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Revista Chilena de Historia Natural ISSN: 0716-078X [email protected] Sociedad de Biología de Chile Chile

REZENDE, ENRICO L.; GOMES, FERNANDO R.; GHALAMBOR, CAMERON K.; RUSSELL, GREGORY A.; CHAPPELL, MARK A. An evolutionary frame of work to study physiological to high altitudes Revista Chilena de Historia Natural, vol. 78, núm. 2, 2005, pp. 323-336 Sociedad de Biología de Chile Santiago, Chile

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative SELECTION, PLASTICITY AND ALTITUDE ADAPTATIONRevista Chilena de Historia Natural323 78: 323-336, 2005

An evolutionary frame of work to study physiological adaptation to high altitudes

Un marco conceptual para estudiar adaptaciones fisiológicas a altas altitudes

ENRICO L. REZENDE1*, FERNANDO R. GOMES1, CAMERON K. GHALAMBOR2, GREGORY A. RUSSELL1, 3 & MARK A. CHAPPELL1

1 Department of Biology, University of California, Riverside, California 92521, USA 2 Department of Biology, Colorado State University, Fort Collins, Colorado 80523, USA 3 University of California White Mountain Research Station, Bishop, California 93514, USA *E-mail for corresponding author: [email protected]

ABSTRACT

How complex physiological systems evolve is one of the major questions in evolutionary . For example, how traits interact at the physiological and genetic level, what are the roles of development and plasticity in Darwinian , and eventually how physiological traits will evolve, remains poorly understood. In this article we summarize the current frame of work evolutionary physiologists are employing to study the evolution of physiological , as well as the role of developmental and reversible in this context. We also highlight representative examples of how the integration of evolutionary and developmental physiology, concomitantly with the mechanistic understanding of physiological systems, can provide a deeper insight on how endothermic vertebrates could cope with reduced ambient temperatures and oxygen availability characteristic of high altitude environments. In this context, high altitude offers a unique system to study the evolution of physiological traits, and we believe much can be gained by integrating theoretical and empirical knowledge from , such as life-history theory or the comparative method, with the mechanistic understanding of physiological processes. Key words: adaptation, evolutionary processes, , life-history, oxygen availability, phenotypic plasticity.

RESUMEN

Una de las preguntas más importantes en fisiología evolutiva es cómo evolucionan los sistemas fisiológicos complejos. Por ejemplo, actualmente sabemos poco sobre la interacción entre varios rasgos a niveles genéticos y fisiológicos, sobre el papel de la plasticidad fenotípica durante distintas etapas del desarrollo y madurez para la evolución fisiológica dentro de un linaje. En este trabajo explicamos el marco conceptual ocupado por fisiólogos evolutivos en la actualidad para estudiar adaptaciones fisiológicas a nivel evolutivo y el papel de la plasticidad dentro de la evolución Darwiniana. Citamos ejemplos de cómo la integración de la fisiología evolutiva y del desarrollo nos permitió un mayor entendimiento de cómo vertebrados endotérmicos pueden “adaptarse” a altas altitudes. Los organismos de alta altitud ofrecen un excelente sistema para estudiar la evolución de rasgos fisiológicos, y hay mucho por aprender en ese contexto al integrarse el conocimiento teórico y empírico de la biología evolutiva, tales como la teoría de historia de vida y el método comparativo, con el conocimiento mecanístico de los procesos fisiológicos. Palabras clave: adaptación, procesos evolutivos, selección natural, historia de vida, disponibilidad de oxígeno, plasticidad fenotípica.

INTRODUCTION pressures involved are known: hypoxia and cold (lower hypobaria or higher atmospheric Studying metabolic adaptations to high altitude radiation seem to be less relevant for birds or (e.g., Rosenmann & Morrison 1975) provides a mammals; Monge & León-Velarde 1991). very interesting and useful model to understand Second, considerable research on the the evolution of complex physiological physiological basis of aerobic performance has systems, for many reasons. First, the selective been done and the transport of O2 from lungs to 324 REZENDE ET AL.

