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The diving physiology of pinnipeds : an evolutionary enquiry Mottishaw, Petra Deigh 1997

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T H E DIVING PHYSIOLOGY OF PINNIPEDS: AN EVOLUTIONARY ENQUIRY by Petra Deigh Mottishaw B.A. Hons. Marine Biology University of California, Santa Cruz, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as/e6nforrniii]g to the required^ sfandard UNIVERSITY OF BRITISH COLUMBIA April 1997 © Petra Deigh Mottishaw, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Hpr 30 DE-6 (2/88) II Abstract During the last century, studies of diving physiology and biochemistry made great progress in mechanistically explaining the basic diving response of aquatic mammals and birds. Key components of the diving response (apnea, bradycardia, peripheral vasoconstriction, redistribution of cardiac output) were generally taken to be biological adaptations, but the evolution of the diving response has not been seriously examined. This study compares several key characters of the diving response in the pinnipeds using phylogenetically independent contrasts. As the traits examined are known to be functionally important for diving, I expected them to correlate positively with diving ability. Instead, some physiological/biochemical characters considered adaptive for diving do not correlate with diving ability. These traits are similar in phocids (true seals) and otariids (sea lions and fur seals) and include diving apnea and bradycardia (and probably also include tissue hypoperfusion, and hypometabolism of hypoperfused tissues). This finding contradicts the generally accepted theory that these traits are adaptations for extending dive time in pinnipeds. The key components of the diving response are more appropriately seen to be ancestral (plesiomorphic) characters. Another group of physiological/biochemical characters was more variable among the taxa examined. These included body weight, spleen weight, whole body hemoglobin content, and blood volume. Increases in these characters correlate with Ill increased diving capacity (defined as maximum recorded diving duration). This correlation of characters, along with functional knowledge of current utility, leads to the conclusion that changes in two traits - spleen size and whole body oxygen carrying capacities - may have been driven by selection for increased diving duration in pinnipeds. iv Table of Contents Abstract ii List of Figures and Tables vi Acknowledgements viii Introduction 1 Before Scholander 3 Since Scholander 5 Some Basic Principles of Evolutionary Biology 7 Phylogenetically Independent Contrasts 9 Comparative Physiology and Evolution 12 Evolution of Diving Physiology 13 The 'Slow-Lane' Phocid Strategy of Diving 14 The 'Fast-Lane' Otariid Strategy of Diving 18 Is the Diving Response Adaptive? 20 Materials and Methods 22 Values, sources, and choice of characters 22 Differences between phocids and otariids 26 Statistical analyses of dive time 27 The phylogeny 28 Phylogenetic analysis 30 Results 33 Physiology, morphology and dive time: the non phylogenetic analysis 33 vi List of Figures and Tables Figure 1. A "worst case" scenario phylogeny 11 Figure 2. Scatter diagrams of a hypothetical data set 11 Figure 3. Phylogeny of pinniped ancestry 15 Figure 4. Composite phylogenetic hypothesis for the pinnipeds 29 Figure 5. Average dive time vs maximum recorded dive time 34 Figure 6. Heart rate and dive time 35 Figure 7. Removal of the effects of body weight on heart rate 36 Figure 8. Body weight and maximum recorded dive time 39 Figure 9. Spleen weight and maximum dive time in phocids and otariids 41 Figure 10. Spleen weight and maximum recorded dive time in pinnipeds 44 Figure 11. Estimated spleen weight and maximum recorded time 46 Figure 12. Spleen weight and average dive time 48 Figure 13. Whole body hemoglobin and maximum dive time 50 Figure 14. Blood and maximum dive time 53 Figure 15. Estimated whole body hemoglobin and dive time 55 Figure 16. Estimated blood volume and dive time 57 Figure 17. Independent contrasts of average vs maximum dive time 59 Figure 18. Lowest heart rate and maximum dive time contrasts 61 Figure 19. Log lowest heart rate and maximum dive time contrasts..... 63 Figure 20. Log lowest heart rate and average dive time contrasts 65 Figure 21. Body weight and dive time contrasts 69 V Bradycardia and diving duration 33 Body size and long diving duration 33 Spleen size and long diving duration 37 Whole body hemoglobin, blood volume, and long diving duration. 42 The phylogeny and sources 51 Phylogenetic analysis 58 Bradycardia and diving duration 58 Body size and long diving duration 58 Spleen size and long diving duration 66 Whole body hemoglobin, blood volume, and long diving duration. 66 Discussion 82 Lack of Correlation of Diving Response Characters 85 Potential Problems and Sources of Error 90 Net Effects of Physiological/Biochemical Adaptations for Diving 91 Emerging Principles of Evolution of Physiological Systems 93 Summary and Conclusions 94 References 96 Appendix 1. Literature values and sources for pinnipeds 109 Appendix 2. Three alternate phylogenetic hypotheses for the pinnipeds 122 vii Figure 22. Body weight and dive time contrasts without the Baikal seal 71 Figure 23. Body weight and dive time contrasts for different phylogenies 73 Figure 24. Spleen weight and maximum recorded dive time contrasts 75 Figure 25. Estimated spleen weight and maximum dive time contrasts 77 Figure 26. Whole body hemoglobin and maximum dive time contrasts 79 Figure 27. Blood volume and maximum dive time contrasts 81 Table 1. Net effects of putative adaptations expressed as ratios of MR 88 Acknowledgements VIII This thesis would not have been possible without the enthusiasm, ideas, and financial support of Dr. Peter Hochachka. I would like to thank him for allowing independent thought and learning through mistakes. I would also like to thank the Hochachka lab, (Gary Burness, Grant McClelland, Sheila Thornton, Gunna Weingartner, Mark Trump, Mark Mossey, Jim Staples, Steve Land, Tim West, and Carole Stanley) for their conversations and insights. I would like to thank Gary, specifically, for the long methodological discussions. Drs. Theodore Garland Jr., Sally Otto, Dolph Schluter and Arne Mooers were instrumental in the development of this thesis. The help and support of Alex Mahon and my parents was invaluable in the preparation of this thesis. I would also like to thank the scientists in marine mammal biology who made unpublished data available for this study. Drs. D. Costa, R. Andrews, M. Hindell, D. Calkins, G. Stenson, G. Kooyman, R. Werner, B. Stewart, J . Bengston, T. Ragen, R. Harcourt, R. Mattlin, K. Kovacs, R. Gentry, I. Boyd, J . Schreer, R. Merrick, D. Boness, M. Fedak, J . Francis, K. Frost, D. Coltman, G. Lento, J . Koskela, T. Eguchi, E. Nordoy, L. Folkow, C. Lydersen, I. Gjertz, N. Gales, O. Wiig, A. Trites, 0 . Bininda-Emonds, A. Berta, K. Ronald, A. Blix, and J . Ford provided indispensable advice, information, and numerical values for species which had little available information on diving, physiology, and phylogeny. 1 Introduction This is the first multi-species evolutionary investigation of a well-studied complex physiological system. The analysis brings an evolutionary approach to one of the most thoroughly explored systems in comparative physiology - 'the diving response'. For decades, comparative physiologists have called the variables that they study in aquatic mammals 'adaptations for diving' (Harrison, 1957; Kooyman, 1973; Andersen, 1966; Snyder, 1983; Castellini, 1991). Until now, no one has examined these assumptions. Evolutionary biologists have recently stressed the need for rigor in the use of the term adaptation by comparative physiologists (Gould and Lewontin, 1979; Leroi et al. 1994. In investigating physiological adaptations to diving , it is necessary to address some of the principles of evolution. The thesis will begin by discussing traditional approaches of comparative physiology, the study of the diving response and diving in pinnipeds, and then review some basic evolutionary principles, and explain the purpose of this study. Comparative physiologists investigate species differences in order to understand the mechanics of how organisms work. The fundamentals of this approach were first voiced by August Krogh (1929), when he stated that for any problem there is an animal or a few animals on which it can be most 2 conveniently studied. This concept was named "the Krogh principle" by Hans Krebs in 1975. The Krogh Principle is well exemplified by the study of diving, which dates back to the early years of modem comparative physiology. In the late 1930's Per Scholander investigated physiological responses to facial immersion by choosing mammals which demonstrate the best ability to endure not breathing - diving mammals (Scholander, 1940). The pioneering studies of Scholander, Lawrence Irving and their colleagues (Irving et al. 1935; Scholander, 1940) revealed the physiological and metabolic mechanisms which conserve oxygenated blood for the brain and heart, and permit an air breathing animal to operate deep into the water column. Their work provided a foundation for diving physiology, which is now known to include three key physiological 'reflexes': (i) apnea, (ii) bradycardia, and (iii) vasoconstriction and thus hypoperfusion of most peripheral tissues. Scholander (1940) referred to these physiological reflexes in combination as the 'diving response', and, in simulated diving under controlled laboratory conditions, he imagined the marine mammal reducing itself to a 'heart, lung, brain machine'. The metabolic representation of this response included the gradual development of oxygen limiting conditions in hypoperfused (ischemic) tissues, with attendant accumulation of end products of anaerobic metabolism (especially lactate and H + ions). Scholander realized that because peripheral tissues were hypoperfused, most of the lactate would remain at sites of formation during the course of a (simulated) dive, and that most of it would not be 'washed out' of the tissues into the blood until perfusion was restored at the end of diving. 3 Scholander's theory was supported by the observation of a small lactate accumulation in blood plasma during diving, and a peak of lactate seen early in recovery from a dive. Initially, Scholander and Irving expected that any energy deficit due to oxygen lack during diving would be made up by anaerobic glycolysis (with concomitant lactate accumulation). However, in their studies on the harbor seal they observed that the post-diving oxygen debt was frequently less than the expected oxygen deficit during diving. Additionally, the amount of lactate accumulated often was substantially less than would be expected if the energy deficit were to be made up by anaerobic glycolysis, which lead to the concept of decreased metabolic rate during diving (Scholander, 1940). Before Scholander Human interest in respiration, or lack of respiration, in diving animals can be traced as far back as the published work of Robert Boyle (1670), better known for his work on the fundamental properties of gases. Zoologists in the 1600s thought that waterfowl had a unique structure around the heart which allowed them to survive without respiring. Boyle experimented with waterfowl and non-aquatic birds, and was the first to demonstrate that ducks could survive longer underwater than hens. Two hundred years after Boyle the French physiologist Paul Bert conducted a set of experiments in the 1860s which investigated the respiratory 4 abilities of ducks and hens. Bert found that ducks have a much larger blood volume than hens, and he attributed their diving ability to the increased oxygen storage capacity that this afforded. He concluded this because he bled the duck until it had the same blood volume as the hen, which unsurprisingly, reduced the ducks diving ability. Bert was also the first to record diving bradycardia. He measured the heart rate of a duck with his hands while he held it under water and found a drop from 100 to 14 beats per minute. Bert's hypothesis was that non-respiring animals died from lack of oxygen, which led him to choose divers as obvious subjects. He catalogued the maximum diving abilities of many species. The French physiologist Charles Richet (1894) did not agree with Bert. He stated that the increase in oxygen stores afforded by the greater blood volume of the duck was not enough to account for the increase in dive time. His experiments showed that submergence in water was an important factor in the abilities of the duck. The animal did not survive as long when it was prevented from respiring in air. Richet also found, from analysis of respired gases, that the metabolism of the duck decreased dramatically during diving. The first comparative analysis of seals was published in 1838 by Burow in Keonigsberg. Burow gave a detailed anatomical description of the circulatory system, and found no major differences in the heart or arteries of seals and terrestrial mammals, but he found a very large venous reservoir in the seal. 5 Since Scholander In the decades since the pioneering studies of the 1940's, many laboratory studies were performed which essentially supported the framework developed by Scholander and Irving (see Zapol et al. 1979; Butler and Jones, 1982; Eisner and Gooden, 1983). The key features which were observed in simulated diving studies over and over again were (i) apnea, (ii) bradycardia, (iii) peripheral vasoconstriction, (iv) low lactate accumulation during diving per se, and (v) a post-dive lactate washout peak appearing in the plasma within 1-4 min of recovery; the term for this combination of processes became fixed in the literature as 'the diving response' (Andersen, 1966; Butler and Jones, 1982). The advent of modern field study technologies, especially of microprocessor-assisted monitoring of aquatic animals while diving voluntarily in their natural environment (Kooyman and Campbell, 1972; Guppy et al. 1986; Hill et al. 1987), has confirmed over the last two decades the existence and plasticity of the overall 'diving response' first elucidated in the 1930s and 1940s and has greatly extended our understanding of how the diving response occurs under natural diving conditions (Kooyman et al. 1980; Ovist et al. 1986; Le Boeuf et al. 1989; DeLong and Stewart, 1991; Castellini ef al. 1992; DeLong et al. 1992; Hindell era/ . 