Physiological correlates of habitat association in East African sunbirds (Nectariniidae)

East African sunbirds (Nectariniidae) vary in the degree to which they use open habitats and forest habitats. Species that use open habitats may experience more extreme temperatures and greater exposure to solar radiation than those in forest habitats. Basal rates of metabolism, body temperature and thermal conductance were compared for open habitat- and forest-associated sunbirds from Kibale National Park, Uganda. Variation in basal rate of metabolism was associated with body mass, but there was no difference between forest and open habitat species. Variation in body temperature was not associated with body mass or habitat. Variation in thermal conductance was associated with body mass and habitat; open habitat species were characterized by significantly lower thermal conductances than forest species. Because reduced thermal conductance may decrease energy expenditure at low ambient temperatures and reduce exogenous heat gain at high ambient temperatures, this difference may optimize energy expenditure when temperatures are highly variable. This suggests a mechanism by which physiological characteristics may influence energetic consequences of habitat selection.     

Competition between Eurasian red and introduced Eastern grey squirrels: the energetic significance of body-mass differences

Daily energy expenditure (DEE) was measured in sympatric populations of red and grey squirrels using the doubly labelled water technique. Grey squirrels had significantly higher DEEs than red squirrels. However, the difference between the species was not separable from the effects of body mass on DEE. The DEEs of both species were in accordance with published allometric predictions incorporating body mass and ambient temperature. The differences in energetic requirements and social dominance, both consequences of body size, may represent means by which grey squirrels exert more interspecific competition on red squirrels than do conspecifics, potentially driving populations below viable levels in some sites.     

Analysis of energy expenditure at different ambient temperatures in mice lacking DGAT1

Mice lacking acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triglyceride synthesis, have increased energy expenditure and therefore are resistant to obesity. Because ambient temperature can significantly affect energy expenditure in mice, we undertook these studies to determine the effects of different ambient temperatures on energy expenditure, food intake, and thermoregulation in DGAT1-deficient [Dgat1(-/-)] mice. Dgat1(-/-) mice had increased energy expenditure irrespective of changes in the ambient temperature. Although core temperature was normal, surface temperature was increased in Dgat1(-/-) mice, most likely reflecting an active mechanism to dissipate heat from increased thermogenesis. Dgat1(-/-) mice had increased food intake at baseline, and this hyperphagia became more pronounced upon exposure to cold. When fasted in a cold environment, Dgat1(-/-) mice developed hypothermia, which was associated with hypoglycemia. These results suggest that the hyperphagia in Dgat1(-/-) mice is a secondary mechanism that compensates for the increased utilization of fuel substrates. Our findings offer insights into the mechanisms of hyperphagia and increased energy expenditure in a murine model of obesity resistance.     

Energy budget during four successive bouts of lactation in striped hamsters exposed to decreases in ambient temperature

A prediction of the seasonal investment hypothesis is that overall energy investment needs to be greater for young being produced at colder temperatures. Then, that energy cost is lower as temperature becomes warmer. To evaluate this assumption, I performed a series of measures of food intake and reproductive output throughout four successive bouts of lactation in striped hamsters (Cricetulus barabensis) exposed to a constant warm temperature (Warm, 21°C) or exposed to consecutive decreases in ambient temperatures from warm to cold (Warm-Cold, 30-0°C). Warm hamsters showed similar asymptotic food intake, litter size and mass over the course of four successive bouts of lactation. Warm-Cold females consumed more food, but raised lighter litters during the third bout than first bout of lactation. Ambient temperatures had significant effects on energy budget and reproductive output, by which resting metabolic rate, nonshivering thermogenesis and activity of cytochrome c oxidase (EC 1.9.3.1) of brown adipose tissue were increased, but reproductive output was decreased with declines of temperatures. These findings suggest that a trade-off occurs between different components of energy expenditure during the successive course of four bouts of lactation. Seasonal hamsters decrease their reproductive output, but increase the energy spent on thermogenesis as the ambient temperature becomes colder. It may also indicate that temperature has a direct effect on metabolism, leading to an increase in overall energy expenditure at lower temperatures.     