tissues have been carefully described implicitly assumed that maximizing O2 delivery (Richardson et al. 1999, Bassett & Howley to the tissues in hypoxic environments is 2000). Third, we now have a better adaptive. However, even if this is the case, understanding of the genetic basis of aerobic ‘adaptation’ and its various definitions must be metabolism and its physiological correlates differentiated because of their underlying through the use of quantitative and causes and levels in which they occur (see also selection experiments (e.g., Dohm et al. 2001, Monge & León-Velarde 1991). Consider the Nespolo et al. 2003). Finally, the tight following hypothetical example: a population association between aerobic capacity and of mammals inhabiting lowlands colonizes sustained locomotor and/or thermoregulatory higher altitudes. Initially, as animals move performance in endotherms makes the former a higher, heart rate and stroke volume increase to likely target of selection, which has been provide more O2 to the tissues, and these recently shown to be the case in wild deer mice changes are perceived as adaptive because in natural habitats (Hayes & O’Connor 1999). without them, animals would have asphyxiated Despite considerable research on the topic, as O2 availability decreased. Phenotypic many questions about how adapt to plasticity would account for this pattern (e.g., high altitudes remain. For example, the relative physiological changes occurred within each contributions of genetic, maternal, individual’s lifetime), and therefore higher developmental and environmental factors cardiac output would be a ‘physiological determining the adult (and how adaptation’ (more precisely, an acclimatory these factors interact) are not fully understood response) to increased altitudes. An animal’s (e.g., Brutsaert 2001, Rupert & Hochachka physiology can change within seconds (e.g., 2001). How animals respond differently to during sprint running), days or months variable altitudes given their inherent nature (acclimatization), or during the course of and evolutionary history (e.g. mammals do not development. have feathers), and which responses are In the same hypothetical population, adaptive in the true Darwinian sense may however, chronically high cardiac output is depend on several factors (e.g., Garland & detrimental in the long-term because of Adolph 1991, Garland & Carter 1994, Feder hypertension and cardiac diseases, and et al. 2000). In light of the current knowledge individuals with this phenotype are selected of evolutionary biology, physiologists against in the course of many generations. In an acknowledge that not all traits are adaptive, and evolutionary perspective, high cardiac outputs past history, genetic structure of the population, would therefore be maladaptive (i.e., among many other factors, can influence for decreasing overall ), and selection would physiological patterns observed in different favor those individuals able to provide enough conditions. Here we attempt to (1) summarize O2 to the tissues without higher workloads. If the current framework on how to study this phenotype has an underlying genetic physiological adaptation, given the background component (and assuming that hypertense physiologists now have from evolutionary individuals had a lower probability of survival biology; (2) demonstrate how this framework before they could pass their genes on), allelic can be applied in the study of altitude combinations allowing for higher metabolic adaptation and; (3) provide incentive to those rates sustained by a lower cardiac output would students interested in physiology to learn ‘not increase in frequency in the population. These only on how animals work, but also how are true adaptations in the Darwinian sense, and physiological systems evolve’. we shall refer to those as ‘genetic adaptations’, which can be defined as ‘changes in the mean Cardiac output and the concept of adaptation phenotype of a population due to changes in gene frequencies as a result of natural Endotherms inhabiting high altitudes face a selection’. ( and founder effects, double challenge: they must thermoregulate also involved in phenotypic evolution through and power activity in an environment where genotypic changes, are considered by both temperature and oxygen partial pressures evolutionary biologists as ‘non-adaptive’ are low. In this context, physiologists have processes; see below). SELECTION, PLASTICITY AND ALTITUDE ADAPTATION 325

GENES AND DARWINIAN ADAPTATION /lineage under selection. Many traits may be present in a lineage not because it is Darwin’s proposition of natural selection as the adaptive to current conditions, but due to past evolutionary process explaining biological events of selection, genetic bottlenecks, drift, adaptation relied on three major observations: etc. Nevertheless, evolutionary history has been species do not grow exponentially through time practically ignored by many comparative as predicted (Malthusian growth), animals from a physiologists (see Garland & Adolph 1994). single population are not all alike (e.g., many Recent advances in the ‘comparative method’ populations have considerable individual (see below), for instance, highlight how variation), and this variation is in part heritable. misleading studying Darwinian adaptation With competition and variation, organisms with without considering the phylogenetic history of traits that would increase their survival and a lineage can be (e.g., Felsenstein 1985, reproductive success (i.e., fitness) would Garland & Ives 2000, Rezende & Garland contribute differentially to the next generation. 2003). After several generations, that trait would be Because of evolutionary history, many more frequent in the population and could physiological responses may be deleterious or eventually become the norm. Although the pathological when colonizing a different concept may seem trivial, ‘the origin of species’ environment. For example, physiologists was a huge collection of information emphasizing initially considered decreased of hemoglobin the complexity of evolutionary processes and (Hb) for O2 affinities and increased red blood patterns (even more when heredity mechanisms cells in as beneficial ‘adaptations’ to were a matter of speculation). Natural selection, high altitudes, whereas now physiologists however, was – and remains (e.g., Nespolo 2003) acknowledge that these responses may simply – the underlying common mechanism responsible not be adaptive (Monge & León-Velarde 1991). for the origin of biological adaptations. Furthermore, it is now acknowledged that high Today, the study of adaptation through Hb-O2 affinity is characteristic of hypoxia natural selection relies on a slightly modified tolerant species, and such pattern has been framework (see Nespolo 2003). First, observed across widely divergent vertebrate phenotypic variation is the result of genetic species (Hopkins & Powell 2001). (additive and non-additive) and environmental The opposite may also be the case, and components, and how they interact (Fig. 1); the some traits already present in a lineage may proportion of the phenotypic variance of a trait facilitate the movement and survival of that due to the (additive) in the lineage in a new niche or environment. These population is the ‘heritability’ of that trait are considered ‘exaptations’ (Gould 1991); (Falconer 1989, Roff 1997, Rupert & Hochachka although they are adaptive (i.e., increasing 2001). Second, the rate of evolution of a trait in overall fitness) to these new conditions, they response to selection will be proportional to the evolved in that lineage before this new additive genetic variance of that trait selective regime was encountered. For (‘fundamental theorem of natural selection’, example, the high Hb-O2 affinity observed in Fisher 1930), and the intensity of selection. With South American camelids (llamas and this framework, modern evolutionary geneticists vicuñas) was initially thought to be an have developed a robust body of knowledge on adaptation to high altitudes. Studies in Old how several traits coevolve, the evolution of life World camels and dromedaries now show histories and complex (see Roff that high Hb-O2 is a common trait in the 1997, 2002 for reviews). In this context, several entire family, being present in the lineage aspects may affect how animals will (or will not) before the colonization of the Andes, adapt to their environment (i.e., high altitude) in suggesting that camelids in South America a Darwinian sense. were ‘preadapted’ to colonize high altitudes (Monge & León-Velarde 1991). (Many Evolutionary history physiologists refer to exaptations as preadaptations, in spite of the misleading Evolutionary responses to a given selective connotation of ‘evolving structures in pressure will first depend on the nature of the anticipation to future need’). 326 REZENDE ET AL.

