Individual Differences in the Phenotypic Flexibility of Basal Metabolic ...

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Individual Differences in the Phenotypic Flexibility of Basal Metabolic Rate in Siberian Hamsters Are Consistent on Short- and Long-Term Timescales* Jan S. Boratyński1,† Małgorzata Jefimow1 Michał S. Wojciechowski2 1 Department of Animal Physiology, Nicolaus Copernicus University, Toruń, Poland; 2Department of Vertebrate Zoology, Nicolaus Copernicus University, Toruń, Poland Accepted 10/12/2016; Electronically Published 11/23/2016

P p 0.005). Finally, the flexibility of BMR in response to changes in Ta was also repeatable on a long-term timescale, that is, among seasons (t p 0.31, P p 0.008). Our results indicate the evolutionary importance of the phenotypic flexibility of energy metabolism and suggest that it may be subject to selection. Keywords: phenotypic flexibility, repeatability, acclimation, acclimatization, thermoregulation, energy metabolism, photoresponsiveness, season.

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ABSTRACT Basal metabolic rate (BMR) correlates with the cost of life in endothermic animals. It usually differs consistently among individuals in a population, but it may be adjusted in response to predictable or unpredictable changes in the environment. The phenotypic flexibility of BMR is considered an adaptation to living in a stochastic environment; however, whether it is also repeatable it is still unexplored. Assuming that variations in phenotypic flexibility are evolutionarily important, we hypothesized that they are consistently different among individuals. We predicted that not only BMR but also its flexibility in response to changes in ambient temperature (Ta) are repeatable on short- and long-term timescales. To examine this, we acclimated Siberian hamsters (Phodopus sungorus) for 100 d to winterlike and then to summerlike conditions, and after each acclimation we exposed them interchangeably to 107 and 287C for 14 d. The difference in BMR measured after each exposure defined an individual’s phenotypic flexibility (DBMR). BMR was repeatable within and among seasons. It was also flexible in both seasons, but in winter this flexibility was lower in individuals responding to seasonal changes than in nonresponding ones. When we accounted for individual responsiveness, the repeatability of DBMR was significant in winter (t p 0.48, P p 0.01) and in summer (t p 0.55,

*This paper is based on a talk given at the ICCPB 2015 symposium, “Phenotypic Flexibility of Energetics in a Seasonal World,” which was sponsored by the Division of Comparative Physiology and Biochemistry, SICB. Marek Konarzewski, David Swanson, and Michał S. Wojciechowski were guest editors for this collection. †Corresponding author; e-mail: [email protected]. Physiological and Biochemical Zoology 90(2):139–152. 2017. q 2016 by The University of Chicago. All rights reserved. 1522-2152/2017/9002-6021$15.00. DOI: 10.1086/689870

Introduction Endothermic animals evolved mechanisms of heat production and conservation that enable them to maintain stable and high body temperatures (Tb) over a wide range of ambient temperatures (Ta; Hayes and Garland 1995). Since basal metabolic rate (BMR) is related to the size and maintenance costs of the metabolic machinery (Kleiber 1961; Daan et al. 1990) and because it correlates with an animal’s total energy expenditure (Ricklefs et al. 1996; but for the most recent discussion of the problem, see Portugal et al. 2016), it reflects the cost of life in endothermic animals (reviewed in Hulbert and Else 2000). Interspecific studies (Nespolo and Franco 2007; Bushuev et al. 2011; Auer et al. 2016) show that in most species whole-animal metabolic rate (MR) is a repeatable trait. Repeatability describes the consistency of among-individual differences in a trait over time and provides information on whether it may be selected for (Lessells and Boag 1987). It may also define the limit of heritability of a trait (Lynch and Walsh 1998; Dohm 2002). BMR and resting metabolic rate have been found to be heritable in several natural and laboratory populations (e.g., Konarzewski et al. 2005; Rønning et al. 2007; Sadowska et al. 2005, 2009; Zub et al. 2012), which indicates that consistent differences among individuals in their MR may be evolutionarily important (Rønning et al. 2005; Szafrańska et al. 2007). Depending on the time of day or year and different environmental conditions, high or low BMR may be beneficial or disadvantageous. Many studies have shown that animal MR is more variable than fixed and that it changes with changing environmental conditions (for reviews, see Piersma and Drent 2003; Lovegrove 2005; McKechnie and Swanson 2010). Changes in the phenotype, which occur during individual life in response to changing environmental conditions, may be irreversible (i.e., plastic) or reversible (i.e., flexible; Piersma and Drent 2003; Pig-

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J. S. Boratyński, M. Jefimow, and M. S. Wojciechowski

liucci 2005). The phenotypic flexibility may occur seasonally (Lovegrove 2005) as a result of predictable changes in environmental conditions (e.g., photoperiodism in mammals; Prendergast 2010; Scherbarth and Steinlechner 2010) or as a result of unpredictable changes that are independent of season (e.g., Ta variations; McKechnie et al. 2007; van de Ven et al. 2013; Boratyński et al. 2016). The range of phenotypes flexibly or plastically produced by a single individual (genotype) in a spectrum of environments is described by the reaction norm (Nussey et al. 2007; Brommer 2013). For instance, animals are able to adjust their heat production via phenotypic flexibility to cope with changes in Ta (McKechnie et al. 2007; van de Ven et al. 2013; Petit and Vézina 2014; Boratyński et al. 2016). High phenotypic flexibility of energetics may correlate with individual growth (Auer et al. 2015) or survival (Wikelski and Thom 2000) and, hence, should affect fitness. However, phenotypic flexibility also bears costs (DeWitt et al. 1998) that result from the maintenance of the sensory and regulatory machineries necessary for the proper function of a flexible phenotype (e.g., Williams and Tieleman 2000; Książek et al. 2009; Stager et al. 2015). Differences in the reaction norms of energy metabolism suggest that phenotypic flexibility may be considered an adaptation to the environments in which animals evolved (Tieleman et al. 2003; Lindsay et al. 2009; van de Ven et al. 2013). The plasticity or flexibility of a trait is adaptive only if reaction norms differ among individuals in a population, indicating the presence of an interaction between the genotype and the environment (Nussey et al. 2007; Brommer 2013). Many studies have shown that morphological, behavioral, and life-history traits show interactions between individuals and their environment (for a review, see Brommer 2013). Similarly, reaction norms of energy metabolism may differ between individuals in response to weather variations (Petit and Vézina 2014). However, whether differences in phenotypic flexibility are consistent among individuals is still unexplored, that is, whether they are repeatable in a population (Araya-Ajoy et al. 2015) and whether they can be a subject for selection. Assuming that individual variations in physiological reaction norms are evolutionarily important, we hypothesized that the flexibility of a trait will show consistent individual differences that eventually might be selected for. To examine this hypothesis, we first tested whether, within and between seasons, BMR and body mass (mb) differ consistently between individuals in a laboratory-raised population of Siberian hamsters (Phodopus sungorus). We predicted that BMR and mb are repeatable within winter and summer and among these seasons. Individuals of Siberian hamsters differ in their responsiveness to seasonal changes in photoperiod (Lynch et al. 1989), and these seasonal changes interact with their flexible responses to changes in Ta (Boratyński et al. 2016). Thus, we asked whether flexibility of BMR is related to other traits, or characteristics, of individuals, such as photoresponsiveness or sex. We expected lower phenotypic flexibility of BMR in winter than in summer, as described by Boratyński et al. (2016), and additionally greater seasonal differences in phenotypic flexibility in phenotypes responding to changes in photoperiod than in nonresponding ones. Finally, to examine our main