Expenditure freeze: the metabolic response of small mammals to cold environments

There is renewed focus on the ecological determinants of animal metabolism and recent comparative analyses support the physiological expectation that the field metabolic rate (FMR) of homeotherms should increase with declining ambient temperature. However, sustained elevation of FMR during prolonged, seasonal cold could be prevented by intrinsic limits constraining FMR to some multiple of basal metabolic rate (BMR) or extrinsic limits on resource abundance. We analysed previous measures of mammalian FMR and BMR to establish the effect of ambient temperature on both traits and found no support for intrinsic limitation. We also measured the FMR of a northern population of red squirrels (Tamiasciurus hudsonicus) exposed to ambient temperatures much colder than all but one previous study of mammal FMR. These measurements revealed levels of energy expenditure that are, unexpectedly, among the lowest ever recorded in homeotherms and that actually decrease as it gets colder. Collectively, these results suggest the metabolic niche space of cold climate endotherms may be much larger than previously recognized.     

Intraspecific differences in the pattern of hibernation in the ground squirrelSpermophilus beldingi

Summary Intraspecific differences in the patterns of heterothermy were found in captive Belding's ground squirrels that hibernated undisturbed at ambient temperatures of 5°C, 10°C, and 15°C. The timing of all entrances into and arousals from hibernation was determined from records of copper-constantan thermocouples that were mounted on the floor of each animal's nest box and connected to continuously recording potentiometers. In the absence of food, large adult males terminated hibernation spontaneously in the spring. In contrast, females and small non-breeding males (yearlings) did not stop hibernating but instead they shortened their bouts of torpor in the spring so that they aroused every three or four days. This interval of frequent arousals, termed the emergence period, lastel until the squirrels became emaciated, and it was only in the 2 or 3 weeks preceeding death (starvation period) that arousal frequency once again decreased towards midwinter values (Fig. 3). These animals terminated hibernation when fed during the emergence or starvation periods, but they were able to resume torpor if that feeding lasted less than a week.Termination of hibernation and reproductive development in males were related to the size of the animals, not their age. Males two years and older which did not deposit normal quantities of fat were like yearlings in that they had open-ended hibernation seasons and showed little testicular enlargement. Likewise, juvenile males that grew to near adult size in their first summer spontaneously terminated hibernation and had well developed testes like most older individuals. In addition, both the frequency and duration of arousals in the emergence period were related, in part, to the extent of the animals' fat reserves, such that large squirrels spent more time at high body temperatures than small individuals (Figs. 5, 6). This trend was most pronounced at high ambient temperatures.These intraspecific differences in hibernation physiology are consistent with the sex and agerelated differences in the timing of emergence above ground in nature. The increase in the time spent euthermic in the spring isinterpreted as an adaptation for increasing the opportunities for environmental assessment. The intraspecific differences in the extent of this euthermia appear to be associated with differences in the balance between the energy supplies available to an animal and its need to accelerate the use of that energy (i.e., arouse) in order to achieve an early and accurately-time emergence from the hibernaculum.     

That’s hot: golden spiny mice display torpor even at high ambient temperatures

Golden spiny mice (Acomys russatus) living in the Judean desert are exposed to extended periods of food and water shortage. We investigated their thermal and metabolic response to three weeks of 50 % food reduction at ambient temperatures of 23, 27, 32 and 35 °C by long term records of metabolic rate and body temperature in the laboratory. At all ambient temperatures, A. russatus responded to starvation by a reduction of daily energy expenditure. At 32 and 35 °C, this metabolic adjustment fully compensated the reduced food availability and they maintained their energy balance at a slightly reduced body mass. At lower ambient temperatures, they could not fully compensate for the reduced food availability and kept a negative energy balance. The reduction of daily energy expenditure was largely achieved by the occurrence of daily torpor. Torpor even occurred at high ambient temperatures of 32 and 35 °C during which metabolic depression was not associated with a marked decrease of body temperature. The results show that the occurrence of daily torpor is not necessarily linked to cold exposure and the development of a pronounced hypothermia, but may even occur as depression of metabolic rate in a hot environment.     

Metabolism and thermoregulation in the springhare (<Emphasis Type="Italic">Pedetes capensis</Emphasis>)