Population structure and genetic background performance in hypoxia (hypoxic exercise tolerance) is consistent with expectations from Knowledge of the structure of a population and a two-locus segregation model. At the its genetic composition is now essential to population level, how information is passed on understand phenotypic change on a in each generation will ultimately determine the microevolutionary scale (e.g., Grant & Grant trajectories of phenotypic evolution. According 1995). Natural selection will act upon the to the fundamental theorem of natural variation already present in the population, and selection, populations lacking additive genetic evolution will occur when that variation is to variance (e.g., high levels of inbreeding) will some extent hereditary. Although optimality have a negligible response to selection. Genetic models have been applied quite successfully to correlations may lead to co-adaptation of study phenotypic evolution (e.g., Alexander several traits in response to a single selective 1982, Roff 2002), evolution ultimately depends pressure, even when these traits do not provide on population structure and the genetic any increased fitness (Lande & Arnold 1983). background of that population. Indeed, As a case study, Rezende et al. (2004a) have quantitative genetic models show that, although shown that rodents from cold environments can correlated traits could eventually evolve to attain higher maximum metabolic rates (MMR), their ‘optimal’, it may take several to many and suggested that basal or resting metabolic generations to attain that optimal in response to rates (BMR) might be also higher in colder selection (Roff 1997). climates because both metabolic indexes were Population structure can affect phenotypic positively correlated. On the other hand, evolution in several ways. First, it ultimately negative correlations (genetic trade-off) may determines how many alleles may be constrain or delay the overall response to ‘immersed’ in the population’s gene pool, selection, and lead to adaptive valleys in the setting an upper limit to allelic variation. (i.e., certain combination of Second, as the effective population size traits would have lower fitness). decreases, genetic drift becomes increasingly important in determining evolutionary Identifying selective pressures trajectories, and it may overcome the effects of natural selection. Third, immigration may High altitude environments impose two major provide enough to counteract the constraints for endothermic organisms: cold effects natural selection. This factor is temperatures, and hence higher thermoregula- particularly important in studies of altitude tory requirements, and low O2 availability. In adaptation. Geographically isolated populations addition, local factors may be influencing phe- at high altitude may be under strong selection, notypic evolution. For example, one would ex- and still not evolve or adapt because additional pect different intensities of selection (and evo- genes are flowing from low lands. In the lutionary rates, if there is enough genetic hypothetical example above, for example, the variation) depending on the predatory regime scenario would be different if there was a high populations encounter (e.g., Reznick & Bryga flow of individuals from the source population 1987). To understand how these selective pres- – many carrying genes potentially detrimental sures are acting in a population is considerably in the long-term – to high lands. According to more difficult, however, and many factors Monge & León-Velarde (1991), ‘natural should be considered. selection does not seem to have operated in How organisms interact with their biotic humans as much as in other high-altitude and abiotic environment (‘’ and/or animals, probably due to their migratory habits’ ‘’, in a broad sense) varies among (see also Brutsaert 2001). individuals, populations and species, and will Genes carry the information from one ultimately determine the nature and intensity of generation to another, and much physiological selection, as well as its spatial and temporal variation has a genetic component. For patterns. For example, although lower example, by crossing two inbred lines of atmospheric pressures may constrain metabolic laboratory mice, McCall & Frierson (1997) power output in any aerobic organism, reduced found that the inheritance of running air density becomes an additional challenge for SELECTION, PLASTICITY AND ALTITUDE ADAPTATION 327 hummingbirds due to their hovering flight genotypes will be represented in subsequent (Altshuler & Dudley 2002). Mortality during generations. winter would vary in intensity depending on Physiology and life history are intimately whether individuals migrate to lower altitudes related to each other because it is largely and warmer environments, hence selection through physiological mechanisms that could account for higher thermogenic selection acts to produce adaptive strategies for capacities or hibernation in gregarious high- allocating limited resources to the competing altitude populations, or increased ability to functions of growth, survival, and reproduction avoid cold temperatures in migrant populations. (Stearns 1992). Physiological adaptations for Behavior plays a crucial role in phenotypic survival can thus place constraints on, or come evolution, therefore, and its importance in the at a cost to, the kinds of life history strategies evolution of physiological systems is now that evolve. From this perspective, the explicitly acknowledged (e.g., ‘the centrality of evolution of physiological systems and life organismal performance paradigm’; Garland & history strategies share the common feature that Carter 1994, p. 593). adaptations are embedded in a complex Selection may also be acting throughout phenotype that incorporates trade-offs between ontogeny, and a single selective pressure may integrated traits (see also Ghalambor et al. affect fitness functions of similar genotypes in 2004). Comparisons of physiology and life many different ways. Many studies have history strategies between low and high altitude reported high mortality rates in avian embryos populations or species provide a particularly relocated to high altitudes, for instance. Beattie good framework for investigating these & Smith (1975) described an overall increase in linkages because both physiological and life egg hatchability from 16 to 56 %, after raising history traits often exhibit predictable patterns chickens from six generations in high altitude, of variation across altitudinal gradients. Yet, highlighting how strong selection may be few attempts have been made to critically during ontogeny and how fast populations may examine the linkages between adaptive changes respond in an evolutionary scale. In this in physiological and life history traits. context, physiology should evolve to decrease Do physiological adaptations to breeding at mortality during development and still lead to high altitudes constrain the kinds of life history viable adult phenotypes, and novel strategies that evolve? While such constraints developmental trajectories may be selected as a can be predicted on theoretical grounds (Hayes new environment is colonized (see et al. 1992), few empirical examples exist, Developmental plasticity section below). although various lines of evidence from endothermic vertebrates suggest linkages Physiology and life-history between physiological and life history strategies. An examination of life history Understanding how selection shapes strategies of birds occupying low and high physiological traits and leads to adaptive altitudes shows a repeated pattern of reduced systems ultimately depends on the linkages fecundity and increased parental care at high between an organism’s physiology and its life altitudes (Badyaev 1997, Badyaev & history. Life history traits are those traits that Ghalambor 2001). The increase in parental care contribute directly to the number of offspring at high altitudes is driven primarily by an an organism produces over the course of its increase in the amount of male contribution to lifetime. Commonly measured life history traits the incubation, nestling, and post-fledging include the size and age when organism begins periods. Badyaev & Ghalambor (2001) argue to reproduce, how often it reproduces, the that this pattern arises because at high altitudes number and size of offspring, and the relative the colder temperatures, reduced food allocation of time and energy to reproduction, availability, and greater climatic versus growth and maintenance (Roff 2002, unpredictability negatively impact juvenile Stearns 1992). Life history traits thus make up survival, thus favoring a life history strategy of the major components of Darwinian fitness and producing fewer offspring of higher quality as variation in life history traits amongst a buffer to these environmental conditions. individuals in a population determine