hypothesis, we tested whether reaction norms of BMR in response to intraseasonal changes in Ta differ consistently among individuals in a population on short (within-season) and long (amongseason) timescales. We predicted that reaction norms of BMR are repeatable both within and among seasons.

Methods Acclimation Protocol This study was done at Nicolaus Copernicus University in Toruń, Poland. All experimental procedures were approved by the Local Committee for Ethics in Animal Research in Bydgoszcz, Poland (decision 4/2014). Six individuals from the initial group of 52 animals were excluded because we were unable to determine their photoresponsiveness status (see below). Finally, we analyzed data from 30 male and 16 female Siberian hamsters (Phodopus sungorus) born from 13 outbred pairs (2–5 individuals per pair) in our breeding colony in late summer 2013. After weaning at the age of 4 wk, hamsters were kept individually in standard rodent cages (model 1246; Tecniplast, Buguggiate, Italy) with access to food (standard rodent diet; Labofeed B; Morawski, Kcynia, Poland) and water ad lib. To induce seasonal changes in hamster phenotypes, 2-mo-old individuals were acclimated for ∼100 d to winterlike conditions (8L∶16D, Ta p 107C; initial acclimation to winter; fig. 1). Thereafter, 40 animals (24 males and 16 females) were divided randomly into two groups (group A: 11 males and 10 females; group B: 13 males and 6 females; fig. 1) and were acclimated for 2 wk to 107 or 287C (under the same short photoperiod) to induce intraseasonal changes in phenotypes. Hamsters were acclimated three times to these Ta’s, always for 14 d. During the first 2 wk, group A animals were exposed to 287C; then, during the second 2 wk, they were exposed to 107C. Finally, during the third acclimation, they were exposed again to 287C (fig. 1). In parallel, group B was exposed to the same Ta’s in reverse order (fig. 1). After completion of winter experiments, all individuals were acclimated to summerlike conditions for ∼100 d (16L∶8D, Ta p 207C; initial acclimation to summer; fig. 1). Thereafter, hamsters were interchangeably acclimated to 287 or 107C for 14 d in each Ta under the summer day length. To compare individual reaction norms between winter and summer, individuals from groups A and B were acclimated to Ta’s in the same order in both seasons (fig. 1). During the entire experiment, six randomly selected males were kept under constant winter- or summerlike conditions to control for any possible time-related changes in their phenotype (fig. 1; Jefimow et al. 2005; Boratyński et al. 2016). Later, after acclimation to winterlike conditions, these control hamsters turned out to be responding to seasonal changes (see below). Data Collection BMR was measured indirectly as the rate of oxygen consump tion (VO2 ) using an open-flow respirometry system. Data were recorded on a PC coupled with an A-D interface (UI-2; Sable Systems International [SSI], Las Vegas, NV) using ExpeData


Repeatability of Phenotypic Flexibility of BMR

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Figure 1. Acclimation protocol for group A (N p 21; white arrows) and group B (N p 19; black arrows) after initial acclimation to winterlike (8L∶16D, Ta p 107C, ∼100 d) and summerlike (16L∶8D, Ta p 207C, ∼100 d) conditions. Hamsters from each group were moved between different ambient temperatures every 2 wk; day length did not change during short acclimations. Control animals (N p 6; dashed lines) were continuously kept under constant winter- or summerlike conditions. For details of the acclimation protocol, see “Methods.”

(ver. 1.43; SSI). We measured 14 individuals at the same time with two parallel respirometry systems. In one system [O2] and [CO2] in the air leaving respirometry chambers were measured with a FoxBox-C integrated O2 and CO2 analyzer (SSI), and in the other system [O2] was measured with a FC-10a O2 analyzer (SSI) and [CO2] was measured with a CA-10 CO2 analyzer (SSI); each system was used to measure the gas exchange of seven animals. Hamsters were measured for up to 7 h during the daytime in 0.85-L chambers constructed of translucent polypropylene containers (HPL 808; Lock & Lock, Hana Cobi, South Korea), which were placed in a temperature-controlled cabinet (ST-1200; Pol-Eko-Aparatura, Wodzisław Śląski, Poland). Temperature in respirometry chambers was measured continuously with type-T thermocouples connected to two 8-channel readers (USB 4718; Advantech Europe, Munich, Germany) and was recorded on a PC with WaveScan (ver. 2.0; Advantech Europe). During measurements, Ta inside chambers ranged between 277 and 307C, which is within the thermoneutral zone (TNZ) of the Siberian hamster (Gutowski et al. 2011; Boratyński et al. 2016). Air was pulled from outside the building using an air pump and compressed in a balloon, then dried and scrubbed of CO2 with a PureGas generator (Puregas, Westminster, CO). After that, air pressure was reduced to slightly exceeding atmospheric pressure (by ∼100 kPa). The air flow (∼400 mL min21) was regulated upstream of the respirometry chambers with precise needle valves and was measured downstream of the chambers with a mass-flow meter (Flow-Bar 4; SSI). Water vapor pressure in the excurrent air was measured using a water vapor analyzer (RH-300; SSI), and it was used to correct air flow measurements (using eq. [8.6] from Lighton 2008). The baseline gas concentrations in air entering chambers were measured in a reference airstream; the airstream

