Introduction

Metabolic rate, i.e., the rate at which organisms acquire and expend energy, is a fundamental aspect of an animal's physiology, forming a link from the first principles of physics and chemistry to the biology of individual organisms (Brown et al. 2004;Lovegrove 2019). For vertebrates, two key boundaries of animal energetics are the maximal rate of oxygen consumption attained during exercise (VO2max) and the resting (RMR) or basal (BMR) metabolic rate (usually also measured as O2 consumption) (Hulbert and Else 2004). In general, these extremes set the upper and lower bounds for energy expenditure (although exceptions exist, e.g., cold-induced summit metabolism in small mammals can exceed VO2max (Chappell and Hammond 2004;Andrew et al. 2019)), and some (e.g., Biro et al. 2018) have argued that the difference between these bounds (i.e., aerobic scope) may constrain variability in the expression of some behaviors. A recent meta-analysis found that various measures of whole-animal metabolic rate (including resting and maximal metabolic rates) were not strongly related to aspects of movement behavior (e.g., activity in familiar environments, exploration of novel environments, dispersal) at the level of individual variation (Wu and Seebacher 2022). From a macroevolutionary perspective, Boratyński (2020) compared 52 species of mammals and found that home range size (corrected for body size) was positively correlated with VO2max but negatively correlated with BMR, thus suggesting that "aerobic scope plays a prominent role in constraining home ranges" (p. 468) (see also Albuquerque et al. 2015). A potential link between metabolic rate and behavior underlies the aerobic capacity model for the evolution of vertebrate endothermy, which posits that directional selection favored high levels of sustained aerobic physical activity, which required an increase in VO2max and, due to unspecified linkages with VO2max, also increased BMR (Bennett and Ruben 1979;Taigen 1983;Hayes and Garland 1995).

On first principles, maximal and resting rates of O2 consumption should be positively correlated, given that the two metabolic states share many organ systems (e.g., cardiovascular), cellular properties (e.g., mitochondrial density), and metabolic pathways (Bennett and Ruben 1979). First principles do not, however, support a 1:1 correlation between VO2max and BMR (as pointed out by Taigen (1983)), given that different tissues account for the bulk of O2 consumption at rest versus during activity. During sustained activity, skeletal muscle is responsible for the bulk of O2 consumption, but has a relatively low metabolic rate when an animal is at rest (Weibel et al. 2004). At rest, O2 is mainly consumed by visceral organs and the brain (Konarzewski and Diamond 1995;Książek et al. 2004;Konarzewski and Książek 2013), whose collective metabolic rates are relatively low during sustained, aerobically supported activity. Nevertheless, the brain and some visceral organs (e.g., heart, liver) may remain quite active during periods of activity and have been consistently correlated with VO2max and/or BMR at the level of individual variation (e.g., Garland 1984;Konarzewski and Diamond 1995;Chappell et al. 1999;Książek et al. 2004;Rezende et al. 2006b;Gębczyński and Konarzewski 2009;Konarzewski and Książek 2013), which should result in some degree of positive correlation between the two metabolic states.

Alternatively, a mechanistic link between VO2max and BMR may stem from cellular properties. Mitochondria consume O2 and produce ATP via a series of protein complexes embedded in its inner membrane. This inner membrane can be "leaky," decoupling O2 consumption and ATP production. This leakiness is a major contributor of BMR (Else and Hulbert 1987;Else et al. 2004). Therefore, higher mitochondrial densities could provide the capacity for higher rates of O2 consumption, but at the cost of a higher resting rate of O2 consumption (Else and Hulbert 1981;Hulbert and Else 1989;Hulbert et al. 2006), although the general application of this as a unifying explanation is debated (Konarzewski and Książek 2013).