which Alternatively, or also contributing to this 328 REZENDE ET AL. pattern, it could be that the increased energetic inherent complexity of physiological systems costs of breeding at high altitude (Weathers et and their multifunctional nature is, therefore, a al. 2002) reduces the amount of energy central component to be considered when available for investment in offspring number. studying adaptation. The cardiovascular Evaluating such physiological constraints will system, for example, must deliver O2, require considerable more research on the hormones and energetic substrates to tissues, physiological variability of birds along remove CO2 to lungs and metabolic wastes to altitudinal gradients. kidneys, etc. Furthermore, regulatory systems In contrast to birds, physiological must ensure adequate oxygenation of different adaptations to high altitude environments are tissues with variable metabolic workloads (e.g., much better known in mammals, whereas life rest or exercise), in a wide range of conditions, history variation is less well understood. responding at different temporal scales (acute However, mammal life histories do appear to or chronic stimulus) – with many different show a similar pattern of reduced fecundity at constraints. higher elevations as observed in birds (e.g., In this context, physics is the first factor Bronson 1979, Zammuto & Millar 1985). One constraining physiological function. A mouse of the few attempts to explore the conflicts cannot have the same absolute cardiac output of between physiological and life history a whale simply because of size, and lungs will strategies is work summarized by Wynne- lose water during respiration through the same Edwards (1998) on the closely related dwarf diffusive process by which O2 eventually hamster species in the genus Phodopus of reaches tissues. In a similar way, many central Asia. One species, P. campbelli occurs physiological processes – such as membrane at higher elevations, experiences colder and potential, or blood pH - will have narrow more arid conditions and has a lower critical functional ranges, potentially constraining maximum temperature compared to a closely increased performance. Constraints also occur related species P. sungorus (Wynne-Edwards when a single physiological system is 1998). The less extreme habitat of P. sungorus responsible for for the different functions (i.e. allows females to rear litters alone, whereas in trade-offs). In very cold and hypoxic proper P. campbelli bi-parental care is necessary to maintenance of environments, for example, alleviate thermoregulatory and water balance ‘mammals attempt to maintain both stresses on the female at higher elevations oxygenation and body temperature, although (Wynne-Edwards 1998). In this case, it appears conflicts can arise because of the respiratory that physiological adaptations for survival to a heat loss associated with the increase in more extreme environment place constraints on ventilation’ (Mortola & Frappell 2000). the kind of reproductive strategies that can be Finally, given the non-linear behavior of many favored by selection (Wynne-Edwards 1998). physiological functions, a response could be Thus, as is observed in birds, male Phodopus adaptive when it is moderate, and detrimental act to alleviate the challenges imposed by high or pathological as it increases. For example, altitudes on females attempting to rear although a higher hematocrit may increase offspring on their own. While it remains blood O2 carrying capacity, abnormally high unknown whether similar physiological values could be pathological (e.g., pathways shape avian life histories at high polycythemia) because of increased blood altitude, examination of the joint evolution of viscosity. physiological and life history strategies offers a useful framework for examining the linkages Non-Mendelian parental effects and constraints between these complex systems. Phenotypic variation will depend on genetic In a more holistic perspective, physiology is and environmental components, and how these always under selection: many physiological factors interact. In this context, the phenotype dysfunctions, at virtually any level of of the offspring may be dependent not only on organization, may compromise its own genotype, but also on the phenotypes of leading to decreased fitness or death their parents (predominantly the mother). (physiologists often take this for granted). The Maternal effects may or may not have a genetic SELECTION, PLASTICITY AND ALTITUDE ADAPTATION 329 component, having different sources: (i) the and often permanent phenotypic changes mitochondria or in cytoplasmatic factors (Spicer & Gaston 1999, Wilson & Franklin inherited in the egg (e.g., more or less yolk to 2002, Spicer & Burggren 2003). In humans, for nurture the embryo), (ii) genetic differences example, several studies support an important underlying variation in parental care, and (iii) developmental component explaining the environmental conditions experienced by the bigger thoracic dimensions observed in mother may affect its contribution to the populations at high altitudes, as well as in offspring’s phenotype (Roff 1997, p. 241). In people growing up with untreated chronic mammals, for example, the capacity of respiratory diseases such as asthma (Monge & providing O2 to the embryos during gestation, León-Velarde 1991). Some authors claim that it and the female’s lactation performance (e.g., is neither the genes themselves nor the discrete phenotype of the mother) may affect drastically adult phenotypes that are main the target of the offspring’s body size (and related life- natural selection, but the interaction between history traits), and probably has a genetic the genes and the environment through the component. However, a starving pregnant ontogenetic trajectory that determine the female cannot nourish her offspring, and this capacity to deal with the environmental source of variation is entirely environmental. contingency (Schlichting & Pigliucci 1993, 1995, McNamara & Houston 1996). The sensitivity of the phenotype to a PHENOTYPIC PLASTICITY: DEVELOPMENT AND particular environmental stimulus changes ADULTHOOD during development (Spicer & Burggren 2003). If specific challenges are presented during Although most of the knowledge in ecological ‘sensitive periods’ of the ontogeny, often called physiology has been built on studies comparing critical windows, the responses can be phenotypes of different individuals, particularly influential on the adult phenotype. populations, or species (see Comparative One example is the large and irreversible Method below), an additional level of variation effects on adult respiratory patterns caused by a must be considered: phenotypic changes within brief exposure to hypoxia during the first few the lifetime of each individual (Fig. 1). Such weeks after birth in rats (Strohl & Thomas changes are possible because the phenotype is a 1997). Maternal effects (above) can be very product of the interaction between genes and important, because the environment the mother the environment. The set of phenotypic ‘provides’ (e.g., an appropriate nesting site in expression of a single genotype in response to egg-laying species) will determine how the naturally occurring (or experimentally embryo develops. In humans, maternal imposed) environmental variation is called residence at high altitude promotes a decrease phenotypic plasticity. An environmental in birth weight when compared to sea-level stimulus can change a phenotype both by (i) pregnancies, possibly due to a reduction of the short-term modulation of the pre-existing uteroplacental and fetal volumetric blood flows physiological and biochemical systems (e.g. (despite of the compensatory response of changing the concentration of modulators of increased placental angiogenesis under such the enzyme-substrate affinity, or altering the conditions). This constraint has been linked to membrane fluidity through changes in its a downregulation of fetal growth reflected in constituents), and/or by (ii) changing gene decreased materno-fetal circulating growth expression. Genetic adjustments usually take a factors, placental nutrient transport, and fetal longer time to be fully expressed and include (Zamudio 2003). (i) altering the concentration of the same During adulthood a short to medium- enzyme isoforms or (ii) expressing different duration exposure to a different environment isoforms with different catalytic properties can also promote phenotypic changes. These (Hochachka & Somero 2002). events of phenotypic plasticity are usually Several studies have described how referred as acclimatization when they occur in environmental stimuli can alter developmental response to environmental changes in nature, trajectories. This developmental plasticity may and as acclimation when they are be crucial, since it tends to produce long-term experimentally induced by environmental 330 REZENDE ET AL.