was switched between animal chambers and a reference line using a computer-controlled multiplexer (MUX; SSI). Approximately 100 mL min21 of air leaving respirometry chambers was sampled for 5 min for each animal, and reference gas readings were sampled at least every 20 min. As a result, gas exchange in each animal was measured every 44 min and was sampled at a rate of 0.5 Hz. Body temperature (Tb) was recorded continuously, every 20 min, with implanted miniaturized thermosensitive data loggers (iButton, model iBBat 22L; Dallas Semiconductors, Dallas, TX) that had an accuracy of 0.57C. Loggers coated in the paraffin wax (final mass: 1.0–1.6 g) were implanted intraperitoneally in all individuals after initial acclimations, at least 1 wk before the first measurement of BMR. Animals were implanted under ketamine (40 mg kg–1; Ketamina 10%; Biowet, Puławy, Poland) and xylazine (8 mg kg–1; Sedazin 2%; Biowet) anesthesia. After completing all measurements in a season, loggers were surgically removed from animals. Each logger was calibrated in a temperaturecontrolled ethylene glycol bath (FBC 635; Fisherbrand, Schwerte, Germany) against a traceable mercury-in-glass thermometer.

Data Analysis

Oxygen consumption and CO2 production (VCO2 ) were calculated from the lowest stable 2 min of a single recording using a steady-state approach (Lighton 2008). BMR was determined as minimum VO2 recorded at TNZ at least 5 h after the last possible meal, that is, in the postabsorptive phase (Gutowski et al. 2011). VO2 and VCO2 were calculated using equations (11.7) and (11.8) from Lighton (2008). MR was calculated in watts assum ing a respiratory exchange ratio (RER; VCO2 /VO2 ) calculated


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from VCO2 and VO2 using an oxyjoule equivalent after Lighton et al. (1987), as follows: MR ðWÞ p

V_ O2 (16 1 5:164 ⋅ RER) , 60

where V_ O2 is the rate of oxygen consumption (mL O2 min21). Torpor Use. The Siberian hamster is a polymorphic species, and after acclimation to winter photo-responding individuals decrease mb and whole-animal BMR, show gonadal regression, molt to white pelage, and develop the capacity to enter daily torpor (Heldmaier and Steinlechner 1981a, 1981b; Puchalski and Lynch 1986; Lynch et al. 1989; Goldman et al. 2000; Jefimow et al. 2004). We differentiated between responding and nonresponding individuals on the basis of their ability to enter torpor (Tb ! 327C; Ruf et al. 1989, 1993; Ruf and Heldmaier 1992). Because several loggers failed during experiments and Tb data were lost, we additionally determined torpor use on the basis of behavioral criteria: inactivity, stereotyped posture, reduced response to tactile stimuli (IUPC Thermal Commission 2001), and whether they felt cold to the touch. This information was collected at least in 1-wk intervals during animal cage cleaning as well as before all metabolic measurements. Since torpor may also occur in response to food restriction or deprivation (Ruby and Zucker 1992; Ruf et al. 1993), after completing the winter series of experiments we deprived animals of food for 24 h at 107C. Hamsters were considered as responding to a short photoperiod (or responders) if they were observed in torpor at least once on the basis of Tb recordings, behavioral observations, or both, while individuals for which Tb never fell below 327C were classified as nonresponding (or nonresponders). In the case where Tb data were lost and hamsters were not observed to commence torpor, they were excluded from the study; this was true for six individuals. To determine individual differences in torpor use, on the basis of available Tb recordings we calculated the percentage of time spent in torpor for 16 individuals (10 males and 6 females); for consistency, this was done for data collected during acclimation to 107C, after hamsters were moved from 287C (see fig. 1 for the acclimation protocol).

Statistical Analyses General Remarks. Unless stated otherwise, we used a repeatedmeasures design in linear mixed-effects (LME) models (IBM SPSS ver. 22, using a Mixed procedure; IBM SPSS Statistics 22 Command Syntax Reference, p. 1093–1106) to compare data and estimate necessary variance components. In all LME analyses, the subsequent measurement was coded as a repeated variable, and the animal ID was set as a random effect. In all LME analyses we used the restricted maximum likelihood method to estimate variance components, with scaled identity set as a repeated covariance type. Assumptions of the linear modeling were checked post hoc by inspecting the distribution of residuals obtained from LME analyses (check of histograms and quantile-quantile plots; Grafen and Hails 2002).

Seasonal Changes in mb and BMR. To investigate seasonal changes in hamster phenotypes, their mb and BMR were compared after initial acclimations. Seasonal differences in mb were tested using an LME analysis in which we included season, sex, and individual responsiveness as fixed factors and all possible interactions between them. Whole-animal BMR was compared in the LME analysis with season, sex, and responsiveness in a full factorial design. Body mass–adjusted BMR was analyzed using the above model with mb as a covariate. Body mass and BMR of individuals from the control group after initial acclimations were compared between seasons using the paired Student’s t-test. Intraseasonal Variations in mb and BMR. Body mass and BMR may change spontaneously due to photorefractoriness, even when animals are kept under a constant photoperiod and Ta (Masuda and Oishi 1995; Jefimow et al. 2005). To test for that in each season separately, mb and BMR of individuals from the control group were compared at four time points corresponding to four acclimations (initial, first, second, and third; fig. 1). This was done using repeated-measures ANOVA (RM-ANOVA) with a pairwise Tukey’s test for post hoc comparisons. Assumptions of the RM-ANOVA were checked with Levene’s test (homogeneity of variance) and the Shapiro-Wilk test (normality of distribution). Because mb and BMR of males and females from experimental groups were not distributed normally (ShapiroWilk’s W ! 0.9), they were compared between the subsequent four acclimations using the Friedman test, with Wilcoxon pairwise comparisons as a post hoc. In winter and summer this was done separately for each experimental group and sex. Phenotypic Flexibility of BMR. Differences in BMRs of the same individuals acclimated to different Ta’s (107 or 287C) between the first, second, and third 14-d acclimations were used to describe phenotypic flexibility (or reaction norm). Specifically, BMR measured after acclimation to 287C was subtracted from BMR measured after acclimation to 107C to estimate the individual response (DBMR) to change in acclimation temperature (DTa). Hence, within each season we obtained for each individual two DBMRs: one between the first and second acclimation and another between the second and third acclimation. Dmb was calculated in the same way. Since animals from groups A and B were acclimated in reverse order, the sequence and direction of DTa were included in the analysis as categorical variables. DBMRs were compared in a single LME analysis including sex, responsiveness, season, and all interactions between them, with the direction of DTa and the subsequent number of the change in Ta as factors, as well as their interaction. To account for the potential effect of changes in mb on DBMR, Dmb was included as a covariate. The relationship between time spent in torpor and DBMR (a measure of phenotypic flexibility) was tested in a general linear model with sex as a fixed factor and Dmb as a covariate. Since Dmb was not significant (P 1 0.05), we excluded it from the model and reran the analysis. Repeatability of mb, BMR, and Phenotypic Flexibility of BMR. Repeatability was estimated as an intraclass correlation coeffi-


