The near constancy of hibernation mass-specific metabolic rate regardless of body size: Insights from ontogeny and comparative physiology

The study of which life history traits primarily affect molecular evolutionary rates is often confounded by the covariance of these traits. Scombroid fishes (billfishes, tunas, barracudas, and their relatives) are unusual in that their mass-specific metabolic rate is positively associated with body size. This study exploits this atypical pattern of trait variation, which allows for direct tests of whether mass-specific metabolic rate or body size is the more important factor of molecular evolutionary rates. We inferred a phylogeny for scombroids from a supermatrix of molecular and morphological characters and used new phylogenetic comparative approaches to assess the associations of body size and mass-specific metabolic rate with substitution rate. As predicted by the body size hypothesis, there is a negative correlation between body size and substitution rate. However, unexpectedly, we also find a negative association between mass-specific metabolic and substitution rates. These relationships are supported by analyses of the total molecular data, separate mitochondrial and nuclear genes, and individual loci, and they are robust to phylogenetic uncertainty. The molecular evolutionary rates of scombroids are primarily tied to body size. This study demonstrates that groups with novel patterns of trait variation can be particularly informative for identifying which life history traits are the primary factors of molecular evolutionary rates.

It is unusual to re-visit a paper a half-century after first reading it, as I have done with the classic publication of Per Scholander, Raymond Hock, Vladimir Walters and Lawrence Irving published in The Biological Bulletin in 1950 (Scholander et al., 1950). My first reading of this paper, and others by Scholander and colleagues on temperature adaptation in aquatic and terrestrial ectotherms (Scholander et al., 1953; Scholander et al., 1957), occurred around 1962, early in my doctoral studies when I trekked to Antarctica to study cold adaptation in fish. These papers provided much of the conceptual and empirical foundation for the work I anticipated doing with notothenioid fishes. My interests in polar birds and mammals at that time were largely confined to their roles as photographic subjects. However, my reading of the 1950 Biological Bulletin paper turned out to be highly important in broadening my scientific horizons and helping me to formulate a perspective on evolutionary adaptation to temperature that spanned the full range of biological (body) temperatures and the adaptive patterns observed among all taxa, whether endothermic or ectothermic. A fresh reading of this paper by scientists of my generation and, even more, a first reading by scientists whose parents may not have been born when the paper first appeared, still has much to offer in teaching us about thermal biology and how scientific research in comparative and evolutionary physiology has evolved.The studies of thermal biology by Scholander and colleagues that were initiated shortly after the end of World War II and conducted with major logistical support from the US military helped in important ways to define the evolutionary strategies used by endothermic homeotherms – mammals and birds – to cope with differences in ambient temperature. Importantly, the focus of these initial studies was on evolutionary adaptation, not phenotypic acclimation or acclimatization to cold, phenomena that later took center stage in analysis of thermal biology in endotherms. The 1950 paper was one in a series from this group that elucidated the roles of what they termed three possible ‘avenues for cold adaptation’ in mammals and birds: variations in the body-to-air thermal gradient, insulation level and basal metabolic rate. In other words, how were heat production and heat exchange altered to allow mammals and birds to maintain a stable and high body temperature? The previous papers by these authors, plus earlier literature cited in the 1950 paper, led to the tentative conclusion that adaptive changes in body temperature to reduce the body-to-air thermal gradient and, thereby, reduce heat flux between animal and environment, are unlikely to play a role in cold adaptation in active adult mammals and birds. To quote the 1950 paper, ‘…there are no signs so far that body temperature of mammals and birds is adaptive to the different climates on earth.’ This well-buttressed conclusion leads to another – and yet unanswered – set of questions: What is the basis for selection of temperatures near 37–40°C for avian and mammalian core body temperatures? Is there something special about this particular range of temperatures? If so, what is it?The remaining two ‘avenues for cold adaptation’ had also been partially explored in earlier studies by Scholander and colleagues. It was clear from their work and that of others that insulation played a critical role. As stated in the 1950 paper, ‘…we established, by insulation measurements, the general rule that arctic mammals have warmer furs than do tropical mammals.’ However, this adaptive mechanism, while of importance, could not at that time be regarded as offering the full story. The final ‘avenue for cold adaptation’, the role of evolved temperature-adaptive differences in basal metabolic rate, remained a possible mechanism of cold adaptation, with animals from cold polar environments possibly exhibiting higher metabolic rates per unit mass than animals from warmer temperate locations. Most existing basal metabolic rate data were from studies on temperate species; there was not an adequate diversity of environments represented in these data sets to fully evaluate whether basal metabolic rates reflected evolutionary thermal history. To resolve this issue, it was necessary to conduct additional studies of mammals and birds from thermal environments that were as different as possible in terms of maximal temperature. Thus the dual focus – Arctic versus tropical – of the studies presented in the 1950 paper. The metabolic rate measurements made with endemic species from Point Barrow, Alaska, and Barro Colorado Island in the Central American tropics essentially put the final nail in the coffin of this potential mechanism of adaptation to cold. The metabolic rate measurements presented in the 1950 paper were made with animals spanning a wide range of sizes and rate measurements were made as closely as possible under ‘resting’ or ‘basal’ conditions and in the absence of acute cold stress. Appropriately, the data gathered by Scholander et al. were analyzed in the context of Francis Benedict's famous ‘mouse to elephant’ relationship of metabolic scaling (Benedict, 1938). The results of these experiments seemed unambiguous: ‘…we may state as a tentative generalization that the basal metabolic rate of terrestrial mammals from tropics to arctic is fundamentally determined by a size relation according to the formula Cal./day = 70 kg3/4 and is phylogenetically nonadaptive to external temperature conditions. Equally nonadaptive is the body temperature, and the phylogenetic adaptation to cold therefore rests entirely upon the plasticity of the factors which determine the heat loss, mainly the fur insulation.’ Thus, natural selection seemed not to have packed more metabolic capacity into a gram of an arctic mammal than into a gram of a similar-sized tropical mammal. At least this seemed to be the case when analyses involved normothermic adult animals, such as those used by Scholander et al. However, as the caveat in the above quotation (‘tentative generalization’) seems to hint, later work showed that there was more to the story.As is the case of many foundational papers, observations and comments made in the 1950 paper seem prescient when examined in the context of what has subsequently been discovered. One such comment is the brief mention that a small mammal, the arctic weasel, showed an extraordinarily high rate of heat production when exposed to cold; this species' metabolic rate was well above the regression line of the ‘mouse to elephant’ curve. Scholander et al. remarked that it seemed ‘somewhat odd’ that the weasel didn't simply increase its insulation when exposed to cold. This ‘oddity’ and similar observations on other small, cold-stressed mammals helped to pave the way for the discovery of the thermogenic role of brown adipose tissue (BAT) (Cannon and Nedergaard, 2004; Smith, 1961). The discovery of this specialized heat-generating tissue casts metabolic adaptation to cold by mammals (no avian equivalent of BAT is known) in a new light. The importance of heat generation by BAT is arguably the most significant extension of our understanding of thermal biology in mammals made since the 1950 paper was published. I'd like to think that the ‘oddity’ noted by Scholander and colleagues puzzled readers enough to catalyze a deeper look at non-basal metabolic capacities, namely those of BAT, the one type of thermogenic tissue known in mammals.A second easily missed point in the 1950 paper is one that is not explicitly indicated by the paper's title. In addition to addressing the adaptations required to withstand cold, Scholander et al. pay heed to the challenges faced by tropical mammals and birds. Thus they state, ‘It seems then that the problem for tropical mammals is neither overheating nor cooling, but, actually, both.’ Moreover, the authors emphasize (presciently, as things are turning out) that, ‘Many parts of the tropics are so hot and humid that a few degrees’ rise in the temperature would mean death for mammals and birds because they cannot adapt to it by raising their body temperatures.' My re-examination of this paper over 50 years since first reading it was done in concert with the appearance in 2010 of articles by Sherwood and Huber and McMichael and Dear that dealt with this threat to low latitude mammals and birds, a danger that would have seemed completely ‘academic’ back in 1950 when concerns about global warming – an expression that seems to have been introduced to the scientific literature only in 1975 in a classic paper by Wallace Broecker in Science (Broecker, 1975) – were still well off in the future (Sherwood and Huber, 2010; McMichael and Dear, 2010). In their analysis of the capacities of birds and mammals to cope with the combination of rising temperatures and humidity, these authors build on (but, regrettably, do not cite) the types of studies found in the 1950 classic by Scholander et al. The 2010 papers point out that, as the need for effective evaporative cooling becomes more critical in a warming world, capacities to evaporate water from the body surfaces becomes increasingly challenging. The basic physics of the situation shows that, when wet bulb temperatures (TW) exceed 35°C, evaporative dissipation of metabolic heat by mammals and birds ceases to be possible. How