J. Zool., Lond. (1991) 225,691-699

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Cold adaptation

ANDREW CLARKE

British Antarctic Survey, High Cross, Cambridge CB3 OET, UK

What is cold adaptation?

In general terms, cold adaptation is straightforward to define: it encompasses all those aspects of an organism's anatomy, physiology, biochemistry and behaviour that allow it to survive in low temperature environments such as polar and alpine regions. Cold adaptation is thus essentially no more than a specific example of the more general adaptation any organism must have to the particular thermal features of its environment, and polar fish are cold adapted in the same sense that fish living on a tropical reef are warm adapted.

Nevertheless we often tend to think of cold adaptation as something special. This is partly because there are certain adaptations specific to low temperatures (for example antifreeze proteins), but also because from an anthropocentric viewpoint it appears somehow easier or more amenable to live in tropical water than a polar ocean. The perception that cold is bad and warm is good has had a profound influence on both the development of thermal biology and also how we view the evolutionary history of marine faunas. Whilst it is undoubtedly true for mammals and birds, its validity for ectothermic (poikilothermic) organisms is questionable (Clarke, 199 I).

The literature of temperature physiology in relation to cold tolerance and cold adaptation is vast. Any review must perforce be selective. I have chosen to touch on most aspects, albeit briefly, combining where possible historically important references with recent reviews.

Some general points about temperature

Polar habitats are by no means universally cold, either in time or space. A fundamental distinction must, however, be drawn between terrestrial and marine habitats.

The polar oceans are at the lower extreme of the temperature range available to marine organisms; close to the Antarctic continent and in the deep Arctic basins sea water temperature reaches - 1.9 "C. Occasionally temperatures are recorded indicating undercooling (- 2.0 ' C or lower, usually under surface ice cover), but the increased salinity resulting from the freezing of sea ice can expose organisms in brine channels to much lower temperatures ( - 7 or - 8 "C). The large thermal capacity of water means that polar oceans have very stable temperatures. At McMurdo Sound (the highest southerly latitude with open water in summer) the annual variation is only f 0.2 "C, making this one of the most thermally stable areas on the planet. Seasonal variations in the Southern Ocean are also small, typically a range of 1.5-2.5 "C. The high Arctic basin is similarly stable but large areas of ocean fringing the Arctic show a seasonal variation from - 1.8 "C to fully temperate. The temperature of the deep sea is dictated largely by the production of cold bottom water at high southern latitudes. The deep sea thus constitutes an enormous habitat where cold temperatures (<4 "C) are combined with high pressure.

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TABLE 1 Types of reniperarurr response (from Clarke. 1991)

Response Definition ~~

Acute

Acclimation Acclimatization*

Adaptation

The adjustment of an organism's physiology to an immediate change in tempcrature; this can include torpor, coma (and death). The adjustment of an organism's physiology to a new temperature in the laboratory. The adjustment of an organism's physiology to changes in environmental temperature. This may be tidal, daily, seasonal or inter-annual. The evolutionary adjustment of an organism's physiology to its environment. This may include adjustment to a seasonal or daily fluctuation in temperature requiring acclimatization.

* In laboratory acclimation i t is usual to modify only a single variable (c.g. temperature) whilst keeping all others constant. An organism undergoing acclimatization in the field I S subject to coincident variation in a large range of environmental variables

Polar terrestrial habitats could not be more different: winter temperatures can reach - 70 "C or below in areas where organisms live, and there are both wide daily and seasonal variations. It is not uncommon for plants at high latitudes to experience a daily temperature range of 50 "C, although under insulating winter snows polar and alpine organisms are exposed for long periods to temperatures of about 0 C. For a recent general description of the Arctic climate see Young (1989).

Clearly the way in which organisms respond to the contrasting thermal features of these two environments will differ. Marine organisms have the opportunity to evolve a fine tuning of their physiology to the narrow range of temperatures they experience, whereas terrestrial polar organisms must be eurythermal and more plastic in their response. The differing responses are dictated primarily by the rate of temperature change and how far it falls; a useful general classification is given in Table I. This classification emphasizes that the processes invoked in short- term responses to low temperature are not necessarily the same as those involved in evolutionary change. We cannot assume that marine and terrestrial organisms cope with the stresses of polar regions in the same way, nor can we assume that laboratory experiments on the responses of eurythermal organisms to temperature acclimation are relevant to the way organisms have adapted over evolutionary time to long-term climate change. Only continued studies of the biology and physiology of polar and tropical organisms can do this.