Empirical studies have tested for a positive correlation between VO2max and BMR at several levels. For example, an allometric comparison indicated that both VO2max and BMR average ~6 times higher in mammals as compared with lizards, with the ratio of VO2max/BMR or SMR being ~9 for both lineages (Garland and Albuquerque 2017, see their Table 1). Among species within lineages, a comprehensive comparative analysis of 176 vertebrate species (including fish, amphibians, reptiles, birds, mammals) found a positive correlation between residual VO2max and BMR or SMR (Nespolo et al. 2017). Among individuals within species, Pough and Andrews (1984) found no correlation between residual exercise VO2 and either standard or resting rates of O2 consumption in the lizard Chalcides ocellatus (see also : Garland 1984), whereas Chappell and Bachman (1995) reported a significant positive correlation between residual VO2max and BMR in the wild rodent Spermophilus beldingi. With respect to quantitative genetics, Dohm et al. (2001) reported a positive additive genetic covariance between residual VO2max and BMR in an outbred strain of laboratory house mice, but only in a reduced model containing additive and environmental variance. Hence, the authors advocated that their results be interpreted with a degree of caution, given potential biases resulting from imposed modelling constraints. Similarly, Sadowksa et al., (2005) found that the additive genetic covariance between VO2max and BMR in bank voles was also positive, and was significant across several models (i.e., potentially more robust). Overall, interspecies comparisons generally report a positive correlation between maximal and resting rates of O2 consumption, whereas comparisons within species are less consistent (Auer et al. 2017;Nespolo et al. 2017).

One fairly direct way to test for correlations among physical activity behavior, wholeanimal metabolic rates, and lower-level traits is through replicated selection experiments, which allow for the study of evolution in real time and in response to well-defined and reproducible selective regimes (Swallow and Garland 2005;Swallow et al. 2009a,b;Storz et al. 2015). Several selection experiments have tested for a positive relationship between VO2max, BMR, and lowerlevel traits. For example, Książek et al. (2004) selectively bred two lines of laboratory mice for high versus low mass-corrected BMR. Starting at generation 7, the between-line difference in mass-corrected BMR increased, and after 19 generations of divergent selection, the between-line difference in BMR was 8.9 mLO2/h, equivalent to ~2.3 phenotypic standard deviations. This increase in BMR (+18%) was accompanied by a larger small intestine, liver, kidneys, and heart in the high-BMR mice. However, the low-BMR mice had significantly higher (+4%) VO2max (elicited by forced swimming) than those from the high BMR group, contradicting the idea that VO2max and BMR are positively related (Książek et al. 2004). Similarly, Gębczyński and Konarzewski (2009) found that 10 generations of selection for high body-mass corrected VO2max (elicited by forced swimming) in laboratory mice resulted in a 12% increase in VO2max, but no change in BMR. Additionally, VO2max was positively correlated with higher masses of gastrocnemius muscles and heart, but not other visceral organs (intestine, stomach, liver, and kidneys). Using a colony of wild-derived bank voles (Myodes glareolus), Sadowksa et al. (2015) conducted a multiway artificial selection experiment meant to mimic an adaptive radiation, with four lines each bred for either high aerobic metabolism during forced swimming, predatory behavior on crickets, or ability to maintain body mass on a low-quality plant diet. After 11 generations, mass-corrected VO2max and BMR were both significantly higher in the four swimming-selected lines as compared with four non-selected control lines, although the magnitude of these increases differed greatly (+49% in VO2max, +7.3% in BMR). Finally, Wone et al. (2015) bred four replicate lines of laboratory house mice for high mass-independent VO2max during forced treadmill exercise, four other lines for high VO2max and low BMR, and maintained four non-selected controls. After eight generations, VO2max significantly increased (+11%) in lines bred for high VO2max, while BMR had not significantly changed (+2.5%). In the antagonistically selected lines, VO2max increased (+5.3%) while BMR decreased (-4.2%, not statistically significant), which, while it does not falsify the notion that VO2max and BMR may be linked, provides support for the independent evolution of the metabolic traits.