1992; Thompson and Fedak, 1992; Guyton era/ . 1995; Hochachka ef al. 1995; Hurford etal. 1995). Scholander and many students following in his path observed that the key features of the diving response were evident in many different kinds of animals. Diving bradycardia was often used as a kind of index or indicator of the diving 6 response. As it was seemingly so universal among vertebrates, Scholander (1963) referred to it as the 'master switch of life'. In his day, the diving response was viewed as an obvious 'physiological adaptation' to diving, even if there was little indication as to how the response evolved through any particular lineage. Since Scholander and Irving's research, other physiological characters have also been called adaptations to diving and these include greater body size, spleen size, blood volume, hemoglobin (Hb) concentration and myoglobin concentration (Butler and Jones, 1982). Body weight influences the total onboard oxygen supplies as well as mass-specific energy demands (Butler and Jones, 1982). The spleen holds and releases oxygenated red blood cells and can be seen as a physiological " S C U B A tank" during diving (Qvist et al. 1986). Finally, blood volume, hemoglobin and myoglobin concentrations are direct measures of oxygen carrying capacity. Current studies implicitly or explicitly consider the above traits as 'adaptations' which help to explain why an elephant seal can survive without breathing for 2 hours while diving (Hindell et al. 1991), and why true seals are better divers than sea lions (Costa, 1991). The study of diving goes back for hundreds of years; yet the approaches thus far have added little insight into how the diving response evolved in any given lineage, or into how physiological characters may be adapted for different life styles and environments. 7 Some Basic Principles of Evolutionary Biology Physiologists often use the term 'adaptation' to mean a process, structure, or behaviour that increases survival. This is a 'current utility' definition of the word: if a character serves a certain function, it is an 'adaptation' for that function. Evolutionary biologists use a different definition of the word 'adaptation.' For the past 20 years the field has become more rigorous in the definition of the term. A well-known definition of 'adaptation' was given by Sober in 1984. "A is an adaptation for task T in population P if and only if A became prevalent in P because there was selection to perform task T." A second definition, given by Coddington (1988), defines adaptation as "apomorphic [evolutionarily derived] function due to natural selection." Both of these definitions require an analysis of the changes in a trait over time. Gould and Lewontin (1979) argued that not all evolutionary changes and not all species traits are adaptive. This anti-optimality approach led to questions about the way that organisms evolve. Gould and Lewontin attest that most species traits exist because of inheritance from ancestors. "The hierarchical nature of speciation," as it is often called, results in closely related animals being more alike. Gould and Vrba (1982) elaborated this concept and noted that, "four legs may be optimal, but we have them due to conservative inheritance, not by selective design." A second category of non-adaptive evolution, and an often cited reason for species differences is pure chance. The less closely related two species are, 8 the more likely they will be different, due to random mutation and genetic drift. This is almost the antithesis of the previous example. If a physiologist studies two traits in a laboratory rat and a desert tortoise, any differences may be due not to 'adaptation to the desert' but to the fact that the two species are likely to be different. The inclusion of history in an analysis is key to understanding the chain of events in the evolution of a series of traits, and is critical in understanding the evolution of a trait. One example is arachnologist's knowledge of the orb web in spiders. For decades it was thought that orb webs had evolved from cob webs as they allowed orb web weavers to catch more flies, and produced a selective advantage. When arachnologists finally developed a phylogeny of spiders, they discovered that the orb web was ancestral to the cob web. The more germane question then became, "what is a cob web an adaptation for?" (Coddington, 1988). Some traits may not serve the function that has been proposed for them or may not have originated for that function. These possibilities cannot be ruled out until the history of the trait and the function of the trait are thoroughly understood. A hypothesis that rabbits are white in the arctic because there is a selective advantage due to camouflage against the snow has not ruled out the possibility that white coats contain thermal properties that may also confer an advantage. As physiologists study mechanisms, this is one area where they can strongly contribute to the knowledge base of evolution. 9 Aside from the above example of current mechanism, many of the other alternate hypotheses for an adaptation may be tested by the investigation of the history of the trait. In order to have a falsifiable hypothesis about the adaptive significance of a trait, it must be possible to trace the history of the trait and its function. If the trait arose before fulfilling its purported function, then it must have been selected for another function or may have been neutral. The evolutionary biology definition of 'adaptation' requires that a trait have originated by selective forces, and/or be selectively maintained for its original function. Phylogenetically Independent Contrasts One of the first errors that develops in an analysis of a character across many species is the failure to acknowledge the problem that arises from the hierarchical nature of phylogenies: species which are more closely related are more likely to be phenotypically similar. This is simply because they have had less time to diverge than species which are farther from them on a phylogenetic tree. As a result, regressions and correlations of observed traits among many species violate the assumption that each point on the graph is independent of the others. Non-phylogenetic analyses run the risk of inflated Type I error rates, because closely related species should not be treated as independent data points in statistical analysis. Non-independence of data results in artificially inflated degrees of freedom and statistical power. Felsenstein (1985) illustrates 10 this problem by demonstrating how two groups of moderately closely related species (Fig. 1) could result in spurious correlations between two measured characters (X and Y) (Fig 2). If X and Y are measured in the 40 species and correlated against each other, differences in the two groups of species' means could result in the appearance of a relationship. To circumvent the problem of non-independence of species, Felsenstein (1985) presented the method of analysis, called phylogenetically independent contrast (PIC), which results in N-1 statistically independent contrasts for N species. The method requires that each species value for a variable is subtracted from its most closely related species or node (branch in the phylogeny). The differences between relatives are "weighted" proportionally to the time since they diverged (or distance on the tree). PIC analysis, therefore, looks at changes in characters over time rather than the actual value for the character. Felsenstein's method of independent contrasts requires a phylogeny with branch lengths in expected units of variance, which in most cases is time. This is based on the null hypothesis that traits evolve randomly (as by Brownian motion) over time. Under this null hypothesis, change in a character should be proportional to the time two species have had to evolve away from each other. Contrasts between less closely related species are likely to be greater in absolute value (characters have had more time to evolve along longer branches), the contrasts are "standardized" by dividing each by the square root of the sum of the branch lengths. The expected variance increases with 11 Figure 1. A "worst case" scenario phylogeny for 40 species, in which there prove to be two groups of 20 close relatives (from Felsenstein, 1985). * * * ww * * * * * «r v «** D O a © ft* • B M Figure 2. A) Scatter diagram of a hypothetical data set that might be used to test the relationship between characters X and Y. B) The same data set, showing that a relationship could potentially be found in the two groups of closely related species (Felsenstein, 1985; Promislow, 1991). 12 increasing branch lengths, therefore the standard deviation is equal to the square root of the branch lengths. Harvey and Paget (1991) give a detailed summary of the methods of independent contrasts and Felsenstein's (1985) mathematical assumptions. Independent contrasts use phylogenetic information to transform data such that they become, in principle, independent and normally distributed. The transformed data (standardized independent contrasts) can then be used in ordinary statistical procedures (Felsenstein, 1985). Comparative Physiology and Evolution The use of independent contrasts in physiology has increased in the past ten years. Studies have been conducted on locomotory performance in reptiles (Garland and Losos, 1994), blood parameters in mammals (Promislow, 1991), and metabolic rates in birds (Reynolds and Lee, 1996), among many other investigations. 'Evolutionary physiology' as a discipline has dramatically expanded over the past decade. The discussion surrounding principles of evolution, proper methods of analysis, and rigor in use of the term 'adaptation,' has sparked a large debate in comparative physiology (Feder et al. 1987; Garland and Adolph, 1994; Garland and Carter, 1994). In evolutionary biology, arguments still continue over the level of rigor necessary in defining adaptations and the relevance of comparative studies in understanding evolution (Leroi et al. 1994; Doughty, 1996). As the number of evolutionary studies which use the comparative method has grown, some 13 evolutionary biologists have maintained that comparative studies can never demonstrate whether a trait is adaptive. Leroi et al. (1994) argue that the potential complexity of the genetic correlations among traits makes understanding the history of a trait impossible. They state that correlations between characters cannot provide enough information to differentiate between many different evolutionary mechanisms. Nonetheless, comparative biologists have been drawing evolutionary conclusions since Darwin, and species differences are integral in our understanding of the relationship between organisms and their environment. Comparative studies use the results of 'natural experiments,' and though we generally cannot follow the value of a trait through time, correlational evidence does provide information about evolutionary processes (Doughty, 1996). Although it remains unclear which evolutionary processes are most likely to result in the correlative patterns we observe, the use of correlational data does provide evidence for adaptation, especially when combined with other methods of analysis (Doughty, 1996). Manipulative experiments and a strong understanding of the function of a character add significantly to an adaptive argument. Evolution of Diving Physiology Since the time of Scholander, in studies of diving physiology little attention has been paid to the criteria of evolutionary biology: that to be defined as adaptive, a character must have arisen and be maintained by natural 14 selection. To assess whether these traits are adaptations for diving in pinnipeds, the ideal outgroup analysis would be to trace the evolution of diving ability between pinnipeds and their closest non-diving ancestors, the bears (Figure 3), and correlate this with changes in the physiological characters. Unfortunately, there have been no studies on diving abilities of bears and weasels, the pinnipeds' closest terrestrial ancestors. If characters are evolutionary adaptations for extending dive time within pinnipeds, it follows that these traits should correlate with maximum dive time. We should see increased evolution of these traits in lineages with especially long dive times. An analysis of these characters throughout pinnipeds should reveal trends in characters, and allow a falsifiable hypothesis on the adaptiveness of these characters. Changes in characters which correlate with changes in dive time are consistent with an adaptive hypothesis. Furthermore, a comparison of the phocids (true seals) with the otariids (sea lions and fur seals) should show changes in physiology which reflect the differences in their diving capacities. The 'Slow-lane' Phocid Strategy of Diving To illustrate the phocid strategy of diving, let us consider the elephant and Weddell seals for which there are good data bases. First of all, the two species of elephant seal currently hold the dive duration record - 2 hours (Hindell et al. 1992) - and dive depth record - 1.5 km (DeLong et al. 1992) - among marine mammals. Northern elephant seals go to sea for months at a time and migrate 15 Sea lions, Figure 3. A phylogeny of the pinnipeds and their closest ancestors. The monophyletic origin for the phocids and otariids has become generally accepted (Sarich, 1969; Berta et al. 1989; Berta and Wyss, 1994). Recent molecular data are consistent with monophyly and