(A) (B)

Fig. 1: Schematic diagram showing the potential effects of plasticity – either through different developmental trajectories, or because of short-term acclimation or acclimatization – on phenotypic differences between species or populations (see also Garland & Adolph 1994). (A) Two populations genetically different (i.e., population 2 is in average smaller than 1) develop in environments with different partial pressures of O2 (PO2). In both hypothetical populations, hypoxia leads to smaller body sizes, either by affecting growth rates during development (often irreversible effects, large arrows), or through modulations in food ingestion and activity levels during adulthood (reversible effects, small arrows with asterisks). (B) Expected distribution of size in each species when pheno- typic measurements are performed completely ‘at random’ (e.g., different ages, sexes, developmen- tal conditions, acclimated to either hypoxia or normoxia, etc). (C) Expected variation when both populations are studied under ‘common garden’ conditions (i.e., at similar PO2; all variance in this case is genetic or within-individual variation, because environmental variance is close to 0). (D) Hypothetical pattern if subsets of the populations developed at different controlled PO2 (N = normoxia, H = hypoxia). Note that specific developmental trajectories may constrain irreversibly the adult phenotype (dotted arrows), and groups developing in N and H within a population do not overlap despite of their genetic similitude (polyphenisms, Huey & Berrigan 1996). (E) Hypothetical diagram of reversible plasticity (solid arrows), when both populations developed under controlled conditions and are submitted to different PO2 when adults (short-term acclimation or acclimatiza- tion experiments). Diagrama representando como la plasticidad –durante el desarrollo o aclimatación y aclimatización– puede afectar compa- raciones entre dos especies o poblaciones. (A) Dos poblaciones genéticamente distintas (i.e., la población 2 es en promedio mayor que la 1) se desarrollan en ambientes con distintos PO2. En las dos poblaciones hipotéticas, la hipoxia tiende a disminuir el tamaño corporal, ya sea cambiando las tasas de crecimiento durante el desarrollo (efectos comúnmente irreversibles, flechas grandes), o al afectar la ingesta o actividad durante la madurez (efectos reversibles, flechas chicas con asteriscos). (B) La distribución de tamaño corporal esperada si las comparaciones entre poblaciones o especies se hace sin controlar ninguna otra variable (e.g., se agrupan distintas edades, sexos, distintos PO2, etc). (C) Variación esperada si se compara las poblaciones en un ambiente controlado similar (“common garden”; toda la varianza fenotípica es genética o intraindividual, ya que la varianza ambiental es cercana a 0). (D) Patrón esperado si muestras dentro de cada población se desarrollan en PO2 contrastantes (N = normoxia, H = hipoxia). Nótese que las trayectorias de desarrollo específicas pueden generar diferencias fenotípicas irreversibles (líneas punteadas). (E) Diagrama hipotético mostrando la variación fenotípica cuando la plasticidad es reversible (líneas sólidas), como en experimentos de aclimatación o aclimatización en animales adultos. SELECTION, PLASTICITY AND ALTITUDE ADAPTATION 331 manipulation in the laboratory. Unlike organismal physiology to , developmental plasticity, such changes can be – can one study these two factors (Fig. 2). We at least partially– reversible. For example, summarize current methods employed to infer reversible seasonal acclimatization can allow adaptive changes in response to selection, an individual to tolerate temperatures in winter highlighting studies in deer mice (Peromyscus), that would be lethal during summer and vice given widespread geographic distribution of this versa. As discussed above, many factors could genus, and the variety of techniques employed to account for non-adaptive plastic responses to study this rodent model’s evolutionary history environmental changes. Furthermore, it is and altitude adaptation. important to remember that the ability of regulatory systems to respond to a particular Comparative method environmental stimulus may have evolved in a context different from the one under study. For From Darwin’s time to the present, comparing example, responses to tissue hypoxia and their species or populations has remained the most underlying regulatory systems may have general technique for addressing questions evolved to deal with exercise instead of high about long-term patterns of evolutionary altitudes, and many of these responses might be change. In five species of Peromyscus reared deleterious when chronic. under common garden conditions, BMR was Rates of acclimation can be highly variable, significantly correlated with habitat primary not only among individuals, but also between productivity (Mueller & Diamond 2001); and different traits in a single individual (e.g., negatively correlated with temperature in 31 Rezende et al. 2004b), and many of the factors deer mice populations (although animals were underlying physiological responses must have a not reared in common garden, MacMillen & genetic component. Whether phenotypic Garland 1989). These results suggest that BMR plasticity can be considered a character itself, has evolved in response to different under genetic control at least partially environmental productivity and temperature independent of the mean phenotypic value, has (which is correlated with altitude). After been a major point of debate. Empirical support controlling for ‘acclimatization effects’ by to the view of phenotypic plasticity as an comparing species in similar thermal independent character has come from studies environments, Hayes (1989) showed that demonstrating independent evolution of trait populations of Peromyscus from high altitude means and plasticities (e.g., Huey & Berrigan have higher BMR than those from low altitude, 1996), as well as from experiments showing that suggesting that metabolism