Comparing Thoracic Morphology and Lung Size in Shallow (Tursiops Truncatus) and Deep (Kogia Spp.) Diving Cetaceans

Home , Bottlenose whale, Kogia, Monodontidae

COMPARING THORACIC MORPHOLOGY AND LUNG SIZE IN SHALLOW (TURSIOPS TRUNCATUS) AND DEEP (KOGIA SPP.) DIVING CETACEANS

Marina A. Piscitelli

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science

Department of Biology and Marine Biology

University of North Carolina Wilmington

2009

Approved By

Advisory Committee

______Dr. Timothy Ballard Dr. Richard Dillaman

______Dr. Stephen Kinsey Dr. Sentiel Rommel

______Dr. D. A. Pabst

Accepted by

______Dean, Graduate School This thesis has been prepared in the style and format

consistent with the journal

Journal of Morphology

TABLE OF CONTENTS Page

ABSTRACT...... iv ACKNOWLEDGEMENTS...... vi DEDICATTION ...... viii LIST OF TABLES...... ix LIST OF FIGURES ...... xi INTRODUCTION ...... 1 MATERIALS & METHODS ...... 15 Specimens……………………………………………….…………….….15 Measures of Lung Size: Mass…………………....……….……….……..16 Measures of Lung Size: Volume...... …………………...……….……...19 Measures of Lung Volume: Whole Body Cross-Sections ...... ….……….22 Gross Morphology of Skeleton………...... …………………….………..27 Mobility of Isolated Thoraxes...... …………………….……….………..28 Thoracic Cavity Volume Models……...... ………………….………...... 32 RESULTS ...... 36 Lung Mass...... ………………………………………..……….…37 Excised Lung Volume....………………………..…………………….….44 In situ Lung Volume Calculated from Whole Body Cross-Sections...... 48 Mobility of Isolated Thoraxes...... …………………………………….…54 Thoracic Cavity Volume Models...... …………………………………....58 DISCUSSION...... 60 CONCLUSION...... 80 LITERATURE CITED ...... 82 APPENDIX...... 95

iii

ABSTRACT

Coastal bottlenose dolphins (Tursiops truncatus) dive to depths of 1-10m while

pygmy and dwarf sperm whales (Kogia breviceps and K. sima) are estimated to dive

between 400-1,000m. These divers will experience vastly different external pressures at

depth that will, according to Boyle’s and Pascal’s gas laws, influence the volume of air

within their lungs and potentially the amount of thoracic collapse they experience. The

goal of this study was to test the hypotheses that lung size will be reduced and/or thoracic mobility will be enhanced in deeper diving cetaceans. T. truncatus and Kogia spp. were compared because relatively large samples of stranded individuals were available.

Thoracic vascular structures were also compared. Lung size was investigated by comparing lung mass (T. truncatus, n = 111; kogiids, n = 18) and lung volume (T. truncatus, n = 5; kogiids, n = 4) to total body mass. One T. truncatus and one K. sima

were cross-sectioned whole to calculate lung (as well as thoracic vasculature and other

organ) volumes as a percent of total thoracic cavity volume. Excised thoraxes were

mechanically manipulated into maximally expanded and collapsed postures (T. truncatus,

n = 3; kogiids, n = 5) to compare changes in thoracic cavity shape and volume.

Kogiid lungs were one-half the mass, and between 20-50% of the volume of those

of similarly-sized T. truncatus. The lung occupied only 15% of the total thoracic cavity

volume in K. sima and 37% in T. truncatus, while thoracic arterial retial tissue occupied

8.9% in K. sima and 4.9% in T. truncatus. The kogiid and bottlenose dolphin thoraxes

underwent similar changes in shape and volume. The only significant difference in

thoracic mobility was at the thoracic inlet; the change in width of the inlet was

constrained in kogiids relative to T. truncatus. Thus, the deeper diving kogiids possess

iv smaller lungs, more voluminous thoracic vasculature, and a similarly mobile thorax as

compared to the shallow diving T. truncatus.

Calculations based upon mass specific metabolic rates and total lung capacities

suggest that the large lung of the shallow diving, “fast” breathing T. truncatus can

provide the oxygen reserves required to meet the metabolic demands of swimming on a

breath-hold. The mobility of the thorax in this species may accommodate the large

changes in air volume routinely experienced during shallow diving, and may function to

permit rapid changes in thoracic volume required during its explosive ventilation. In

contrast, the deep diving, “slow” breathing kogiids possess relatively small lungs, and

will, thus, experience reduced pressure-induced changes in both lung and thoracic

volumes at depth.

A broader phylogenetic comparison demonstrated that the ratio of lung mass to

total body mass in kogiids, physeterids, ziphiids and mysticetes is similar to that of

terrestrial mammals, while delphinids and phocoenids possess relatively large lungs.

Thus, small lung size in deep diving odontocetes may be a plesiomorphic character,

rather than a specialization for diving. The large lung size of delphinids and phocoenids

appears to be a derived condition that may permit the lung to function as a site of

respiratory gas exchange during a dive in these relatively short-duration, shallow divers.

v ACKNOWLEDGEMENTS

This study on every level required a collaborative effort. There are many people to thank at multiple institutions. First and foremost, I would like to acknowledge the tremendous amount of assistance, both mentally and physically, provided by Dr. Ann

Pabst, Bill McLellan, and Dr. Sentiel Rommel. I would first like to especially thank my wonderfully patient and genuine advisor, Dr. Ann Pabst. Without her guidance, inspiration, enthusiasm and willingness to go above and beyond what is required, I would not have been able to complete this project.

I would also like to express my endless appreciation to Bill McLellan for his masterful ability in meeting every challenge in this study, from excising whole thoraxes to inflating lungs, and for his creative ideas and thoughtful discussions. I would also like to express my sincere appreciation to Dr. Sentiel “Butch” Rommel for all the encouragement along the way, and for his thoughtful critiques and ideas that have led to a better understanding of the mechanisms at hand.

I would like to acknowledge all of my unbelievably wonderful lab mates, with special thanks to Brian Balmer, Peter Nilsson, Ryan McAlarney, Sarah Pagentine, and

Laura Bagge, who were always willing to lend a hand at a moment’s notice and provided endless critiques. I would like to thank Dr. Andrew Westgate for his insights into pressurized systems and access to his pressure vacuum module, and Dr. James Blum in the Department of Mathematics and Statistics at UNCW, for his help with analysis of multiple data sets. I would also like to thank Mark Gay for his patience and repeated assistance with both histology and image analysis, and Jim Moravansky, computer tech master, for his fruitful efforts in rescuing data from multiple crashed hard drives.

vi For sample collection, I would like to thank the Virginia Aquarium Stranding

Response team, especially Sue Barco, for open access to their database and facility, and

for always having such willingness to collect samples. I would especially like to thank

Dr. James Mead at the Smithsonian Institute for the conception of the idea of creating

these databases, and all the employees and volunteers, both at VAQS and UNCW Marine

Mammal Stranding Programs, for their diligence over the past two decades in ensuring they are kept up to date and accurate. I would also like to extend my gratitude to

Gretchen Lovewell, NIMFS NOAA Beaufort and Dr. Craig Harms, NC State University for their wonderful support in sample collection. Much appreciation is also expressed to

Dr. Dave Rotstein, NIMFS NOAA, for his wonderful insights into Kogia lungs, and Alex

Costidis for the insightful discussions on Kogia vasculature. I also offer much gratitude to the Florida Marine Mammal Pathology Laboratory, especially Andy Garrett, for access to their facility for whole body cross-sectioning.

Finally, I would like to thank those that have willingly served on my committee,

Drs. Timothy Ballard, Richard Dillaman, Stephen Kinsey, and Sentiel “Butch” Rommel.

All of who have provided valuable insights, critiques, and thoughtful questions at every

step in producing this final product.

vii DEDICATION

I dedicate this thesis to my family, especially my parents Deborah and Eugene

Piscitelli, Jr., who have supported me through myriad opportunities and challenges.

Thank you for always nurturing a curious, creative, and adventurous attitude towards life.

