / FREE Full Text ↵ Fitzpatrick LC . 1973 . Energy allocation in the Allegheny Mountain salamander, Desmognathus ochrophaeus . Ecological Monographs . 43 ( 1 ): 43 – 58 . doi: 10.2307/1942158 OpenUrl CrossRef ↵ Crespi EJ . 2001 . Adaptation of reproductive strategies: Endocrine and ecological factors associated with the evolution of terrestrial breeding in amphibians [Ph.D.]. University of Virginia . ↵ Gatten Jr . RE , editor Finkler MS , Cullum KA . 2002 . Sex-related differences in metabolic rate and energy reserves in spring-breeding small-mouthed salamanders (Ambystoma texanum) Gatten Jr . RE , editor. Copeia . 2002 ( 3 ): 824 – 829 . doi: 10.1643/0045-8511(2002)002[0824:SRDIMR]2.0.CO;2 OpenUrl CrossRef ↵ Takahashi M. 2002 . Comparisons in morphology, reproductive status, and feeding ecology of Plethodon cinereus at high and low elevations in West Virginia. [M.S. Thesis]. Marshall University . ↵ Gillette JR . 2003 . Population ecology, social behavior, and intersexual differences in a natural population of red-backed salamanders: A long-term field study [Ph.D.]. University of Louisiana at Lafayette . ↵ Gergits WF , Jaeger RG . 1990 . Field observations of the behavior of the red-backed salamander (Plethodon cinereus): Courtship and agonistic interactions . Journal of Herpetology . 24 ( 1 ): 93 . doi: 10.2307/1564298 OpenUrl CrossRef ↵ Dyal LA . 2006 . Novel courtship behaviors in three Small eastern Plethodon species . Journal of Herpetology . 40 ( 1 ): 55 – 65 . doi: 10.1670/20-05A.1 OpenUrl CrossRef ↵ Leclair MH , Levasseur M , Leclair R. 2008 . Activity and reproductive cycles in northern populations of the red-backed salamander, Plethodon Cinereus . Journal of Herpetology . 42 ( 1 ): 31 – 38 . doi: 10.1670/06-234R2.1 OpenUrl CrossRef ↵ Jaeger RG , Gollmann B , Anthony CD , Gabor C. 2016 . Behaviorial ecology of the Eastern red-backed salamander: 50 years of research . Oxford university press . ↵ Fisher-Reid MC et al. 2024 . Eastern red-backed Salamanders: A comprehensive review of an undervalued model in evolution, ecology, and behavior . Herpetological Monographs . 38 ( 1 ): 74 – 121 . doi: 10.1655/HERPMONOGRAPHS-D-21-00003.1 OpenUrl CrossRef ↵ Sayler A. 1966 . The reproductive ecology of the red-backed salamander, Plethodon cinereus, in Maryland . Copeia . 1966 ( 2 ): 183 . doi: 10.2307/1441125 OpenUrl CrossRef ↵ Werner JK . 1969 . Temperature-photoperiod effects on spermatogenesis in the salamander Plethodon cinereus . Copeia . 1969 ( 3 ): 592 . doi: 10.2307/1441939 OpenUrl CrossRef ↵ Nagel JW . 1977 . Life history of the red-backed salamander, Plethodon cinereus, in northeastern Tennessee . Herpetologica . 33 ( 1 ): 13 – 18 OpenUrl ↵ Takahashi MK , Pauley TK . 2010 . Resource allocation and life history traits of Plethodon cinereus at different elevations . American Midland Naturalist . 163 ( 1 ): 87 – 94 . doi: 10.1674/0003-0031-163.1.87 OpenUrl CrossRef ↵ Petranka JW . 2010 . Salamanders of the United States and Canada . Smithsonian Books . [accessed 2023 Sep 7 ]. https://www.journals.uchicago.edu/doi/10.1086/393117 ↵ Feder ME . 1985 . Acclimation to constant and variable temperatures in plethodontid salamanders—I . Rates of oxygen consumption. Comparative Biochemistry and Physiology Part A: Physiology . 81 ( 3 ): 673 – 682 . doi: 10.1016/0300-9629(85)91046-1 OpenUrl CrossRef ↵ Bernardo J , Spotila JR . 2006 . Physiological constraints on organismal response to global warming: mechanistic insights from clinally varying populations and implications for assessing endangerment . Biol Lett . 2 ( 1 ): 135 – 139 . doi: 10.1098/rsbl.2005.0417 OpenUrl CrossRef PubMed Web of Science ↵ Gillette JR , Peterson MG . 