close are we to this situation of ‘melt down’, the exclusion of habitats from occupancy by endothermic homeotherms due to TW values >35°C? Currently, TW never exceeds 31°C, even in the hottest climates (Sherwood and Huber, 2010). However, TW is predicted to rise with global mean temperature at a ratio of 3°C for every 4°C of global warming. The models used to predict rates of global warming offer a range of estimates and are typically limited for extrapolating trends beyond the current century. However, as Sherwood and Huber emphasize, using models based on realistic assumptions about release of greenhouse gases and extending these models beyond 2100, within two or three centuries the temperatures of some regions may increase by ~7°C and, therefore, have combinations of heat and humidity that result in TW near 35°C. Because such a large fraction of the human population resides at low latitudes, Sherwood and Huber conjecture that global warming of 11–12°C would lead to intolerable values of TW in the regions where most of the human population is currently found (Sherwood and Huber, 2010). Moreover, these low latitude regions are zones where the greatest biodiversity is commonly found. The early studies of Scholander and colleagues help to put into sharp relief the challenges faced by endothermic homeotherms in confronting rising temperatures and humidity. Birds and mammals are ‘stuck’, evolutionarily, with high mass-specific metabolic (heat generation) rates. It seems inconceivable that they can evolve a physiological solution that would allow them to cope with the threats posed by TW values greater than 35°C.In summary, reading (or re-reading) this classic paper offers a number of rewards. First, in terms of its primary question about fundamental evolutionary strategies for cold adaptation, we can come to appreciate how the principal ‘avenue’ taken by birds and mammals for maintaining high and stable body temperatures regardless of ambient temperature was elucidated. Second, we find a nice example of how an experimental ‘oddity’ can lead to further work that, in this case, helped open up the broad field of BAT physiology. Third, and certainly without the intention of the authors, the paper has provided a valuable context for evaluating the challenges posed to endothermic homeotherms by global climate change. And, lastly, there are rewards from (re)visiting this work that deal less with specific scientific discoveries than with the ways in which doing science have evolved. These lessons seem especially valuable for younger scientists for whom the practice of reading literature that is more than a year or two old may not exist. In our (usually futile) obsession to stay on top of the current literature, the classics of the past – the formative publications that built the foundation for our field – are too often neglected or just downright forgotten entirely. As Sydney Brenner recently commented, ‘…most scientists are too busy working in the present and thinking anxiously about the future and have no time to view their work in the context of what has gone before’ (Brenner, 2012). Disregarding the past is a shame, for several reasons. One is that, by neglecting the foundational literature of one's field, one is apt to lack an appreciation of how the shaping principles (‘paradigms’) of one's discipline have originated and subsequently evolved. Another benefit of examining the older literature is to get a sense of how different it was to do science at a time when major questions were first being addressed experimentally. For comparative physiology, the first two or three decades after World War II were an era of both intellectual and geographical explorations of exciting new territory. These were halcyon days when opening up entirely new lines of study could be done with simple instruments, minimal needs for costly reagents, and, therefore, relatively tiny budgets (with the exception of travel costs for exploring distant and exotic lands). And, as the working and writing styles of early, intrepid explorers of comparative physiology such as Per Scholander and his collaborators, Knut Schmidt-Nielsen, and others suggest, doing research was probably a lot more fun and adventurous back then. Writing could be a bit more colorful too, compared with the prose found in the space-limited pages of contemporary journals. Can you think of a recent paper in which there occurs something equivalent to a statement that the sample size was low because the sled dogs ate one-third of the fish that were caught (Scholander et al., 1957)? In these incredibly busy times, it is worthwhile to pause, read some of the classics and contemplate what life was like back in the days when the ratio of novelty of discovery to grant dollars expended was arguably relatively high compared with the present. Classic papers such as those published by Scholander and colleagues over a half-century ago give their readers a clear sense of not only how the foundational discoveries of our field were made, but also of the adventure that was associated with these early intellectual and geographical explorations. Having been his faculty colleague for two decades, I think I can safely say that when Per Scholander chose the title for his autobiography, Enjoying a Life in Science (Scholander, 1990), he was at once giving an honest summary of a great career and challenging his readers to follow an example that, while increasingly tough to meet, should still be our goal.

