DNA replication

Introduction High-altitude environments pose numerous challenges to animal life. The physical environment changes dramatically on ascent, with declines in oxygen availability, temperature, air density and humidity. Despite these challenges, many animals live successfully in the high mountains. Birds are particularly diverse in montane regions – many live at over 4000m above sea level and some surmount the world’s highest mountain peaks during their migration (Fig.1). Although some species are unique to high elevation, others are found across broad elevational gradients (McCracken et al., 2009b).

The decreases in total barometric pressure (hypobaria) and O2 partial pressure (hypoxia) at high altitude are inescapable, unlike elevational declines in temperature and humidity, which can be buffered by local climatic variation. Hypobaria has unique consequences for flying animals, because the mechanical power output needed to sustain lift increases in thin air (Altshuler and Dudley, 2006). This amplifies the already high metabolic rates needed for flapping flight (Chai and Dudley, 1995) in an environment where the O2 available to fuel metabolism is limited. According to Tucker, “some birds perform the strenuous activity of flapping flight at altitudes in excess of 6100m, an altitude at which resting, unacclimated man is in a state of incipient hypoxic collapse” (Tucker, 1968). How then can O2 supply processes meet the high O2 demands of flight at high altitudes? What unique physiological characteristics allow the highest-flying species (Fig.1) to sustain the most metabolically costly form of vertebrate locomotion at elevations that can barely support life in many other animals?

In order to properly address these questions, one must consider the properties of the pathway that transports O2 from the environment to the sites of O2 demand throughout the body. This pathway is composed of a series of cascading physiological ‘steps’ (Fig.2): (1) ventilation of the lungs with air; (2) diffusion of O2 across the pulmonary gas-exchange surface, from the air to the blood; (3) circulation of O2 throughout the body in the blood; (4) diffusion of O2 from the blood to mitochondria in tissues (the pectoralis muscle is the primary site of O2 consumption during flight); and (5) metabolic utilization of O2 to generate ATP by oxidative phosphorylation. Although not a strict component of the O2 transport cascade, properties of intracellular ATP turnover will also have important consequences for matching O2 supply and O2 demand. Not surprisingly, the answer to how birds fly at high altitudes lies, at least partly, in the characteristics of this pathway.

The objective of this Commentary is to review the importance of both ancestral and derived characteristics in the O2 transport pathway of birds that fly at high altitudes. Many features of birds in general probably endowed high fliers with numerous exaptations (also known as pre-adaptations), but many uniquely derived and presumably adaptive traits also appear to be important for high- altitude flight.

The benefits of being avian The hypoxia tolerance of birds has frequently been suggested to be greater than that of mammals. Although some ectothermic vertebrates are even more tolerant of hypoxia, birds possess a relatively high tolerance when considering the increase in

The Journal of Experimental Biology 214, 2455-2462 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jeb.052548

COMMENTARY

Elevated performance: the unique physiology of birds that fly at high altitudes

Graham R. Scott Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada and School of Biology,

University of St Andrews, St Andrews, Fife KY16 8LB, UK scottg2@mcmaster.ca

Accepted 9 May 2011

Summary Birds that fly at high altitudes must support vigorous exercise in oxygen-thin environments. Here I discuss the characteristics that help high fliers sustain the high rates of metabolism needed for flight at elevation. Many traits in the O2 transport pathway distinguish birds in general from other vertebrates. These include enhanced gas-exchange efficiency in the lungs, maintenance of O2 delivery and oxygenation in the brain during hypoxia, augmented O2 diffusion capacity in peripheral tissues and a high aerobic capacity. These traits are not high-altitude adaptations, because they are also characteristic of lowland birds, but are nonetheless important for hypoxia tolerance and exercise capacity. However, unique specializations also appear to have arisen, presumably by high-altitude adaptation, at every step in the O2 pathway of highland species. The distinctive features of high fliers include an enhanced hypoxic ventilatory response, an effective breathing pattern, larger lungs, haemoglobin with a higher O2 affinity, further augmentation of O2 diffusion capacity in the periphery and multiple alterations in the metabolic properties of cardiac and skeletal muscle. These unique specializations improve the uptake, circulation and efficient utilization of O2 during high-altitude hypoxia. High-altitude birds also have larger wings than their lowland relatives to reduce the metabolic costs of staying aloft in low-density air. High fliers are therefore unique in many ways, but the relative roles of adaptation and plasticity (acclimatization) in high- altitude flight are still unclear. Disentangling these roles will be instrumental if we are to understand the physiological basis of altitudinal range limits and how they might shift in response to climate change.

Key words: avian, bar-headed goose, oxygen cascade, respiratory physiology, pulmonary diffusion, capillary, mitochondria.