I would also like to acknowledge the strong support of my brothers, Noah Mehrkam and

Eugene “Anthony” Piscitelli, III, who have always enthusiastically encouraged me to pursue a profession with passion and verve.

viii

LIST OF TABLES

Table Page

1. Cetacean species utilized in the broader phylogenetic comparison for which previously published values of lung and total body mass were available ...... 7

2. Lung variables compared to total body mass (kg) in terrestrial and marine mammals...... 17

3. Measurements used to calculate total body volume (Vb)...... 26

4. Summary of allometric relationships between organ mass and total body mass...... 38

5. Lung volume measurements for kogiids and T. truncatus...... 45

6. Mean lung air volume (± S.E.) measures for T. truncatus and Kogia spp. compared to P. phocoena (n = 4) (*data from Kooyman and Sinnett 1979)...... 46

7. A comparison of estimated total lung capacity (TLC), based upon measurements from this study, and those predicted from a variety of existing allometric relationships based upon total body mass (TBM) ...... 49

8. Absolute (L) and relative (%) volumes of organs, vascular tissues and specific cavities within the entire musculoskeletal thorax calculated from whole body cross-sections of a single K. sima and T. truncatus ...... 50

9. The mean (± S.D.) percent difference between the maximal cranially expanded to caudally collapsed postures (averaged across ribs 1-5) for thoracic dimensions measured for each species (* denotes significant differeces) ...... 55

10. Cranial thoracic cavity volume (L) calculated from whole body cross-sections and calculated via four geometric models ...... 59

11. Cranial thoracic cavity volumes (L), calculated using geometric Model 1, for cranially most expanded and caudally most collapsed postures...... 61

12. Common diving capacity measures for a variety of odontocetes for which a corresponding lung mass and total body mass ratio was available...... 63

13. Summary of allometric relationships between lung mass and total body mass for diving behavior and myoglobin content...... 66

14. Myoglobin values for a variety of odontocetes for which a corresponding lung mass and total body mass were available ...... 67

Page

15. The estimated contribution of lung oxygen stores to meeting metabolic costs of resting and swimming in T. truncatus and kogiids...... 73

16. Heart mass (kg) vs. total body mass (kg) ratios for a variety of cetaceans...... 75

17. Liver mass (kg) vs. total body mass (kg) ratios for a variety of cetaceans...... 78

x LIST OF FIGURES

Figure Page

1. The relationship between gas volume within the lung and dive depth, based on Pascal’s and Boyle’s gas laws ...... 4

2. The range of thorax mobility in T. truncatus (A-D) ...... 9

3. Schematic of the custom-built apparatus used to inflate excised lungs [container used was a 50 or 100 gallon Rubbermaid® water trough (Newell Rubbermaid Inc., Atlanta, GA, USA)]...... 20

4. Lung volume-pressure relationships for species used in this study...... 23

5. Each excised thorax was (A) suspended from a stable frame and manipulated into the defined maximum cranially expanded posture ...... 30

6. Example of one of four geometric models of the thoracic cavity used to calculate volumes at the extreme cranially expanded and caudally collapsed positions...... 33

7. Four geometric models of the thoracic cavity used to estimate cavity volume changes between a maximally cranially expanded and a maximally caudally collapsed posture ...... 34

8. Log lung mass (kg) vs. log total body mass (kg) for all age classes and body conditions of T. truncatus and Kogia spp ...... 39

9. Ratio of lung mass (kg) vs. total body mass (kg) against total body length (cm) across all age classes and body conditions of T. truncatus and Kogia spp ...... 40

10. Log lung mass (kg) vs. log total body mass (kg) for sub-adults and adults of T. truncatus and Kogia spp. compared to adult terrestrial mammals...... 42

11. Log lung mass (kg) vs. log total body mass (kg) for Families Delphinidae and Kogiidae ...... 43

12. Log lung mass (kg) vs. log total body mass (kg) for species pooled in Families Delphinidae and Phocoenidae (D-P) and Kogiidae, Physeteridae, Ziphiidae (K-P-Z) ...... 47

13. Log of calculated total lung capacity (TLC, in liters) vs. log of total body mass (kg) for T. truncatus and Kogia spp...... 51

xi Page

14. Whole body cross-sections at the level of the heart in a (A) T. truncatus and (B) K. sima ...... 52

15. In (A) T. truncatus the lungs overlap the heart ventro-laterally ...... 53

16. Whole body cross-sections at the level of the inlet to the thorax in a (A) T. truncatus and (B) K. sima...... 56

17. The range of thoracic mobility in Kogia spp ...... 57

18. The ratio of lung mass vs. total body mass is mapped upon a phylogeny for cetaceans...... 64

19. Log lung mass (kg) vs. log total body mass (kg) of species within five families (Kogiidae, Physeteridae, Ziphiidae, Delphinidae and Phocoenidae)...... 68

20. Log lung mass (kg) vs. log total body mass (kg) of species within five families (Kogiidae, Physeteridae, Ziphiidae, Delphinidae and Phocoenidae)...... 69

21. Log heart mass (kg) vs. log total body mass (kg) for T. truncatus and Kogia spp. compared to adult terrestrial mammals ...... 76

22. Log liver mass (kg) vs. log total body mass (kg) for T. truncatus and Kogia spp. compared to adult terrestrial mammals ...... 79

xii INTRODUCTION

All cetaceans are breath-hold divers, but the depths to which species routinely

dive vary immensely. For example, coastal bottlenose dolphins (Tursiops truncatus) dive

to depths of 1-10m while, on average, Cuvier’s beaked whales (Ziphius cavirostris) dive

to over 1000m (Mate et al. 1995, Barros and Wells 1998, Nowacek 1999, Connor et al.

2000, Reeves et al. 2002, Young and Phillips 2002, Torres et al. 2003, Tyack et al.

2006). These two divers will experience vastly different external pressures at depth that

will, according to Boyle’s and Pascal’s gas laws, influence the volume of air within their

lungs and potentially the amount of thoracic collapse they experience (reviewed in

Scholander 1940, Ridgway et al. 1969, Taylor 1994, Skrovan et al. 1999). This study

investigated lung size and thoracic mobility in the shallow diving bottlenose dolphin (T.

truncatus) and the deeper diving pygmy and dwarf sperm whales (Kogia breviceps and

Kogia sima). These species, chosen because relatively large samples of stranded

individuals were available, were used to test the hypotheses that lung size will be reduced and/or thoracic mobility will be enhanced in deeper divers.

Marine mammals possess a suite of anatomical and physiological specializations that enhance diving ability, including large body size, increased on-board oxygen stores, reduced metabolic costs, and energy-saving locomotor strategies (e.g. Scholander 1940,

Kooyman and Andersen 1969, Ridgway 1971, Kooyman 1989, Schreer and Kovacs

1997, Kooyman et al. 1999, Skrovan et al. 1999, Williams et al. 1999, Williams et al.

2000, Nowacek et al. 2001, Miller et al. 2004). Both dive depth and duration can be extended by increasing on-board oxygen stores (Scholander 1940, Kooyman 1973,

Kooyman 1985, Kooyman et al. 1999, reviewed in Pabst et al. 1999, Noren and Williams 2000, Duffield et al. 2003). It has been demonstrated in pinnipeds, for example, that

deeper diving Weddell seals (Leptonychotes weddellii) (common depth 150-400m, max

depth 500-740m; Kooyman 1966, Kooyman et al. 1971, Castellini et al. 1992, Williams

et al. 2000) possess a relatively larger blood volume, higher hematocrit, and higher

hemoglobin and myoglobin concentrations than does the more shallow diving California

sea lions (Zalophus californianus) (common depth 60-65m; max depth 250-480m; Gentry

et al. 1986, Feldkamp et al. 1989, Melin et al. 1993, Orr and Aurioles-Gamboa 1995)

(Kooyman 1985). The role of the lung as an oxygen store appears to be reduced in both

species, relative to human divers. Alveolar collapse has been demonstrated to occur at

between 30-80m depth in pinnipeds (Scholander 1940, Kooyman et al. 1970, Kooyman et al. 1971, Kooyman et al. 1973, Denison et al. 1971, Williams et al. 2000, Falke et al.

