Glycogen, not dehydration or lipids, limits winter survival of side-blotched lizards (Uta stansburiana).

Climate change is causing winters to become milder (less cold and shorter). Recent studies of overwintering ectotherms have suggested that warmer winters increase metabolism and decrease winter survival and subsequent fecundity. Energetic constraints (insufficient energy stores) have been hypothesized as the cause of winter mortality but have not been tested explicitly. Thus, alternative sources of mortality, such as winter dehydration, cannot be ruled out. By employing an experimental design that compared the energetics and water content of lizards that died naturally during laboratory winter with those that survived up to the same point but were then sacrificed, we attempt to distinguish among multiple possible causes of mortality. We test the hypothesis that mortality is caused by insufficient energy stores in the liver, abdominal fat bodies, tail or carcass or through excessive water loss. We found that lizards that died naturally had marginally greater mass loss, lower water content, and less liver glycogen remaining than living animals sampled at the same time. Periodically moistening air during winter reduced water loss, but this did not affect survival, calling into question dehydration as a cause of death. Rather, our results implicate energy limitations in the form of liver glycogen, but not lipids, as the primary cause of mortality in overwintering lizards. When viewed through a lens of changing climates, our results suggest that if milder winters increase the metabolic rate of overwintering ectotherms, individuals may experience greater energetic demands. Increased energy use during winter may subsequently limit individual survival and possibly even impact population persistence.

Postglacial range expansion from northern refugia by the wood frog, Rana sylvatica.

Although the range dynamics of North American amphibians during the last glacial cycle are increasingly better understood, the recolonization history of the most northern regions and the impact of southern refugia on patterns of intraspecific genetic diversity and phenotypic variation in these regions are not well reconstructed. Here we present the phylogeographic history of a widespread and primarily northern frog, Rana sylvatica. We surveyed 551 individuals from 116 localities across the species' range for a 650-bp region of the NADH dehydrogenase subunit 2 and tRNA(TRP) mitochondrial genes. Our phylogenetic analyses revealed two distinct clades corresponding to eastern and western populations, as well as a Maritime subclade within the eastern lineage. Patterns of genetic diversity support multiple refugia. However, high-latitude refugia in the Appalachian highlands and modern-day Wisconsin appear to have had the biggest impact on northern populations. Clustering analyses based on morphology further support a distinction between eastern and western wood frogs and suggest that postglacial migration has played an important role in generating broad-scale patterns of phenotypic variation in this species.

Postfreeze reduction of locomotor endurance in the freeze-tolerant wood frog, Rana sylvatica.

Considerable study has focused on the physiological adaptations for freeze tolerance in the wood frog, Rana sylvatica, a northern species that overwinters within the frost zone, but little attention has been paid to the associated costs to organismal performance. Here we report that freezing causes transient impairment of locomotor endurance and adverse changes in exercise physiology that persist for at least 96 h. Wood frogs frozen at -2 degrees C for 36 h exhibited normal behaviors and hydro-osmotic status and near-normal metabolite (glycogen, glucose, and lactate) levels within 24 h after thawing began. However, when exercised to exhaustion on a treadmill, these frogs showed a 40% reduction in endurance as compared to sham-treated (unfrozen) controls, a reduction that persisted for at least 96 h. Previously frozen frogs exhibited higher rates of lactate accumulation during exercise than controls, suggesting that prior freezing forces greater reliance on the glycolytic pathways of energy production to support exercise. Given that this species breeds in late winter, when subzero temperatures are common, freezing may result in reduced fitness by hampering their ability to reach the pond, avoid predators, and successfully obtain mates.

Metabolism and cold tolerance of overwintering adult mountain pine beetles (Dendroctonus ponderosae): evidence of facultative diapause?

We sought evidence for a distinct diapause in adult overwintering mountain pine beetles (Dendroctonus ponderosae Hopkins) by measuring metabolic rate and supercooling ability of field collected beetles throughout the year. Metabolic rates measured at 0, 5, and 10°C declined significantly from October through November, then rose slowly, reaching levels as high as those recorded in October by late May. From December to February metabolic rates were not correlated with minimum weekly phloem temperatures (R(2)=0.0%, P=0.592), but were correlated with phloem temperatures as winter advanced to spring (R(2)=44.8%, P=0.010), a pattern consistent with progression through the maintenance and termination phases of diapause. Supercooling points were also significantly lower in winter compared to fall and spring (F((8,143))=32.6, P<0.001) and were closely correlated with metabolic rates (R(2)>79% for all three temperatures). Dry mass declined linearly with winter progression (F((8,150))=8.34, P<0.001), explained by catabolism of metabolic reserves, with a concomitant accumulation of metabolic water (F((8,147))=35.24, P<0.001). The strong mid-winter metabolic suppression correlated with improved supercooling ability, coupled with their lack of response to variation in environmental temperature, are evidence of possible diapause in adult overwintering mountain pine beetles.

Energy and water conservation in frozen vs. supercooled larvae of the goldenrod gall fly, Eurosta solidaginis (fitch) (Diptera: Tephritidae).

Insects that tolerate severe cold during winter may either supercool or tolerate ice forming within the tissues of the body. To compare the relative advantages of freezing and supercooling, we measured rates of CO(2) production and water loss in frozen and supercooled goldenrod gall fly larvae (Eurosta solidaginis). As an important first step, we measured the time required for ice content and metabolic rate to stabilize upon freezing. Ice content stabilized after only three hours of freezing at -5 degrees C, whereas CO(2) production required 12 hours to stabilize. Subsequent experiments found that freezing greatly reduced both water loss and metabolic rate. Comparisons of supercooled and frozen larvae at -5 degrees C indicated that CO(2) production fell 47% with freezing and water loss decreased 35%. As temperature decreased to -10 and -15 degrees C, CO(2) production fell exponentially and was no longer detectable at -20 degrees C with our measurement system. Our results demonstrate that freezing significantly reduces energy consumption during the winter and may therefore improve winter survival and spring fecundity. The advantages of freezing over supercooling would drive selection toward insect freeze tolerance and also toward higher supercooling points to increase the duration of freezing each winter.

Geographic variation in energy storage and physiological responses to freezing in the gray treefrogs Hyla versicolor and H. chrysoscelis.

The physiological responses supporting freeze tolerance in anurans are well known, but the evolution of this trait remains little studied. This is the first common-garden study of geographic variation in cryoprotective responses to freezing and the degree of freeze tolerance. We studied the gray treefrogs (Hyla versicolor and H. chrysoscelis) from sympatric sites in Minnesota, Indiana and Missouri. Patterns in the literature suggest that northern frogs produce more cryoprotectants upon freezing, but we found no geographic variation in cryoprotective responses or degree of freeze tolerance. The concentration of glucose produced upon freezing was higher than previously reported for this species (liver: 475 micro mol g(-1) dry mass). Unfrozen frogs had high levels of glycerol (liver: approx. 150 micro mol g(-1) dry mass), and did not produce more upon freezing. Liver glycogen content (concentration multiplied by liver mass) was highest in frogs from Minnesota and Missouri, and was stored in preference to lipids in Minnesota frogs, possibly to provide energy for the longer northern winters. Minnesota frogs accumulated more ice (53.4+/-1.8%) after freezing to-2.5 degrees C than Indiana frogs (45.5+/-3.3%). The two species differed in body size but not in any of the physiological parameters measured. We conclude that these populations show no adaptive variation in freeze tolerance and that comparing published studies may be misleading because of different acclimation and feeding regimes.