BODY SIZE AND METABOLIC RATE

The hypothesis that sperm competition should favour increases in sperm size, because it results in faster swimming speeds, has received support from studies on many taxa, but remains contentious for mammals. We suggest that this may be because mammalian lineages respond differently to sexual selection, owing to major differences in body size, which are associated with differences in mass-specific metabolic rate. Recent evidence suggests that cellular metabolic rate also scales with body size, so that small mammals have cells that process energy and resources from the environment at a faster rate. We develop the 'metabolic rate constraint hypothesis' which proposes that low mass-specific metabolic rate among large mammals may limit their ability to respond to sexual selection by increasing sperm size, while this constraint does not exist among small mammals. Here we show that among rodents, which have high mass-specific metabolic rates, sperm size increases under sperm competition, reaching the longest sperm sizes found in eutherian mammals. By contrast, mammalian lineages with large body sizes have small sperm, and while metabolic rate (corrected for body size) influences sperm size, sperm competition levels do not. When all eutherian mammals are analysed jointly, our results suggest that as mass-specific metabolic rate increases, so does maximum sperm size. In addition, species with low mass-specific metabolic rates produce uniformly small sperm, while species with high mass-specific metabolic rates produce a wide range of sperm sizes. These findings support the hypothesis that mass-specific metabolic rates determine the budget available for sperm production: at high levels, sperm size increases in response to sexual selection, while low levels constrain the ability to respond to sexual selection by increasing sperm size. Thus, adaptive and costly traits, such as sperm size, may only evolve under sexual selection when metabolic rate does not constrain cellular budgets.

We present an alternative to the traditional classroom lecture on the topics of metabolic scaling, allometric relationships between metabolic rate (MR) and body size, and reasons for rejecting Rubner's surface "law," concepts that students have described as challenging, counterintuitive, and/or mathematical. In groups, students work with published data on MR and body size for species representing all five vertebrate groups. To support the exercise, we developed a worksheet that has students define the concept in their own words, compare different measures of MR, and evaluate plots of MR and mass-specific MR vs. body mass for both homeotherms and poikilotherms. Students also attempt to explain why selected species have exceptionally high or low MR values for their body sizes. Student feedback indicated that active learning is an effective way to learn the concepts of metabolic scaling and allometric relationships and that the opportunity to work in groups with real data stimulates interest and an appreciation for the importance of metabolic scaling to the understanding of animal physiology.NEW & NOTEWORTHY Here we describe a worksheet that we designed for a group exercise in which students study real data to learn about metabolic scaling in different groups of vertebrates, understand that metabolic rates are allometric functions of body size, and consider why physiologists now reject Rubner's surface "law." We used this exercise in a course in animal physiology in place of the traditional lecture approach to teaching the concept of metabolic scaling.