2008). Thus, deeper divers do not rely upon their lungs as an oxygen store at depth

(Kooyman 1985, Schmidt-Nielsen 1984).

The total on-board oxygen stores in cetaceans have not been less well studied, but

it is known that deeper diving species do have higher myoglobin concentrations in their

locomotor muscles and larger blood volumes than do shallow divers (Ridgway and

Johnston 1966, Ridgway and Harrison 1986, Noren and Williams 2000, Dolar et al.

1999). Cetaceans also experience alveolar collapse during a dive. The depth at which alveolar collapse occurs was first predicted from intramuscular nitrogen tensions measured in two trained bottlenose dolphins as they completed a series of repetitive dives to 100m (Ridgway and Howard 1979). The results suggested alveolar collapse was

complete at approximately 70m (Ridgway and Howard 1979). A free-swimming bottlenose dolphin, trained to wear a time-depth recorder and video camera, also

2 appeared to experience alveolar collapse at a depth of approximately 80m (Skrovan et al.

1999). At this depth, the dolphin began gliding downwards, rather than actively swimming. This change in locomotor behavior, which has been measured in other diving cetaceans, has been ascribed to reduce whole-body buoyancy due to compression of lung air volume (e.g. Skrovan et al. 1999, Williams et al. 1999, Williams et al. 2000,

Nowacek et al. 2001, Miller et al. 2004, Tyack et al. 2006).

Based on Boyle’s and Pascal’s laws (Resnick and Halliday 1966), as depth increases air volume within the lung decreases (Figure 1; reviewed in Taylor 1994).

Thus, the relative contribution of the lung as an oxygen storage site is inversely related to dive depth. Because deeper divers do not rely upon their lungs as an oxygen store at depth, Scholander (1940) suggested that lungs of deeper diving cetaceans should be smaller than those of shallow diving species.

Lung size has been investigated in a number of cetacean species. Lung size can be reported in multiple ways, including total lung mass (Laurie 1933, Crile and Quiring

1940b, Scholander 1940, Crile 1941, Quiring 1943, Omura 1950, Slijper 1958, Kooyman et al. 1971, Smith and Pace 1971, Bryden 1972, Leith et al. 1972, Perrin and Roberts

1972, Tarasoff and Kooyman 1973, Miyazaki et al. 1981, Lockyer and Waters 1986,

McLellan et al. 2002), total lung volume (Scholander and Irving 1941, Ridgway et al.

1969, Olsen et al. 1969, Denison et al. 1971, Kooyman 1973, Kooyman and Sinnett

1979, Bergey and Baier 1987, Schmidt-Nielson 1997, Falke et al. 2008) and with a variety of air capacity measurements (Irving et al. 1941, Kenyon 1961, Spencer et al.

1967, Kooyman et al. 1970, Kooyman et al. 1973, Kooyman et al. 1975, Ridgway and

Howard 1979, Kooyman and Cornell 1981, Watson and Gaskin 1983, Ridgway and

100 10 L Surface 90

60 5 L 10 m 50

Percent Volume 40

30 Percent volume of gas in lung (%) 20 1 L 100 m 1000 m 0.1L 10

0 0 100 200 300 400 500 600 700 800 900 1000 DepthDepth (m) (m)

Figure 1. The relationship between gas volume within the lung and dive depth, based on Pascal’s and Boyle’s gas laws. The fill in each box to the right of the curve represents a lung volume of 10L at the surface. Note that air volume decreases by 50% within the first 10m of descent; below 100m the rate of change in air volume decreases dramatically (reviewed in Taylor 1994).

Harrison 1986, Kooyman 1989, Shaffer et al. 1997). Thus, evidence of differences in

relative lung size does exist across clades of cetaceans.

Slijper (1958), for example, reported that the average lung mass of selected

delphinids (Delphinus delphis, Grampus griseus, and Lagenorhynchus acutus) and harbor

porpoises (Phocoena phocoena) were larger than those of similarly-sized terrestrial mammals. Mysticetes (Megaptera novaeangliae, Balaenoptera borealis, B. physalus, B.

musculus, Eschrichtius robustus, and Eubalaena glacialis) and sperm whales (Physeter

macrocephalus) possess relative lung masses that are smaller than those of similarly-

sized terrestrial mammals (Slijper 1958). Miyazaki et al. (1981) reported that the lung

mass to total body mass ratios of stenellids (Stenella attenuata, Stenella coeruleoalba)

were larger than those reported for sperm whales and at least some of the mysticetes

reported by Bryden (1972).

Kooyman (1973) reported that lung volumes of a variety of cetaceans were

slightly larger than those of similarly-sized terrestrial mammals. Delphinids (D. delphis,

G. griseus, and L. acutus) and phocoenids (P. phocoena) appear to possess a larger lung

capacity than do similarly-sized terrestrial mammals (Irving et al. 1941, Slijper 1958,

reviewed in Bryden 1972, Kooyman and Sinnett 1979). In contrast, Irving et al. (1941)

reported that lung capacities of rorquals (M. novaeangliae, B. borealis, B. physalus, B.

musculus), sperm whales (P. macrocephalus), and bottlenose whales (Hyperoodon

ampullatus) were smaller than those of similarly-sized terrestrial mammals (reviewed in

Slijper 1958). Thus, existing data suggest that some deep divers, including sperm whales

and bottlenose whales, possess relatively small lungs, but so do a diversity of mysticetes,

which include shallow diving species. In addition, to date, the number of individuals in

which lung size has been investigated for any given species is relatively small (Table 1;

but see Miyazaki et al. 1981 and McLellan et al. 2002).

It has also been hypothesized that deep diving cetaceans possess enhanced

thoracic mobility to accommodate pressure-induced reductions in lung volume at depth

(Scholander 1940, Ridgway et al. 1969, Hui 1975, Rommel 1990). That is, during a dive,

as the air volume within the lungs compress and total lung volume is reduced, the thorax

must also simultaneously collapse (Scholander 1940, Kooyman and Andersen 1969,

Ridgway et al. 1969, Ridgway 1972, Ridgway and Harrison 1986). Only two cetacean

studies, both on relatively shallow diving delphinids, have described thoracic collapse

under pressure (Ridgway et al. 1969, Hui 1975). Thoracic shape change was observed to

be a gradual process in a free-diving U.S. Navy bottlenose dolphin named “Tuffy”. At

10m Navy divers observed changes in thoracic shape, and by 60m the collapse of the

thorax was reported to be very apparent, especially in the area behind the pectoral flipper

(Ridgway et al. 1969). The only published photographic evidence of this thoracic

collapse is of “Tuffy” at 300m (Ridgway et al. 1969). These qualitative observations

suggested that the flexible thorax of a bottlenose dolphin continuously changed shape

during a dive (Ridgway et al. 1969).

Hui (1975) investigated the compressibility of the dolphin thorax, by placing a

carcass of a short-beaked common dolphin (D. delphis) in a supine position (i.e. ventral

surface up) in a hyperbaric chamber and subjecting it to two simulated dives to

approximately 70m. Hui (1975) observed a decrease in both thoracic height and width

during the simulated dives. Hui (1975) hypothesized that during thoracic compression the

vertebral rib-sternal rib joints became more acute, and that both the vertebral and sternal

Table 1. Cetacean species utilized in the broader phylogenetic comparison for which previously published values of lung and total body mass were available.