Repeatability of Phenotypic Flexibility of BMR cient (t) based on variance components obtained from LME analyses (see “General Remarks” above). We followed Lessells and Boag (1987) and used the following equation: jA tp , jA 1 jW where jA is among-individual variance and jW is within-individual variance. We estimated t’s of mb, BMR, and DBMR separately within each season (short-term scale) and among seasons (longterm scale). Short-term t’s of mb and BMR were estimated separately within each season, on the basis of data obtained after three short acclimations (first, second, and third; fig. 1). Intraseasonal t’s of mb and BMR were calculated in an LME analysis in which data for both experimental groups were incorporated in a modeling procedure. In these models we accounted for sex, responsiveness, subsequent number of the acclimation, and acclimation Ta as fixed factors. In models analyzing mb-adjusted BMR, mb was included as a covariate. Since Ta can affect mb and BMR, being an additional source of within-individual variation, to quantify the effect of changing Ta on the short-term repeatability of mb and BMR we applied a two-step approach. The first model included sex, responsiveness, and subsequent acclimation as factors. The second model accounted for sex, responsiveness, subsequent acclimation, and acclimation Ta as fixed factors. Longterm t’s of mb and BMR were calculated on the basis of variance components obtained from LME analyses describing seasonal differences in hamster phenotypes (see “Seasonal Changes in mb and BMR” above). As indicated at the beginning of this section (see “General Remarks” above), in all models animal ID was included as a random effect, while the subsequent measurements were included as repeated variables. Short-term t’s of DBMR within each season were obtained from LME analyses where sex, responsiveness, direction of change in Ta, and its sequence were included as fixed factors. This model also accounted for interactions between sex and responsiveness and between the direction of DTa and its sequence. Repeatability of Dmb-adjusted DBMR was estimated in a model with corresponding changes in mb as a covariate. Long-term t’s of DBMR and Dmb-adjusted DBMR were calculated on the basis of variance components obtained from an LME analysis comparing individual response to acclimation Ta (see “Phenotypic Flexibility of BMR” above). Additionally, to determine the effect of photoresponsiveness on the repeatability of DBMR, we ran similar models for responding and nonresponding hamsters separately. The statistical significance of among-individual variance of a trait, which is indicative of the statistical significance of t, was tested with the Wald x2 test. The t’s were presented with 95% confidence intervals (CIs) estimated on the basis of CIs obtained for among- and within-individual variances. In all analyses, statistical significance was accepted at P ≤ 0.05, and data were reported as estimated marginal means and their upper and lower 95% CIs. We also considered P ! 0.1 to indicate a nonsignificant trend.

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Results Seasonal Changes in mb and BMR Body mass of hamsters after initial acclimation to winterlike conditions (28.35 g [95% CI: 27.02–29.69 g]) was ∼18% lower than that after acclimation to summerlike conditions (34.53 g [95% CI: 33.20–35.87 g]; F1, 36 p 63.58, P ! 0.001). Overall, females (25.88 g [95% CI: 24.21–27.56 g]) were 30% lighter than males (37.00 g [95% CI: 35.56–38.44 g]; F1, 36 p 104.24, P ! 0.001). There was also a significant interaction between sex and season (F1, 36 p 6.61, P p 0.014), and from winter to summer males increased their mb by 19.9%, while females increased it by 15.0%. On average, after acclimations to winter and summer there was no difference in mb between hamsters that were or were not responding to changes in photoperiod (F1, 36 p 0.32, P p 0.578). However, in summer responders increased mb by 25.5% (fig. 2A), while nonresponders increased it by only 9.7% (fig. 2B; season # responsiveness interaction: F1, 36 p 14.27, P p 0.001); there were no interactions between sex and responsiveness (F1, 36 p 0.10, P p 0.760) or between sex, responsiveness, and season (F1, 36 p 3.60, P p 0.066). Hamsters assigned to control groups were ∼17% heavier after initial acclimation to summerlike conditions than after acclimation to winterlike conditions (t p 3.54, P p 0.017; table 1). Whole-animal BMR was ∼5% lower in winter (247 mW [95% CI: 237–257 mW]) than in summer (260 mW [95% CI: 250–270 mW]; F1, 36.0 p 5.51, P p 0.025). However, seasonal changes in BMR positively correlated with changes in mb of individuals (F1, 68.7 p 52.97, P ! 0.001; fig. 2). After accounting for mb, sex and individual responsiveness BMR was ∼6% higher in winter (267 mW [95% CI: 257–276 mW]) than in summer (250 mW [95% CI: 242–258 mW]; F1, 53.1 p 8.56, P p 0.005). Whole-animal BMR was ∼16% higher in males (276 mW [95% CI: 265–287 mW]) than in females (231 mW [95% CI: 218–244 mW]; F1, 36.0 p 30.19, P ! 0.001), and this difference was not affected by season (sex # season interaction: F1, 36.0 p 1.36, P p 0.252). Despite the lack of difference in mb-adjusted BMR between males and females (F1, 63.1 p 0.60, P p 0.442), in males it decreased from winter to summer by 12.2%, while in females it did not change (0.4%; sex # season interaction: F1, 38.5 p 14.53, P ! 0.001; fig. 2). On average, responders and nonresponders did not differ in either whole-animal (F1, 36.0 p 1.67, P p 0.205; fig. 2) or mbadjusted (F1, 35.9 p 1.33, P p 0.256; fig. 2) BMR. Responding hamsters increased (∼17%) and nonresponding hamsters decreased (∼8%) their whole-animal BMR from winter (responders: 225 mW [95% CI: 213–238 mW]; nonresponders: 269 mW [95% CI: 254–285 mW]) to summer (responders: 271 mW [95% CI: 259–284 mW]; nonresponders: 249 mW [95% CI: 233– 264 mW]; season # responsiveness interaction: F1, 36.0 p 36.77, P ! 0.001; fig. 2A). Nonresponders decreased their mb-adjusted BMR during acclimation to summerlike conditions (12.9%; fig. 2B), while responders increased it slightly (1.2%; season # responsiveness interaction: F1, 41.5 p 19.33, P ! 0.001; fig. 2A). In models analyzing whole-animal BMR there were no interactions between sex and responsiveness (F1, 36.0 p 1.07, P p 0.308) or among sex,