None of the aforementioned selection experiments directly tested the specific scenario proposed by the aerobic capacity model (Bennett and Ruben 1979), which has been more broadly interpreted as suggesting a fundamental link between physical activity behavior, VO2max, and BMR that is of general applicability to vertebrates (Taigen 1983;Hayes and Garland 1995). We address this scenario with a well-established mouse model in which four replicate High Runner (HR) lines have been bred for high voluntary wheel-running behavior and are compared with four non-selected Control (C) lines (Swallow et al. 1998a;Garland 2003;Wallace and Garland 2016).

In a sample of females from generations 88 and 96, we measured maximal and basal rates of O2 consumption, and recorded the mass of the kidneys, spleen, liver, brain, heart (ventricles), and lungs. We hypothesized that: (1) selection for high voluntary exercise would have resulted in an increased VO2max for HR mice, (2) that HR mice would also have an increased BMR, and (3) that some organs (e.g., brain, heart, liver), which are quite active during aerobic exercise as well as under basal conditions, would be increased in HR mice. Although several previous studies have reported elevated VO2max in the HR lines (e.g., Rezende et al. 2005Rezende et al. , 2006b,a;,a;Kolb et al. 2010;Dlugosz et al. 2013b), only one previous study reported BMR (of aged individuals), finding no statistical difference between the HR and C lines (Kane et al. 2008).

Materials and methods (a) Mouse model

For logistical reasons, we sampled from two generations of an ongoing selection experiment for high voluntary wheel-running behavior (Swallow et al. 1998a;Garland 2003;Careau et al. 2013;Wallace and Garland 2016): 50 females from generation 88 and 22 from generation 96. Only females were used because: 1) the number of mice per day that could be tested for VO2max and BMR was limited, such that a smaller sample size or lengthier testing period were required; and 2) females can be housed four per cage as adults, whereas males often need to be individually housed to prevent fighting. The delay between generations was approximately 2 years and was primarily the result of COVID-19-related restrictions on personnel and research. Individuals were measured for VO2max, BMR, and organ masses, but were not exposed to wheels at any time; thus, they represent "baseline" or untrained conditions. Additionally, we collected wheel-running data from siblings that were part of the routine selective breeding procedures. For each generation of the selection experiment, mice are housed four per cage by sex from weaning (21 days of age) until ~6-8 weeks of age, when they are housed individually with access to an activity wheel (1.12-m circumference). Over six days, wheel revolutions are recorded in 1-minute intervals. For the four replicate HR lines, the highest-running male and female from each family are chosen as breeders for the next generation, with no sibling pairings allowed. For the four replicate C lines, breeders are chosen without regard to wheel running (Swallow et al. 1998a;Careau et al. 2013). Animals were maintained in accordance with NIH guidelines, and all procedures were approved by the IACUC of UCR, which is accredited by AAALAC.

The original base population of mice used to start the selection experiment included individuals with hindlimb muscles that were ~50% smaller than normal-muscle individuals (Garland et al. 2002;Houle-Leroy et al. 2003). This "mini-muscle" phenotype is caused by a single nucleotide polymorphism that acts as a Mendelian recessive and was present at a frequency of ~7% in the base population (Kelly et al. 2013). The phenotype was only ever observed in one C line and in two HR lines. The phenotype eventually disappeared from the C line, became fixed in HR line 3, and remains polymorphic in HR line 6 (Hiramatsu et al. 2017;Cadney et al. 2021;Castro et al. 2021). Of the 72 mice used here, all 14 in HR line 3 had the mini-muscle phenotype (as expected) and 5 of the 22 mice in HR line 6 had the mini-muscle phenotype.

Although the mice used within this study never had access to a running wheel, we did have the wheel-running data from their siblings, which were part of the overall selection experiment.

Briefly, mice are housed with access to an exercise wheel (1.12-m circumference) for six days.