support the bears as the closest ancestor (Lento et al. 1995). v 16 thousands of km per year, during which they spend approximately 90% of the time submerged (Le Boeuf et al. 1989; 1992). One of the major food items for these phocids appears to be midwater vertically migrating squid. The prey are pelagic and so the northern elephant seal too becomes a pelagic mammal. While the natural histories of the southern elephant seals (McConnell et al. 1992) are different, their diving capabilities are similarly large (Hindell et al. 1992). The natural history of the Weddell seal differs even more from that of the northern elephant seal. Weddell seals operate in seas that freeze overwinter. By maintaining breathing holes in these areas, these seals are able to exploit a niche and food resource, again well into the pelagic water column, that would otherwise be unavailable. Not surprisingly, the diving capacities of the Weddell seal (in terms of depth and duration) are also impressive (Castellini et al. 1992; Hochachka, 1992). Many of the biological and physiological adjustments underpinning the making of a mesopelagic marine mammal are now well understood (Hochachka, 1986; 1992; Kooyman, 1985). Excluding obvious morphological adaptations for pelagic performance (flippers vs feet, collapsible pulmonary system, etc.), the major functional characteristics for such sensational diving capabilities include: (1) apnea, with exhalation upon initiation of diving (for minimizing buoyancy and other pressure-related problems) (2) bradycardia, (3) peripheral vasoconstriction and hypoperfusion (in order to conserve oxygen for the central nervous system (CNS) and heart), 17 (4) hypermetabolism of (vasoconstricted) ischemic tissues (also in order to conserve oxygen and plasma borne fuels for the C N S and heart). (5) an enhanced oxygen carrying capacity (enlarged blood volume, expanded red blood cell mass within the blood volume - i.e. higher hematocrit (Hct), higher hemoglobin concentration ([Hb]) in red blood cells, and possibly higher myoglobin concentration ([Mb]) in muscles and heart), and (6) an enlarged spleen (for regulating the Hct so that a very high % of R B C s need not be circulated under all physiological conditions). Additionally (7) , it should be noted that, for really outstanding diving, all of the above characteristics (i) are incorporated with a large body weight (in order to maximize the amount of oxygen that can be carried while minimizing mass-specific energy demands during diving by allometric effects), and (ii) are coupled with slow swimming speed while at sea (to minimize the cost of locomotion while maximizing submergence, hence foraging, time). The evidence for these overall patterns arises from studies of several phocid species (for example, see Kooyman et al. 1980; Guppy et al. 1986; Ovist et al. 1986; Castellini et al. 1992; Hindell et al. 1992; Thompson and Fedak, 1992; Reed et al. 1994; Hochachka et al. 1995; Hurford et al. 1995; Guyton et al. 1995). This combination of characters likely enables phocids to dive longer than otariids. 18 The 'Fast-Lane' Otariid Strategy of Diving Several sea lions and fur seals, for which there are good data bases (see Gentry and Kooyman, 1986; Ponganis et al. 1990; Costa, 1991; 1993), can be used to illustrate the characteristics of this lineage. First of all, these are animals that do not spend as much time diving, either expressed as a fraction of each year or as a fraction of each day when they are foraging. Average dive durations are on the order of a few minutes. Maximum duration dives observed so far are only 15-20 min and maximum depths are only in the 300-400 m range. Diet consists largely of inshore prey species. Swim speeds in terms of body lengths per second are substantially higher in otariids than in phocids (see Williams et al. 1991; Ponganis et al. 1990). Again, many of the physiological characteristics underpinning this otariid diving behaviour are now well understood (for literature in this area, see Butler and Jones, 1982; Gentry and Kooyman, 1986; Costa, 1991; 1993; Ponganis et al. 1990; Williams et al. 1991). They include: (1) apnea (initiated on inhalation, in contrast to phocids) and a gas exchange system that does not completely collapse (so the lung probably continues to function as a gas exchange organ during most diving), (2) bradycardia, (3) peripheral vasoconstriction, with propulsive muscle microvasculature presumably more relaxed than in phocids (because of the higher oxygen and fuel demands of otariid swimming) relative to other less-energy demanding peripheral tissues, 19 (4) hypometabolism of ischemic tissues (the longer the dive, the lower the metabolic rate (Hurley, 1996), presumably to conserve oxygen for the C N S and the heart, as in diving phocids). (5) an oxygen carrying capacity intermediate between that of the large phocids and terrestrial mammals (this includes blood volume, red blood cell mass or Hct, [Hb] in RBCs, and [Mb] in muscles and heart), (6) a modest-sized spleen, not much larger as a percentage of body weight than found in terrestrial animals, and, additionally, (7) a relatively small and hydrodynamically sleek body consistent with a higher speed predator life style. The operational tradeoff seems to require sacrificing extended foraging time for energy needed for higher speed swimming (Costa, 1991). The diving strategy of otariids is more energetically demanding relative to that of the phocids, perhaps as a result of being a predator targeting fast swimming inshore fish as a major food source. Therefore, the energy dissipative diving strategy of the otariids probably results in lower selection for long duration dives than the energy conserving diving strategy of the phocids. 20 Is the Diving Response Adaptive? Ideally, for tracing the evolution of the diving response in these lineages, one would like to be able to compare all of the above metabolic and physiological characters in numerous phocid and otariid species. However, in reality, detailed information on complex characters such as tissue specific regional hypoperfusion is available for only a few species. For multiple species comparisons, the analysis is unavoidably restricted to only a few data sets (or characters) that have been quantified for many species. On the basis of data availability for sufficient numbers of species, maximum recorded diving duration was analyzed as a function of (i) maximum bradycardia during diving, (ii) body weight, (iii) spleen weight, (iv) whole body blood volume and (v) whole body Hb (defined as the content of Hb in the entire blood volume of the organism). Data for other components of the diving response, such as tissue specific regional hypoperfusion, muscle myoglobin concentrations and myoglobin saturation, are available for only a few (or even a single) species and were not analyzed. It is implicit in much of the literature on diving that all five characters play an adaptive role in diving. This is the first comparative attempt to quantify the physiological characteristics that confer diving ability within pinnipeds. These characters were investigated in the seals, sea lions and furs seals, as they are some of the world's best divers, and because there is a large data base of published physiological, morphological, and diving behaviour values available. 21 The current study addresses how these mechanisms evolved by looking at the trends in these characters across all pinnipeds, and comparing the phocids with the otariids. The hypothesis is that characters which are adaptations for increasing dive time in pinnipeds will positively correlate with increases in diving ability. By using a historical context for analyzing differences in diving mechanisms, this study may be able to develop a better understanding of their evolution. 22 Materials and Methods Values, sources, and choice of characters Literature values were used for diving variables (maximum and 'average' dive times), body weights, and the physiological/ morphological characters associated with the diving response for which enough data were available (maximum bradycardia, spleen weight, hemoglobin concentration [Hb], and blood volume). Variables were collected for the species and subspecies of pinniped which were available in the literature or from unpublished studies. Separate diving data and physiological values from different subspecies were included whenever possible, as separating distinct populations and including them in the phylogenetic analysis often adds power to the analysis. Variables were correlated against maximum recorded dive time, as selection may act on the maximum capacity (to allow increased foraging time), and 'average' dive time was assessed in a less consistent manner across studies. 'Average' dive time includes mean or modal dive times presented in the different studies on pinnipeds, and was dependent on the settings of the dive recorders (minimum depth of measured dives and maximum capacity of the recorder). The variables used in dive time analysis were chosen because they have been called 'adaptations for diving' by physiologists (Harrison, 1957; Kooyman, 1973; Andersen, 1966), and there were enough published values to perform the analysis. 23 Dive times available for pinnipeds are given in Appendix 1. Also included are the weight of animals, number of measured animals of each species, the method used in measuring dive time, and the sources. Only values obtained using remote sensing technologies attached to the animal were used. No published values of direct observations or forced dives were used in order to maintain consistence. How close the published maximum recorded dive time comes to the true maximum dive time of the species depends on how many dives have been observed in the species, where the animals were diving, age of the animal, what time of year, and many other factors. In recent years, dive times have been monitored in a wide range of species, and the study of diving patterns does not seem to be correlated with the diving ability of the species. The error introduced by these potential inconsistencies did not appear to depend on the branch of the phylogeny that the species was in. Therefore, the error should not result in spurious correlations. Furthermore, 'average' dive times depended on the types of recording devices (mostly TDRs or time depth recorders), what their minimum 'dive' setting was, and the methods of dive time analysis (mean or modal dive times). In spite of this, there was a strong correlation between maximum recorded dive time and 'average' dive time (r = 0.77, P < 0.001). As more dive data were available on females, only dive times of females were used in otariids and elephant seals, where a large sexual dimorphism encouraged the use of only one sex. Including both sexes as separate points violates the statistical assumption that each point is independent of the others, 24 and using females eliminates possible sex differences in diving within each species. Mean weight of the adult animals used to gather the diving data were used when available. If weights were not given for these individuals, mean weight of an adult of the same sex, in sexually dimorphic species, was used and the source given. Age, time of year, fasting state, and reproductive state varied for measurements of individuals in different species. The values and sources of maximum bradycardia, spleen weight, hemoglobin concentration, and blood volume are given in appendix 1, along with the method of measure, and number, age, and weight of the animals when it was given. Comparable measures of the physiological characters were also used, whenever possible. Measures of lowest heart rate during a dive (bradycardia) were taken from literature on forced dives, trained dives, and free dives as there were not enough measurements available to choose only one of these methods. Most spleen weight data were taken from dissections. Methods of estimating spleen weight by in vivo imaging of volume resulted in much higher estimates of weight than direct measurement, and were not included, as dissection data for those species were available. Blood volumes gathered by different methods were included, as no large differences were seen, and not enough values were available from one method, as with the bradycardia measurements. Differences in measuring techniques did not appear to be associated with dive time measurements, and therefore, were likely to introduce more noise to the correlations in this analysis. 