Species Family n Citation Lockyer and Waters 1986, Balaenoptera borealis Balaenopteridae 22 Omura 1950, Leith and Lowe 1972* Balaenoptera musculus Balaenopteridae 1 Laurie 1933

Lockyer and Waters 1986, Balaenoptera physalus Balaenopteridae 14 Quiring 1943, Leith and Lowe 1972* Miyazaki et al. 1981, Perrin and Stenella attenuata Delphinidae 60 Roberts 1972

Stenella coeruleoalba Delphinidae 40 Miyazaki et al. 1981

Stenella longirostris Delphinidae 4 Perrin and Roberts 1972

Crile and Quiring 1940b, Phocoena phocoena Phocoenidae 9 Kooyman and Sinnett 1979, McLellan et al. 2002***

Crile and Quiring 1940b, Delphinapterus leucas Monodontidae 3 Ridgway and Harrison 1986 Physeter macrocephalus Physeteridae 10 Omura 1950

Kogia sp. Kogiidae 1 Scholander 1940

Berardius bairdii** Ziphiidae 1 Balcomb 1989**

Hyperoodon ampullatus Ziphiidae 1 Scholander 1940

Quiring 1943, Kenyon 1961, Ziphius cavirostris Ziphiidae 1 reviewed in Bryden 1972

* body mass estimated from published length - weight data ** body mass estimated by Sleptsov 1961 = 8.848 metric tons *** values used from this source were means from each of four life history categories (n = 122 total individuals)

ribs rotated caudally to decrease thoracic width. Physical manipulations of isolated

bottlenose dolphins thoraxes support these hypotheses (Cotten et al. 2008). Thoracic cavity volume was observed to decrease by the rotation of the vertebral rib-sternal rib joints medially (decrease in width) and dorso-caudally (decrease in height) (Figure 2).

These changes in thoracic shape have been attributed to the enhanced flexibility

of the musculoskeletal thorax (Scholander 1940, Ridgway et al. 1969, Hui 1975, Rommel

1990). To date, though, no functional morphological data exist on potential thoracic

shape change in any deep diving cetacean. Interestingly, deep diving cetaceans lack the

specialized thoracic morphologies observed in the flexible bottlenose dolphin thorax (e.g. bony sternal ribs and associated mobile joints) (Ridgway et al. 1969, Hui 1975, Rommel

1990, Cotten et al. 2008), and instead possess cartilaginous sternal ribs (Benham 1902,

Nagorsen 1985) that may limit thoracic collapse.

Although it has been hypothesized that deeper divers possess enhanced thoracic mobility to accommodate reductions in lung volume at depth, recent data from free- swimming Weddell seals (Leptonychotes weddellii) suggests such collapse may be relatively limited (Falke et al. 2008). This study demonstrated that the seal’s thoracic circumference decreased by less than 2% when measured at 196m. Falke et al. (2008) hypothesized that thoracic shape may be maintained at depth because as the lungs collapse, vascular structures within the thorax may become engorged. Phocid seals possess elaborate venous structures within their thoracic cavity that would accommodate such a response (Harrison and Tomlinson 1956, Ponganis et al. 2006, Falke et al. 2008).

Two mechanisms that may aid in this process are intrathoracic blood pooling and the

A C

B D

Figure 2. The range of thorax mobility in T. truncatus (A-D). The body outline in each of the lateral views (C-D) is fixed between positions. (A) and (C) depict the cranially most expanded posture, while (B) and (D) depict the caudally most collapsed posture. These changes in posture occur due to movement at joints between the vertebrae and vertebral ribs, vertebral and sternal ribs, and sternal ribs and sternum. (Image from Cotten et al. 2008)

9 thoracic (respiratory) pump (Wislocki 1929, Craig 1968, Schaefer et al. 1968, Fanning and Harrison 1974, Ponganis et al. 2006, Falke et al. 2008).

Intrathoracic blood pooling has been observed in several studies in human breath- hold divers (Craig 1968, Schaefer et al. 1968, reviewed in Ferrigno and Lundgren 1999).

As one component of the dive response, systemic vasoconstriction occurs and blood is redistributed from the periphery into the thorax (Scholander 1940, Craig 1968). This redistribution of blood volume may help collapse the lungs, and thus contribute to maintaining pressure equilibrium across the thoracic wall (Craig 1968). Schaefer et al.

(1968) estimated that in a human breath-hold diver, 1.4 to 1.7L of blood was redistributed into the thorax. This volume represents 25-30% of an average adult human’s total blood volume (approximately 5.5L; Saladin 2007).

The thoracic (respiratory) pump is a mechanism that aids the flow of venous blood from the abdominal to the thoracic cavity during ventilation (Saladin 2007,

Ponganis et al. 2006). During inhalation, the thoracic cavity expands and the internal pressure drops. Simultaneously, the caudal movement of the diaphragm raises pressure in the abdominal cavity. As a result of this pressure differential, blood is squeezed cranially, via the caudal vena cava, from the abdominal to the thoracic cavity (Saladin 2007).

Cetaceans are known to inhale prior to a breath-hold dive (Scholander 1940). Thus, the thoracic pump may bring venous blood into the thorax just prior to diving and intrathoracic blood pooling may occur as part of the dive response once submerged at depth.

These two vascular responses, which are well-documented in humans, suggest that deeper diving cetaceans, which likely have larger blood volumes than do shallow

divers (Ridgway and Johnston 1966, Ridgway and Harrison 1986), may redirect a large

amount of blood into the thoracic cavity during a dive. If this hypothesis is true, then one

would expect a deeper diving cetacean to possess more voluminous vascular structures

within its thoracic cavity (Melnikov 1997), as well as mechanisms of blood movement from the periphery to the thorax.

Scholander (1940) referred to vascular structures that could dynamically change

shape as “swelling” and “displacement” organs. There are a number of vascular

structures within the cetacean thoracic cavity that may function in this manner. These

structures include the thoracic arterial rete, venous plexuses within the trachea and

bronchi of the lung, and the pericardial venous plexus (Scholander 1940, Harrison and

Tomlinson 1956, Ridgway 1972, Hui 1975, Vogl and Fisher 1981, Vogl and Fisher 1982,

Melnikov 1997, Ninomiya et al. 2005, Ponganis et al. 2006).

The thoracic arterial rete lies dorsally between the ribs and extends into the neural

canal to form the epidural rete (reviewed in McFarland et al. 1979,Vogl and Fisher 1981,

Vogl and Fisher 1982, reviewed in Pabst et al. 1999, Melnikov 1997). Hui (1975)

suggested that the thoracic rete may affect the degree and pattern of thoracic collapse in a

diving common dolphin (Delphinus delphis). However, Scholander (1940) hypothesized

that the volume of this vascular structure was too small in Phocoena phocoena to permit

it to function as a significant blood reservoir.

Additionally, extensive plexuses of veins, arteries and arterioles, collectively

referred to as retial tissue, are present within the trachea (between the elastic membrane

enclosing the tracheal hyaline cartilage and the smooth muscle layer) of many marine

mammals (Wislocki 1929, Fanning and Whitting 1969, Fanning and Harrison 1974,

Smodlaka et al. 2006). In T. truncatus and Baird’s beaked whale (Berardius bairdii), a

venous plexus in the mucosa that lines the airways of the lung, extending from the extra-

pulmonary bronchi to the terminal bronchi, has also been described (Wislocki 1929,

Fanning and Harrison 1974, Ninomiya et al. 2005). This periarterial venous plexus is

supplied by pulmonary veins and surrounds the pulmonary arteries up to the terminal

airways. Ninomiya et al. (2005) hypothesized that these veins may become engorged

with large volumes of oxygenated blood, which in turn may be transported to the tissues

to allow for longer aerobic dive duration.

Finally, a pericardial venous plexus has been described in the harbor porpoise

(Phocoena phocoena), harbor seal (Phoca vitulina), gray seal (Halichoerus grypus), elephant seal (Mirounga spp.), and leopard seal (Hydrurga leptonyx) (Burne 1910,

Harrison and Tomlinson 1956, Ponganis et al. 2006). This plexus, a continuous venous ring surrounding the caudal base of the pericardium, connects the caudal vena cava dorsally and the phrenic veins ventrally. It can possess leaf-like projections into the pleural cavity formed by thick-walled veins (0.2-0.3mm) surrounded by lobules of brown fat (Harrison and Tomlinson 1956). However, Scholander (1940) hypothesized that the volume of these venous plexuses were also too small to function as a “swelling” organ within the thoracic cavity of Phocoena phocoena.