Mammals and birds

Mammals and birds maintain a relatively constant internal temperature. The precise value varies with species, but is generally between 35 and 40 'C. Because the heat to maintain this temperature is generally derived from metabolism, and hence is internal, these organisms are referred to as endotherms or homeotherms (for a succinct discussion of these terms and their etymology see Schmidt-Nielsen, 1979).

Homeostatic physiological mechanisms enable mammals and birds to maintain normal body temperature over a range of ambient temperatures (often called the thermoneutral zone). Below the lower critical temperature (which marks the lower bound of this zone) extra heat needs to be supplied to offset that lost to the environment; mechanisms include non-shivering thermogenesis in brown adipose tissue, and shivering. When circumstances permit, behavioural mechanisms are

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also employed (for example the huddling of roosting birds). These topics are discussed by Schmidt- Nielsen (1979), Cossins & Bowler (1987) and Marchand (1987).

Cold adaptation in mammals and birds consists essentially of shifting the thermoneutral zone to lower temperatures. This is achieved by a variety of means including thicker or more extensive insulation (fur in terrestrial species, blubber in marine mammals), a reduced sensitivity to low temperature in extremities such as ears and limbs, and counter-current circulatory mechanisms to reduce heat loss. Polar animals tend to be white, though this may have as much to do with camouflage as thermal constraints (Arctic seal pups, which are eaten by a variety of predators are white; in the Antarctic seal pups have no predators on land and are born with dark fur). It has long been felt that clinal variation in body size with latitude had its explanation in considerations of thermal balance but recent data indicate that at high latitudes this effect (Bergmann's Rule) is obscured by factors such as overall resource limitation (Geist, 1987). However Allen's Rule (a reduced relative size of extremities) does appear to have stood the test of time. Also for small mammals the insulated subnivean habitat offers a protection against extreme low temperature as well as a refuge from predation.

For many mammals and birds the polar winter poses problems of food availability as much as cold. Most species thus migrate to lower latitudes (or in at least one spectacular instance, the Arctic tern, Sterna paradisaea, to the opposite polar ocean). A few species hibernate but, contrary to popular belief, hibernation is not particularly a polar phenomenon. Bears (Ursidae) are not true hibernators and the Arctic has the lowest incidence of hibernating species (as percentage of the total mammalian fauna) in North America.

For most invertebrates and many fish migration is not an option, and cold hardiness is necessary for survival. An important component of this is the ability either to withstand or prevent freezing.

Freezing

Most work on the ecology and biochemistry of freezing tolerance has involved arthropods (especially insects) or fish. However, there is also an extensive literature discussing the survival of

x

> 0 0 a, [r

& 50

0.1 1 10 100

Cooling rate (OC/rnin) FIG. 1. Relationship between cooling rate (Tjmin) and recovery from freezing to - 196 "C (%) in the snow alga

Chlamydomonas nivalis. Note that cooling rate is plotted logarithmically, and that survival never reaches loo'%, as some cells are always killed by freezing to liquid nitrogen temperatures. Redrawn from Morris, Coulson & Clarke (1979).

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unicellular organisms or cell cultures subject experimentally to freezing temperatures, and these studies form an essential background against which to view the ecological data.

A typical result is shown for the unicellular chlorophyte alga Chlamydomonas nivalis (Fig. 1). This species (snow alga) is found in free water on snow in both polar and alpine regions and is subject naturally to cycles of freeze/thaw in its normal habitat. However, survival of the cells from freezing depends critically on cooling rate and in this experiment optimum survival was obtained at a cooling rate of 2 C/min, more cells being killed at both slower and faster rates. A similar pattern is usually found in heterotrophic cells (a good example is Amoeba: McLellan et al., 1984). A complete explanation is lacking but damage at fast rates of cooling is thought to be caused by intracellular ice formation which (at least at temperatures encountered in the wild) is always lethal. At slow rates of cooling the bathing medium increases in osmolality (as water is withdrawn to ice) and the cells shrink because water is drawn into the extracellular medium osmotically. Osmotic and ionic effects are believed to be a major cause of freezing damage at slow rates of cooling. This emphasizes the parallel between the stresses of freezing and dehydration, and many drought- resistant cells are also tolerant of freezing in the external medium. Experiments with isolated cells or unicellular organisms thus suggest two general results: that the formation of intracellular ice is always lethal, and that survival from freezing of the extracellular medium depends on cooling rate.