Springhares are large, nocturnally active, diurnally fossorial rodents that typically inhabit arid and semi-arid areas. This lifestyle means that they need to balance excessive heat loss when foraging at night against insufficient heat loss in a potentially warm, humid burrow and both of these against the need to minimize water turnover and energy requirements. In this study we investigated metabolism and thermoregulation in these animals. Basal metabolic rate averaged 8.62+/-1.37 J g(-1) h(-1) and minimum thermal conductance 0.386+/-0.062 J g(-1) h(-1) degrees C(-1). These were higher and lower than expected, respectively. This, along with a relatively low, lower critical temperature and broad thermal neutral zone indicate that springhares are physiologically well suited to the low night-time temperatures, which they typically encounter. Body temperatures were quite labile but springhares became hyperthermic at temperatures above 30 degrees C suggesting that they are poor thermoregulators at high temperatures. This is attributed to their seldom, if ever, encountering temperatures in this range. Insufficient heat loss under normal resting conditions does not appear to be a problem, as springhares inhabit deep burrows in which the temperature never exceeds the upper critical temperature. Excess heat generated during vigorous underground exercise is presumably stored and dissipated to the cool night air or the cooler soil when subsequently resting. Water turnover and energy expenditure are presumably adequately addressed by other physiological and behavioural characteristics.     

Annual cycle of energy and time expenditure in a golden-mantled ground squirrel population

Summary We have analyzed seasonal shifts of energy and time allocation in a population of golden-mantled ground squirrels (Spermophilus saturatus) by directly measuring total daily energy expenditure (DEE) with an isotopic technique (doubly labeled water=dlw), and by estimating components of total DEE through an integration of field behavioral observations with laboratory-measured rates of energy expenditure (oxygen consumption) associated with major behavioral and physiological states. Hibernation laster about 7 1/2 months, and the 4 1/2-month activity season consisted of mating, a 28-d gestation of 3–5 young, 5 1/2 weeks of postnatal growth building to a peak in lactation just before the young emerged above ground, an additional 2–3-week period of maternal care before dispersal, and finally restoration of body mass preceding hibernation. Although the hibernation season comprised nearly two-thirds of the year, it involved only 13–17% of annual energy expenditure, leaving about 85% of energy expenditure for the active season. Ground squirrels were actually present on the surface for only about 11% of the year's time, and the foraging time required to obtain the total annual energy supply amounted to only about 2% of the year's time. The squirrels fed mainly on herbs in the early season and hypogeous fungi later; both were used extensively during peak lactation when female energy expenditure and demand were maximal. Average daily foraging time increased steadily throughout the season to a maximum of 28% of aboveground time as availability of greens diminished and fungus predominated in the diet; time availability did not limit foraging since the animals sat on average for 65% of the daily surface time of about 7 h. Timing of reproduction is apparently optimized such that peak reproductive energy demands are matched with maximal food availability and moderate thermal conditions that minimize energy demand. Despite the greater body mass of males, the greatest total DEE (measured by dlw) of any squirrels at any time of year was that of females during peak lactation. For production of young and lactation through above-ground emergence of an average litter of 2.7, females required a total energy increase of 24% above annual nonreproductive metabolism. Yearling females all bred and performed similarly to older females, yet some costs were greater because the yearlings began and ended hibernation at smaller mass, compensated by giving birth later, and finally showed a greater absolute increase in body mass over the active season than older females. Annual metabolic energy expenditure of breeding males was about 18% greater than that of females, due to greater male body mass. Yet the annual energy intake requirement for both sexes was essentially identical (about 42MJ) due to the greater reproductive export by females in the form of newborn and milk. During the mating season males showed wide-ranging exploratory behavior and social interactions, including aggression, that involved considerable locomotory energy expenditures. Although we did not directly account for the energetics of these specific reproductive behaviors, they are critical to male reproductive success and on a daily basis they probably involved much greater energy expenditure than sperm production. Some yearling males avoided these costs by foregoing testicular development, yet they allocated four times as much energy to growth as older males, thereby increasing somatic condition for the future.     

Probing,Assessment, and Management during Interactions between Ground Squirrels and Rattlesnakes

The predator-prey relationship between California ground squirrels (Spermophilus beecheyi) and northern Pacific rattlesnakes (Crotalus viridis) is a useful system for exploring conflict and assessment. Rattlesnakes are major predators of ground squirrel pups, but pose a less significant threat to adult squirrels. Adults approach, harass, and even attack rattlesnakes in defense of their pups. Two factors that may influence risk to both squirrel and snake during encounters are the size and body temperature of the rattlesnake. We used high-speed video to analyze the strikes of rattlesnakes of various sizes tested at different body temperatures. Results indicate that warmer snakes are more dangerous because they strike with higher velocity, greater accuracy, and less hesitation. Similarly, larger snakes are more dangerous because they can strike farther and at higher speeds, and keep their fangs embedded longer. Thus, ground squirrels would benefit from extracting information about a rattlesnake's size and temperature. The converse of our results is that cooler, smaller rattlesnakes may be more vulnerable. These snakes could mitigate their risk by avoiding dangerous adversaries and minimizing cues that divulge their weaknesses. Such tactics might explain the active probing that squirrels direct at rattlesnakes, which may function to overcome a snake's resistance to disclosing its vulnerability.     