has evolved with phenotypic plasticity is responsive to selection altitude. (Schlichting & Pigliucci 1998, for a review). The comparative method has radically changed in recent years (e.g., Garland & Adolph 1994) and with the development of APPROACHES TO STUDY HIGH ALTITUDE analytical methods that incorporate ADAPTATIONS phylogenetic information and use explicit models of character evolution to allow Multiple levels of organization – from genes to statistical inference (see Rezende & Garland ecosystems – must be considered when studying 2003, Garland et al. in press, for reviews). Darwinian evolution and adaptation. Although Importantly, many additional questions can ‘proving’ that a particular trait is an adaptation now only be conceived in a phylogenetic may be logistically difficult (i.e., by rejecting all context. Randomization methods developed to other alternative hypotheses), physiologists now estimate ‘phylogenetic signal’ (the tendency of have tools to approach evolutionary problems more related species to resemble each other, more rigorously. The fundamental problem lies Blomberg et al. 2003), for instance, can provide in demonstrating that (1) allelic changes insights on how geographical factors may have underlying a particular trait were the result of affected‘historical patterns of and selection, and (2) the correlation between that vicariance. Among 57 species of rodents, trait, performance and ultimately fitness. Only altitude did not show significant signal by combining different techniques, from (whereas latitude signal was highly significant) 332 REZENDE ET AL. and was positively correlated with MMR Measuring selection in nature (Rezende et al. 2004a); which suggests that (i) altitude was not a major geographical barrier to Studying natural selection in action in the migration or colonization at that temporal scale field, and subsequent phenotypic evolution (e.g., thousands to millions of years of (e.g., Grant & Grant 1995), is an ultimate goal divergence), and (ii) there is selection for of evolutionary physiology. Very few studies, increased MMR at higher altitudes, probably however, have attempted to measure whether due to lower environmental temperatures. selection acts on individual variation in physiological traits and overall performance in Selection experiments and natural populations (e.g., Jayne & Bennett 1990). The basic protocol to study selection in Quantitative genetics studies how quantitative performance is as follows. Some estimate of traits are inherited in a population, and performance (e.g., running speed or ultimately the role of the genetic background in endurance) is measured in a cohort of phenotypic evolution (Roff 1997). Evolutionary individuals of known age, which is then physiologists are now studying, for instance, released in the field and the survivors are the underlying genetics of hypoxic tolerance in recaptured some time later. Several statistical laboratory strains of Mus (see above), as well procedures allow determining whether the as aerobic performance and subordinate traits probability of survivorship is correlated with a in laboratory and wild rodents (Dohm et al. particular estimate of performance. Using this 2001, Nespolo et al. 2003, Bacigalupe et al. approach, Hayes & O’Connor (1999) reported 2004). These studies have reported very low significant directional selection for higher heritabilities and additive genetic variance for MMR in a high altitude population of BMR and MMR (during cold or exercise), and Peromyscus, during one particularly cold no genetic correlations between traits (although winter. (Note that recapture rates are not new results suggest a heritability of about 0.6 necessarily a good index of survivorship or for MMR in a population of‘Phyllotis darwini, fitness, if emigration occurs). Nespolo personal communication). These An alternative approach consists of studies require very large sample sizes and comparing allelic or allozyme variation in complex breeding designs. Another approach different population (i.e., comparative study) relies on measurements of individual to infer selection from patterns of geographic consistency (repeatability) of a trait in a variation. In this case, candidate genes to be population (e.g., how consistent are the under selection are first identified, their differences between individuals, Hayes & contribution to phenotypic variation and Jenkins 1997). Repeatability is a prerequisite ultimately fitness would then be addressed for natural selection to affect trait variation, (Fig. 2). For instance, Peromyscus show an and it may set the upper limit on the narrow array of Hb polymorphisms, which are sense heritability of the trait (Dohm 2002). inherited basically as two different Selection experiments allow researchers to haplotypes (i.e., similar to what is expected study evolution of complex phenotypes in with Mendelian inheritance of a single locus action, under controlled conditions. Major with two alleles), and haplotype frequencies advantages of selection experiments over are correlated with altitude (Snyder et al. comparative studies include: (i) evolutionary 1988). Further research showed that inferences do not rely only on a correlational individuals with the ‘high-altitude haplotype’ approach, and (ii) environmental factors can be had increased Hb-O2 affinities, and higher either controlled or included in the design MMR during cold-exposure or forced (genotype by environment interaction). exercised when measured at high altitudes, Because previous authors have already whereas the opposite was observed in the reviewed how selection experiments can be ‘low-altitude haplotype’ (Chappell & Snyder employed for studying physiological evolution 1984). Again, different lines of evidence and adaptation to hypoxic environments (e.g., support that the correlation between allelic Garland 2001, 2003), we will not address the frequencies and altitude may be due to local topic in more detail here. adaptation. SELECTION, PLASTICITY AND ALTITUDE ADAPTATION 333