Early observations of cold hardiness in polar arthropods suggested that organisms adopted one of two strategies; they either tolerated formation of ice in body fluids (but not within cells), or they attempted to avoid ice formation completely by undercooling (Table 11). Experimental work on whole organisms in the laboratory has generally ignored the problem that survival may depend on cooling rate, by standardizing on a rate of (usually) - 1 ‘C/min. In theory this might result in either misclassifying the type of response or missing a range of responses. Cooling rates to which organisms are exposed in the wild can vary greatly and in some species of arthropod experiments have shown that survival depends critically on cooling rate (Miller, 1978; Baust & Nishino, 1991). Nevertheless, the large corpus of experimental work has produced a coherent view of cold hardiness in invertebrates, and laboratory studies using ecologically meaningful cooling rates generally match the strategies observed in the wild.

Terrestrial organisms have to withstand far lower temperatures than any marine species (other than some intertidal forms). Freezing is a stochastic process, dependant on the formation of nuclei sufficiently large to trigger bulk freezing: the lower the temperature the greater the chance of spontaneous freezing. This was shown clearly in the overwintering larvae of the wheat stem sawfly Cephus cinctus in an elegant early study by Salt (1966). Many insects have now been shown to

TABLE I1 Straregies of cold hardiness

Strategy Features ~~~ ~~~

1. Freezing tolerance Extracellular ice allowed to form. Ice nucleator proteins may be present to initiate freezing at high sub-zero temperatures. Thermal hysteresis proteins (antifreezes) may also be present. Polyols often present as cryoprotectants. Ice within cells lethal. Some arthropods. Many intertidal invertebrates. Some amphibians but no fish.

2. Freezing avoidance No ice allowed to form. Thermal hysteresis agents (antifreezes) present in arthropods and marine fish. In some arthropods clearance of gut nucleators essential. Polyols often present as cryoprotectants. Ice within cells lethal. Many arthropods and other terrestrial invertebrates. Fish.

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produce thermal hysteresis proteins (THPs) which reduce the chances of spontaneous nucleation at any given temperature. Many species also clear the gut of potential ice nucleators as winter approaches, for despite the presence of THPs these organisms are still undercooled and hence in an unstable thermodynamic state. In those insects which have adopted the strategy of freezing extracellular water at high sub-zero temperatures (usually above - 10 "C), ice nucleator proteins are often present. The cells of these insects do not freeze, but they must be tolerant of osmotic stress. Somewhat paradoxically some of these insects also contain THPs. The function of these may be to prevent freezing whilst ambient temperature is still relatively high in autumn or spring (Duman & Horwath, 1983), or to prevent cellular damage by limiting recrystallization in the frozen winter period or during thawing (Knight & Duman, 1986). For a recent review of insect ice nucleator and thermal hysteresis (antifreeze) proteins, see Duman et al. (1 991).

In both freezing-tolerant arthropods and those that undercool, substantial quantities of polyhydric alcohols and/or sugars are produced over winter. Although often referred to as antifveezes in the early literature, it is now clear that their physiological roles are primarily cryoprotective: that is they serve to protect proteins and cellular organelles against low temperature damage such as denaturation or subunit disaggregation. Nevertheless, they can be present at such concentrations that they will also have a colligative effect in depressing the equilibrium freezing point and the associated high viscosity may also slow ice crystal growth. For recent reviews of insect cold-hardiness see Block (1 990) and Lee & Denlinger ( 1 99 I).

Most work on terrestrial cold-hardiness has concerned arthropods but in the early 1980s several species of wood frog that overwinter in forest litter were shown to survive freezing of their extracellular fluids (Schmid, 1982). These are notable in being the only truly freeze-tolerant vertebrates known to date (numerous apocryphal reports for goldfish notwithstanding!). Intertidal marine animals also have to tolerate low air temperatures, coupled with a regular tidal emersion. These have been little studied in comparison with terrestrial arthropods or polar fish but the presence of thermal hysteresis proteins has been shown in Mytilus edulis (Theede, Schneppenheim & Bevess, 1976), although the level of antifreeze protection they impart is low and their function may be primarily to limit recrystallization damage. Many species also use mucus as a mechanical barrier to prevent penetration of ice crystals (Kanwisher, 1955) and the freeze-tolerant intertidal gastropod Melampus bidentatus produces an ice nucleating protein in winter (Loomis, 1987). In those species that do freeze, it is again only the extracellular fluids that form ice.