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metabolic demands associated with endothermy. Early work showed that lowland house sparrows (Passer domesticus) behaved normally, and could even fly for short periods in a wind tunnel, at a simulated altitude of 6100m (Tucker, 1968). In contrast, mice were comatose and unable to maintain body temperature at the same simulated altitude. Comparisons of the few species for which tolerance (survival) data are available also support the suggestion that birds are more tolerant of hypoxia than mammals (Thomas et al., 1995). However, this issue has yet to be addressed with rigorous phylogenetic comparisons that incorporate species in both groups that are adapted to hypoxia.

The O2 transport pathway of birds has several distinctive characteristics that should support a greater capacity for vigorous activity and aerobic metabolism during hypoxia (Fig.2). Increases in breathing (i.e. ventilation) are an important response of the respiratory system to hypoxia, and the magnitude of this response is dictated primarily by the partial pressures of O2 and CO2 and the pH of arterial blood (Scott and Milsom, 2009). The decline in arterial O2 tension (hypoxaemia) drives the increase in ventilation, whose secondary consequence is an amplification of CO2 loss to the environment. This causes hypocapnia (low partial pressure of CO2 in the blood), which reflexively inhibits breathing and causes an acid–base disturbance. It has been suggested that birds have a higher tolerance of hypocapnia than mammals (Scheid, 1990), which could arise from an ability to rapidly restore blood pH in the face of CO2 challenges (Dodd et al., 2007) and from the hypocapnic insensitivity of the brain vasculature (see below). The significance of this tolerance is that it would allow birds to breathe more before

depletion of CO2 in the blood impairs normal function, thus enhancing O2 transport to the gas-exchange surface.

The structure and function of the lungs is perhaps the best- known advantage of avian respiratory systems. The many distinctive features of bird lungs are the subject of an extensive literature that unfortunately can be dealt with only briefly here (Piiper and Scheid, 1972; Scheid, 1990; Maina, 2006). Air flows in one direction through the gas-exchange units of avian lungs (parabronchioles) and the arrangement of airway and vascular vessels creates a functionally cross-current gas exchanger (Fig.3A). This differs substantially from the lungs of most other terrestrial vertebrates, in which gases flow in and out of terminal gas-exchange units (alveoli in mammals) such that capillary blood equilibrates with air having a uniform gas composition (uniform pool gas exchanger; Fig.3B). The important consequence of this difference is that avian lungs can attain a superior efficiency for gas exchange in normoxia and moderate hypoxia (as explained in Fig.3), although their advantage diminishes as hypoxia becomes severe (Scheid, 1990). The capacity for pulmonary O2 diffusion is also greater in birds because of the exceptional thinness and large surface area of the exchange tissue. Nevertheless, the diffusion barrier appears to be mechanically stronger in birds than in mammals, so pulmonary blood flow and pressure can increase without causing stress failure (West, 2009). Each of these distinctive features of avian lungs should improve O2 loading into the blood during hypoxia.

The capacity for delivering O2 throughout the body in the systemic circulation may be higher in birds than in other vertebrates. Birds have larger hearts and cardiac stroke volumes than mammals of similar body size (Grubb, 1983), suggesting that birds are capable of higher cardiac outputs. If this were indeed the case, birds would have an enhanced capacity for convective delivery of O2 in the blood during hypoxia. Cardiac output increases sevenfold to eightfold during flight (Peters et al., 2005) and threefold or more during hypoxia at rest (Black and Tenney, 1980), but maximum cardiac output has yet to be determined in birds, particularly during flight in hypoxia.

The distribution of blood flow throughout the body has consequences for hypoxia tolerance, and the mechanisms regulating this distribution are altered in birds compared with mammals. Hypoxaemia per se causes a preferential redistribution of O2 delivery towards sensitive tissues like the heart and brain and away from more tolerant tissues (e.g. intestines). However, increases in O2 delivery to the brain are offset in mammals at high altitudes because of the respiratory hypocapnia induced by increases in breathing. This causes a constriction of cerebral blood vessels that can completely abolish the hypoxaemic stimulation of cerebral blood flow. In contrast, the cerebral vessels of birds are insensitive to hypocapnia, such that blood flow is allowed to increase and O2 delivery is maintained (Faraci, 1991). This and possibly other distinctive features of the avian cerebral circulation (Bernstein et al., 1984) should improve brain oxygenation during hypoxia. Coupled with the inherently higher tolerance of avian neurons to low cellular O2 levels (Ludvigsen and Folkow, 2009), the central nervous system of birds appears to be well protected from cellular damage induced by a lack of O2. Nevertheless, an intriguing question that has yet to be addressed is whether heightened blood flow increases intracranial pressure in birds, as frequently occurs in humans (Wilson et al., 2009). If so, birds may face the secondary challenge of avoiding or tolerating cerebral oedema and other neurological syndromes that can result from excessive intracranial pressure in mammals.