Stress Proteins in Mammalian Hibernation

Many organisms whose body temperatures (T_b) vary when they areexposed to a wide range of environmental temperatures exhibitdifferential expression of stress (heat shock) proteins, presumablyto minimize protein damage during thermal stress. In contrast,we know relatively little about natural variation in stressproteins in homeotherms (i.e., birds and mammals), perhaps dueto the relatively constant T_b that is the hallmark of this vertebrategroup. The significant changes in T_b and metabolism characteristicof mammalian hibernators suggest they could provide insightinto the ecological relevance of stress proteins in mammals.Here we examined differential expression of stress proteinsin several tissues of active and torpid 13-lined ground squirrels.There were few significant differences in expression of inducibleHsp70 protein in liver, kidney, heart, intestine, and skeletalmuscle between active and torpid squirrels, and neither fastingin active squirrels nor torpor bout length in hibernators significantlyaltered Hsp70 abundance in these tissues. However, Hsp70 proteinwas lower in brown adipose tissue (BAT) of torpid compared withactive squirrels. In contrast, abundance of GRP75, the mitochondria]form of Hsp70, was greater in liver, skeletal muscle and intestineof torpid compared with active squirrels, with the greatestchange in intestine. Because GRP75 has been shown to be inducedby non-thermal stressors including glucose deprivation and oxidativestress, these results suggest that the ecological significanceof stress proteins for hibernators may be more closely associatedwith the metabolic demands of heterothermy rather than thermalstress per se.     

Wheel-running activity and energy metabolism in relation to ambient temperature in mice selected for high wheel-running activity

Interrelationships between ambient temperature, activity, and energy metabolism were explored in mice that had been selectively bred for high spontaneous wheel-running activity and their random-bred controls. Animals were exposed to three different ambient temperatures (10, 20 and 30°C) and wheel-running activity and metabolic rate were measured simultaneously. Wheel-running activity was decreased at low ambient temperatures in all animals and was increased in selected animals compared to controls at 20 and 30°C. Resting metabolic rate (RMR) and daily energy expenditure (DEE) decreased with increasing ambient temperature. RMR did not differ between control and selected mice, but mass-specific DEE was increased in selected mice. The cost of activity (measured as the slope of the relationship between metabolic rate and running speed) was similar at all ambient temperatures and in control and selected mice. Heat generated by running apparently did not substitute for heat necessary for thermoregulation. The overall estimate of running costs was 1.2 kJ/km for control mice and selected mice.     

Increased heat loss affects hibernation in golden-mantled ground squirrels

During hibernation at ambient temperatures (T(a)) above 0 degrees C, rodents typically maintain body temperature (T(b)) approximately 1 degrees C above T(a), reduce metabolic rate, and suspend or substantially reduce many physiological functions. We tested the extent to which the presence of an insulative pelage affects hibernation. T(b) was recorded telemetrically in golden-mantled ground squirrels (Spermophilus lateralis) housed at a T(a) of 5 degrees C; food intake and body mass were measured at regular intervals throughout the hibernation season and after the terminal arousal. Animals were subjected to complete removal of the dorsal fur or a control procedure after they had been in hibernation for 3-4 wk. Shaved squirrels continued to hibernate with little or no change in minimum T(b), bout duration, duration of periodic normothermic bouts, and food intake during normothermia. Rates of rewarming from torpor were, however, significantly slower in shaved squirrels, and rates of body mass loss were significantly higher, indicating increased depletion of white adipose energy stores. An insulative pelage evidently conserves energy over the course of the hibernation season by decreasing body heat loss and reducing energy expenditure during periodic arousals from torpor and subsequent intervals of normothermia. This prolongs the hibernation season by several weeks, thereby eliminating the debilitating consequences associated with premature emergence from hibernation.     