Based upon Pascal’s and Boyle’s gas laws, the lungs of deep diving cetaceans will undergo much larger changes in volume than those of shallow diving species. These differences likely influence the size of the lung and thoracic vascular structures, as well as the range of mobility of the thorax. Although lung size has been measured in many species, few studies have investigated explicitly the relationship between lung size and

dive depth. It has been hypothesized that the thorax of a deep diver is capable of

undergoing large shape changes and that shape change may occur due to presence of

highly mobile joints within the thoracic skeleton. However, no data exist that compares

the mobility of the thorax in deep and shallow diving cetaceans. Thoracic vascular

structures may potentially affect thoracic shape change, but to date the relative size of

these thoracic vascular structures in a deep versus shallow diving cetacean has not been

investigated.

Thus, the goals of this study were to compare lung size and thoracic mobility

between the shallow diving coastal bottlenose dolphin (T. truncatus) and the deeper

diving pygmy (K. breviceps) and dwarf (K. sima) sperm whales. An additional goal was

to provide insight into the relative volume of thoracic vascular structures across these

cetaceans. These species were chosen because access to a relatively large sample set was

possible through stranding programs in the mid-Atlantic.

The coastal bottlenose dolphin morphotype is found in relatively shallow

estuarine and near shore waters (within 7.5km of shore), and feeds on shallow water prey

(Mate et al. 1995, Mead and Potter 1995, Barros and Wells 1998, Nowacek 1999, Connor

et al. 2000, Young and Phillips 2002, Torres et al. 2003, Gannon and Waples 2004).

Mean dive durations range from 20 to 40sec, and dive depths range between 1 – 10m

(Würsig 1978, Irvine et al. 1981, Shane et al. 1990, Bassos 1993, Mate et al. 1995,

Barros and Wells 1998, Nowacek 1999, Connor et al. 2000, Reeves et al. 2002, Young and Phillips 2002).

In contrast, pygmy and dwarf sperm whales are rarely sighted at sea and when observed are usually seen “logging” (resting) at the surface (Allen 1941, Yamada 1953,

Nagorsen 1985, Caldwell and Caldwell 1989, Scott et al. 2001). A radio-tracked juvenile pygmy sperm whale displayed surface intervals ranging from 4sec (77% of surfacings) to

11min (Scott et al. 2001), and a maximum dive duration of 18min (Scott et al. 2001).

Stomach contents and stable isotope analyses demonstrate that kogiids forage primarily on deep sea squid (Ross 1979, Martins et al. 1985, Klages et al. 1989, McAlpine et al.

1997, Plön et al. 1999, Barros 2003, Santos et al. 2006, Beatson et al. 2007). Kogiids have been sighted typically in waters 400-1,000m off the eastern coast of North America

(Scott et al. 2001, Fulling 2003, NOAA Stock Report 2005, Dunphy-Daly et al. 2008).

These species will be used to specifically test three hypotheses. The deeper diving kogiids are hypothesized to possess smaller lungs and/or enhanced thoracic mobility, and to possess more voluminous thoracic vascular structures than the shallow diving bottlenose dolphin. Lung size will be investigated by comparing lung mass and volume relative to total body size. Thoracic mobility will be investigated by mechanically manipulating excised thoraxes. The static volumes of thoracic vascular structures will be compared using whole body cross-sections. To broaden the phylogenetic scope of this study lung mass relative to total body mass will also be investigated across five families of odontocete cetaceans. Although sample sizes for individual species in this broader comparative analysis are relatively small, these data permit comparisons across clades that display different dive depths and durations.

MATERIALS & METHODS

Specimens

Carcasses of Atlantic bottlenose dolphins (Tursiops truncatus) (n = 116), and

pygmy (Kogia breviceps) and dwarf (Kogia sima) sperm whales (n = 21) that stranded or were incidentally killed in fishing operations were utilized in this study (Appendix A).

For all analyses, K. sima and K. breviceps are combined and reported as Kogia spp.

These specimens were collected by the University of North Carolina Wilmington

(UNCW) Marine Mammal Stranding Program, the Virginia Aquarium Stranding

Response Program (VAQS), and the Cetacean and Sea Turtle Team at the National

Marine Fisheries Service Laboratory in Beaufort, North Carolina. Each specimen was

placed into a life history category as defined by Caldwell and Caldwell (1989), Mead and

Potter (1990), Struntz et al. (2004), and Dunkin et al. (2005). All specimens used in this

study were in fresh to moderate condition (Smithsonian Institute Code 1 through 3;

Geraci and Lounsbury 2005). Carcasses were either freshly dissected or frozen within

hours of death and remained frozen until dissection at a later date.

To examine relative lung size across a broader phylogenetic sample of cetaceans, the stranding archive databases at UNCW and VAQS were searched. All specimens, for which both a total lung mass and total body mass were available, were included in this analysis (Appendix B). Similar data were also obtained from previously published reports

(see Table 1 for species and references). These combined data sets provided at least two

individual records for each genus, with the exception of a single individual record for

Hyperoodon ampullatus, Ziphius cavirostris, and Berardius bairdii. In total, these

combined data sets yielded lung mass and total body mass for 352 individuals, from 28

species, representing seven families.

Measures of Lung Size: Mass

To test the hypothesis that deeper divers possess relatively small lungs, lung masses and total body masses were measured. During dissection, lungs were excised whole from the carcasses. Each lung was dissected free at the level of the primary

bronchus. Left and right lungs were weighed separately using digital scales and their

masses were added together to obtain total lung mass (n = 111 for T. truncatus and n = 18 for Kogia spp.; Appendix A).

The total data set for each species included a broad range of age classes (neonates through adults) and individual body conditions (robust to emaciated). To determine whether body condition influenced the relationships between lung mass and total body mass for the total data set, a regression analysis was performed both with and without emaciated specimens included. Because there was no significant difference found in the resulting allometric relationships for either species (see Results), emaciated individuals were included in the total dataset. A subset of these individuals, sub-adults and adults only (n = 84 for T. truncatus and n = 17 for Kogia spp.), were also analyzed separately and compared to a known allometric equation for adult terrestrial mammals (Table 2).

Using similar methods, total lung mass and total body mass were recorded for a variety of other cetacean species (Appendix B). This dataset was supplemented with lung masses and total body masses from previously published reports (Table 1).

Table 2. Lung variables compared to total body mass (kg) in terrestrial and marine mammals. y = axb ± 95% C.I., where x = total body mass (kg), y = organ variable, a = y-intercept, and b = slope. (NR = not reported)

Animal Actual Measurement n a b ± 95% C.I. Citation Category Measurement Brody 1945, Stahl 1965, Stahl 1967, adult terrestrial Lasiewski and Calder 1971, reviewed Lung Mass (kg) > 100 0.0113 0.986 ± 0.012 mass mammals in Calder 1984, reviewed in Peters 1993 Lung Volume terrestrial Stahl 1967, reviewed in Schmidt- total lung 333 53.5 1.06 ± 0.02 (mL) mammals Nielsen 1984 capacity

Lung Volume marine Kooyman 1973; reviewed in total lung 17 0.135 0.92 (L) mammals Kooyman and Sinnett 1979 capacity mammals Tenney and Remmers 1963, reviewed Lung Volume (shrew to incld. inflated lung 26 56.7 1.02 in Schmidt-Nielsen 1984, reviewed in (mL) whale and tissue volume Peters 1993 porpoise) Lung Volume Gehr et al. 1981; reviewed in Peters inflated lung 27 66.1 0.986 ± 0.017 wild mammals (mL) 1993 tissue volume Lung Volume Maina and Settle 1982, reviewed in NR 41.92 1.041 mammals NR (mL) Schmidt-Nielsen 1984

Heart Mass (kg) 568 0.006 0.98 mammals Stahl 1967, reviewed in Calder 1984 mass

Liver Mass (kg) 175 0.033 0.87 mammals Brody 1945, reviewed in Calder 1984 mass

To compare T. truncatus and Kogia spp., relative lung size was investigated in a number of ways. The allometric relationship between lung mass and total body mass was computed for (1) all specimens (i.e. the total data set) and (2) a subset of adult and sub- adult animals, which could be compared with a known allometric relationship for adult terrestrial mammals (y = 0.0113x0.986; Brody 1945, Stahl 1967, Lasiewski and Calder

1971, reviewed in Calder 1984 and Peters 1993). Total lung masses and body masses

were log-transformed and plotted for each species. An allometric equation was generated

for T. truncatus and Kogia spp. [y = axb, where a = y – intercept, b = slope, x = total body mass (kg), and y = total lung mass (kg)]. The slopes and y-intercepts of the species lines were compared using an ANCOVA multiple regression analysis with a pre-determined α

(alpha) set at 0.05. To compare relative lung size across a broader phylogenetic sample of

cetaceans, these analyses were repeated for the entire comparative data set (see Appendix

B). Allometric equations were generated for each family of cetaceans. All statistical

analyses for this study were made using SAS (SAS Institute, Inc., Cary, NC, USA) and

SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA, USA) statistical software.