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Figure 2. Relationship between basal metabolic rate and body mass in female (circles) and male (triangles) Siberian hamsters that were classified as responding (A) or not responding (B) to seasonal changes in photoperiod. Graphs present data obtained after initial acclimation to winterlike (filled symbols) and summerlike (open symbols) conditions.

responsiveness, and season (F1, 36.0 p 0.50, P p 0.483); the same was true for mb-adjusted BMR (sex # responsiveness interaction: F1, 35.8 p 2.29, P p 0.139; sex # responsiveness # season interaction: F1, 37.2 p 0.58, P p 0.449). Hamsters assigned to the control group had ∼11% lower whole-animal BMR in winter than in summer (t p 2.57, P p 0.050; table 1). Intraseasonal Variations in mb and BMR During winter experiments control and experimental hamsters increased their mb slightly (tables 1, 2). However, during summer experiments control hamsters did not change their mb (table 1), while experimental animals did (table 3). After 14 d of exposure to 287C mb was lowest, but it increased after exposure to 107C (table 3). There were no intraseasonal differences in BMR of control hamsters in either winter or summer (table 1). On the contrary, experimental animals exposed for 2 wk to 107C increased their BMR, and they decreased it when exposed to 287C (tables 2, 3). Phenotypic flexibility, defined as DBMR in response to intraseasonal changes in Ta, was 24% higher in summer (47 mW [95% CI: 41–53 mW]) than in winter (36 mW [95% CI: 30–42 mW];

F1, 114.0 p 11.55, P ! 0.001). Although DBMR correlated positively with concomitant Dmb (F1, 147.6 p 22.71, P ! 0.001), this seasonal pattern did not change after accounting for changes in mb; Dmb-adjusted DBMR was ∼18% smaller in winter experiments (37 mW [95% CI: 31–43 mW]) than in summer experiments (45 mW [95% CI: 39–51 mW]; F1, 116.2 p 5.74, P p 0.018). Male DBMR (48 mW [95% CI: 41–55 mW]) was 29% higher than female DBMR (34 mW [95% CI: 26–43 mW]; F1, 35.0 p 6.52, P p 0.015). Similarly, females had 33% lower Dmb-adjusted DBMR (33 mW [95% CI: 25–41 mW]) than males (49 mW [95% CI: 42–56 mW]; F1, 35.5 p 9.77, P p 0.004; fig. 3), and season did not affect the difference between sexes in DBMR (sex # season interaction: F1, 114.0 p 0.62, P p 0.804) and in Dmb-adjusted DBMR (sex # season interaction: F1, 113.6 p 0.34, P p 0.562). On average, responders did not differ from nonresponders in either DBMR (F1, 35.0 p 1.49, P p 0.230) or Dmb-adjusted DBMR (F1, 35.3 p 0.82, P p 0.373). There was, however, a significant interaction between season and animal responsiveness for both DBMR (F1, 114.0 p 10.19, P p 0.002) and Dmb-adjusted DBMR (F1, 113.3 p 12.16, P p 0.001): namely, BMR of responders was less flexible in winter than in summer, while in nonresponders such a difference was absent (fig. 3).

Table 1: Body mass (mb; g) and basal metabolic rate (BMR; mW) of six Siberian hamsters assigned to the control group during winter and summer experiments Season, variable Winter: mb BMR Summer: mb BMR

Initial

First

Second

Third

F

P

34.77 5 7.65A 264 5 44

35.37 5 8.92 268 5 53

36.32 5 9.67 267 5 66

38.08 5 9.46A 284 5 68

3.33 .98

.048 .427

41.67 5 9.55 297 5 42

41.88 5 8.73 272 5 42

42.18 5 9.68 285 5 54

41.50 5 9.86 285 5 63

.6 2.35

.624 .114

Note. These hamsters were classified as responding to seasonal changes. See the text for more details on the classification criteria. Values were compared using repeated-measures ANOVA with the Tukey test for post hoc comparisons. Values are presented as means 5 SD. F p value of the test statistics. Superscript letters indicate statistical differences within seasons at P ≤ 0.05.



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Table 2: Body mass (mb; g) and basal metabolic rate (BMR; mW) in male and female Siberian hamsters assigned to experimental groups (A or B) during winter experiments Group, sex, variable Group A: Females: mb BMR Males: mb BMR Group B: Females: mb BMR Males: mb BMR

Initial 107C

First 287C

Second 107C

Third 287C

N

x2

P

23.46 5 2.74A,B,C 219 5 33A

24.73 5 3.25A,D 210 5 18

26.21 5 3.46B,D 226 5 44B

25.91 5 3.60C 202 5 33A,B

10 10

20.8 4.68

!.001 .197

32.73 5 4.09A,B 271 5 34A

33.37 5 4.03C 241 5 40A,B

34.69 5 4.79A 286 5 38B,C

35.22 5 4.19B,C 256 5 33C

11 11

9.3 16.76

.026 !.001

24.05 5 1.54A,B,C 217 5 21A,B

25.93 5 1.86A 229 5 18A,C

26.12 5 2.16B 184 5 28B,C,D

26.62 5 3.10C 220 5 34D

6 6

10.8 14.7

31.43 5 5.06A,B,C 263 5 37A

32.85 5 5.16A 272 5 39B,C

32.81 5 5.93B 223 5 40A,B,D

33.02 5 5.96C 257 5 33C,D

13 13

15.22 20.58

.006 .001 .002 !.001

Note. Values were compared using the Friedman test with the Tukey test for post hoc comparisons. Values are presented as means 5 SD. x2 p value of the test statistics. Superscript letters indicate statistical differences at P ≤ 0.05.