G. R. Scott

Fig.1. Although most birds live and fly at relatively low altitudes, species from several avian orders live, migrate or occasionally ascend much higher. These include multiple species of raptor, waterfowl, crane, passerine, hummingbird and others. The highest flight altitudes reported from various sources in the literature are shown here (Eastwood and Rider, 1965; Swan, 1970; Faraci, 1986; Faraci, 1991; del Hoyo et al., 1999; Kanai et al., 2000; McCracken et al., 2009b).

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The capacity for O2 to diffuse from the blood into the tissues is higher in birds compared with mammals and other vertebrates. The best evidence for this difference is the systematically higher ratio of capillary surface area to muscle-fibre surface area in the flight muscle of birds compared with the locomotory muscles of mammals (Mathieu-Costello, 1990). At least two factors account for this difference: (1) the tight mesh of capillaries surrounding avian muscle fibres, due to a high degree of branching between longitudinal vessels, and (2) the smaller aerobic fibres of birds compared with similar-sized mammals (Mathieu-Costello, 1990). The heart and brain also have higher densities of capillaries in birds compared to mammals (Faraci, 1991). Diffusion of O2 from the blood to the mitochondria in various tissues should therefore be higher in birds than in other vertebrates during hypoxaemia.

Although these distinctive characteristics of birds should enhance hypoxia tolerance by improving the overall capacity for O2 transport, being avian is not in itself sufficient for flight at high altitudes. The flight muscle of birds has a very high aerobic capacity, by virtue of fast-contracting oxidative fibres (type IIa) that have abundant mitochondria (Mathieu-Costello, 1990; Scott et al., 2009b), and the high rates of metabolism during flight are supported primarily by lipid fuels (Weber, 2009). Lipid oxidation is essential for supporting long-duration flight, but it amplifies the amount of O2 required to produce a given amount of ATP when compared with carbohydrate oxidation. The metabolic demands of flight are further intensified at high altitudes by hypobaria, which requires that birds flap harder to produce lift (Chai and Dudley, 1995). The implication of these factors is that high-altitude flight requires very high rates of O2 transport when very little O2 is available. This is clearly not possible for most lowland birds – many species cannot tolerate severe hypoxia (Black and Tenney, 1980) and some fly long distances to avoid high-elevation barriers during their migration (Irwin and Irwin, 2005). What then are the uniquely derived attributes that differentiate the high fliers?

The unique attributes of high fliers The physiology of birds that fly at high altitudes differs in many ways from that of lowland birds. The basis for this conclusion comes largely from studying the bar-headed goose [Anser indicus

(Latham 1790)], a species that can tolerate severe hypoxia [~21Torr or ~2.8kPa (1Torr133Pa), equivalent to 12000m elevation] (Black and Tenney, 1980) and has been seen flying over the Himalayas at nearly 9000m elevation during its migration between South and Central Asia (Swan, 1970) (Fig.1). Studies of bar-headed geese have revealed many important insights into the physiological basis for high-altitude flight and, when coupled with comparative phylogenetic approaches, its evolutionary origins. My discussion of the unique attributes of high fliers will focus largely – out of necessity – on this species, but will also highlight work in other species when possible. Most of the previous work looking for inherent differences between high- and low-altitude birds compared animals in a common environment at sea level. This will be the case in the following discussion unless otherwise stated.

It is useful to begin this discussion by outlining the most influential steps in the O2 transport pathway during exercise in hypoxia. We have assessed this issue in waterfowl using theoretical modeling to calculate the physiological control coefficient for each step in the pathway (Fig.4) (Scott and Milsom, 2006; Scott and Milsom, 2009). This approach allows physiological traits to be altered individually so that their influence on the whole O2 pathway can be assessed without compensatory changes in other traits. A physiological trait with a larger control coefficient will have a greater influence on flux through the pathway, so an increase in the capacity of this trait will have a greater overall benefit. Interestingly, the proportion of control vested in each step was dependent on the inspired O2 (Fig.4). At sea level (inspired O2 tensions ~150Torr) and in moderate hypoxia (~90Torr, equivalent to ~4500m elevation), circulatory O2 delivery capacity (which incorporates both maximum cardiac output and blood haemoglobin concentration) and the capacity for O2 diffusion in the muscle retained most of the control over pathway flux (Fig.4). In contrast, ventilation and the capacity for O2 diffusion in the lungs became much more influential in severe hypoxia (~40Torr, roughly 9000m), whereas muscle diffusion remained important and circulatory O2 delivery capacity became less so (Fig.4). These results suggest that every step in the O2 transport pathway can be influential and that the relative benefit of each step changes with altitude.