Mass-specific metabolic rate, the rate at which organisms consume energy per gram of body weight, is negatively associated with body size in metazoans. As a consequence, small species have higher cellular metabolic rates and are able to process resources at a faster rate than large species. Since mass-specific metabolic rate has been shown to constrain evolution of sperm traits, and most of the metabolic activity of sperm cells relates to ATP production for sperm motility, we hypothesized that mass-specific metabolic rate could influence sperm energetic metabolism at the cellular level if sperm cells maintain the metabolic rate of organisms that generate them. We compared data on sperm straight-line velocity, mass-specific metabolic rate, and sperm ATP content from 40 mammalian species and found that the mass-specific metabolic rate positively influences sperm swimming velocity by (a) an indirect effect of sperm as the result of an increased sperm length, and (b) a direct effect independent of sperm length. In addition, our analyses show that species with higher mass-specific metabolic rate have higher ATP content per sperm and higher concentration of ATP per μm of sperm length, which are positively associated with sperm velocity. In conclusion, our results suggest that species with high mass-specific metabolic rate have been able to evolve both long and fast sperm. Moreover, independently of its effect on the production of larger sperm, the mass-specific metabolic rate is able to influence sperm velocity by increasing sperm ATP content in mammals.

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)

The Relationship between Summated Tissue Respiration and Metabolic Rate in the Mouse and Dog

Gaseous Metabolism and Water Relations of the Zebra Finch, Taeniopygia castanotis

Metabolic scaling describes the relationship between metabolic rate and body size, and can be calculated using the equation Metabolic Rate = a(Body Mass)B. Juvenile mammals have been reported to have higher exponent (B) values until they reach adult body size experience a critical switch, after which B decreases and scaling is hypometric. This study measured and compared metabolic rate and mass in 13-lined ground squirrels (Ictidomys tridecemlineatus) through a wide range of developmental stages to investigate how mass specific metabolic rate (MSVO2) change throughout parturition (change in metabolism with no change in weight), as well as how MSVO2 scale as pups gain weight though development (weight gain due to growing tissue). Metabolic rates of 6 litters were found to increase roughly 150% upon birth. Developing I. tridecemlineatus pups then displayed a switch from hyper- to hypometric scaling at 20-26 days of age, as marked by a decrease in the exponent B value from 1.20 to 0.76 (adj R2=0.90, p p=2.70E-03; adj. R2=0.67, p=2.84E-02). The critical switch in B was found to occur before reaching adult size, thus indicating factors other than body size may be more influential for scaling during development.

Body Size and Metabolic Rate in Salamanders

The capacity to extract oxygen from the environment and transport it to respiring tissues in support of metabolic demand reportedly has implications for species' thermal tolerance, body size, diversity and biogeography. Here, we derived a quantifiable linkage between maximum and basal metabolic rate and their oxygen, temperature and size dependencies. We show that, regardless of size or temperature, the physiological capacity for oxygen supply precisely matches the maximum evolved demand at the highest persistently available oxygen pressure and this is the critical PO2 for the maximum metabolic rate, Pcrit-max For most terrestrial and shallow-living marine species, Pcrit-max is the current atmospheric pressure, 21 kPa. Any reduction in oxygen partial pressure from current values will result in a calculable decrement in maximum metabolic performance. However, oxygen supply capacity has evolved to match demand across temperatures and body sizes and so does not constrain thermal tolerance or cause the well-known reduction in mass-specific metabolic rate with increasing body mass. The critical oxygen pressure for resting metabolic rate, typically viewed as an indicator of hypoxia tolerance, is, instead, simply a rate-specific reflection of the oxygen supply capacity. A compensatory reduction in maintenance metabolic costs in warm-adapted species constrains factorial aerobic scope and the critical PO2 to a similar range, between ∼2 and 6, across each species' natural temperature range. The simple new relationship described here redefines many important physiological concepts and alters their ecological interpretation.