Overall, there were no differences in hamster DBMR (F1, 114.0 p 1.46, P p 0.230) and Dmb-adjusted DBMR (F1, 113.7 p 0.77, P p 0.381) between subsequent changes in Ta (fig. A1, available online). However, Dmb-adjusted DBMR was 17.8% higher when Ta was changed from 107 to 287C (fig. 3) than when it was changed in the reverse order (F1, 35.0 p 5.56, P p 0.020; fig. 3). There was no such difference when mb was not included in the analysis (F1, 114.0 p 0.97, P p 0.325). We found no interaction between subsequent change in Ta and the direction of change for either DBMR (F1, 35.0 p 1.06, P p 0.310) or Dmb-adjusted DBMR (F1, 117.3 p 1.66, P p 0.206). The phenotypic flexibility of BMR (DBMR) resulting from transfer from 287 to 107C was positively related to the proportion of time that photoresponsive hamsters spent in torpor (F1, 13 p 7.80, P p 0.014; fig. 4), and sex did not affect this relationship (F1, 13 p 2.02, P p 0.661). Repeatability of mb, BMR, and Phenotypic Flexibility of BMR Body mass, when accounting for different sex, responsiveness, and season, tended to be repeatable between initial acclimations to winter- and summerlike conditions (t p 0.33 [95% CI: 0.21–0.47]; x2 p 1.89, P p 0.062). Both whole-animal BMR (t p 0.39 [95% CI: 0.29–0.50]; x2 p 2.16, P p 0.031) and mb-adjusted BMR (t p 0.48 [95% CI: 0.41–0.55]; x2 p 2.60, P p 0.009) were repeatable between initial acclimations to winter- and summerlike conditions when sex, responsiveness, season, and their interactions were taken into account. Within each season mb was highly repeatable (table 4), and neither in winter nor in summer did t’s for mb estimated from models adjusted or not adjusted for different acclimation Ta’s differ between each other (table 4). Whole-animal BMR was repeatable in both seasons; however, the t’s were highest when acclimation Ta’s were taken into account (table 4). Body mass– adjusted BMR was repeatable only when acclimation Ta was

included in models as a factor (table 4), whereas when different acclimation Ta’s were ignored, t for mb-adjusted BMR was not statistically different from 0 (table 4). When individual responsiveness, sex, subsequent change in acclimation Ta’s and the direction of DTa, and the defined interactions were taken into account, DBMR (in other words, its phenotypic flexibility) was repeatable in both winter (t p 0.37 [95% CI: 0.26–0.49]; x2 p 2.05, P p 0.041) and summer (t p 0.56 [95% CI: 0.50–0.62]; x2 p 2.90, P p 0.004; figs. 5, A1). Likewise, Dmb-adjusted DBMR was repeatable in both winter (t p 0.48 [95% CI: 0.41–0.56]; x2 p 2.58, P p 0.010) and summer (t p 0.55 [95% CI: 0.48–0.61]; x2 p 2.82, P p 0.005; figs. 5, A1). On the long-term scale, when all factors and defined interactions were controlled for in the analyses, both flexibility of BMR (DBMR; t p 0.29 [95% CI: 0.20–0.41]; x2 p 2.54, P p 0.011; fig. 5) and DBMR adjusted for concomitant Dmb (t p 0.31 [95% CI: 0.22–0.42]; x2 p 2.64, P p 0.008; fig. 5) were significantly repeatable. However, additional analysis revealed that long-term repeatability of phenotypic flexibility differed slightly between responders and nonresponders. While DBMR was not repeatable in responders (t p 0.18 [95% CI: 0.08–0.37]; x2 p 1.48, P p 0.139), it tended to be repeatable in nonresponders (t p 0.46 [95% CI: 0.31–0.61]; x2 p 1.87, P p 0.061). Dmb-adjusted DBMR showed nonsignificant trends toward repeatability for both responders (t p 0.27 [95% CI: 0.15–0.43]; x2 p 1.86, P p 0.062) and nonresponders (t p 0.40 [95% CI: 0.25–0.57]; x2 p 1.74, P p 0.083). Discussion BMR is a labile trait (see Futuyma and Moreno 1988), and its within-individual variations may sometimes exceed differences observed among individuals. This is why its evolutionary importance has been questioned, at least in some species (Bozinovic


146


Table 3: Body mass (mb; g) and basal metabolic rate (BMR; mW) in male and female Siberian hamsters assigned to experimental groups (A or B) during summer experiments Group, sex, variable Group A: Females: mb BMR Males: mb BMR Group B: Females: mb BMR Males: mb BMR

Initial 207C

First 287C

Second 107C

Third 287C

N

x2

P

28.00 5 2.59A,B 240 5 26

28.82 5 2.99C,D 220 5 25A

30.83 5 3.12A,C,E 251 5 19A,B

29.66 5 3.47B,D,E 214 5 25B

10 10

17.41 14.64

!.001 .002

43.51 5 6.36A,B 296 5 43A

44.64 5 6.22A 288 5 37B

45.23 5 6.48B 348 5 45A,B,C

44.66 5 6.43 289 5 46C

11 11

10.22 17.94

.017 !.001

28.13 5 1.79A,B 244 5 12A

30.32 5 1.57A 261 5 20B

29.00 5 2.48 213 5 18A,B,C

30.12 5 1.39B 260 5 19C

6 6

9.36 12.6

.017 .001

40.10 5 3.73A 276 5 29A.B,C

40.47 5 3.87B 300 5 24A,D

40.12 5 4.87C 249 5 26B,D,E

41.61 5 4.25A,B,C 300 5 26C,E

13 13

10.41 26.52

.015 !.001

Note. Values were compared using the Friedman test with the Tukey test for post hoc comparisons. Values are presented as means 5 SD. x2 p value of the test statistics. Superscript letters indicate statistical differences at P ≤ 0.05.

2007; Russell and Chappell 2007; Cortés et al. 2015). We found that in Siberian hamsters both BMR and its flexibility were repeatable in summer and winter and that both were repeatable on a longterm scale, that is, among seasons. To the best of our knowledge, these results provide the first experimental evidence that not only BMR but also its adjustments in response to varying thermal conditions (i.e., phenotypic flexibility or reaction norm) consistently differ among individuals and thus may be a subject for phenotypic selection. Our results show that despite slight differences between males and females, the individual responsiveness to photoperiod did not differ between sexes (fig. 2). Although most of the seasonal variations in BMR were explained by seasonal changes in mb, mbadjusted BMR was slightly higher in winter than in summer. Moreover, the increase in mb-adjusted BMR in winter occurred mainly in nonresponders (fig. 2). The photoperiod is the main cue

for seasonal changes in physiology, morphology, and behavior of Siberian hamsters (Heldmaier and Steinlechner 1981a, 1981b; Scherbarth and Steinlechner 2010), yet some individuals in a population do not undergo seasonal changes (Lynch et al. 1989). In the studied population, more than half of individuals entered torpor at least once during the whole study and thus were classified as responders (fig. 2). They increased both mb and wholeanimal BMR during summer acclimation (fig. 2A). Nonresponders, in turn, despite a slight increase in mb between winter and summer decreased their whole-animal BMR (fig. 2B). Nevertheless, these differences do not reflect divergent responses to photoperiod. We argue that nonresponders represent a much more flexible phenotype than responders and that their winter increase in whole-animal BMR is a product of phenotypic flexibility (see “Introduction”) in response to cold during winter acclimation.