25 Analysis of weight also presented a problem in the analysis of the physiological parameters. A number of the published spleen measurements had been collected from immature seals or pups, and a few studies gave the spleen weight as a percentage of body weight, without giving the weight of the animals or the organ itself. Two thorough papers on spleen anatomy in harbor seals (Sokolov, 1966) and southern elephant seals (Bryden, 1971) showed that the relative size of the spleen did not drastically change between seals of different body weights. Put another way, the slope of a plot of log spleen weight vs. log body weight of these animals is 1. Spleen weights were therefore estimated for animals of the same body weight as the divers. This was done by dividing the average measured spleen weight by the average measured weights of the animals in the spleen weight experiments, which resulted in the fraction of body weight accounted for by the spleen. This number was multiplied by the average weight measurements in the diving experiments, which resulted in an estimate of the spleen weight of the diving animals. The analysis was performed for measured and estimated weights. Measured and estimated analyses were also conducted on blood volume and whole body hemoglobin content (calculated as the measured concentration of hemoglobin in the blood multiplied by the blood volume). In order to perform regressions on a number of physiological variables, they had to correlate with animals of the same weight in each species. Estimates of spleen weights, blood volumes, and whole body hemoglobin 26 contents (for an animal the size of the animal that made the dive) allowed comparisons of the physiological/morphological variables with dive times. Although values for the walrus are included in Appendix 1, the walrus was not included in the analysis, as the dive data were not comparable to the other species (one dive recorder strapped to the tusk of a large male in a shallow area for a short time). The Baikal seal had such a long dive time for its size that it changed the relationship between dive time and body weight. It has been suggested that Baikal seals have evolved significantly longer dive times and gotten smaller as a result of the pressures of human hunting on the iced over lake Baikal (the world's deepest lake). The correlation of maximum recorded dive time and body weight was done with and without the Baikal seal. The physiological parameters were analyzed with the Baikal seal included, as this thesis predicts that the Baikal seal should be able to dive longer due to these characters. Differences between phocids and otariids If, as decades of diving literature claims, the phocids are longer divers than the otariids, and the physiological variables in this study extend dive time, these variables should differ predictably between the phocids and otariids. Differences between phocid and otariid characters were analyzed with one tailed t-tests, or in the case of dive time and spleen weight, Mann-Whitney rank sum tests. The significance level used for all tests was 0.05. No multiple 27 comparisons correction was performed. The null hypothesis was that there were no significant differences between phocids and otariids. Statistical analyses of dive time All variables, except bradycardia, which showed little variation and was normally distributed, were log-io transformed to meet the assumptions of the statistical analyses. To remove the influence of body weight, dependent variables were regressed on body weight using ordinary least squares linear regression, and residuals were generated (as in Bennett, 1987). (Residuals answer questions such as 'do animals with larger spleens than predicted for their body weight have longer dive times than predicted for their body weight?' This is done by correlating the distance each species falls from the regression line of spleen weight versus body weight with the distance each species falls from the regression line of dive time versus body weight.) Many studies attempt to remove body weight from biological characters by dividing by the weight of the animal and expressing the character as a ratio or percentage of weight. Metabolic rate, for instance, is expressed in the literature as ml oxygen per kg per minute, and is consequently presented as such in Table 1. This may result in erroneous conclusions if the relative size of the biological character changes with the size of the animal (if the slope of the regression of the character versus body weight is not 1 on a log-log plot). Residuals were taken from regression analysis on each physiological, morphological, and dive time variables versus body weight. Correlations were 28 conducted for the residuals of each physiological and morphological variable vs. body weight and the residuals of dive time vs. body weight. The residuals generated from the regression lines were correlated using Pearson product moment correlations. Correlations and regressions were analyzed with one tailed tests, due to predictions from previous functional studies. The null hypothesis was that the diving behaviour traits would not correlate with the physiological, biochemical, and morphological values that are functionally associated with dive time in the literature. The phylogeny Sources of the pinniped phylogenies available in the literature and used in this study are given in figure 4 and Appendix 2. Four phylogenetic hypotheses were constructed from the literature phylogenies. The phylogenies were constructed using both morphological and molecular information from the literature. Figure 4 is the phylogeny that is agreed upon by the largest number of pinniped biologists, and is used for the majority of the contrast analyses. The pinnipeds are very well studied, and many analyses have been conducted on the relationships of these species. Figure 4 has been supported by recent morphological and molecular studies. The remaining three phylogenies (Appendix 2) each represent an area of debate among pinniped biologists. The branch lengths of the four phylogenies were based on time (dated nodes). 29 Figure 4. One composite phylogenetic hypothesis for the pinnipeds based on molecular and morphological data (compiled from Arnason et al. 1995; Berta and Demere, 1986; Berta and Wyss, 1994; Burns and Fay, 1970; Lento et al. 1995; Muizon, 1982; Perry et al. 1995). Some branches of the tree are supported by both molecular and morphological evidence, while other areas, such as the fur seal species, are not fully resolved. 30 Phylogenetic analysis Phylogenetically independent contrasts were calculated for each variable in the manner described in Garland, et al 1993. Contrasts were generated using the computer programs P D T R E E (Garland et al. 1992) and CAIC (Purves and Rambaut, 1995). Contrasts were "standardized" by dividing by the square root of the branch lengths (which is an estimate of the standard deviation expected among the contrasted species). Dividing contrasts by their standard deviation should result in values that are independent of the phylogeny they were generated from. P D T R E E was used to determine that the branch lengths shown in figure 4 resulted in adequate "standardization." This was done by plotting the absolute value of the standardized contrasts against their standard deviations (Garland et al. 1992). This is conceptually the same as plotting any ratio versus its denominator to determine whether scaling effects of the denominator have been effectively removed (Garland et al. 1992). Generating contrasts is done by random direction of subtraction of the values for each variable, which results in indeterminate signs for contrast values. Therefore, no scatterplot of contrasts from two variables are unique. By convention, as more than one graph can be generated, the independent variable is made positive and the dependent variable is changed as needed ("positivised"). All contrasts were positivised, and all regressions were run through the origin, as required by PIC analysis. 31 Prior to computation of independent contrasts, all variables except maximum bradycardia were log 1 0 transformed. Calculating contrasts resulted in n-1 number of values for n species. The transformed data (standardized independent contrasts) can then be used in the same ordinary statistical procedures as the non-phylogenetic analysis. All regressions were run through the origin, as required by PIC analysis. Each variable had been measured in a different number of species. Therefore, contrasts were generated for each physiological or morphological variable, along with the corresponding body weights and maximum recorded dive times. Maximum dive time and maximum bradycardia contrasts were regressed on body weight contrasts, and residuals were generated by ordinary least squares linear regression through the origin. To obtain the relationship between dive time and bradycardia, independent of body weight, the resulting residuals were regressed through the origin, as required by independent contrast analyses, and analyzed with one-tailed t-tests. Contrasts were taken for log 1 0 maximum recorded dive time (min), log 1 0 spleen weight (g) and log 1 0 body weight (kg) for 14 phocid and 6 otariid species. Contrasts were also taken for log 1 0 maximum recorded dive time (min), log 1 0 blood volume (I), log™ whole body hematocrit (g), and log 1 0 body weight (kg) for 13 phocids and 3 otariids. Maximum dive time, spleen weight, blood volume, and whole body hemoglobin contrasts were regressed on body weight contrasts. 32 The residuals were also regressed through the origin, as required by PIC analysis (Garland etal. 1992). 33 Results Physiology, morphology, and dive time: the non-phylogenetic analysis The relationship between maximum recorded dive time and mean dive time is shown in Figure 5. Bradycardia and diving duration Values of maximum bradycardia during diving periods show no consistent phylogenetic patterns or relationship with diving duration (Figure 6). There was very little variation in the values for bradycardia between or within phocids and otariids (minimum value: 3.5, maximum value: 12, mean value: 6.5), and when bradycardia was logged and regressed on body weight (as heart rate should correlate with body weight (Schmidt-Nielsen, 1984)), no correlation was found with maximum recorded dive time. No correlation was seen between maximum observed bradycardia and maximum dive time in pinnipeds after the removal of body weight (r = 0.19; P = 0.29) (Fig. 7). Body Size and Long Diving Duration Based on species for which data were available (Appendix 1, Tables 1 and 2), phocids are significantly larger than otariids (phocid mean weight: 151 kg, otariid mean weight: 66 kg; P < 0.001), and phocid maximum recorded dive duration is significantly longer than otariid maximum recorded dive duration 34 Figure 5. Regression of log average dive time (min) on log maximum recorded dive time (min) for 17 species of phocid and for 14 species of otariid. For pinnipeds R 2 = 0.59, P < 0.001. 35 o • phocid maximum bradycardia phocid resting heart rate otariid maximum bradycardia otariid resting heart rate 2 H t o o • • 0 - J - 1 1 —r 1 1——i 1 —I 0 20 40 60 80 100 120 140 • .  • , Heart rate (bpm) Figure 6. Heart rate and dive time. Relationship between lowest observed diving heart rate (maximum bradycardia) (bpm) and login maximum recorded dive time (min) is given for 9 species of phocids (closed circles) and for 2 species of otariids (open circles). The correlation of lowest heart rate and dive time for pinnipeds was not significant (r = -0.44; P = 0.09). The correlation was also not significant within phocids (r = -0.03; P= 0.47). Any correlation (presumably within phocids) between resting heart rate and dive time would be due to allometric effects of body weight. The data illustrate that a small seal with a high resting heart rate and a large seal with a low resting heart rate are able to lower heart rate similarly during diving. Otariids are also able to lower their heart rate to a comparable degree, and differences seen between phocids and otariids are due to differences in maximum recorded dive times, not heart rates. o 1.0 1.5 2.0 2.5 3.0 Log pinniped weight (kg) 0.4 E 1.0 1.5 2.0 2.5 3.0 Log pinniped weight (kg) E -H CO o E - Q 0 > I I 1 « 2>=5 0.0 •0.3 -0.4 r = 0.19 P=0.29 • • • c • • ° • o -4 residuals of maximum bradycardia vs body weight Figure 7. Removal of the effects of body weight (kg) on heart rate (bpm). The regression of logio body weight (kg) on log-|Q maximum recorded dive time (min) for 9 species of phocid (closed circles) and for 2 species of otariid (open circles) is given in panel A. The regression of lowest observed diving heart rate (maximum bradycardia) (bpm) on log body weight is given in panel B. (Panel C) Residuals of log body weight (kg) vs maximum bradycardia (bpm) are plotted as the x-axis; residuals of log maximum dive time (min) vs log body weight (kg) as the y-axis. The correlation of lowest heart rate and dive time for pinnipeds, independent of body weight (Panel C) was not significant (r = 0.19; P - 0.29). The correlation was also not significant within phocids (r = 0.03; P = 0.47). 37 (phocid median duration: 20 min; otariid median duration: 8 min; P < 0.001). The relationship between body weight and maximum recorded diving time is shown in Figure 8A for phocids, and otariids, and pinnipeds. Fig. 8B shows the regression of average dive time on body weight. The general trend is essentially the same in both lineages: bigger animals tend to be able to dive for longer time periods, but the trend for maximum dive time is statistically significant only for the phocids ( R 2 = 0.37; P < 0.001) and for pinnipeds as a whole (R 2 = 0.33; P < 0.001). Removing the Baikal seal (Ps) from the analysis increases the coefficient of determination between the variables (R 2 = 0.46; P < 0.001). Spleen Size and Long Diving Duration The phocid spleen weights used in this study are significantly larger than otariid spleen weights (phocid mean spleen weight: 457 g; otariid mean spleen weight: 158 g; P = 0.043). If the relative spleen weight is estimated as percentage of body weight in both lineages, the difference in spleen size is also significant (median phocid spleen: 0.33%; median otariid spleen: 0.21%; P = 0.013). The measures of spleen weight were conducted on animals of all ages and sizes. Estimated spleen weight correlated with maximum dive time (r = 0.74; P < 0.0001). Spleen weight is a function of body weight for both groups (Figure Figs. 9A and 9B). 38 Figure 8. Body weight and maximum recorded dive time. (Panel A) Regression of log maximum recorded dive time (min) on log body weight (kg) for 17 phocid species (closed circles) and 15 otariid species (open circles). For the pinnipeds, R 2 = 0.33, P < 0.001 (without the Baikal seal (Phoca sibirica (Ps)) R 2 = 0.46, P < 0.001). For phocids (R 2 = 0.37, P < 0.01), and for otariids the regression is not statistically significant: R 2 = 0.12, P = 0.11. (Panel B) Regression of log average dive time (min) on log body weight (kg) for 17 phocids and 15 otariid species. For the pinnipeds, R 2 = 0.59, P < 0.001 (without the Baikal seal {Phoca sibirica (Ps)) R 2 = 0.65, P < 0.001). 