Marine invertebrates do not generally freeze as their body fluids are isosmotic or hyperosmotic to sea water. The only exceptions are those sessile forms that become encased in anchor ice (ice which forms from undercooled water at the seabed). Fish, however, are hyposmotic to sea water; their body fluids are usually 300-400 mosm, corresponding to a freezing point of - 0.6 to - 0.8 T. Although polar fish have an increased ionic concentration which imparts a small degree of colligative freezing point depression, the presence of an antifreeze was first demonstrated in Arctic fish by Scholander et al. (1957) and Gordon, Amdur & Scholander (1962). The first antifreeze to be isolated was that of Pagothenia (then Trematomus) borchgrevinki from McMurdo Sound, and was shown to be a glycoprotein by DeVries (1971). Subsequent research has shown that most notothenioid fish (a group endemic to southerly latitudes) have a range of antifreeze glycoproteins of differing molecular weights. An exception is Notothenia kempi which lives permanently undercooled in a layer of warmer water at depths where contact with ice is unlikely. Other Antarctic species and many Arctic fish have peptide antifreezes similar in composition to the subsequently isolated thermal hysteresis proteins of polar insects. For reviews of fish antifreezes see Clarke (1 983) and DeVries (1988).

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It is now clear that antifreeze peptides (and probably glycopeptides) have evolved several times. They are found in a wide range of body fluids, including the intestinal contents and cerebrospinal fluid. Their mode of action is still not fully understood, but it is believed to involve binding of the antifreeze molecule to the growing face of the ice crystal nucleus, thereby reducing significantly the rate of further crystal growth. As with insects. the presence of antifreezes promotes undercooling. However, since the sea water temperature rarely drops below - 2 ‘C, fish can live for up to 100 years or more without freezing.

Metabolic compensation

The various physiological processes involved in preventing death from freezing are survival mechanisms; they allow a fish or terrestrial invertebrate to continue existence. They are thus resisrmce adaptations in the terminology advocated by Cossins & Bowler (1987). However, polar organisms do not need only to survive at high latitudes, they must also be able to move, grow and reproduce. They thus need some way o counteracting the thermodynamic tendency for all reactions to slow down in the cold: they must also have evolved compensation for the effects of temperature (also referred to as capacity adaptation: Cossins & Bowler, 1987).

The equations describing any reaction involving a change in free energy (and hence all physiological processes) include terms for the effects of temperature and pressure: all such processes slow down as the temperature falls and usually also slow with increasing pressure. It is usual for ecologists and laboratory physiologists working on terrestrial or shallow-water organisms to ignore the pressure term, although in marine organisms living below surface waters the etTects of pressure on physiology are extremely significant. For abyssal organisms the effects of the low temperature are confounded by the effects of the enormous pressure; however, severe technical difficulties mean that these have unfortunately been little studied so far.

A change in temperature affects many of the physical parameters that influence the functioning of organisms, including viscosity, solubilities of gases and solutes, and diffusion. If an organism is to cope with a change of environmental temperature, all cellular processes must evolve some degree of compensation for the effects of this change in temperature. At the cellular level this includes all aspects of gene expression and regulation, contractile protein function, ion pump activity, ATP and macromolecule synthesis. membrane function, and intermediary metabolism. At higher levels of integration, temperature compensation may also involve neural and muscular activity, circulatory and respiratory processes, growth, reproduction and development.

It is in the area of metabolic compensation that i t is most necessary to distinguish clearly the effects of rate. Organisms that need to cope with rapid changes of temperature (for example polar terrestrial arthropods, or marine organisms undertaking diurnal vertical migrations through the thermocline) may have evolved quite different mechanisms from those organisms that have tracked the slow change in sea water temperature at high latitudes since the Tertiary. We must extrapolate from the temperate laboratory to the polar environment with care.