Figure 3. Changes in basal metabolic rate of male and female Siberian hamsters that were classified as responders (A; N p 25; 16 males and 9 females) or nonresponders (B; N p 15; 8 males and 7 females) to seasonal changes in photoperiod during winter (filled bars) and summer (open bars) experiments. Graphs present absolute changes in basal metabolic rate as a result of a short 14-d acclimation to 287C (W) or 107C (C). Boxes indicate the lower quartile, median, and upper quartile, and whiskers indicate the range of changes (minimum to maximum).



Figure 4. Relationship between percentage of time spent in torpor by male (triangles) and female (circles) photo-responding hamsters during 14 d of cold exposure and their phenotypic flexibility of basal metabolic rate in winter.

Phenotypic flexibility may bring about significant benefits to animals in the face of predicted increasing climate unpredictability (Visser 2008; Canale and Henry 2010). A number of studies have focused on the flexibility of BMR in response to short-term changes (from days to weeks) in Ta, in both birds (McKechnie et al. 2007; Barceló et al. 2009; van de Ven et al. 2013) and mammals (Li et al. 2001; Russell and Chappel 2007; Książek et al. 2009; Chi and Wang 2011), and quantified their reaction norms in response to acclimations to different Ta’s. In contrast to seasonal changes, slight changes in Siberian hamster mb within each season do not seem to offer a biologically meaningful explanation for intraseasonal flexibility of BMR (present results and Boratyński et al. 2016). This is further

supported by the general lack of differences between results obtained from models with and without mb as a covariate and suggests that most of the flexible changes in hamster phenotype were due to changes in metabolic performance on the cellular level. In general, the effect of changing mb on phenotypic flexibility seems to vary among taxa; depending on the species, changes in BMR correlated positively with changes in mb (Chi and Wang 2011; van de Ven et al. 2013) or did not (Nespolo et al. 2002; del Valle et al. 2004; Novoa et al. 2005; McKechnie et al. 2007). This is likely a result of species-specific mechanisms underlying the reaction norms of energy metabolism. Phenotypic flexibility of BMR may result from discreet adjustments of the metabolic performance of all tissues composing the body (Piersma and van Gils 2011) and not solely from changes in the mass of metabolically active organs (but see Williams and Tieleman 2000). For example, cell proliferation, mitochondrial efficiency, and enzyme activities may change in different tissues without changes in an animal’s mb (Li et al. 2001; Nespolo et al. 2002; del Valle et al. 2004; Chi and Wang 2011). In this study, mb of hamsters from control and experimental groups increased throughout winter experiments, while in the previous study it decreased under similar conditions (Boratyński et al. 2016). Although it is rather unlikely, we cannot exclude the possibility that slight differences in the duration of experiments (here the whole schedule was ∼2 wk longer than the previous one; Boratyński et al. 2016) resulted in the beginning of photorefractoriness in hamsters (Jefimow et al. 2005). This is why changes in mb were included as a covariate in the analyses of the phenotypic flexibility of BMR. In winter and summer mb varied considerably among individuals (fig. 2) and only slightly within individuals (tables 1–3), and hence it was highly repeatable within each season (t p ∼0.9). Since the repeatability of mb almost did not differ between models that did and did not account for different Ta’s during 14-d acclima-

Table 4: Repeatability coefficients (t), confidence intervals (CIs), and values of the test statistics (x2) for among-individual variances in body mass (mb), basal metabolic rate (BMR), and mb-adjusted BMR, adjusted for sex, responsiveness, and subsequent acclimation in linear mixed-effects models in which different ambient temperatures (Ta’s) during acclimation were or were not included as a factor Winter Trait, model mb: Not Ta adjusted Ta adjusted BMR: Not Ta adjusted Ta adjusted mb-adjusted BMR: Not Ta adjusted Ta adjusted

147

Summer t (95% CI)

x2

4.13*** 4.13***

.94 (.93–.95) .96 (.95–.96)

4.22*** 4.24***

.40 (.32–.50) .68 (.63–.72)

2.81** 3.68***

.32 (.22–.44) .78 (.75–.81)

2.42* 3.92***

.06 (.00–.61) .43 (.35–.52)

.55NS 2.88**

.03 (.00–.95) .50 (.43–.58)

.29NS 3.12**

t (95% CI)

x

.89 (.87–.90) .89 (.88–.91)

2

Note. NS p not significant. *P ! 0.05. **P ! 0.01. ***P ! 0.001.


148


Figure 5. Individual variations in absolute changes in basal metabolic rate obtained after the first (squares) and second (diamonds) change in acclimation temperature in winter (filled symbols) and summer (open symbols). Individuals on the graph were sorted from the most to least flexible on the basis of average change in basal metabolic rate obtained for each hamster.

tions (table 4), we conclude that in Siberian hamsters mb is not flexibly adjusted in response to unpredictable, abrupt changes in the environment. This result also indicates that hamsters defended their mb according to the actual setpoint that is determined by photoperiod (e.g., Mrosovsky and Fisher 1970; Steinlechner et al. 1983). It is possible that this lack of flexibility also explains why mb was found to be highly repeatable (0.5 ! t ! 0.9) in free-living mammals and birds, even though animals experienced variable environmental conditions during the course of studies (Bozinovic 2007; Szafrańska et al. 2007; Broggi et al. 2009; Cortés et al. 2015). In comparison to mb, the whole-animal BMR was less repeatable, but the repeatability estimates were still higher than that of mb-adjusted BMR. This agrees with data from the literature (Konarzewski et al. 2005), since approximately onethird of interindividual variation in BMR was related directly to different mb of individuals. Repeatability of mb-adjusted BMR was significant in Merriam’s kangaroo rats Dipodomys merriami (Hayes et al. 1998), bank voles Myodes glareolus (Labocha et al. 2004), weasels Mustela nivalis (Szafrańska et al. 2007), zebra finches Taeniopygia guttata (Rønning et al. 2005), and great tits Parus major (Broggi et al. 2009) but not in deer mice Peromyscus maniculatus (Russell and Chappell 2007), leaf-eared mice Phyllotis darwini (Bozinovic 2007), or European stonechats Saxicola rubicola (Versteegh et al. 2008). Additionally, the repeatability of MR in laboratory populations was slightly but significantly higher than that in wild-living ones (Auer et al. 2016). This is plausible, because wild-living animals frequently face fluctuations in environmental conditions (Cortés et al. 2015). It is also supported by experiments in which different acclimation Ta’s resulted in low repeatability of BMR in birds (McKechnie et al. 2007; van de Ven et al. 2013). In line with that, mb-adjusted BMR of hamsters, when calculated from models not adjusted for different Ta’s, was not repeatable, in either winter (t p 0.06) or summer (t p 0.03). This changed considerably when Ta was included in the models, and as a consequence repeatability coefficients increased, in both winter (t p 0.43) and summer (t p 0.50). The differences between repeatability obtained from the