During this period, we record the wheel revolutions in each 1-minute interval over a period of 23 hours. We then compute the total number of revolutions (i.e., daily running distance), the number of 1-minute intervals that had at least one revolution (i.e., minutes of wheel activity), the mean revolutions per minute (i.e., average running speed), and the maximum revolutions per minute (i.e., maximum running speed) (Koteja and Garland 2001;Hiramatsu and Garland 2018). A measure of wheel freeness is also included as a covariate in analyses of wheel running (e.g., Girard et al. 2007;Kolb et al. 2010).

(b) Maximal O 2 consumption

VO2max was measured in an enclosed wheel metabolic chamber (effective volume 900 mL; ~15 cm diameter), as described in (Dlugosz et al. 2013a). Briefly, an upstream mass flow controller set incurrent air flow to ~2,000 mL/min. Excurrent air was subsampled at ~150mL/min, scrubbed of H2O and CO2 by Drierite and soda lime, respectively, and directed to an O2 sensor. Data from an O2 analyzer (Applied Electrochemistry Inc., S-3A) were recorded in 1-second intervals on a computer equipped with a National Instruments A-D converter and LabHelper software (M.A.

Chappell, Warthog Systems, www.warthog.ucr.edu).

Each mouse was tested twice, with a day of rest between trials. The repeatability of VO2 achieved in forced running trials was determined by performing a paired Student's t-test using the raw VO2 values, which tests for differences in the average values from one day to the next. In addition, after regressing the VO2 of each trial on its corresponding body mass and age, we performed a paired Student's t-test of the residuals. Pearson's correlation and the associated t-and p-values for these tests are reported below. Mice averaged 58 days of age (range 50-66 days) at the start of testing, were randomized with regard to time of day and testing order, and all tests were performed at 22-25°C during the photophase. Each test was less than 10 minutes and consisted of: 1) 1-minute reference reading of incurrent air at the start; 2) ~1-minute adjustment period after the mouse was placed in the chamber; 3) testing period wherein (a) the wheel was manually propelled by one of two researchers across all measurements, (b) the initial speed was used to elicit a walking pace from the mouse, (c) the researcher accelerated the wheel (by hand) approximately every 30 seconds, (d) which continued until VO2 did not increase for ~3 minutes or the mouse could not continue running, 4) ~1-minute recovery period before removal from chamber; and 5) 1-minute reference reading. The same protocol was applied to all mice in this study, and so any measurement error should be comparable across individuals. Values reported here are similar to those previously reported for these mice, both when using a treadmill (Rezende et al. 2005 p. e.g.,; Kolb et al. 2010) or the wheel apparatus (e.g., Claghorn et al. 2017;Cadney et al. 2022).

After every trial, each mouse was given an objective score of exhaustion, indicated by the number of seconds after the trial before the mouse began walking again. These data were analyzed on a scale of 1 to 5, where an exhaustion of 1 indicated that 1 second had elapsed and an exhaustion of 5 indicated that 5 or more seconds had elapsed. Additionally, each mouse was given a subjective score of overall cooperativity, indicated by whether the mouse consistently ran with the direction of wheel rotation, or sometimes ran in the opposite direction. This was also on a scale of 1 to 5, where a cooperativity score of 1 indicated the mouse did not run when prompted, and a cooperativity score of 5 indicated the mouse attempted to run even at the highest speeds. In cases of uncooperative mice (e.g., cooperativity scores of 1 or 2) or technical difficulties, a third trial was conducted (N = 16) and used to replace the poor trial.

Warthog LabAnalyst software recorded %O2 and flow rate of incurrent air. LabAnalyst was used to smooth metabolic data via a nearest-neighbor algorithm, and the 'instantaneous' transformation was used to resolve rapid changes in respiration (Bartholomew et al. 1981). VO2 was calculated as:

where V is flow rate (mL/min STP; standard temperature and pressure), and FIO2 and FEO2 are the fractional O2 concentrations in the incurrent and excurrent air, respectively. For each mouse, the highest 1-minute continuous average for each trial was calculated and the highest VO2 of any trial was used as VO2max for subsequent analyses.