The percentage of total body mass invested in lung tissue was also investigated using the total data set for T. truncatus and kogiids. For this analysis, the ratio of lung mass (kg) to total body mass (kg) was plotted against total body length (cm). The slopes

and y-intercepts of these linear relationships were compared using similar statistical

methods as described above.

Measures of Lung Size: Volume

During necropsy, on a subset of individuals (n = 5 for T. truncatus and n = 4 for

Kogia spp.) the intact respiratory tree was dissected free of the carcass at the level of the trachea, wrapped in plastic to prevent desiccation, and frozen (Appendix A). Before inflation experiments, lungs were removed from the freezer and thawed to room temperature. Volumes of both un-inflated and inflated lungs were measured using the water displacement method (Figure 3). Lungs were inflated using a custom built system.

Each lung was attached to an inflation and pressure monitoring system via rubber tubing and all connections were wrapped to ensure complete sealing. Each lung was then submerged and secured loosely below the water’s surface with nylon netting. The initial un-inflated lung volume was measured and the lung was subsequently inflated using a 1L super syringe (Hamilton Company, Reno, NV, USA) (Figure 3).

Pressure changes within the lung were measured and recorded using a pressure vacuum module (PV350, FLUKE®, WA, USA) connected to a multimeter (87 III True

RMS Multimeter, FLUKE®, WA, USA), which was connected in-line with the inflation system. Air was added in increments of 100mL and an equilibration period of 30sec was allowed between incremental additions (Denison et al. 1971, Bergey 1986, Bergey and

Baier 1987). The corresponding pressure (psi) at each additional air increment was recorded. At every 500mL air increment, the total lung volume (mL) was re-measured by the water displacement method.

While the un-inflated lung volume could be unambiguously defined, a definition of maximum lung volume had to be developed. Comparative studies on a variety of terrestrial and marine mammals have defined “inflated” lung volume at + 30cm H2O

P S

Air Outlet Ruler

Figure 3. Schematic of the custom-built apparatus used to inflate excised lungs [container used was a 50 or 100 gallon Rubbermaid® water trough (Newell Rubbermaid Inc., Atlanta, GA, USA)]. Each lung was attached to the inflating (S) and pressure monitoring (P) system via rubber tubing. The lung was then submerged underwater and loosely secured with nylon netting. The water height was recorded on a metric ruler. The lung was inflated using a 1L super syringe (S) in 100mL increments of air. The un- inflated and inflated lung volumes were recorded using the water displacement method. Changes in lung volume were measured by removing known volumes of water (via valve, V), until water height returned to original height on ruler.

(0.42psi) of pressure (Denison et al. 1971, Dennison and Kooyman 1973, Weibel 1973,

Kooyman and Sinnett 1979, Bergey 1986, Bergey and Baier 1987). The lungs of T. truncatus reached this pressure during inflation experiments, but the kogiid lungs did not.

Alternatively, the point at which the volume-pressure curve plateaus has been defined as the static compliance point of the lung (Denison et al. 1971, Dennison and Kooyman

1973, Kooyman and Sinnett 1979, Bergey 1986, Bergey and Baier 1987). The pressure at which this point was reached differed between T. truncatus (0.40 - 0.45psi) and kogiids

(0.20 – 0.30psi). Thus, the inflated static compliant volumes reported here are those at which these species-specific pressures were reached. However, the lungs of both species reached their static compliance at minimal inflation volumes (usually less that 1.0L), which did not appear to accurately represent total lung capacity (TLC, maximum amount of air that the lungs can contain).

Scholander (1940) warned that inflating excised, cannulated lungs could also result in over-inflated lung volumes. The experimental approach utilized here confirmed this concern. Kogiid lungs, for example, could be inflated to six times their un-inflated volume without an appreciable increase in pressure. Visual inspection of the resulting lung, though, revealed air emboli, suggesting that tissue damage had occurred.

Because neither the volume at a standard inflation pressure nor the static compliance inflation volume were useful in estimating TLC, and over-inflation of lungs was of concern, an approach described by Kooyman and Sinnett (1979) in their study of

Phocoena phocoena lungs was used. They recognized that each un-inflated lung represented a unique start point, because each contained a variable initial volume of trapped air, termed the minimum air volume (MAV; the amount of air within the lung

before the first artificial inflation). To estimate MAV, Kooyman and Sinnett (1979) first

weighed each un-inflated lung, and assuming a tissue density of 1kg/L, calculated lung

tissue volume. These authors then compared this lung tissue volume to that of the whole

un-inflated lung volume, determined via the water displacement method. The difference

in these values represented MAV. In P. phocoena MAV represented 0-17% of the final

TLC measured. A conservative estimate of TLC was thus calculated for each T. truncatus

and kogiid lung by dividing MAV by 0.17. These methods assume that the lungs of both

species are mechanically similar to those of P. phocoena. This assumption likely

introduces error as the pressure-volume curves suggest that kogiid and T. truncatus lungs

reach different maximum pressures (Figure 4).

All lung volumes (L) – un-inflated lung volume, inflated static compliant volume,

MAV, and TLC – for each specimen were reported and each was normalized relative to

total body mass (kg).

Measures of Lung Volume: Whole Body Cross-Sections

To further investigate lung volume, relative to total thoracic cavity volume and

post-cranial body volume, whole body cross-sections were utilized. For T. truncatus,

archived scaled images of cross-sections (approximately 3-5cm thick) through a frozen,

small bottlenose dolphin (“no number”, female, 170.9cm) were used (Pabst 1990). A

small Kogia sima carcass (VAQS 20081002, male, 160.0cm) was frozen and then serially

cross-sectioned whole using a commercial grade Hobart© meat saw (Model #5801;

Hobart Corporation, Troy, Ohio, USA) into approximately 2-3cm thick sections at the

Marine Mammal Pathology Laboratory in St. Petersburg, Florida. Scaled, digital images

0.6

0.5

0.4

0.3

0.2

Lung Pressure (psi) Lung 0.1

0.0 T. truncatus Kogia spp.

0 500 1000 1500 2000 2500 Air Volume (mL) B

0.6

0.5

0.4

0.3

0.2

0.1 Mean Lung Pressure (psi) Lung Mean

0.0 T. truncatus Kogia spp.

0 200 400 600 800 1000 1200 Air Volume (mL)

Figure 4. Lung volume-pressure relationships for species used in this study. (A) Example of individual records for T. truncatus, WAM 633 and K. breviceps, VAQS 20071081. (B) The mean (± S.D.) pressure (psi) of all the specimens used in this part of the study.

(TIF format, D100 Nikon camera) were taken of each section on a photo stand. All

images were analyzed using Adobe Photoshop CS3 Extended© (Adobe Systems

Incorporated, San Jose, CA, USA) and Image ProPlus Software (Media Cybernetics,

Baltimore, MD, USA).