Should an animal extending its range into a cooler climate rely most on pelage or on body size change to minimize its mass-specific metabolic rate? The various examples of animals following Bergmann's rule support the latter. The fact that an increase in size will result in an increase in total metabolic rate (though coupled to the decrease in the mass-specific metabolic rate) suggests that increases in the insulative value of the pelage would be the preferred strategy. We used a thermal simulation model to compare the relative effects of increasing body mass versus increasing pelage insulative properties on the mass-specific metabolic rate. We found that even the fur of summer-adapted small mammals from temperate climates is extremely dense compared with that of larger mammals and is near the point at which increases in density increase, rather than decrease, heat loss as a result of the high conductivity of individual hairs compared with the layer of still air that it encloses. Small mammals also have lower fur depths, presumably as a result of biomechanical constraints. Seasonal changes in pelage observed in small mammals have very modest effects on mass-specific metabolism. Summer-adapted temperate large mammals, however, are less heavily insulated and, consequently, have substantial latitude for increasing insulation as a means of minimizing mass-specific metabolism. Thus, Bergmann's rule should be more relevant to small mammals than to large ones.

ROSENMANN, MARIO, AND PETER MORRISON. Maximum oxygen consumption and heat loss facilitation in small homeotherms by He-02 . Am. J. Physiol. 226(3) : 490495. 1974.-The high thermal conductance of an 80% He-20y0 02 atmosphere was used to elicit maximum metabolism (Mmax) in moderate cold in species ranging from 7-g pygmy mice (Baiomys taylori) to 250-g white rats, including redpolls (Acanthis J?ammea), two vesper mice (Calomys d&la, C. callosus), tundra voles (Microtus oeconomus), and four strains of Mus musculus. Values slightly exceeded those in similar animals using other methods to confirm the low metabolic ratio (Mmax/M, in) in rodents (4-8 X). Submaximal values at higher temperatures defined thermal conductance in He-02 and air. In different species the ratios of these conductances ranged from 1.4 to 2.6, differences which relate to the extent and quality of the respective insulation. M,,, was obtained at 13-70°C warmer in He-02 than required in air for the same metabolic effort. Avoidance of low-temperature technology and freezing injury, elimination of treadmills and training in running, prompt attainment of M,,., (3-10 min after He-02 exposure), and obviation of shaving or wetting procedures are advantages of the present technique.

Organismal metabolic rate is linked to environmental temperature and oxygen consumption, and as such, may be a useful predictor of extinction risk. This is especially true during major climate-driven extinctions, given the tightly linked stressors of warming and hypoxia. However, metabolic attributes can be quantified in different ways, highlighting differing aspects of organisms’ ecology. Here, we estimate resting whole-body and mass-specific metabolic rates in post-Carboniferous bivalve taxa using body size, seawater paleotemperature, and a taxon-specific adjustment factor to assess how metabolic rate correlates with survival both during and outside intervals of rapid climate warming, or “hyperthermals.” Accounting for the effects of geographic range size, we find a pattern of preferential extinction of bivalves with lower total calorific needs, consistent with increasing body size and the postulated ramping up of ecosystem energetics over the Meso-Cenozoic. Contrary to expectations, extinction selectivity based on total calorific needs, which emphasizes body size, does not differ between hyperthermals and other time intervals. However, a higher metabolic rate per gram of tissue—which is more strongly determined by environmental temperature than by body size—consistently increases the probability of extinction during hyperthermals relative to baseline conditions, particularly within the paleotropics. This serves to highlight the potential significance of environmental temperature on metabolic performance, particularly in organisms that are already living close to their thermal limits. In tandem with previously documented patterns of extinction selectivity based on relative activity levels, including motility and feeding style, these results enhance our understanding of the role of metabolic rate through time and during climate-driven extinctions. When standardized by mass, metabolic rate may represent a useful metric through which to predict the effects of anthropogenic climate change on modern marine faunas.