above models were estimated to be 37% of total phenotypic variance in winter and 47% in summer. Thus, irrespective of season, ∼40% of the observed variance in BMR was related to changes in Ta itself, which indicates that BMR is highly flexible in Siberian hamsters. Our previous study showed that flexibility of BMR in male Siberian hamsters was lower in winter than in summer (Boratyński et al. 2016), and this was true in the present study as well. However, we also found that only responders decreased their phenotypic flexibility in winter, whereas nonresponders did not (fig. 3). This was true for both sexes, yet females were generally less flexible than males. Our criterion for individual responsiveness assumed torpor use; thus, one could suggest that responders changed their BMR less than nonresponders because they spent relatively less time coping with cold at high normothermic Tb’s. Since torpor is more effective in cold (Geiser 2004), this could explain why changes in BMR adjusted for Dmb were smaller when Ta decreased. Heterothermy is the most effective energy-saving strategy (Lyman et al. 1982; Boyer and Barnes 1999), and it is considered an adaptation to living in unpredictable climates (Canale and Henry 2010). Thus, it may be considered an alternative for expensive flexible adjustments of the phenotype (Boratyński et al. 2016). However, the present results indicate that in responding hamsters there was a positive relationship between the time spent in torpor and the phenotypic flexibility of BMR, and they suggest that torpor use itself does not explain low phenotypic flexibility of responding hamsters in winter (fig. 4). Nevertheless, this result does not contradict the hypothesis of the interaction between phenotypic flexibility and seasonal changes in the phenotype that are driven by photoperiod (Boratyński et al. 2016). Slight differences in the long-term repeatability of the phenotypic flexibility of BMR between responders and nonresponders may support this hypothesis. Nonresponders, in which phenotypic flexibility tended to be more repeatable, seem to maintain the character of their response to changes in temperature irrespective of season.


Repeatability of Phenotypic Flexibility of BMR In winter, photo-responding individuals regress their gonads and, as a result, cease reproduction (Prendergast 2010). Hence, depending on winter conditions, responders and nonresponders should achieve different fitness (Goldman et al. 2000 and references therein). Still, thanks to genetic polymorphism, the two phenotypes are retained in a population, with one of them being able to reproduce regardless of day length, when environmental conditions permit (Heideman and Bronson 1991; Goldman et al. 2000; Sharp et al. 2015). However, many authors have suggested that the flexibility of energy metabolism also affects animal fitness and is under Darwinian selection (Tieleman et al. 2003; Terblanche et al. 2009; van de Ven et al. 2013; Petit and Vézina 2014; Bartheld et al. 2015; Auer et al. 2016). Thus, under suitable environmental conditions, the highly flexible nonresponding phenotype may possibly be selected for and favored over the less flexible photo-responding one. Since a number of climate models predict a continued increase in weather anomalies with an increasing probability of warm weather in winter (IPCC 2007; for Siberia, see Gong and Ho 2002), it is possible to expect a decrease in the frequency of photo-responding individuals in natural populations. However, although selection may act only on repeatable traits (Lessells and Boag 1987; Dohm 2002), no study to date has shown that individual flexibilities of energy metabolism consistently differ among individuals (ArayaAjoy et al. 2015). We found that the flexibility of BMR was highly repeatable in both winter (t p 0.48) and summer (t p 0.55). More importantly, we found that phenotypic flexibility was also repeatable on a long-term axis (t p 0.31) when animal responsiveness, sex, and season were accounted for. Additionally, phenotypic flexibility tended to be more repeatable in nonresponders, but in responders its estimate increased by ∼10% when changes in mb were controlled for. This suggests that in photoresponding hamsters changes in mb associated with the development of winter phenotype or with photorefractoriness may interact with the phenotypic flexibility of energy metabolism in response to varying thermal conditions. Nevertheless, for our population of Siberian hamsters, the long-term repeatability of phenotypic flexibility was comparable to that obtained for BMR, and both were within the range reported in the literature for repeatability of basal or resting MRs (Szafrańska et al. 2007; Nespolo and Franco 2007; Auer et al. 2016). The most important conclusion, however, is that selection may act on individual reaction norms in response to changes in environmental conditions. Since repeatability may define the upper limit for heritability of a trait (Dohm 2002), it is likely that the phenotypic flexibility of energy metabolism is also heritable in Siberian hamsters as well as in other taxa. Acknowledgments This study was funded by a grant from the National Science Center in Kraków, Poland (2011/01/B/NZ8/00049) awarded to M.S.W. We thank Berry Pinshow and two anonymous reviewers whose comments improved the manuscript. Joanna Jankowicz, Jakub Wiśniewski, and Anna Przybylska helped us during the experiments. Results of this study were presented

149

at the symposium “Phenotypic Flexibility of Energetics in a Seasonal World” of the 9th International Congress of Comparative Physiology and Biochemistry, which was supported by the Division of Comparative Physiology and Biochemistry, Society for Integrative and Comparative Biology.

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Appendix 1

A

B

C

D

Figure A1. Relationship between first (abscissa) and second (ordinate) change in basal metabolic rate which occurred in response to first or second change in acclimation temperature in winter (filled symbols) and in summer (open symbols), in male (triangles) and female (circles) Siberian hamsters, which responded (black symbols) or not (grey symbols) to changes in photoperiod. Note that hamsters assigned to group A (panel A and C) and group B (panel B and D) were presented on different panels. See Material and Methods section for details of acclimation procedure.