39 slope = 0.60 R2 = 0.33 P< 0.001 7 / P s 0 r 1 hocids otariids 2 3 Log pinniped weight (kg) 1 A 0 H slope = 0.75 R2 = 0.59 B P< 0.001 Ps c9f^^ phocids 5 o o o otariids Log pinniped weight (kg) 40 Figure 9. Spleen weight and maximum recorded dive time in phocids and otariids. (Panel A) Regression of log spleen weight (g) on log body weight (kg) for 14 phocids. (Panel B) Regression of log spleen weight (g) on log body weight (kg) in 6 otariid species. For phocids R 2 = 0.72, P < 0.001, and for otariids R 2 = 0.98, P < 0.001. (Panel C) Residuals generated from the regressions of phocid spleen weight and dive time on body weight show a significant correlation (r = 0.8.1; P < 0.001); the correlation between spleen weight and maximum dive time residuals for otariids is not significant. 41 "O © "E o u s> E _ E 1 I I -o E o o> o o .c _l o X -I "5 E 3 E T3 •«= CO CD CD > OC =6 0.6 0.4 H 0.2 0.0 -0.2-^ -0.4 H -0.6 r = 0.81 P< 0.001 c • • • • • -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Residuals of Log Phocid spleen mass (g) vs. Log mass (kg) "2 8 a> t_ E _ 3 qj E 1 85 E E == O) Si -I o I 3 E •g •» CO CD CD > OC =6 0.6 0.4 H 0.2 H 0.0 -0.2 H -0.4 4 -0.6 r = 0.22 P=0.68 D •• 9 i i -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Residual of Log Otariid spleen mass (g) vs. Log mass (kg) 42 Residuals of phocid spleen vs body weight are plotted against residuals of maximum recorded dive vs log body weight (Figure 9C). With the effect of body weight removed, these data clearly show that for phocids, diving capacity is strongly correlated with spleen size (r = 0.81; P < 0.0005), while it is not in otariids (although sufficient data are lacking) (Fig 9D). For pinnipeds, the relationship is also significant (Fig. 10) (r = 0.73; P < 0.001). When estimated spleen weights are used, (Fig. 11) the relationship between spleen weight and body weight is R 2 = 0.80; P > 0.001. The relationship between estimated spleen weight and dive time, independent of body weight is also significant (r = 0.74; P < 0.001). Species with large spleens for their body weight have longer dive times. For comparison, spleen weight residuals were also correlated with average dive time, and were found to be significant (Fig. 12)(r = 0.49; P < 0.001). Whole Body Hemoglobin. Blood Volume, and Long Diving Duration The third character we examined in this manner was the total or whole body hemoglobin (Fig. 13) content (calculated from data on hemoglobin concentration in g per 100 ml of blood and total blood volume in litres). Phocids have a significantly higher [Hb] than otariids (phocid mean [Hb]: 20.8g/100ml; otariid mean [Hb]: 16.4 g/100ml; P = 0.003) (Appendix 1, Table 5). As data for blood volume were available in only 3 otariid species, the power of comparing means of blood volume and whole body Hb was greatly reduced (phocid mean estimated whole body Hb: 4898 g; otariid mean: 1698 g; P = 0.04). 43 Figure 10. Spleen weight and maximum recorded dive time in pinnipeds. (Panel A) Regression of and log spleen weight (g) on log body weight (kg) for 14 phocids and 6 otariid species. For the pinnpeds R 2 = 0.89, P < 0.001. (Panel B) Regression of log maximum dive time on log pinniped weight for spleen weight pinnipeds R 2 = 0.25, P = 0.012. (Panel C) Residuals of log body weight (kg) vs log spleen weight (g) are plotted as the x-axis; residuals of log maximum dive time (min) vs log body weight (kg) as the y-axis. r = 0.73, P < 0.001. 44 O) "CD c CD _g> Q . CO D) O slope = 1.15 R 2 = 0.80 P< 0.001 3 H 2 H 0 1 phocids otariids ~T~ 2 3 Log pinniped weight (kg) c I E •4—» CD > I' E o> o slope = 0.52 ^ B R 2 = 0.25 P= 0.012 • phocids % • • • otariids Log pinniped weight (kg) CD CO •> CD E CD > E E "8 E »*—. o U) CO 3 •g CO CD 1.0 0.5 H 0.0 •0.5 H •1.0 r = 0.73 P< 0.001 oo phocids o otariids T T T -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 residuals of spleen weight vs. pinniped weight 45 Figure 11. Estimated spleen weight and maximum recorded dive time. (Panel A) Regression of log estimated spleen weight (g) on log body weight (kg) for 14 phocids and 6 otariid species. For the pinnpeds R 2 = 0.80, P < 0.001. (Panel B) Residuals of log body weight (kg) vs log estimated spleen weight (g) are correlated with residuals of log maximum dive time (min) vs log body weight (kg), r = 0.74, P < 0.001. 1.0 0.5 H 0.0 -0.5 •1.0 r = 0.74 P< 0.001 • B • phocids £ • O - O • • o otariids u • • • -0.4 -0.2 0.0 0.2 0.4 residuals of estimated spleen weight vs. pinniped weight 47 Figure 12. Spleen weight and average dive time. (Panel A) Regression of log average dive time (min) on log body weight (kg) for 14 phocids and 6 otariid species. For the pinnpeds R 2 = 0.42, P < 0.001. (Panel B) Residuals of log body weight (kg) vs log spleen weight (g) are correlated with residuals of log average dive time (min) vs log body weight (kg) (r = 0.49, P < 0.001). c "E, CD E > T3 CD O) CO I_ CD > co o o slope = 0.59 R 2 = 0.42 P < 0.001 phocids O otariids Log pinniped weight (kg) £ 1.0 'CD CO > ' CD E CD > CD O) CO U. CD > CO O J O CO 3 •g "co CD 0.5 H 5 0.0 -0.5 H  -1.0 r = 0.49 P = 0.015 o o B phocids O o otariids T T T -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 residuals of spleen weight vs. pinniped weight 49 Figure 13. Whole body hemoglobin and maximum dive time. (Panel A) The regression of log maximum dive time (min) on body weight (kg) in 13 phocid and 2 otariid species for which hemoglobin and blood volume values were available. (Panel B) The regression of log whole body hemoglobin (g) on log body weight (kg). (Panel C) Residuals of log whole body hemoglobin vs log body weight plotted as the x-axis; residuals of log maximum recorded dive time vs log body weight plotted as the y-axis (r = 0.72, P < 0.001). (In the correlation of residuals generated from regressions only on phocids r = 0.82, P < 0.01. For otariids there are not enough data available). 50 C E o> •I2 > E Z3 .1 1 X CO E CO o slope = 0.55 R 2 = 0.27 A P= 0.025 • • o # CD C o CO o E CD >% "D O X) O o x: CO o £ 4 H 3 A 2 H 1 slope = 1.06 B R 2 = 0.89 P< 0.001 1.0 1.5 2.0 2.5 3.0 Log pinniped weight (kg) 0.5 1.0 1.5 2.0 2.5 3.0 Log pinniped weight (kg) CO 1.0 -"O o JO 0.5 -to > E > E E x 03 E £ -1.0 o 03 3 •g CO 0.0 § -0.5 H r = 0.72 P = 0.001 * C # phocids • • • otariids o • — i — • • » -0-4 -0.2 0.0 0.2 0.4 residuals of whole body hemoglobin vs body weight 5"1 Blood volume (Fig. 14) increases significantly with body weight in phocids (R 2 = 0.66, P < 0.01) and in pinnipeds (R 2 = 0.95, P < 0.0001). Whole body hemoglobin content also increases significantly with body weight in pinnipeds (R 2 = 0.89, P < 0.001) and in phocids (R 2 = 0.67; P < 0.01). As before, plotting residuals indicates that independent of body weight effects, there is a statistically significant relationship between whole body blood volume and maximum diving duration (Fig. 14). Similarly, plotting residuals shows that whole body Hb content and maximum diving duration are significantly correlated (Fig. 13). Results for the estimated blood volumes and estimated whole body hemoglobin stores are given in figures 15 and 16. Data are available for only three species of otariids, so an analysis cannot be made with confidence within this lineage. The phylogeny and sources The pinniped phylogeny used for the primary analysis is given in figure 4 along with the sources for important branches. The alternate phylogenies are given in appendix 2. This phylogeny is the most generally accepted relationship between the pinnipeds. Certain areas are more controversial and less well supported than others. Very little is known about the relationships between the Arctocephaline fur seal species. 52 Figure 14. Blood volume and maximum dive time. (Panel A) The regression of between blood volume (I) on measured body weight (kg). (Panel B) Residuals of log blood volume vs log body weight correlated with residuals of log maximum recorded dive time vs log body weight (r = 0.81, P < 0.001). (In correlation of residuals generated by regressions only on phocids r = 0.81, P < 0.001. For otariids there are not enough data available). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Log pinniped weight (kg) r = 0.81 P= 0.0001 B * ^phocids • • • otariids 9 * , 1 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 residuals of blood volume vs body weight 54 Figure 15. Estimated whole body hemoglobin and dive time. (Panel A) The regression of estimated whole body hemoglobin on body weight for 13 phocid and 3 otariid species for which hemoglobin and blood volume values were available. (Panel B) Residuals of log estimated whole body hemoglobin vs log body weight correlated with residuals of log maximum recorded dive time vs log body weight (r = 0.80, P < 0.001). 55 E CO -3 1.0 1.5 2.0 2.5 3.0 Log pinniped weight (kg) r = 0.80 B P= 0.0002 • • • • • • - m • o ° • Q . • i i -0.4 -0.2 0.0 0.2 0.4 residuals of estimated whole body hemoglobin vs body weight 56 Figure 16. Estimated blood volume and dive time. (Panel A) The regression of estimated blood volume on body weight for 13 phocid and 3 otariid species for which hemoglobin and blood volume values were available. (Panel B) Residuals of log estimated blood volume vs log body weight plotted as the x-axis; residuals log maximum recorded dive time vs log body weight plotted as the y-axis (r = 0.81, P < 0.001). 57 E 3 O > •o o o - Q •D <D h—< C3 E *-» CO d) CO o 2H 1 H o H -1 slope = 1.04 A R2 = 0.90 P< 0.0001 1.0 1.5 2.0 2.5 Log pinniped weight (kg) 3.0 £ . " 1 . 0 o 03 =3 •g CO 0.5 A CO "O 0 .O CO > 0) 1 0.0 E E x 03 E -0.5 H •1.0 r = 0.81 P< 0.0001 • B • • • • • % . ° • -• -0.2 -0.1 0.0 0.1 0.2 residuals of estimated blood volume vs body weight 58 Phylogenetic analysis Figure 17 shows the relationship between the standardized phylogenetically independent contrasts of log 1 0 average dive time and log 1 0 maximum recorded dive time. Bradycardia and diving duration PIC analysis indicates that within pinnipeds, the lowest heart rate observed during diving showed little variation and did not correlate with dive time directly (r = 0.02, P = 0.49), or independently of body weight (r = 0.25, P = 0.15) (Figure 18). For comparison, Log 1 0 maximum bradycardia contrasts, independent of body weight, were correlated with maximum recorded dive time contrasts, independent of body weight (Fig. 19)(r = 0.34, P = 0.16). No correlation was seen between the residuals of the regressions of maximum observed bradycardia and average dive time on body weight in pinnipeds (r = 0.15; P < 0.33) (Fig 20). Body Size and Long Diving Duration From basic principles of biology it is clear that a larger seal should have a longer dive time, as it will be able to hold more oxygen on its body and use up the oxygen at a lower rate (Butler and Jones, 1982). If a seal is larger than its closest relative, it should also have a longer dive time (16 phocid and 14 otariid species; R 2 = 0.04, P < 0.05) (Fig 21). The relationship between the standardized independent contrasts of body weight and dive time is shown in 59 0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 Log maximum recorded dive time contrasts Figure 17. Regression of the standardized phylogenetically independent contrasts of log average dive time (min) on the standardized phylogenetically independent contrasts log maximum recorded dive time (min). Closed circles (•) represent contrasts from 17 phocid species, the squares (•) represent contrasts from 14 otariid species and the open circle (O) represents the root node, or contrast between phocids and otariids. For the pinnipeds the relationship is R 2 = 0.16, P = 0.01. 60 Figure 18. Lowest heart rate contrasts and maximum dive time contrasts. (Panel A) The regression of contrasts of log-| Q maximum recorded dive time (min) on contrasts of log io body weight (kg) is given in panel A . Circles (•) represent contrasts from 9 phocid species, the square (•) represents a contrast from 2 otariid species and the diamond ( • ) represents the root node, or contrast between phocids and otariids. The regression of contrasts of lowest observed diving heart rate (maximum bradycardia) (bpm) on contrasts of log body weight is given in panel B. (Panel C) The residuals of lowest heart rate contrasts vs log body weight contrasts are plotted on the x-axis and the residuals of log dive time contrasts on log body weight contrasts are plotted on the y-axis. The correlation was not significant (r = 0.25; P = 0.15). 61 CO CO 03 l_ c 8 E E > E 3 E x 03 E CO o 0.10 0.08 -0.06 -0.04 -0.02 0.00 -0.02 -0.04 -0.06 -0.08 slope = 0.71 R 2 = 0.32 ^P=0.04 # i 0.00 0.02 0.04 0.06 Log pinniped weight (kg) contrasts CO k. •*-> c o £ 0 . 5 E Q . ^o.o H | o .5 H 2 JO 1-1 E x 03 E slope = -3.80 R 2 = 0.18 P=0.1 B n 1 1 r~ 0.00 0.02 0.04 0.06 Log pinniped weight (kg) contrasts RESIDUALS 0.08 - i 0.06 -0.04 -E 0.02 -> E 0.00 -3 E -0.02 -X 03 E -0.04 --0.06 --0.08 -r = 0.25 P=0.15 •1.0 -0.5 0.0 0.5 maximum bradycardia 1.0 62 Figure 19. The regression of contrasts of log<|rj maximum bradycardia (bpm) on contrasts of log-]rj body weight (kg) is given in panel A. Circles (•) represent contrasts from 9 phocid species, the square (•) represents a contrast from 2 otariid species and the diamond ( • ) represents the root node, or contrast between phocids and otariids. (Panel B) The residuals of log lowest heart rate contrasts vs log body weight contrasts are plotted on the x-axis and the residuals of log dive time contrasts on log body weight contrasts are plotted on the y-axis. The correlation was not significant (r = 0.34; P = 0.16). CO -•—» CO CO •4—» c 8 IT Q . . Q CO s " O CO L_ . Q E ZD E x CO E CO o 0.06 -r 0.04 -0.02 -0.00 --0.02 --0.04 --0.06 -Slope = -0.24 R2 = 0.13 P=0.14 — i : r — : ~ i 0.000 0.025 0.050 0.075 Log pinniped weight (kg) contrasts RESIDUALS 0.08 0.06 0.04 0.02 0.00 -0.08 r = 0.34 P=0.16 • • B • -1 • • i • -0.04 -0.02 0.00 0.02 0.04 Log maximum bradycardia 0.06 64 Figure 20. The regression of contrasts of log-|rj average dive time (min) on contrasts of log-jo body weight (kg) is given in panel A. Circles (•) represent contrasts from 9 phocid species, the square (•) represents a contrast from 2 otariid species and the diamond ( • ) represents the root node, or contrast between phocids and otariids. (Panel B) The residuals of log lowest heart rate contrasts vs log body weight contrasts are plotted on the x-axis and the residuals of log average dive time contrasts on log body weight contrasts are plotted on the y-axis. The correlation was not significant (r = 0.15; P = 0.33). 