(c) Basal O 2 consumption

Basal metabolic rate (BMR) was determined by measuring O2 consumption at rest in postabsorptive mice at ~32°C (within their thermal neutral zone: Lacy and Lynch 1979). The setup for recording BMR was similar to that for VO2max, except incurrent air flow was ~500mL/min, mice were in plastic respiration chambers (10cm x 7.5cm x 7.5cm), and excurrent air was subsampled at ~100mL/min.

Mice were separated into two groups, and tests began at either 1200-or 1600-hours PST.

Mice averaged 68 days of age (range 57-80 days) at the start of testing. Food was removed four hours prior to testing, which is adequate for obtaining a postabsorptive state in mice (Jensen et al. 2013). Mice were tested over a period of 4 hours, wherein excurrent air was subsampled for 45 minutes, then incurrent air was subsampled for 15 minutes. Two mice were measured at a time using separate channels (Sable Systems International., Oxzilla). Four mice were tested each day, over a period of 13 days. Analysis of BMR was the same as for VO2max, except data were recorded in 2-second intervals and the lowest 5-minute continuous average was used. For each of the lowest values, we verified that the trace was stable, thus indicating that animals were at rest.

(d) Dissection

Mice were euthanized by decapitation without anesthesia (average age 76 days, range = 70-81) and blood samples were immediately collected from the trunk via heparinized micro-hematocrit tubes, then spun in a micro-hematocrit centrifuge for 5 minutes. Approximately four samples were collected for each mouse and readings were averaged. The whole brain, heart ventricles, kidneys, liver, lungs, spleen, and the triceps surae muscle group were each collected and weighed.

(e) Whole-body, lean, and fat mass All mice were weighed at weaning, before VO2max, before and after BMR trials (average used), and before dissection. Body composition was measured by non-invasive quantitative magnetic resonance (EchoMRI-100; Echo Medical Systems LLC, Houston, Texas, USA), which independently calculated fat and lean mass, after the first VO2max trial, after the BMR trial, and before dissection.

(f) Statistical analyses

Statistical analyses were performed using SAS Proc Mixed v15 (SAS Institute, Cary, NC, USA).

HR and C lines were compared by a mixed model, using the restricted maximum likelihood (REML) method, with linetype and mini-muscle status as fixed effects. Replicate line (4 HR and 4 C) was nested within linetype as a random effect using the containment method for d.f., such that the d.f. for linetype were always 1 and 6. We tested the significance of the random effect of the replicate lines using the COVTEST option. This yields the estimates, standard errors, and statistical significance of any covariance parameters, which were restricted to non-negative covariance estimates. The effect of replicate line was never statistically significant for any measured trait (Tables 1,2). For wheel-running traits, separate variances were allowed for HR and C lines, as previous studies have established a greater variability in wheel-running behavior among HR lines (Garland et al. 2011). We also checked for any interactions between body mass and linetype for all measured traits (i.e., heterogeneity of slopes). None were statistically significant, and thus were not included in the final model (i.e., slopes for body mass were assumed to be homogenous). Generation was included as a random effect in preliminary analyses, but was never statistically significant, and thus was removed from final analyses. Additionally, some values were removed due to known problems (e.g., loss of tissue during dissection, equipment malfunction) prior to analysis. Outliers were removed when the standardized residual was greater than ~3 standard deviations and/or the difference from the next value was greater than ~1 standard deviation. Least Squares Means (LSMs) and associated standard errors are presented to compare HR with C lines and mini-muscle versus normal mice.

Correlations between VO2max, BMR, and relevant organ masses were calculated in two ways: (1) for individual mice, using the standardized residuals for each trait, derived from each of the SAS Proc MIXED analyses with linetype and mini-muscle status as fixed effects, and line nested within linetype as a random effect; (2) for line means, using the Least Squares Means (LSMs) derived from SAS Proc MIXED analyses with "line" (N = 9, separating mini-and normalmuscle mice in Line HR6) as a fixed effect. Finally, a multiple regression analysis was performed (listwise deletion of data; p to enter = 0.05) to test for combined predictors of VO2max.