Thermoregulation by an Australian murine rodent, the ash-grey mouse (Pseudomys albocinereus)

We examine here the thermal physiology of the ash-grey mouse, as there is a paucity of data to explain how Australian rodents meet thermoregulatory demands. Most ash-grey mice remained normothermic over a range of ambient temperatures (10°C to 30°C), although they became hyperthermic at high ambient temperatures. One individual entered torpor at ambient temperatures of 20°C and 25°C, with minimal body temperatures of 24.5°C and 28.4°C respectively, before spontaneously arousing. This is the first evidence of torpor use by an Australian murine rodent. Our data suggest that although ash-grey mice have the physiological ability to use torpor, it is used rarely, presumably due to other behavioural and physiological adaptations. Their higher-than-expected basal metabolic rate (1.56±0.25mLO(2)g(-1)h(-1)) indicates that ash-grey mice do not have a frugal approach to energy expenditure. Other standard physiological variables were typical of a generalised rodent. A readily-available omnivorous diet, nocturnal activity, semi-fossorial habit and social behaviour presumably allow a high energy lifestyle. A reluctance to use torpor, despite an apparent physiological ability to do so, supports the idea that the use of torpor reflects a net balance between the costs and benefits of a heterothermic thermoregulatory strategy.     

Weather-Related Indices of Autumn– Winter Dabbling Duck Abundance in Middle North America

ABSTRACT Research on effects of key weather stimuli influencing waterfowl migration during autumn and winter is limited. We investigated relationships between changes in relative abundances of mallard (Anas platyrhynchos) and other dabbling ducks (Anas spp.) and weather variables at midlatitude locations in North America. We used waterfowl survey data from Missouri Conservation Areas and temperature and snow cover data from the Historical Climatology Network to evaluate competing models to explain changes in relative abundance of ducks in Missouri, USA, during autumn-winter, 1995–2005. We found that a cumulative weather severity index model (CumulativeWSI; calculated as mean daily temp - degrees C + no. of consecutive days with mean temp ≤ 0° C + snow depth + no. of consecutive days with snow cover) had the greatest weight of evidence in explaining changes in relative abundance of ducks. We concluded the CumulativeWSI reflected current and cumulative effects of ambient temperatures on energy expenditure by ducks, and snow cover and wetland icing, on food availability for ducks. The CumulativeWSI may be useful in determining potential changes in autumn-winter distributions of North American waterfowl given different climate change projections and associated changes in habitat conservation needs. Future investigations should address interactions between CumulativeWSI and landscape habitat quality, regional waterfowl populations, hunter harvest, and other anthropogenic influences to increase understanding of waterfowl migration during autumn-winter.     

Effects of ambient temperature on adaptive thermogenesis during maintenance of reduced body weight in mice

We showed previously that, at ambient room temperature (22°C), mice maintained at 20% below their initial body weight by calorie restriction expend energy at a rate below that which can be accounted for by the decrease of fat and fat-free mass. Food-restricted rodents may become torpid at subthermoneutral temperatures, a possible confounding factor when using mice as human models in obesity research. We examined the bioenergetic, hormonal, and behavioral responses to maintenance of a 20% body weight reduction in singly housed C57BL/6J +/+ and Lep(ob) mice housed at both 22°C and 30°C. Weight-reduced high-fat-fed diet mice (HFD-WR) showed similar quantitative reductions in energy expenditure-adjusted for body mass and composition-at both 22°C and 30°C: -1.4 kcal/24 h and -1.6 kcal/24 h below predicted, respectively, and neither group entered torpor. In contrast, weight-reduced Lep(ob) mice (OB-WR) housed at 22°C became torpid in the late lights-off period (0200-0500) but did not when housed at 30°C. These studies indicate that mice with an intact leptin axis display similar decreases in "absolute" energy expenditure in response to weight reduction at both 22°C and 30°C ambient temperature. More importantly, the "percent" decrease in total energy expenditure observed in the HFD-WR compared with AL mice is much greater at 30°C (-19%) than at 22°C (-10%). Basal energy expenditure demands are ～45% lower in mice housed at 30°C vs. 22°C, since the mice housed at thermoneutrality do not allocate extra energy for heat production. The higher total energy expenditure of mice housed at 22°C due to these increased thermogenic demands may mask physiologically relevant changes in energy expenditure showing that ambient temperature must be carefully considered when quantifying energy metabolism in both rodents and humans.     