65 CO CO 2 c 8 E CD E CD . > T3 CD CO CO i_ CD > CO CO o 0.08 0.06 0.04 0.02 0.00 -0.02 -0.04 -0.06 slope = 0.75 R2 = 0.49 P- 0.009 0.00 0.02 0.04 0.06 Log pinniped weight (kg) contrasts RESIDUALS CD E CD > CD CO 2 CD > CO 0.08 0.06 0.04 0.02 0.00 -0.02 -0.04 -0.06 -0.08 r = 0.15 P=0.33 • B • • • • 1 • • -0.04 -0.02 0.00 0.02 0.04 Log maximum bradycardia 0.06 66 Figure 21. Removing the Baikal seal from the analysis increases the coefficient of determination between the variables (for maximum recorded dive time: R 2 = 0.16; P = 0.015) (Fig. 22). Figure 23 shows that using alternate phytogenies (appendix 2) does not appear to have a large effect on the relationships between body weight and maximum or average dive times. Spleen Size and Long Diving Duration Fig. 24 clearly shows that a pinniped with a larger spleen for its size than its closest relative displays a greater diving capacity for its size than its closest relative (r = 0.54, P < 0.007). (Residuals of log spleen weight contrasts vs log body weight contrasts are plotted against log maximum recorded dive contrasts vs log body weight contrasts). With the effect of body weight removed, these data clearly show (Fig. 25) that diving capacity is also strongly correlated with estimated spleen size (r = 0.69; P < 0.001). Species with large spleens for their body weight have longer dive times. Species with larger spleens (without the effect of body weight removed) also had longer dive times (r = 0.49; P < 0.02) (data not shown). Whole Body Hemoglobin and Long Diving Duration For whole body hemoglobin (g), the general finding (for the relationship between residuals of contrasts for maximum dive time vs residuals of contrasts for whole body hemoglobin) is illustrated in Fig. 26 (r = 0.44, P < 0.05). This PIC analysis shows that pinnipeds with a larger whole body hemoglobin content for a given 67 body weight display longer maximum duration diving capacities than their closest relative species, and the same is true for blood volume (r = 0.60, P < 0.01) (Fig. 27). This means that for pinnipeds, independent of body size, an increase in blood volume occurs with an increase in the maximum diving capacity; similarly, an increase in the whole body Hb content corresponds with greater maximum diving duration. 68 Figure 21. Body weight contrasts and dive time contrasts. (Panel A) Regression of contrasts of log maximum recorded dive time (min) on contrasts of log body weight (kg). Closed circles (•) represent contrasts from 17 phocid species, the square (•) represents contrasts from 15 otariid species and the open circle (O) represents the root node. For the pinnipeds the relationship is R 2 = 0.04, P = 0.14. (Panel B) ) Regression of contrasts of log average recorded dive time (min) on contrasts of log body weight (kg). The relationship is R 2 = 0.07, P = 0.08. 69 CO "55 0.15 03 c 8 E 0.10 <j 0.05 a> > E 3 E x 03 E CO o 0.00 H >< -0.05 H -0.10 0.15 o.io H 0.05 H CO *-» CO 2 c 8 E Q) •I o.oo H a) > (D CO 5 CD > (0 CO o -0.05 H -0.10 H -0.15 slope = 0.26 A R 2 = 0.04 • • • P=0.14 • • • • • • i • • • • • • • T ' 1 0.00 0.02 0.04 0.06 0.08 Log pinniped weight (kg) contrasts 0.10 slope = 0.28 R2 = 0.07 P=0.08 B o 0.00 0.02 0.04 0.06 0.08 Log pinniped weight (kg) contrasts 0.10 70 Figure 22. Body weight and dive time contrasts without the Baikal seal (Phoca sibirica (Ps)). (Panel A) Regression of contrasts of log maximum recorded dive time (min) on contrasts of log body weight (kg). Closed circles (•) represent contrasts from 17 phocid species, the square (•) represents contrasts from 15 otariid species and the open circle (O) represents the root node. For the pinnipeds, R 2 = 0.16, P = 0.015. (Panel B) Regression of contrasts of log average recorded dive time (min) on contrasts of log body weight (kg). R 2 = 0.10, P = 0.045. 71 3 0-15 CO 0.10 H c 8 E aT 0.05 E % 0.00 E | -0.05 CO E CO o -0.10 slope = 0.61 R2 = 0.16 P = 0.015 — i — : — i 1 1 r r r r~ 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 Log pinniped weight (min) contrasts 0.15 CO +-> CO 2 c 8 E •I 0.00 H > CD O) CO i_ CD > CO CO o 0.10 H 0.05 -\ -0.05 H •0.10 H -0.15 slope = 0.40 B R2 = 0.10 • P= 0.045 • • • • • • # • • r 0.00 0.02 0.04 0.06 Log pinniped weight (min) contrasts 0.08 72 Figure 23. Body weight contrasts and dive time contrasts, without the Baikal seal, generated from phylogeny 2 and phylogeny 3 (appendix 2). (Panel A) Regression of contrasts of log maximum recorded dive time (min) on contrasts of log body weight (kg). Using phylogeny 2, R 2 = 0\17, P = 0.01. (Panel B) Regression of contrasts of log average dive time (min) on contrasts of log body weight (kg). R 2 = 0.12, P = 0.01 for phylogeny 2. (Panel G) Regression of contrasts of log maximum recorded dive time (min) on contrasts of log body weight (kg). Using phylogeny 3, R 2 = 0.16, P = 0.016. (Panel B) Regression of contrasts of log average dive time (min) on contrasts of log body weight (kg). R 2 = 0.12, P = 0.04 for phylogeny 3. 73 in 2 0.15 c 8 E E a) > E ZJ E x 03 . E CO o Phylogeny 2 0.10 H 0.05 H 0.00 H •0.05 H -0.10 slope = 0.63 R2 = 0 .17 # 0.00 0.03 0.06 Log pinniped weight (kg) contrasts CO -4—» CO 03 5 0.15 0.10 -0.05 -0.00 -c 8 Q) E o -0.05 H > o -0.10 H CO 03 l_ O) > 03 CO o -0.15 B slope = 0.44 R 2 = 0.12 P=0.01 T 0.00 0.03 0.06 Log pinniped weight (kg) contrasts Phylogeny 3 CO CO 03 c 8 E a> E Q) > E E x 03 E CO o 0.15 0.10 0.05 -0.10 slope = 0.63 R 2 = 0.16 # 1 P= 0.016 T 0.00 0.03 0.06 CO CO 2 c 8 E E 0.15 0.10 H 0.05 0.00 H O) > 03 CO O 2 -0.05 H g)-0.10 H 03 -0.15 slope = 0.44 R 2 = 0.12 P=0.04 0.00 0.03 0.06 Log pinniped weight (kg) contrasts Log pinniped weight (kg) contrasts 74 Figure 24. Spleen weight and maximum recorded dive time contrasts. (Panel A) Regression of log maximum dive time contrasts (min) on log body weight contrasts (kg). Closed circles (•) represent contrasts from 14 phocid species, the square (•) represents contrasts from 6 otariid species and the open circle (O) represents the root node. For the pinnipeds, R 2 = 0.04, P = 0.21. (Panel B) Regression of contrasts of log spleen weight (g) on contrasts of log body weight (kg). R 2 = 0.88, P < 0.0001. (Panel C) Correlation between residuals of spleen weight contrasts (g) vs. log body weight contrasts (kg) and contrasts of log maximum recorded dive time (min) vs. log body weight contrasts (kg). The correlation coefficient is r = 0.54, P = 0.007. CO -•—» co 2 •*-> c 8 E CD E +-< > E, E X CO E CO o 0.15 - r 0.10 -0.05 -0.00 --0.05 --0.10 --0.15 slope = 0.23 R2 = 0.04 ' P=0.21 _ 0.20 <o _ . _ "to 0.15 co 75 C 8 _ l ] ! 1 ! 0.000.020.040.060.080.10 Log pinniped weight (kg) contrasts 0.10 H CO r 0.05 CO | 0.00 c CD a -0.05 Q . CO c?-o.io H -0.15 slope = 1.24 R2 = 0.88 P< 0.0001 1 i 1 I -0.10-0.05 0.00 0.05 0.10 0.15 Log pinniped weight (kg) contrasts jO CO 2 -t-> c 8 •*-> .c co CD •o c CO CD E 0.15 CD > •o o w CO 3 •g CO CD a: o.io H 0.00 £ -0.05 H -0.10 H •0.15 r = 0.54 P= 0.007 V c • • o • * • • • • • • • -0.10-0.08-0.06-0.04-0.02 0.00 0.02 0.04 0.06 0.08 0.10 Residuals of spleen weight and weight contrasts 76 Figure 25. Estimated spleen weight and maximum recorded dive time contrasts. (Panel A) Regression of log estimated spleen weight contrasts (g) on log body weight contrasts (kg). Closed circles (•) represent contrasts from 14 phocid species, the square (•) represents contrasts from 6 otariid species and the open circle (O) represents the root node. For the pinnipeds R 2 = 0.77, P < 0.0001. (Panel B) Correlation of residuals of estimated spleen weight contrasts (g) vs. log body weight contrasts (kg) and contrasts of log maximum recorded dive time (min) vs. log body weight contrasts (kg). The correlation coefficient is r = 0.69, P = 0.001. 77 to 03 c 8 0.20 0.15 A r o.io a 0.05 A 0.00 H O) C o 0) Q . CO "O O) 03 •I -0.10 CO <D g>-0.15 -0.05 slope = 0.90 R2 = 0.77 P< 0.0001 o I I I — : 1 1— 0.00 0.02 0.04 0.06 0.08 Log pinniped weight (kg) contrasts 0.10 0.15 CO •4—' CO c o o <D E •*-> > »«— o w 03 •g CO or 0.05 A 0.00 -0.15 -0.05 A 5 -0.10 H r = 0.69 P< 0.001 • • • B f • • • • • • -0.10-0.08-0.06-0.040.020.00 0.02 0.04 0.06 0.08 0.10 Residuals of estimated spleen weight contrasts 78 Figure 26. Whole body hemoglobin and maximum dive time contrasts. (Panel A) The regression of maximum dive time contrasts (min) on body weight contrasts (kg) in 13 phocid and 2 otariid species for which hemoglobin and blood volume values were available (the open circle (O) represents the root node). (Panel B) The regression of whole body hemoglobin (g) on body weight contrasts (kg). (Panel C) Residuals of log whole body hemoglobin (g) vs log body weight (kg) contrasts plotted as X-axis; residuals log maximum recorded dive time (min) vs log body weight (kg) contrasts plotted as y-axis (r = 0.44, P < 0.05). 79 0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15 ^slope = 0.22 R2 = 0.04 m P=0.25 CO o 0.00 0.04 0.08 to to 2 0.15 o 0.10 3 ¥ 0.05 -O O co 0.00 o §-0.05 >» "D -O _Q Q) O sz CO o 0.10 •0.15 A •0.20 slope = 1.06 R 2 = 0.91 P< 0.0001 T -0.2 -0.1 0.0 0.1 Log pinniped weight (kg) contrasts Log pinniped weight (kg) contrasts i2 0.15 to 2 CD .1 0.05 CD > E 3 E x CO E 0.00 U -0.05 H o _to (0 3 •g to CD -0.10 H -0.15 r = 0.44 P<0.05 • * c • • • • • -0.04 -0.02 0.00 0.02 0.04 residuals of whole body hemoglobin contrasts 80 Figure 27. Blood volume and maximum dive time contrasts. (Panel A) The regression of blood volume contrasts (I) on body weight contrasts (kg). (Panel B) Residuals of log blood volume vs log body weight contrasts plotted as x-axis; residuals log maximum recorded dive time vs log body weight contrasts plotted as y-axis (r = 0.60, P < 0.01). 81 £ CO 03 i_ •*-» c 8 0.15 0.10 0.05 0.00 H •0.05 •0.10 a> E O > "O o o "co -0.15 H o -0.20 slope = 1.09 R2 = 0.97 P< 0.0001 •0.2 -0.1 0.0 0.1 Log pinniped weight (kg) contrasts 0.15 CO -•—' CO 2 c 8 E = 0.05 H > E E x 03 E >»-o <n 03 13 •g CO •0.15 0.00 r = 0.60 P<0.01 • • # f B • • i • • -0.04 •0.02 0.00 0.02 0.04 residuals of blood volume contrasts vs weight contrasts Discussion 82 Taken together, the above characters reflect most of the known components of the diving response (either directly, as in the case of bradycardia, or indirectly, as in the case of spleen weight). It is clear that a number of diving characters show little variation in pinnipeds, such as diving apnea and bradycardia. As changes in these characters do not correlate with increases in dive time, they are not adaptations for extending dive time within seals and sea lions. The universality of some diving response traits, and the fact that they can be elicited in terrestrial mammals including man, raises the possibility that their occurrence in diving animals is less 'an adaptation' for diving than it is a plesiomorphic or ancestral trait that simply allowed air breathing vertebrates to deal with a variety of stressful situations, including diving. As I did not compare these traits with values from pinniped's non-diving relatives, I can only say that within pinnipeds these appear to be ancestral traits. In contrast, the physiological characters that do vary, not only differ between the pinniped lineages in the expected direction, but also correlate with dive time. It is important to mention that correlated evolution in these characters does not prove that they have been selected for increased dive time. Even though comparisons among species have been used to show the relationship between an organism's features and its environment (Garland and Adolph, 1994; Doughty, 1996), comparative studies are limited in demonstrating processes that 83 happened in the past (Leroi et al. 1994). The traits that correlated with increased dive time are not necessarily causes of that ability. It seems likely that these traits allow the differences in dive times observed in pinnipeds, but proving this, and identifying the relative contribution of each physiological character to dive time variation is difficult. The correlation of traits makes it highly unlikely that these relationships arose by chance alone. The possibilities of correlation with another unmeasured trait or origin for another function remain (Leroi et al. 1994). Nevertheless, decades of research on the function of the characters associated with the diving response strengthens the adaptive argument. Further manipulative experiments would add to the demonstration that these characters are adaptations for increasing dive time. Nevertheless, the analysis demonstrates that at least three factors (i) spleen weight (independent of body weight) (ii) whole body Hb (independent of body weight) and (iii) blood volume (also independent of body weight) are consistent with the hypothesis that increased expression is an adaptation for extending diving duration in pinnipeds. If each of these traits contributed separately to increasing dive time, then evolution in very different regulatory characters (organogenesis, angiogenesis, erythropoesis) would be required. Studies of other mammalian species suggest that these are independent; for example, in mammalian hypobaric hypoxia responses, hemoglobin concentration and red blood cell mass are independent from blood volume, and neither is necessarily associated with spleen size 84 (Winslow and Monge, 1987). How these characters have co-evolved in pinnipeds remains an unexplored problem. The use of independent contrasts produced very similar results to the non-phylogenetic analysis in this investigation. One unexpected result was the large difference in R 2 values for the regressions of dive time on body weight between the different analyses (non-phylogenetic analysis of maximum dive time R 2 = 0.33 with the Baikal seal, R 2 = 0.46 without the Baikal seal; phylogenetic analysis showed R 2 = 0.04 with the Baikal seal; R 2 = 0.16 without the Baikal seal). (Regressions were