Similarly, population geneticists can now determine candidate genes affected by selection, by comparing frequencies across several loci. As summarized by Storz & Nachman (2003), the basic idea is that, ‘if allelic variation at most loci is simply tracking stochastic demographic processes, loci under selection should produce a detectable signal against the genome-wide backdrop of neutral variation’. After a multilocus survey of allozyme variation in A B Peromyscus populations across a steep altitudinal gradient, Storz and colleagues have suggested that the albumin locus (or perhaps a closely linked gene) is a candidate for local adaptation. Nevertheless, albumin is associated with several physiological processes, and how variation at this locus translates in differences in physiology, performance and fitness, is not clear (Storz & Dubach 2004).

Developmental physiology and acclimation Fig. 2: Diagram showing three of the major approaches used to study evolutionary physio- experiments logy. (1) The ‘gene to phenotype’ approach consists in screening for candidate genes affec- Because the period of development is such a ted by natural selection, and then studying the complex series of processes that results in a physiological and ecological mechanisms ex- fully-functioning individual, it is important to plaining the observed differences in allelic fre- understand how environmental perturbations quencies for that gene. (2) The ‘phenotype to can affect organisms during this period (Spicer gene’ approach initially identifies which phe- & Gaston 1999). Challenges occurring early in notypes have evolved as true adaptations (e.g., development may have quite different effects comparative method, selection experiments), than those occurring at later life stages, since approaching the problem at lower levels of or- different organ systems have different ‘critical ganization until the identification of the genetic architecture underlying these adaptations. (3) windows’ during which environmental Factorial experiments, studying the interaction perturbations will exert maximal effects between genotypes (in this example, being the (Dzialowski et al. 2002). Similarly, the amount same) and the environment, can be used to stu- of plasticity allowed for during ontogeny can dy plasticity during both development and define an individual’s physiological capacities adulthood. (All approaches can be applied at early during growth and development, which different levels of organization, from whole in- may have consequences for later survival (e.g., dividual performance to lower levels such as Tracy & Walsberg 2001). Hb-O2 affinity, cardiac output, etc). In this context, factorial designs are another Esquema mostrando tres metodologías para estudiar la useful tool to study the contributions of evolución de rasgos fisiológicos. (1) Del ‘gen al fenotipo’ se refiere a aquellos estudios donde primero se buscan ge- developmental and reversible plasticity in the nes potencialmente afectados por la selección natural, se- adult phenotype. In this sense, many guidos por estudios fisiológicos y ecológicos que explica- experimental designs may be employed to rían mecanísticamente los cambios alélicos en esos genes. (2) Del ‘fenotipo al gen’ se refiere a estudios que primero address different questions. Specific designs intentan determinar los fenotipos que evolucionaron como can provide insights about critical windows of adaptaciones Darwinianas, para después elucidar la estruc- development, whereas other approaches may tura genética que conlleva a esos fenotipos. (3) Experi- mentos factoriales, que estudian la interacción entre el ge- allow partitioning of the phenotypic variance notipo (que es constante en nuestro ejemplo) y el on a population in developmental and ambiente, pueden ocuparse para estudiar plasticidad feno- reversible components, and study their relative típica durante el desarrollo o madurez. (Cualquiera de esos métodos puede ser aplicado para estudiar rasgos fisiológi- contributions to overall plasticity during cos a distintos niveles de organización). adulthood. 334 REZENDE ET AL.