Synchronization of circannual cycles: a cold spring delays the cycles of thirteen-lined ground squirrels

Summary Ground squirrels, show circannual cycles with periods normally less than a year when kept under laboratory conditions. The way in which environmental factors synchronize these cycles with the geophysical year under natural conditions is not known. We tested the possibility that cold temperatures can cause long-term phase delays in circannual cycles of thirteen-lined ground squirrels.Two groups of animals were kept in the cold (4 °C) for either 8.5 or 13 months, after which they were returned to the warm (21 °C) and kept there until they had completed at least one additional cycle. A third group was kept in the warm for the entire experiment. Most of the males in the cold room groups became arrested in the spring phase of their cycles while they were in cold. When returned to the warm, these males resumed cycling. When animals showed prolonged spring phases, their cycles were phase-delayed and continued to reflect this delay even after they were returned to the warm. Both body weight cycles and molt cycles were delayed. In contrast to the males in the cold room, none of the females in the cold room groups and none of the warm room animals of either sex showed this response.Our results demonstrate that cold temperatures can phase-delay both body weight and molt cycles and that the spring phase is a critical stage in this effect. We suggest that spring temperatures are largely responsible for the seasonal synchronization of circannual cycles in ground squirrels and are, therefore, a possible Zeitgeber for these cycles.     

Nuclear DNA phylogeny of the squirrels (Mammalia: Rodentia) and the evolution of arboreality from c-myc and RAG1

Although the family Sciuridae is large and well known, phylogenetic analyses are scarce. We report on a comprehensive molecular phylogeny for the family. Two nuclear genes (c-myc and RAG1) comprising approximately 4500 bp of data (most in exons) are applied for the first time to rodent phylogenetics. Parsimony, likelihood, and Bayesian analyses of the separate gene regions and combined data reveal five major lineages and refute the conventional elevation of the flying squirrels (Pteromyinae) to subfamily status. Instead, flying squirrels are derived from one of the tree squirrel lineages. C-myc indels corroborate the sequence-based topologies. The common ancestor of extant squirrels appears to have been arboreal, confirming the fossil evidence. The results also reveal an unexpected clade of mostly terrestrial squirrels with African and Holarctic centers of diversity. We present a revised classification of squirrels. Our results demonstrate the phylogenetic utility of relatively slowly evolving nuclear exonic data even for relatively recent clades.     

Seasonal effects on thermoregulatory responses of the Rock Kestrel, Falco rupicolis

Thermoregulatory responses are known to differ seasonally in endotherms and this is often dependent on the environment and region they are resident. Holarctic animals are exposed to severe winters and substantial seasonal variation in ambient temperature. In contrast, those in the Afrotropics have less severe winters, but greater variation in temperature, rainfall and net primary production. These environmental factors place different selection pressures on physiological responses in endotherms. In this study, metabolic rate (VO₂) and body temperature (T_b) were measured in captive bred Rock Kestrels (Falco rupicolus) from the Afrotropics after a period of summer and winter acclimatisation. Resting metabolic rate was significantly lower after the winter acclimatisation period than after the summer acclimatisation period, and there was a shift in the thermoneutral zone from 20–33 °C in summer to 15–30 °C in winter. However, no significant difference in basal metabolic rate between summer and winter was found. The results show that Rock Kestrels reduce energy expenditure at low ambient temperatures in winter as expected in an Afrotropical species.     

THE IMPACT OF TREE SQUIRRELS (TAMIASCIURUS) ON LIMBER PINE SEED DISPERSAL ADAPTATIONS

Tree squirrels (Tamiasciurus) are important selective agents on conifer reproductive strategies (Smith 1970, 1975). Although this is well established for wind-dispersed pines, the impact of tree squirrels on bird-dispersed pines has been largely ignored. I assessed the impact of tree squirrels on the allocation of reproductive energy in the bird-dispersed limber pine (Pinus flexilis) by comparing its cone and seed traits from three sites in the Rocky Mountains where tree squirrels (Tamiasciurus) are present to those from three mountain ranges in the Great Basin where tree squirrels are absent. As predicted, differences between the two regions in individual cone and seed traits are consistent with the hypothesis that tree squirrels are important selective agents on these traits. In the absence of tree squirrels, limber pine allocates more than twice as much energy to kernel compared with that invested in putative seed defenses (cone, resin, and seed coat) as does limber pine where tree squirrels are present. Such a large difference is particularly striking, because tree squirrels may have become extinct in the Great Basin in only the last 12,000 yr. Although many factors influence the allocation of energy to cones and seeds, no single factor other than the presence of tree squirrels is compatible with the large and consistent differences between limber pine in the Rocky Mountains and Great Basin. These results show that tree squirrels are an important constraint on the evolution of cone and seed traits that promote the dispersal of seeds by birds.