The entire musculoskeletal thorax is divided into the thoracic cavity (cavum

thoracis) and intrathoracic abdominal cavity (Nickel et al. 1986, Schaller 1992, Nomina

Anatomica Veterinaria 1994). The thoracic cavity, whose caudal border is defined by the

diaphragm, contains the coelomic pleural (cavum pleurae) and pericardial (cavum pericardii) cavities (Nickel et al. 1986, Schaller 1992, Nomina Anatomica Veterinaria

1994). The lungs are contained within the pleural cavity, while the heart is located with the pericardial cavity. The cupula pleurae are the blind cranial ends of the pleural cavity

located at the thoracic inlet (Schaller 1992, Nomina Anatomica Veterinaria 1994). The

intrathoracic abdominal cavity, whose cranial border is defined by the diaphragm,

contains the abdominal organs (i.e. liver) (Nickel et al. 1986, Schaller 1992, Nomina

Anatomica Veterinaria 1994). Thus, both the thoracic and the intrathoracic abdominal

cavities are bounded by the musculoskeletal thorax.

For each species, in situ measurements of the cross-sectional areas of thoracic

viscera, including the lung (with and without surrounding air space), heart, thoracic

arterial rete, and intra-thoracic abdominal viscera, including the liver, were measured for

each cross-section through the thorax. Although this study could not address dynamic

changes in vascular tissues, it could assess the differences in static vascular tissue

volumes. In addition, the entire musculoskeletal thorax, and pleural and intra-thoracic

abdominal cavities were similarly measured. Using Adobe Photoshop CS3 Extended©

and Image ProPlus the desired structure to be measured was outlined and its cross-

sectional area calculated using a calibration value determined from the fixed scale bar

within the image.

For each section, the reported cross-sectional area (cm2) for each structure was the

mean of three repeated measures. To assess measurement error for each structure across

the cross-sections, its mean cross-sectional area and mean standard deviation were

calculated. The percent error for each structure was defined as the mean standard

deviation/mean cross-sectional area*100. The percent error ranged from 0.13% for the

intra-thoracic abdominal cavity, the largest space within the thorax, to 3.5% for the

thoracic arterial rete, the smallest structure measured within the thorax. For each section,

the volume of each visceral organ and cavity measured was then calculated by

multiplying its cross-sectional area (cm2) by the thickness of that cross-section (cm).

These volumes (cm3) were then summed across all cross-sections to get a total organ or cavity volume.

Volume of the entire musculoskeletal thorax was reported relative to the

specimen’s post-cranial body volume (L). The total post-cranial body volume was

estimated by modeling it as a cylinder (from nuchal crest to anus) in series with a

truncated cone (from the anus to fluke insertion) (Dunkin et al. 2005). Measurements

required for this model, including appropriate lengths and girths were garnered from

standard morphometrics datasheets completed during necropsy (see McLellan et al. 2002;

Table 3). This model of the post-cranial body excludes the area of the head cranial to the

eye and thus is an underestimate of total body volume. Because the volume of the

musculoskeletal thorax en toto represented a similar percentage of total post-cranial body

Table 3. Measurements used to calculate total body volume (Vb).

Length Total Average Fluke Total Length (anus to Anus Body V Girth (eye Insertion Field ID Species Length b (eye to fluke Girth Mass (L) to anus, n = Girth (cm) anus) (cm) insertion) (cm) (kg) 7) (cm) (cm) (cm) VAQS 20081002 a K. sima 160.0 77.0 64.9 87.8 34.6 90.3 74.0 28.4 WAM 637 b K. sima 198.0 134.0 122.7 124.0 36.5 106.0 82.0 41.0 VAQS 20081003 b K. sima 218.4 161.5 142.9 122.4 51.4 113.0 94.2 35.2 CLP001 b K. breviceps 224.0 158.0 150.3 128.5 46.0 115.4 83.5 35.0 WAM 634 b K. breviceps 237.0 219.0 208.4 149.0 42.0 127.5 88.5 45.5 VAQS 20071081 b K. breviceps 275.0 234.5 205.0 150.0 56.5 123.9 92.5 45.5 "No ID" a T. truncatus 170.9 62.5 58.2 95.4 47.6 82.0 58.0 24.4* BRF 090 b T. truncatus 182.0 79.0 74.7 99.0 41.0 92.9 61.5 25.5 WAM 633 b T. truncatus 244.0 180.0 155.2 141.5 52.0 112.2 75.0 34.0 PBN 003 b T. truncatus 246.0 173.0 159.8 145.0 55.5 113.3 72.5 30.0

* measurement not reported on morphometric datasheet; predicted from linear equation of girth at fluke insertion (cm; y-variable) and total length (cm; x-variable): y = 0.109 x + 5.747, r2 = 0.804 a specimen used in Measures of Lung Size: Whole Body Cross-Sections b specimen used in Mobility of Isolated Thoraxes

volume in both species (see Results), volume measurements for each organ and cavity

were reported relative to the volume of the entire musculoskeletal thorax.

The cross-sectional images from both species were also used to examine the in

situ gross morphology of the organs, diaphragm trajectory, and thoracic shape. This

information was used to help choose the best geometric thoracic cavity volume model in

each species (see “Thoracic Manipulations” below).

Gross Morphology of Skeleton

The thoracic skeleton of the bottlenose dolphin has been well described (Rommel

1990, Cotten et al. 2008), but there exist only fragmented published reports on the kogiid thoracic skeleton, which are usually focused on rib counts (Haast 1873, Benham 1902,

Allen 1941, Yamada 1953, Omura et al. 1984, Nagorsen 1985). Thus, on a subset of individual kogiids (n = 5), gross thoracic dissections were conducted to investigate the morphologies and articulations of skeletal elements, including cervical and thoracic vertebrae, vertebral ribs, sternal ribs, and sternum. A single K. breviceps thorax (WAM

637) was osteologically prepared (manure and ammonia treatment) and the skeletal elements re-articulated. The length and width of each cartilaginous sternal rib (these elements do not survive osteological preparation) were measured before re-articulation.

Two other skeletons (K. breviceps, NMNH 504737 and K. sima, NMNH 504221), curated at the Smithsonian Institution’s National Museum of Natural History, were also investigated. Scaled, digital images were taken of the re-articulated and curated skeletons

(Nikon D100, TIFF image files). These images were imported into EasyCAD (Evolution

Computing, Phoenix, Arizona; e.g. Figure 2), scaled, and then used as references to draw

scaled illustrations of kogiid osteology (methods similar to Rommel 1990 and Cotten et al. 2008). These drawings were later used to illustrate the skeletal postures resulting from the thorax manipulations (see more detail of the thorax drawing under “Mobility of

Isolated Thoraxes”).

Mobility of Isolated Thoraxes

To test the hypothesis that the thorax of deep divers may undergo relatively larger changes in shape, the mobility of the isolated thorax was investigated on a subset of individual T. truncatus (n = 3), K. breviceps (n = 4), and K. sima (n = 1). Each isolated thorax, defined here as consisting of articulated vertebral elements [all cervical (C), all thoracic (T), and first 1-5 lumbar (L) vertebrae], vertebral ribs, sternal ribs, sternum, and associated muscle (internal and external intercostals, transverse thoracic, hypaxialis bisected at L3, and rectus abdominis bisected at sternum), was excised whole during dissection. Isolated thoraxes were wrapped in plastic to prevent desiccation and frozen for later mechanical tests. Before tests were conducted, each thorax was removed from the freezer, and thawed to room temperature. The thorax was kept moist using physiological saline throughout the physical manipulations.

The goal of the physical manipulations was to document and compare the ranges of thoracic mobility between T. truncatus and kogiids. For each thorax, forces were imposed, as described below, to create both a maximally expanded and maximally collapsed posture (methods similar to those of Cotten et al. 2008). The differences in metrics of thoracic size between these two extreme postures were used to represent the range of thoracic mobility.

To create the maximally expanded posture, the isolated thorax was suspended from a metal frame that consisted of a horizontal bar, connected via two uprights, to a stable base (Figure 5). To secure the thoracic unit, cable ties were wrapped around the vertebral column at four positions (between C5-6, T6-7, T12-13, and L4-5). Each of these cable ties was then connected to the metal frame to secure the vertebral column in place.