Results (a) Sibling wheel-running behavior

To avoid any training effect on the comparison of VO2max, BMR, or organ mass, focal mice did not receive wheel access. However, for their female siblings, HR mice ran ~3-fold more revolutions/day than C mice (Figure 1A, Table S1). This increase in wheel-running behavior was caused primarily by a significant increase in average running speed (+142%: Figure 1C, Table S1), accompanied by a non-significant increase in running duration (+27%: Figure 1B, Table S1).

Additionally, maximum running speed was significantly higher in HR mice (+94%: Figure 1D, Table S1).

(b) Body, lean, and fat mass HR mice did not significantly differ from C mice for whole-body, lean, or lean-adjusted fat mass when measured at VO2max, BMR, or dissection, although HR mice did have consistently less lean-adjusted fat mass at each measurement (Tables 1 and2). Mini-muscle mice were significantly smaller when measured at VO2max, BMR, and dissection, due to decreased lean mass, but also had increased lean-adjusted fat mass at VO2max, BMR, and dissection (Tables 1 and2).

(c) Maximal and basal rates of O 2 consumption

The VO2 achieved in forced running trials was repeatable (r = 0.821, P < 0.0001), although the latter of the VO2 trials was consistently higher (t = 2.554, P = 0.013). Additionally, after regressing each value on its respective body mass, residual VO2 was also repeatable (r = 0.751, P < 0.0001).

The mass corrected VO2max (higher of the two VO2 values) of HR mice was ~13.6% higher than that of C mice (Figure 2A, Table 1). However, mass-corrected BMR did not significantly differ between HR and C mice, although HR mice had +6.5% higher mass-corrected BMR than C mice (Figure 2C, Table 1). Mini-muscle status did not significantly affect either VO2max or BMR (Figure 2A andC, Table 1). However, when lean mass was used as a covariate (see above), VO2max was ~4.6% higher in mini-muscle mice (Figure 2B, Table 1). Mini-muscle mice were also significantly more exhausted after VO2max (Table 1).

(d) Organ masses HR mice did not significantly differ from C mice for any mass-adjusted organ mass, nor did they differ in hematocrit; however, they tended to have smaller lungs than C mice (Figure S1, Table 2).

Mini-muscle mice had ~50% less hindlimb muscle mass, as expected (Garland et al. 2002;Houle-Leroy et al. 2003), had significantly larger livers and lungs, and tended to have larger kidneys and spleens (Figure S1, Table 2).

(e) Correlations

VO2max and BMR were not significantly correlated at the level of residual (individual) variation or for line means (with HR line 6 split into normal and mini-muscle individuals, i.e., based on nine values) (Table 3). However, residual VO2max and ventricle mass were significantly positively correlated among all individuals and for line means (Table 3). BMR and ventricle mass were significantly positively correlated among line means, but not among individuals (Table 3). Masscorrected VO2max and BMR were not significantly correlated with any other lower-level trait (Table 3). In a forward regression analysis (listwise deletion of data; p to enter = 0.05), only residual ventricle mass entered (N = 65). Correlations among organ masses are presented in Supplemental Table S2.

Discussion

Several alternative, though not necessarily mutually exclusive, hypotheses have been proposed to explain the often-observed positive relationship between VO2max and BMR, at the level of proximate and/or ultimate causation. The aerobic capacity model (Bennett and Ruben 1979) suggests that, with respect to ultimate causation, selection for high levels of sustained aerobic activity would require an increase in VO2max, and that an increase in BMR would also occur due to hypothetical mechanistic linkages (proximate causation). Although originally proposed in the context of the evolution of avian and mammalian endothermy, this model might also apply more generally. As outlined in the Introduction, empirical studies have provided mixed support for this model at several levels. However, a direct test of the primary assertions of the aerobic capacity model has not previously been conducted.