The central concept of compensation is that physiological processes should proceed at the same speed and efficiency in polar, temperate and tropical organisms. Since cold generally slows physiological rates this means that processes in polar organisms are generally faster than in temperate organisms cooled to polar temperatures. This has led to the idea that physiological rates must be elerared in polar organisms in order to maintain function, an idea which originated with the seminal work of Krogh (1916) who examined the metabolism of goldfish in relation to temperature. The concept of elevated rates (often referred to as metabolic cold adaptation) has

f

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influenced thinking about polar physiology for over 70 years. Although useful for processes that can be examined at the molecular level, i t can be extremely misleading when applied to higher level processes such as growth or respiration (Clarke, 1980, 1983, 1991).

To date few cellular processes have been looked at in detail in relation to temperature compensation. Although examples of genes being switched on by temperature change are well known, for example, we know almost nothing of how the complex processes involved in gene expression are affected by temperature (if they are at all). Detailed studies have concentrated on contractile protein function and, in the case of microtubule function (Detrich, Prasad & Luduena, 1987) and fish white muscle (Johnston, 1988), we have an idea of adaptation at the molecular level. Although microtubule function shows a high degree of compensation for temperature (that is, microtubules in polar fish operate as effectively as those in mammals or temperate fish), current evidence suggests that temperature places an upper bound on muscle performance. Although polar fish are active, they cannot quite achieve the levels of performance found in tropical species (Johnston, 1988). For neural activity, compensation may be even poorer (Macdonald, 198 1).

No clear picture has emerged from studies of enzymes, possibly because of the difficulties of reproducing in vitro the conditions inside the cell (Clarke, 1991). All other things being equal, enzyme-catalysed reactions proceed more slowly at low temperatures, and there is a tendency for enzymes to denature by subunit disaggregation and/or unfolding (Franks, 1985). It is currently unclear whether evolutionary compensation for temperature involves changes in the primary structure of all cellular enzymes (which seems intuitively unlikely), changes only to those enzymes important in regulating flux through metabolic pathways, or whether there may be a mechanism of whole-cell homeostasis. Regulation of cell pH has been suggested (the alphastat hypothesis: Hazel, Garlich & Sellner, 1978) but no convincing evidence of a general role for pH in temperature compensation has been forthcoming (Clarke, 1987). Much attention has been paid to the role of lipid composition in membrane adaptation to temperature. Apart from a widespread tendency for membrane lipids to increase in unsaturation at low temperature, no clear picture has emerged from these studies. The major difficulties are the bewildering complexity of membrane lipids, and designing a properly controlled experiment (Morris & Clarke, 1987). However, there has also been a tendency to extrapolate from studies of acclimated eurythermal organisms to evolutionary adaptation in polar organisms, and to confuse adaptation of membrane function to temperature with tolerance to freezing. The way cells cope with these differing stresses may not necessarily be the same (Clarke, 1987).

Although the ideas of temperature compensation have frequently been applied to high level processes such as growth or respiration, this has not proved helpful. Growth of polar organisms is frequently slow for reasons of resource limitation rather than temperature. Also, since respiration is not a single process (‘metabolism’) but rather the sum of those processes requiring ATP, application of ideas of compensation to respiratory data is particularly misleading (Clarke, 1980, 1991).

Euolutionary considerations

The polar regions have not always been cold. A variety of evidence indicates that sea water at high Southern latitudes has cooled more or less steadily since the Tertiary. The marine fauna has thus had to evolve continually to track the change in mean sea water temperature (Clarke & Crame, 1989). Although there have been perhaps two or three periods when temperature fell quite rapidly, these rates (and those during past climatic cooling) are several orders of magnitude slower

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than those that many marine organisms can tolerate without stress (Clarke, 1990). There is thus an interesting dichotomy between the invocation of climatic cooling (‘deterioration’) as a cause of widespread extinction in the seas, and the physiological capabilities of living organisms. This difficulty may be resolved partly by viewing temperature (which is reasonably easily determined or inferred for fossil assemblages) as simply a marker for more wide-ranging ecological change (which is not). A final point is that to an organism whose physiology has adjusted to a particular temperature over evolutionary time, any change in average temperature will require adjustment. Climatic ‘amelioration’ (warming), although often seen by palaeontologists as beneficial, is just as much a problem, though different in kind, as climatic ‘deterioration’ (that is, cooling). Indeed, the low costs of basal metabolism at low temperatures indicate that living in cold water confers some energetic advantages (Clarke, 1991). We must continue to beware of anthropocentric judgements in discussing evolutionary and physiological adjustment to low temperature.

I am grateful to Dr W. C. Block and Professor J. Davenport for helpful comments on an early draft of this review.

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