We emphasize that, although we have some unique system to study the evolution of knowledge of general effects of environmental physiological traits and fortunately, several variables in a phenotype, much remains to be tools such as the phylogenetically corrected done. For example, studies of reaction norms in comparative analyses, quantitative genetic physiological traits across many different analyses, selection and factorial experiments environments, addressing physiological among others, are now available to achieve responses to combined stimulus (e.g., how this goal. would mice acclimate to different temperatures as O2 availability changes?), or determining the effects of the maternal environment in the ACKNOWLEDGEMENTS offspring’s phenotype, are scarce in the literature. The ‘costs’ of phenotypic plasticity, We are particularly grateful to the staff of the as well as the adaptive character of many White Mountains Research Station at Barcroft physiological responses, must be tested for their constant support throughout the years; rigorously (Huey & Berrigan 1996). several studies cited here and performed by some of us would not be possible without their help. GAR also acknowledges a mini-grant CONCLUSIONS from WMRS. ELR and CKG thank C del Agua for insights and support. This work was The fields of physiology and evolutionary supported in part by NSF IBN-0111604 (KA biology have long functioned as separate Hammond and MAC). Finally, thanks to Mario disciplines, with each providing their own Rosenmann for the great tutor he was, always important contributions to science, but without sharing his knowledge and enthusiasm about consistent attempts to examine their natural science and life. Profe, muchas gracias por interrelationships. Comparative and todo, y vamos a extrañarte. environmental physiologists have been particularly successful describing (and demonstrating the functional importance of) LITERATURE CITED patterns of physiological variation associated to life in challenging environments, such as ALEXANDER RM (1982) Optima for animals. Edward Arnold, London, United Kingdom. 112 pp. high altitudes. However, a comprehensive ALTSHULER DL & R DUDLEY (2002) The ecological understanding of how organisms adapt to and evolutionary interface of hummingbird flight different environments requires robust physiology. Journal of Experimental Biology 205: 2325-2336. knowledge of the genetic, developmental and BACIGALUPE LD, RF NESPOLO, DM BUSTAMANTE evolutionary mechanisms underlying these & F BOZINOVIC (2004) The quantitative genetics patterns of phenotypic variation. To achieve of sustained energy budget in a wild mouse. Evolution 58: 421-429. this goal, evolutionary physiologists need to BADYAEV AV (1997) Avian life history variation along embrace conceptual and methodological altitudinal gradients: an example with cardueline advances provided by evolutionary biology. finches. Oecologia 111: 365-374. BADYAEV AV & CK GHALAMBOR (2001) Evolution Factors such as previous evolutionary history, of life histories along elevational gradients: trade- population genetic structure, developmental off between parental care and fecundity. Ecology plasticity, acclimatization, performance and 82: 2948-2960. BASSETT DR & ET HOWLEY (2000) Limiting factors of fitness consequences of physiological maximum oxygen uptake and determinants of variation, and the interactions of physiological endurance performance. Medicine and Science in systems with aspects of behavior and life- Sports and Exercise 32: 70-84. BEATTIE J & AH SMITH (1975) Metabolic adaptation of history traits need to be properly investigated the chick embryo to chronic hypoxia. American in order to avoid the circular reasoning typical Journal of Physiology 228: 1346-1350. of the ‘adaptationist program’ (Gould & BLOMBERG SP, T GARLAND & AR IVES (2003) Testing for phylogenetic signal in comparative data: Lewontin 1979). Similarly, evolutionary behavioral traits are more labile. Evolution 57: 717- biologists will benefit from the quantitative 745. mechanistic understanding of fitness- BRONSON MT (1979) Altitudinal variation in the life- history of the golden-mantled ground-squirrel determining performance traits provided by (Spermophilus lateralis). Ecology 60: 272-279. physiological methods. High altitude offers a BRUTSAERT TD (2001) Genetic and environmental SELECTION, PLASTICITY AND ALTITUDE ADAPTATION 335

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Associate Editor: Francisco Bozinovic Received December 2, 2004; accepted March 1, 2005