Here, we used an ongoing artificial selection experiment wherein mice are bred for high voluntary exercise behavior during days 5 and 6 of a 6-day exposure to wheels (Swallow et al. 1998a;Garland 2003;Wallace and Garland 2016). After 10 generations of selection, mice from the HR lines ran, on average, ~75% more revolutions per day than those from the C lines, and had ~7% higher body mass-corrected VO2max (Swallow et al. 1998a,b), though BMR was not measured. In later generations, HR mice reached a selection limit at which they ran approximately three-fold more than C mice on a daily basis (Careau et al. 2013), which has remained true across tens of generations (e.g., see Singleton and Garland 2019;Cadney et al. 2021;McNamara et al. 2022;present study). HR mice also have higher activity in home cages when housed individually without wheels (Malisch et al. 2009), and higher food consumption, both with and without wheels (Copes et al. 2015; see also Rezende et al. 2009), as compared with C mice. Several additional studies have reported VO2max, and most have verified higher values for HR lines (Rezende et al. 2005(Rezende et al. , 2006a,b;,b;Kolb et al. 2010;Dlugosz et al. 2013b). However, the only study of BMR found no significant effect of selection, did not measure VO2max or any organ masses, and used mice that were far older (~22.5 months) than the normative wheel-testing age (~2 months) for the selection experiment (Kane et al. 2008). Thus, more information has been needed to determine whether the HR mouse model supports the aerobic capacity model of vertebrate energetics. In the present study, HR mice had significantly higher VO2max (+13.6%, Table 1, Figure 2A), but did not have significantly higher BMR (+6.5%, Table 1, Figure 2C). Additionally, VO2max and BMR were not correlated at any level (e.g., among individuals or replicate lines, Table 3). Finally, aside from the positive correlation between ventricle mass and VO2max among individuals (consistent with a previous study: (Rezende et al. 2006b)) (but not among replicate lines; Table 3), and between ventricle mass and BMR among replicate lines (but not among individuals; Table 3), VO2max and BMR were not correlated with any other organs. Thus, the two metabolic states do not appear mechanistically linked through the lower-level traits measured here. Overall, our results offer limited support for the aerobic capacity model, consistent with the three rodent selection experiments that targeted VO2max and/or BMR (see Introduction).

Beyond the aerobic capacity model, our results, and the HR selection experiment as a whole, may offer insights into other hypotheses regarding links between VO2max, BMR, and other traits (Hayes and Garland 1995;Hillman et al. 2013;Careau et al. 2015;Auer et al. 2017).

For example, under the assimilation capacity model (Koteja 2000), selection favors high-intensity parental care, especially the feeding of juveniles, which requires higher daily energy expenditure (e.g., due to foraging (see also Farmer 2000)), and thus an increased rate of energy processing.

Here, BMR increases as a correlated response to the increased capacity of the alimentary tract, as these organs are a primary contributor toward BMR (Konarzewski and Diamond 1995;Książek et al. 2004;Konarzewski and Książek 2013). HR mice in the present study did not have statistically larger internal organs (e.g., liver, kidney) (or BMR) and have not been shown to have a larger alimentary tract (Kelly et al. 2017), although mini-muscle mice (a subset of the HR mice) have higher stomach dry mass and longer small intestines (Kelly et al. 2017). Additionally, Koteja (2000) proposed that high daily energy expenditure was driven by high-intensity parental care, which has not been found to differ in an important way between HR and C lines (Girard et al. 2002;Keeney 2011).

In closing, we note that the HR mouse selection experiment is relevant to the "behavior evolves first" model (e.g., see Blomberg et al. 2003;Huey et al. 2003;Rhodes and Kawecki 2009). Specifically, our results demonstrate that selection for a behavioral trait can result in very large changes (in our case, an ~ 3-fold increase in daily running distance; Table S1, Figure 1), without large modifications to underlying morphological or physiological traits (here, only a 13.6% increase in VO2max (Table 1, Figure 2A), a 6.5% increase in BMR (Table 1, Figure 2C), and no statistically detectable changes in organ masses or hematocrit (Table 2, Figure S1).