used in order to generate residuals, not to imply a causal relationship). The result for body weight reflects the problems discussed in figure 2. Figure 8 shows that the two groups of species (phocids and otariids) have different mean values for body weight and dive time, and as a result, the regression of maximum dive time on body weight is heavily influenced by the differences in the two groups. The phylogenetic analysis reveals that body weight does depend, to a high degree, on phylogeny. Changes in body weight were not as closely associated with changes in dive time. The lower correlation of body weight and dive time than spleen weight and dive time may be a result of difficulty in weighing a large animal. Nevertheless, the fact that the relationship between spleen weight and dive time was not removed by a phylogenetic analysis, while the relationship of body weight and dive time decreased, demonstrates that the error in measurement is not the reason for the low correlation in the phylogenetic analysis. This is an unexpected result, as body weight has been called one of the most important 85 influences on dive time in the literature (Butler and Jones, 1982). Many other potential influences on body weight in pinnipeds must be considered, however (Costa, 1991). The true effect of the weight of an animal (which has fluctuating levels of fat stores and tissues with low levels of metabolic activity) on dive time may not be as large as previously expected. None of the measured variables correlated with maximum dive time in otariids. The apparent lack of significant correlations may be a simple artifact of the available data. The tentative conclusions are (i) that there was not enough data for otariid species to reach the statistical power required, (e.g. body weight vs. dive time), (ii) that variation in otariid maximum diving duration is not large enough for the relatively insensitive diving response characters to reveal any adaptive trends, otariids are more closely related than phocids (see Figure 4) and may be less variable as a consequence, and/or (iii) that the evolution of the otariids has been 'driven' more by factors (such as reproductive requirements (Costa, 1991)) other than requirements for long duration diving. Lack of Correlation of Diving Response Characters The lack of correlation between dive time and maximum bradycardia could mean that heart rate is too crude a measure of circulatory control during diving and recovery (and its measure is consistent with what Scholander knew all along - the basic reflex is nearly universally present in some form in all vertebrates). Heart rate may be selectively constrained due to its role in so many different biological systems that any adaptational changes for extending 86 diving are too modest to detect with the crude physiological criteria thus far utilized. The ability to decrease heart rate could have been selected for (to some limit) during the invasion of the ancestral pinniped into the water. These possibilities would result in the lack of variation in maximum bradycardia. Alternately, as the number of species for which bradycardia data are available is much smaller than the other variables, the result could be due to a lack of statistical power. Different methods of heart rate collection could also introduce error to this analysis. Both of these problems seem unlikely to affect the result, however, as there was so little variation in the values, and any differences observed did not appear to correspond with a particular method of heart rate measure. Interestingly, a lack of correlation with dive time seems to hold for another physiological character - diving hypometabolism - which was initially expected to vary with dive time. In earlier studies (Guppy et al. 1986; Hochachka and Guppy, 1987; Hochachka and Foreman, 1993; Le Boeuf et al. 1989; Costa, 1991; Hindell et al. 1992) it was explicitly or implicitly assumed that the impressive diving performance of large seals depended in large part on an 'energy conserving' physiology and diving strategy. Central to this was some concept of diving hypometabolism. Subsequent research has uncovered two potential underlying mechanisms: (i) One hypothesis is that hypoperfusion (vasoconstriction) of nonworking peripheral muscles and other tissues is the proximate cause of hypometabolism, with reduction in tissue metabolic rate being a direct function of the reduction in oxygen delivery, a relationship also 87 observed in terrestrial mammals (Hochachka, 1992; Guyton et al. 1995; Hochachka et al. 1995); (ii) an alternate postulate is that regional hypothermia contributes to low metabolic rates, with metabolic suppression being a function of tissue cooling (Hill et al. 1987; Andrews et al. 1994). While these two mechanisms are not mutually exclusive, it was at first thought that the physiological characteristic of hypometabolism would be largely restricted to the large seals, or at least to phocids. However, recent careful experimental studies with sea lions (animals trained to remain submerged and relatively inactive for defined time periods) indicate that the metabolic rate declines as a direct function of diving duration; the metabolic rate for seven minute diving periods (water temperature at about 15°C) falls to about 50% of resting metabolic rate (RMR). Since the times involved are so short, it is unlikely that regional hypothermia plays a significant role in this metabolic suppression (Hurley, 1996). I interpret this to mean that activation of the diving response automatically leads to hypoperfusion of some tissues/organs and subsequently to diving hypometabolism. On its own, then, this physiological character, like bradycardia, would appear to be general among pinnipeds and thus could not be expected to vary (and indeed did not vary (Table 1) in any systematic way with diving capacity). 88 Table 1. Net e f f e c t s of putative p h y s i o l o g i c a l and biochemical adaptations for diving i n phocids compared to o t a r i i d s expressed as the r a t i o s of diving metabolic ra t e s / r e s t i n g metabolic rates. Species Weight RMR DMR DMR/RMR Source Phocids Weddell seal 355 4.1 elephant seal 156 7.6 grey seal 189 7.6 harp seal 140 3.7 harbor seal 42 6.1 Ota r i i d s sea l i o n 85 northern fur seal 37 Antarctic fur seal 39 5.0 (S) 1.20 1 3.4 (L) 0.82 1 4.5 a l l 1.10 1 8.2 1.13 2 7.9 (S) 1.04 3 3.5 (L) 0.46 3 5.2 a l l 0. 68 3 3.5 (S) 0.95 4 2.9 (L) 0. 78 4 3.2 a l l 0.88 4 6.75 a l l 1.16 5 4.80 6 6.0 6 7.0 6 Body weight i n kg; for phocids, r e s t i n g metabolic rates (RMR) and diving metabolic rates (DMR) are i n mis oxygen x kg" 1 x min - 1. Elephant seal DMRs estimated using doubly l a b e l l e d water for animals at sea while RMRs were for seals on shore; a l l other phocid metabolic rates are based on studies with free diving seals using i n d i r e c t calorimetry. RMR values are not the same as basal metabolic rates or 89 BMRs; by d e f i n i t i o n , the l a t t e r have to be taken at s p e c i f i e d times and conditions (post-absorptive, quiescent, thermoneutral) and are r a r e l y available for phocids. The RMR of f a s t i n g (about 345 kg) elephant seals on shore averaged over a 32 day period was 4.64 ml oxygen per kg per min (Worthy et al. 1992) s l i g h t l y lower than the value used above for younger seals; i n t h i s case, the DMR/RMR would increase to about 1.7. O t a r i i d metabolic data i n Costa (1991) are a l l given i n kJ x m i n - 1 and are based on 'at sea 1 estimates using the doubly l a b e l l e d water technique. S - data for short dives; L - data for long dives; a l l - data for a l l dives studied. Data Sources: (1) C a s t e l l i n i et al. (1992) . (2) Andrews R.D. et a l . (1994). (3) Reed et al. (1994). (4) G a l l i v a n (1981). (5) Craig and Pasche (1980). (6) Costa (1991). 90 Potential problems and sources of error Some of the potential errors that present themselves in this study include 1) differences in methods of variable collection, 2) number of values available for each variable, 3) differences in condition and annual cycle of species during variable measurement, 4) choice of variables, 5) and choice of phylogeny. The first two categories are addressed in the first two sections of this discussion. The third and fourth categories may be the largest source of error in this study. Using an average value for body weight of a species fails to address consistency in the state of the animals (age, sex, time of year, fasting state, reproductive state). As stated in the materials and methods, only females were used in sexually dimorphic species, but differences in the other variables may introduce error into the body weight character. The fact that spleen weight, uncorrected for body weight, correlates more strongly with dive time than body weight, may demonstrate the greater amount of error present in the body weight character than in spleen weight. This may be due to the difficulty in weighing large animals accurately. Variation in dive time with all of the potential differences listed above (location of the studies, differences in data recorders, and length of measurements) also may have introduced error into recordings of maximum dive times. Obviously, dive times measured on well studied species are likely to be far closer to the true maximum dive time than those of less well studied species. The close correlation of average dive time with maximum recorded dive time, 91 and the correlation of all measured variables with average dive time shows, however, that either measurement would be acceptable. All of the potential sources of error in the variables discussed above would increase the probability of type II errors (failure to reject the null hypothesis). The positive correlations between body weight, spleen weight, whole body hemoglobin, and blood volume could not have resulted from these potential errors. The final potential error discussed in this study is inaccuracy in the phylogeny. Theoretically, using an inaccurate phylogeny is superior to not using any phylogeny in the analysis (Felsenstein, 1985). As the four phylogenetic hypotheses resulted in similar correlations in the characters, and since altering the branch lengths did not appear to change the results, it is unlikely that errors occurred from discrepancies in the phylogeny. Net Effects of Physiological/Biochemical Adaptations for Diving One way to evaluate the biological meaning of these putative adaptations, is to compare known differences in energetic costs in phocids and otariids. In phocids, the net effect of these characters is an 'at sea' or 'free diving' metabolic rate (DMR) which is similar in magnitude to the 'routine' or resting metabolic rate (RMR) on land, or, in the case of other seals, RMR at the breathing hole (Table 1). In well controlled experimental studies of the grey seal, DMR/RMR ratios, determined by direct oxygen consumption measurements, were found to be less than 0.5 for long dives (indicating significant diving hypometabolism) but these 92 values were near 1 for short dives (Table 1). Field oxygen consumption studies of the Weddell seal also indicate values of DMR/RMR of close to 1 (Table 1). Using doubly labelled water technique, field studies of northern elephant seals indicate that the ratios of DMR/RMR range from fractionally less than 1 to about 1.3 (Andrews et al. 1994), similar to values estimated from time-depth diving data for elephant seals at sea (Costa, 1991). (Boyd et al. (1995) reported that direct gas exchange estimates of average 24-hour metabolic rates were somewhat lower than estimates based on the doubly labelled water measurements. Both methods measure the same biochemical reactions, which makes it unclear which is the more reliable. The differences were small enough not to affect the current analysis). Thus it appears that for these species, the diving response is so effective that foraging at sea is no more costly than staying relatively inactive on shore. The same conclusions derive from independent studies (Table 1) of harp seals (Gallivan, 1981) and of harbor seals (Craig and Pasche, 1980). Another way of evaluating the net effects of these diving characteristics is to compare diving and interdive intervals at sea. In phocids, the overall diving response is so effective that diving duration can greatly exceed interdive periods of recovery and recharging of oxygen stores. For elephant seals, these surface intervals are in the 2-4 min range (Hindell era/ . 1992; Le Boeuf e ra / . 1989). In these large seals, ratios of diving duration to interdive periods can thus range from about 15 during routine diving to as high as 40 - 50 during maximum observed diving times (Le Boeuf et al. 1989; 1992; Hindell e ra / . 1992). Similar 93 data are available for several other phocids (see Kooyman et al. 1980; Fedak, 1986; Reed et al. 1994). Although qualitatively some of the diving response characteristics are similar in sea lions and in seals, they function differently in each lineage. The most striking way in which to contrast the two diving strategies is to compare DMR/RMR for the two kinds of pinnipeds. While in the phocids this ratio is about 1, or even less than 1, in otariids it is in the 4-7 range (Table 1), either because of the high cost of swimming or thermogenesis, or both (Butler et al. 1995). Not surprisingly, the ratios of diving duration to interdive time for recharging oxygen stores are much lower in sea lions than in elephant seals. Instead of values between 15-50 noted for the large seals, in sea lions the values are about 3 for routine dives and approach 10 for maximum duration dives. Emerging Principles of Evolution in a Physiological System From this inquiry into the variability of the diving response in pinnipeds, the following conclusions seem to be valid: 1. A number of physiological/biochemical characters, considered adaptations in diving animals, do not vary with dive duration in pinnipeds. These traits, which are necessarily similar in phocids and otariids, include diving apnea and bradycardia (and probably tissue hypoperfusion). At this stage in the understanding of diving physiology and biochemistry, this analysis is unable to detect any correlation between these characters and diving capacity. 