Fig.2. The transport of O2 occurs along several steps of a cascading physiological pathway from atmospheric air to the mitochondria in tissue cells (e.g. muscle fibres). The effectiveness of this pathway at transporting O2 during hypoxia is imperative for flight at high altitudes, which depends upon several distinctive characteristics of birds in general and many unique features that have evolved in high fliers. The properties of O2 utilization and ATP turnover in the flight muscle are also important to consider in high fliers, such as how ATP equivalents are moved between sites of ATP supply and demand [which can occur via phosphocreatine (PCr) by virtue of the creatine kinase shuttle; see text]. Cr, creatine.

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High capacities at several steps in the O2 transport pathway have been shown to distinguish high-flying birds from their lowland cousins (Fig.2), confirming the theoretical predictions. The first step of this pathway, ventilation, appears to be enhanced in high- altitude birds to improve O2 uptake into the respiratory system. Bar- headed geese breathe more than low-altitude waterfowl when exposed to severe hypoxia (inspired O2 tensions ~23–35Torr or ~3.1–4.7kPa) (Black and Tenney, 1980; Scott and Milsom, 2007) and the magnitude of their ventilatory response is greater than in any other bird species yet studied (Scott and Milsom, 2009). Bar- headed geese also breathe with a more effective breathing pattern, taking much deeper breaths (i.e. higher tidal volumes) than low- altitude birds during hypoxia. There are at least two mechanistic causes for these differences: (1) ventilatory insensitivity to

respiratory hypocapnia and (2) a blunting of the metabolic- depression response to hypoxia (Scott and Milsom, 2007; Scott et al., 2008). These differences increase the amount and partial pressure of O2 that ventilates the pulmonary gas-exchange surface during hypoxia. Bar-headed geese also have enlarged lungs (Scott et al., 2011), as do numerous other highland species sampled at high altitudes (Carey and Morton, 1976), which should enhance the second step of the O2 transport pathway by increasing the area of the gas-exchange surface. The respiratory system of high-altitude birds therefore seems capable of loading more O2 into the blood during hypoxia than that of lowland birds.

The circulatory delivery of O2 throughout the body is also enhanced in high-altitude birds. The most pervasive mechanism for sustaining the circulation of O2 in hypoxia is an alteration in the O2- binding properties of haemoglobin in the blood. Numerous high- altitude birds, such as the bar-headed goose (Fig.5), Andean goose (Chloephaga melanoptera) (Black and Tenney, 1980), Tibetan chicken (Gallus gallus) (Gou et al., 2007) and Ruppell’s griffon (Gyps rueppellii) (Weber et al., 1988), are known to possess haemoglobins with an increased O2 affinity. This can dramatically increase O2 delivery and pulmonary O2 loading in hypoxia by increasing the saturation of haemoglobin (and thus the O2 content of the blood) at a given O2 partial pressure (Fig.5A), and can, in doing so, greatly improve flux through the O2 transport pathway (Scott and Milsom, 2006). The genetic and structural bases for haemoglobin adaptation to high altitude have been resolved in many species. For example, the bar-headed goose possesses a major (HbA) and minor (HbD) form of haemoglobin, whose  subunits contain four (A) (Fig.5B) and two (D) uniquely derived amino-acid substitutions, respectively (McCracken et al., 2010). One of the substitutions in A (Pro-119 to Ala) (green in Fig.5B) is thought to cause a large increase in O2 affinity (Jessen et al., 1991) by altering the interaction between

G. R. Scott

A

B

Fig.3. Schematics of (A) the cross-current model of gas exchange in the avian lung and (B) the uniform pool model of gas exchange in the lungs of mammals and most other terrestrial vertebrates. In the cross-current model, inspired air flows through rigid parabronchioles that are oriented perpendicular to blood capillaries. The partial pressure of O2 (PO2) in the parabronchioles (PPO2) declines along their length as O2 diffuses into the blood, such that capillaries leaving the exchanger near the entrance of airflow (right side of figure) take up more O2 than capillaries leaving near the exit (left side). The contents of all capillaries mix to dictate the PO2 of arterial blood (PaO2), which can have a higher PO2 than expired air (PEO2). In the uniform pool model, gas flows in and out of terminal alveoli. Capillary blood flowing past these alveoli extract O2, such that capillary PO2 rises and alveolar PO2 (PAO2) declines uniformly to less than the PO2 of gas that entered the alveoli. Arterial blood leaving the lungs has a PO2 that is at best equal to PAO2 (but is generally slightly less), which is less than the average PEO2. The cross-current model is therefore considered to be more efficient at gas exchange than the uniform pool model (Piiper and Scheid, 1972). PIO2, inspired PO2; PVO2, PO2 of venous blood.

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