2001 . The benefits of transparency: Candling as a simple method for determining sex in red-backed salamanders (Plethodon cinereus) . ↵ Homyack JA , Haas CA , Hopkins WA . 2010 . Influence of temperature and body mass on standard metabolic rate of eastern red-backed salamanders (Plethodon cinereus) . Journal of Thermal Biology . 35 ( 3 ): 143 – 146 . doi: 10.1016/j.jtherbio.2010.01.006 OpenUrl CrossRef ↵ Lighton JRB . 2019 . Measuring Metabolic Rates: A Manual for Scientists . 2nd edition. Oxford University Press . ↵ R Core Development Team . 2020 . R: A language and environment for statistical computing. R Foundation for Statistical Computing. [published online ahead of print] ↵ Bates DM , Maechler M , Bolker B , Walker S. 2015 . Fitting linear mixed-effects models using lme4 . Journal of statistical software . 67 ( 1 ): 1 – 48 . doi: 10.1088/1742-6596/43/1/292 OpenUrl CrossRef ↵ Lenth RV et al. 2022 . Package ‘emmeans’: Estimated marginal means, aka least-squares means . R Package version 340. [published online ahead of print] [accessed 2022 Jun 17 ]. doi: 10.1080/00031305.1980.10483031 OpenUrl CrossRef ↵ Howard JS , Maerz JC . 2022 . A model for estimating final clutch size from follicle counts in plethodontid salamanders . Journal of Herpetology . 56 ( 2 ): 191 – 195 . doi: 10.1670/21-015 OpenUrl CrossRef ↵ Kiss ACI , de Carvalho JE , Navas CA , Gomes FR . 2009 . Seasonal metabolic changes in a year-round reproductively active subtropical tree-frog (Hypsiboas prasinus) . Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology . 152 ( 2 ): 182 – 188 . doi: 10.1016/j.cbpa.2008.09.011 OpenUrl CrossRef PubMed ↵ Moss J , Borthwick Z , Wapstra E , While GM . 2023 . Thermal plasticity in behavioral traits mediates mating and reproductive dynamics in an ectotherm . American Naturalist . 201 ( 6 ): 851 – 863 . doi: 10.1086/724381 OpenUrl CrossRef PubMed ↵ Riddell EA et al. 2019 . Thermal cues drive plasticity of desiccation resistance in montane salamanders with implications for climate change . Nat Commun . 10 ( 1 ): 4091 . doi: 10.1038/s41467-019-11990-4 OpenUrl CrossRef PubMed ↵ Feder ME . 1983 . Integrating the ecology and physiology of plethodontid salamanders . Herpetologica . 39 ( 3 ): 291 – 310 OpenUrl Welcker J et al. 2015 . Resting and daily energy expenditures during reproduction are adjusted in opposite directions in free-living birds . Functional Ecology . 29 ( 2 ): 250 – 258 . doi: 10.1111/1365-2435.12321 OpenUrl CrossRef Web of Science ↵ Church DR , Okazaki RK . 2002 . Seasonal variation of plasma testosterone titres in male redback salamanders, Plethodon cinereus . Amphibia Reptilia . 23 : 93 – 97 . doi: 10.1163/156853802320877654 OpenUrl CrossRef ↵ Giacometti D , Tattersall GJ . 2025 . Seasonal plasticity in the thermal sensitivity of metabolism but not water loss in a fossorial ectotherm . Oecologia . 207 ( 5 ): 67 . doi: 10.1007/s00442-025-05711-6 OpenUrl CrossRef PubMed ↵ Sears MW , Riddell EA , Rusch TW , Angilletta MJ . 2019 . The world still is not flat: Lessons learned from organismal interactions with environmental heterogeneity in terrestrial environments . Integrative and Comparative Biology . 59 ( 4 ): 1049 – 1058 . doi: 10.1093/icb/icz130 OpenUrl CrossRef PubMed ↵ Riddell EA , Sears MW . 2015 . Geographic variation of resistance to water loss within two species of lungless salamanders: implications for activity . Ecosphere . 6 ( 5 ): art86 . doi: 10.1890/ES14-00360.1 OpenUrl CrossRef ↵ Tannenbaum C et al. 2019 . Sex and gender analysis improves science and engineering . Nature . 575 ( 7781 ): 137 – 146 . doi: 10.1038/s41586-019-1657-6 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 30, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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