94 2. A number of physiological/biochemical characters are clearly correlated with long duration diving and prolonged foraging at sea among the pinnipeds. These characters include body weight, spleen weight, and whole body oxygen carrying capacity. The larger each of these characters are, the greater the diving capacity (defined as diving duration). Since the relationships between diving capacity and spleen weight or between diving capacity and whole body oxygen carrying capacities are evident even when corrected for body weight and analyzed using independent contrasts, it is reasonable to suggest that the two traits - large spleens and large whole body oxygen carrying capacities - extend diving duration. That is, in contrast to traits such as bradycardia, these characters (and presumably other similar ones, such as tissue specific metabolic organization (Hochachka and Foreman, 1993)) have evolved to enable prolonged dive times. I conclude that increased spleen size and 0 2 carrying capacity are likely to be physiological adaptations for increased diving duration in pinnipeds. Summary and Conclusions Whole body hemoglobin content, blood volume, and spleen weight are correlated in pinnipeds with prolonged duration diving. It is worth emphasizing that the positive correlations are evident even when corrected for body weight and analyzed using phylogenetically independent contrasts. It is reasonable to suggest, given our knowledge of their function and the present analysis of their 95 evolution, that increases in spleen size and whole body oxygen carrying capacity extend diving duration. I conclude that these two traits are likely to represent physiological adaptations for increased diving duration. Other traits that have been generally accepted as adaptations for increased diving capacity, such as bradycardia, are more appropriately viewed as plesiomorphic (ancestral) characters in pinnipeds. 96 References Andrews, R.D., D.R.Jones, J.D:Williams, D.E.Crocker, D.P.Costa, and B.J.Le Boeuf (1994) Thermoregulation and metabolism in freely diving northern elephant seals. FASEBJ.8,A2. Arnason, O., K. Bodin, A. Gullberg, C. Ledje, and S. 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Physiol. 47, 968-973. 1 s l 2 I •isi| j G W « O § 3 § eo<o 3, i 1 $ s 1 « S I 1 •s "a -s I a - § -s 1 H Si a ft * E g 1 | S 3 § s »i a « | § g 5 8 8. 3."2i"8 w s > a 43 .2 '"3 a .8 S § g I ti II 2 "8 u S C/L OL OL PPP t-J P P P P P l i P P P V O O O s — ' 0 0 — I T t T t T j -,C - vq ^ - - -» 00 Ov rt TJ- <S 00 <S 00 o* CO s s I Ov CN 27.1 11.5 6.4 00 o rs CO «/-> t* >o <n CN CO CO VO <s Ox © VO CN 00 vq 00 CN <S 00 0 0 a; 00 © <s CN CN o ts CN co CN oo o CN ~' cs CN CO 00 CN CO 00 VO r~" o OO t-; CO 3 6 3 W U ' « £ « £ « § 109 V9 o 0 1 > 1 c 00 ' w u « 3 S « 8 00 s •a s s E 5 f g> J •3 •« ?> ?> -§ § S" 5 "S £ . 1 3 o C s >» 6 0 a " •§ 3 a, a: a, 1.5 1 5 ^ 43 •8 « •a § 5 S « I y s * 3 3 ' "111 a i l vVl SO 3 3 3 3 1111311 "8 I op g cS m S ^ oo H H H H o o H H H rn «-i cs H </i t> r » 00 ""4 Ov <s ._, o * p «" O !Z £ E" l3 l>$ <5 111 Table 2. Body weights of pinniped species used in the diving studies. Common names and abbreviation of Latin names for each species are given. Body weight (kg) was taken from the same sources as the diving data. If body weight was not given, data were requested from the authors (given as pers. comm.), or mean weight was used from other sources. common name abrv weight source Northern elephant seal Ma Kg 361 Le Boeuf et al 1989 Southern elephant seal Ml 387 Hindell etal 1991 Weddell seal Lw 355 Castellini et al 1992a Ross seal Or 173 Ray 1981 Crabeater seal Lc 182 Nord0y et al 1995a Hawaiian monk seal Ms 195 Ragen pers. comm. Bearded seal Eb 283 Gjertz pers. comm. Hooded seal Cc 235 Folkow pers. comm. Harp seal Pg 123 Lydersen and Kovacs 1993 Grey seal Hg 210 Thompson and Fedak 1993 Baikal seal Ps 29 Stewart et al 1996 Ringed seal Phi 73 Kelly and Wartzok 1996 Saimaa seal Phs 63 Hyvarinen et al 1995 Spotted seal Pla 85 Frost pers. comm. Harbor seal E Atl Pw 110 Fedak et al 1988 Harbor seal E Pac Pvr 93 P. Olesiuk pers. comm. Harbor seal W Atl Pvc 107 Coltman et al 1995 Northern fur seal Cu 37 Kooyman et al 1976 Antarctic fur seal F Ag 41 Boyd and Croxall 1992 South African fur seal Ap 75 Kooyman and Gentry 1986 Guadalupe fur seal At 50 Gallo-Reynoso pers. comm. Juan Fernandez fur seal Aph 48 Francis and Boness in prep New Zealand fur seal Af 38 Harcourt et al 1995, Mattlin pers. comm. South American fur seal Aa 35 Trillmich et al 1986 Galapagos fur seal Aga 29 Kooyman and Trillmich 1986a Southern sea lion Of 126 Werner and Campagna 1995 Australian sea lion Nci 84 Costa et al 1989 New Zealand sea lion Pho 150 Gentry et al 1987 Steller sea lion Ej 268 Calkins pers. comm. California sea lion Zc 111 Odell 1981 Galapagos sea lion Zcw 70 Kooyman and Trillmich 1986b Walrus Oro 1500 Wiig et al 1993 112 S ? « 00 S 8- >. 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A g £ £ £ £ <5 3 2 p?a J 1 5 C B PO 5 2 115 1 ! 1 § 8 2* " S i •a ••a — A A =5 £ ON © 00 g g C> oo oo 1 S T j - O O O O T f i O r r N O T t i O C S O N J2* 00 NO g E E 116 Appendix references Andrews, R.D., Jones, D.R, Williams, J.D., Crocker, D.E., Costa, D.P., and Le Boeuf, B.J. (1995). Metabolic and cardiovascular adjustments to diving in northern elephant seals (Mirounga angustirostris). Physiol. Zool. 68: 105. Banish, L. D., and Gilmartin, W. G. (1988). Hematology and serum chemistry of the young Hawaiian monk seal (monachus schauinslandi). J. Wild. Diseases. 24: 225-230. Bengston, J. L., and Stewart, B. S. (1992). Diving and haulout behavior of crabeater seals in the Weddell Sea, Antarctica, during March 1986. Polar Biol.. 12: 635-644. Bengston, J. L., and Stewart, B. S. (1997). Diving patterns of a Ross seal (Ommatophocarossii) near the eastern coast of the Antarctic Peninsula. Polar Biol. In Press. Blessing, M. H., Ligensa, K., and Winner, R. (1972). 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Zool.. 74: 1547-1555. 119 Kooyman, G. L., Gentry, R. L., and Urquhart, D. L. (1976). Northern fur seal diving behavior: a new approach to its study. Science. 193: 411-412. Kooyman, G. L., and Gentry, R. L. (1986). Diving behavior of South African Fur Seals. In Fur Seals: Maternal Strategies on Land and at Sea (ed. R. L. Gentry and G. L. Kooyman), pp. 142-152. Princeton: Princeton University Press. Kooyman, G. L., and Trillmich, F. (1986a). Diving behavior of Galapagos Fur Seals. In Fur Seals: Maternal Strategies on Land and at Sea (ed. R L. Gentry and G. L. Kooyman), pp. 186-195. Princeton: Princeton University Press. Kooyman, G. L., and Trillmich, F. (1986b). Diving behavior of Galapagos Sea Lions. In Fur Seals: Maternal Strategies on Land and at Sea (ed. R L. Gentry and G. L. Kooyman), pp. 209-219. Princeton: Princeton University Press. Koskela, J. T., Hyvarinen, H., and Kunnasranta, M. (1995). Movements and habitat use of radio-tagged Saimaa ringed seals. 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(1969). Respiratory function of blood of the adult and fetus weddell seal Leponychotes weddelli. Am. J. Physiol.. 216: 1595-1597. Lenfant, C , Johansen, K., and Torrance, J. D. (1970). Gas transport and oxygen storage capacity in some pinnipeds and the sea otter. Resp. Physiol.. 9: 277-286. Lydersen, C. (1991). Monitoring ringed seal (Phoca hispida) activity by means of acoustic telemetry. Can. J. Zool.. 69: 1178-1182. Lydersen, C , and Kovacs, K. M. (1993). Diving behaviour of lactating harp seal, Phoca groenlandica, females from the gulf of St. Lawrence, Canada. Animal Behav.. 46: 1213-1221. Mattlin, R H. (1993). Seasonal behavior of the New Zealand fur seal, Arctocephalus forsteri. Abstracts of the 10th Biennial Conference on the Biology of Marine Mammals: 74. McConnell, L. C , and Vaughan, R W. (1983). Some blood values in captive and free-living common seals (phoca vitulina). Aq. Mamm., 10: 9-13. Merrick, R. L., Loughlin, T. R, Antonelis, G. A., and Hill, R. (1994). Use of satellite-linked telemetry to study Steller sea lion and northern fur seal foraging. Polar Res.. 13: 105-114. 120 Needham, D. J., Cargill, C. F., and Sheriff, D. (1980). Haematology of the Australian sea lion, Neophoca cinerea. J. Wild. Diseases. 16: 103-107. Nordoy, E. S., Folkow, L., and Blix, A. S. (1995a). Distribution and diving behavior of crabeater seals (Lobodon carcinophagus) off Queen Maud Land. Polar Biol.. 15: 261-268. Nordoy, E. S., Folkow, L. P., Potelov, V., Prichtchemikhine, V., and Blix, A. S. (1995b). Distribution and dive behavior of white sea harp seals, between breeding and moulting. Abstracts of the 11th biennial conference on the biology of marine mammals: 83. Odell, D. K. (1981). California Sea Lion, Zalophus califomianus (Lesson, 1828). . In Handbook of Marine Mammals Volume 1: The Walrus. Seal Lions. Fur Seals, and Sea Otter (ed. S. H. Ridgway and J. H. Harrison), pp. 67-97. London: Academic Press. Pasche, A., and Krog, J. (1980). 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London: Academic Press. Reeves, R. R., Stewart, B. S., and Leatherwood, S. (1992). The Sierra Club Handbook of Seals and Sirenians. San Francisco: Sierra Club Books. Ridgway, S. H. (Ed.). (1972). Mammals of the Sea. Biology and Medicine. Springfield: Charles C. Thomas. Ronald, K: (1970). Physical blood properties of neonatal and mature harp seals. International Council for the Exploration of the Sea C M . . 5: 1-4. Scheffer, V. B. (1960). Weights of organs and glands in the northern fur seal. Mammalia. 24: 477-481. Scholander, P. F. (1940). Experimental investigations on the respiratory function in diving mammals and birds. Hvalrad. Skr.. 22: 1-131. Shustov, A. P., and Yablokov, A. V. (1968). Comparative morphological characteristics of the harp and ribbon seals. Trans. Ser. Fish. Res. Bd. Can.. 1084: 1-19. Simpson, J. G., Gilmartin, W. G., and Ridgway, S. H. (1970). Blood volume and other hematologic values in young elephant seals (Mirounga angustirostris). Am. J. Vet. 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Seasonal movements and dive patterns of juvenal Baikal seals, Phoca sibirica. Mar. Mamm. Sci.. 12: 528-542. Thompson, D., and Fedak, M. A. (1993). Cardiac responses of grey seals during diving at sea. J. Exp. Biol.. 174: 139-154. Trillmich, F., Kooyman, G. L., Majluf P., and Sanchez-Grinan, M. (1986). Attendance and diving behavior of South American Fur Seals during el Nino in 1983. In Fur Seals: Maternal Strategies on Land and at Sea (ed. R. L. Gentry and G. L. Kooyman), pp. 153-167. Princeton: Princeton University Press. Vaz-Ferriera (1981). South American Sea Lion Otaria flavescens (Shaw, 1800). In Handbook of Marine Mammals Volume 1: the Walrus. Sea Lions. Fur Seals, and Sea Otter (ed. S. H. Ridgway and J. H. Harrison), pp. 39-65. London: Academic Press. Wells, R. M. G. (1978). Observations on the haematolbgy and oxygen transport of the New Zealand fur seal, Arctocephalus forsteri. New Zealand J. Zool.. 5: 421-424. Werner, R., and Campagna, C. (1995). Diving behaviour of lactating southern sea lions (Otaria flavescens). Can. J. Zool.. 73: 1975-1983. Wiig, 0., Gjertz, I., Griffiths, D., and Lydersen, C. (1993). Diving patterns of an Atlantic walrus Odobenus rosmarus rosmarus near Svalbard. Polar Biol.. 13: 71-72. 122 Appendix 2. Three alternate phylogenetic hypotheses for the pinnipeds. .a to Figure 1. Phylogeny 2. A composite phylogenetic hypothesis for the pinnipeds based on molecular and morphological data (compiled from Arnason et al. 1995; Berta and Demere, 1986; Berta and Wyss, 1994; Burns and Fay, 1970; Lento et al. 1995; Muizon, 1982; Perry et al. 1995). Some branches of the tree are supported by both molecular and morphological evidence, while other areas are not fully resolved. De Muzion's (1982) cladistic phylogeny and Perry et a/.'s rrdtochodrial DNA tree (1995) present the harp (Phoca groenlandica) and hooded (Cystophora cristata) seals as having diverged through a common ancestor, while Arnason et a/.'s (1995) tree and Burns and Fay's (1970) well established systematic studies support the earlier divergence of Cystophora. 123 Cj> CD o o T3 • O o o u 2 2 2 •I ^  1 § CD C 5 s c _ o 5 T J „ o 2 -o •5 O X £• e i 1 CD £ & f S3 | £ I I o •o •a CD eg I i" -S °- Q ™ ™ S S S 8 - g 8 8 _ _ _ _ _ S £ < ! £ ! £ £ £ £ £ £ £ B 2 « - Q I 5 8 o CO 3 c » o o oo x: ca O or co u o in o CA w CO a> o> O 04 j2 1 o CM Figure 2. Phylogeny 3. A composite phylogenetic hypothesis for the pinnipeds based on molecular and morphological data (compiled from Arnason et al. 1995; Berta and Demere, 1986; Berta and Wyss, 1994; Burns and Fay, 1970; Lento et al. 1995; Muizon, 1982; Perry et al. 1995). Some branches of the tree are supported by both molecular and morphological evidence, while other areas are not fully resolved. Berta and Wyss's cladistic analysis (1994) and Lento et a/.'s mitochodrial DNA tree (1995) present the Northern for seal (Callorhinus ursinus) as having diverged from the otariids before the Arctocephaline for seal species, while the majority of the literature places Callorhinus with the for seals (Arctocephalus). 124 Figure 3. Phylogeny 4. A composite phylogenetic hypothesis for the pinnipeds based on molecular and morphological data (compiled from Arnason et al. 1995; Berta and Demere, 1986; Berta and Wyss, 1994; Burns and Fay, 1970; Lento et al. 1995; Muizon, 1982; Perry et al. 1995). Some branches of the tree are supported by both molecular and morphological evidence, while other areas are not fully resolved. Very few studies have been conducted on the fur seal (Arctocephalus) species. Lento (pers. comm.) supports a common divergence of the New Zealand fiir seal (Arctocephalus forsteri) and the Australian fur seal (Arctocephalus pusillus). 

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