Before applying a load, white string was placed along the caudal margin of each vertebral and sternal rib. These strings provided high-contrast markers for the skeletal elements as they changed positions during the physical manipulations. To manipulate the thorax into a maximally expanded posture, hi-test nylon fishing line was tied to cranial skeletal elements at the (1) first vertebral rib-sternal rib joint, (2) manubrium, and (3) vertebral rib 1, at mid-shaft. These lines were then manually pulled cranially to expand the thorax. A 5 or 10kg spring scale (Pesola®, Baar, Switzerland), attached in series to the

nylon fishing lines, was used to measure the applied load, so that equivalent forces were

applied across multiple manipulations within the same posture. The maximal cranially

expanded posture was defined at that load at which no further shape change was grossly

observed to occur.

To create the maximally collapsed posture, the thorax was secured in a supine

position (Figure 5). The neural spines were placed between two wooden blocks, which

supported the vertebral column and maintained a stable supine position. In this position, the thorax naturally collapsed under its own weight, and this posture was defined as the maximal caudally collapsed posture.

At these two postures, the shape and size of the isolated thorax and the changes in position of the skeletal elements (vertebral ribs, sternal ribs, and sternum) were recorded.

A B

Dorsal

DorsalDorsal

Figure 5. Each excised thorax was (A) suspended from a stable frame and manipulated into the defined maximum cranially expanded posture. The lines attached to the cranial portion of the thorax represent the lines used to apply the known load to cranially expand the thorax. Arrows denote the direction of thorax movement during manipulations. (B) The isolated thorax was secured in a supine position, and allowed to collapse under its own weight into the defined maximum caudally collapsed position, as denoted by the arrow. White strings were placed along the caudal margin of each vertebral and sternal rib, which provided high contrast markers for the skeletal elements as they changed positions during the physical manipulations. Methods were similar to those used by Cotten et al. (2008).

Circumferences were measured with a flexible measuring tape; heights, depths, widths, and lengths were measured with anthropometers (Haglof ®, Sweden) (see Appendix C and Appendix D for detailed descriptions of measurements and datasheet). All reported measurements were the means of three repeated measures for each measurement. All values were reported as percent change from the cranially expanded to the caudally collapsed posture. A parametric two-sample t test (pre-determined α = 0.05) was utilized to test for differences among measurements between species.

Digital, scaled photographs were taken with a D100 Nikon camera in the dorsal, ventral, lateral, and frontal views and at a position perpendicular to the joint faces. To facilitate CAD rendering of the skeletal elements in each posture, the skeleton was flensed of all muscles except the internal intercostals, which revealed the ribs, while maintaining the integrity of the thorax (n = 1; VAQS 20081003 K. sima).

Lateral and frontal perspectives of anatomically correct, scaled skeletal figures were drawn in the cranially expanded and caudally collapsed postures using EasyCAD software and digital skeletal templates (see “Gross Morphology of Skeleton” above). The skeletal template for T. truncatus consisted of a vertebral column and skull, and was provided by Dr. Sentiel Rommel (modified from Rommel 1990; Cotten et al. 2008). For the kogiid skeletons, digital images taken during the thoracic manipulations were imported into EasyCAD, along with images of re-articulated and “re-aligned” skeletons

(NMNH 504221 K. sima and WAM 637 K. breviceps; see above). The skeletal template for the Kogia spp. consists of a vertebral column, thorax and skull, and was the result of a collaborative drawing effort with Dr. Sentiel Rommel. The re-articulated images were used as a reference for kogiid anatomy, while the thoracic manipulation images and

31 measurements were used as references for the shape change and range of mobility of the kogiid thorax.

Thoracic Cavity Volume Models

Measurements of thoracic shape (circumference, length, width, depth, and height) taken during the manipulations of isolated thoraxes were used to estimate volume changes between extreme postures. The external circumference and lateral width measurements were adjusted, by subtracting the thickness of the thoracic wall, and used to calculate internal thoracic cavity volumes (see model descriptions below). For each specimen, thoracic cavity volume (L) in each posture and percent change of thoracic cavity volume between postures were reported with respect to the specimen’s total body mass (kg) or total post-cranial body volume (L) (Table 3). Thoracic cavity volume calculated here refers only to the thoracic cavity proper (cavum thoracis) and does not include the intrathoracic abdominal cavity. The thoracic cavity was further sub-divided in this study into the (1) cranial thorax, defined as the space bounded by vertebral ribs that attach directly to the sternum via a sternal rib (vertebral ribs 1-5), and (2) caudal thorax, defined as that space caudal to rib 5 and bounded by the diaphragm. The shapes of the cranial and caudal thoraxes differ dramatically and, thus, were modeled with a different geometric shape (Figure 6).

To calculate thoracic volume, four models were investigated (Figure 7 and

Appendix E). Cranial thoracic volume was modeled using two alternative cross-sectional shapes, a circle and a cardioid (heart-shape). These geometric shapes were chosen based upon examination of photographs of cross-sections through the bottlenose dolphin thorax

LL LL L L L L L L D H D S H Li Li

Figure 6. Example of one of four geometric models of the thoracic cavity used to calculate volumes at the extreme cranially expanded and caudally collapsed positions, where r = radius of circle at the cross-section of rib 1, R = radius of a circle taken at the cross-section of rib 5, l = length between ribs 1-5, L = length between rib 5 and L3, and θ = angle of approach of the diaphragm to L3 (Sandifer and Moshos 1996; see Figure X for explanation of all four models). (B) Cross-sectional photographs of the thoracic cavity of a T. truncatus corresponding to positions within the cone model at ribs 1, 5, and 8. (L=lung, H = heart, S = stomach, Li = liver, D = diaphragm) (Pabst 1990) (C) Position of the diaphragm (thick black line) within the thoracic cavity of a T. truncatus (modified from Rommel 1990). 33

A B

C D

Figure 7. Four geometric models of the thoracic cavity used to estimate cavity volume changes between a maximally cranially expanded and a maximally caudally collapsed posture. (A) Model 1 represents a frustum of a right circular cone in series with a complete slanted cone. (B) Model 2 represents a circular cylinder in series with a complete slanted cone. (C) Model 3 represents the frustum of a cardioid cone in series with a complete slanted cone. (D) Model 4 representes a cardioid cylinder in series with a complete slanted cone. In (A) r = radius of circle at the cross-section of rib 1; in (B) r = average radius of circle from cross-section of ribs 1-5; in (C) r = the radius of a cardioid at the cross-section of rib 1 (measured from the ventral aspect of the centrum of V1 laterally to the angle in the rib); in (D) r = average radius of a cardioid at the cross-section of ribs 1-5 (measured from the ventral aspect of the vertebra centrum laterally to the angle in the rib). In (A) and (B), R = radius of a circle taken at the cross-section of rib 5, while in (C) and (D) R = radius of cardioid taken at the cross-section of rib 5. In all models (A) – (D), l = length between ribs 1-5, L = length between rib 5 and L3, and θ = angle of approach of the diaphragm to L3 (Sandifer and Moshos1996; see Appendix E for cardioid proofs of area and volume).

and morphology of the diaphragm (Figure 6). Height and width measurements taken on

cross-sectional images of both species in EasyCAD demonstrate that the cranial thorax of

T. truncatus is circular in shape (thorax is as tall as it is wide), while the kogiid thorax is

cardioid in shape (thorax is twice as tall as it is wide). The cross-sectional images also demonstrate that the thoracic cavity volume between ribs 1-5 increases gradually. Thus, for each cross-sectional shape the cranial thorax was modeled as either a right cylinder or a frustum of a right cone.

Cross-sectional images of both species also demonstrated that caudal to rib 5, thoracic cavity volume decreased rapidly due to the presence of the diaphragm and concomitant increasing volume of the abdominal cavity (Figure 6). The relaxed diaphragm of T. truncatus runs virtually horizontally from its dorso-caudal origin at

lumbar vertebrae 2-3 to the level of thoracic vertebra 6 (Dearolf 2003). At thoracic vertebra 6 the diaphragm changes its trajectory to become vertically oriented. The diaphragm runs ventro-cranially and inserts on the distal ends of vertebral ribs 7-13, on the vertebral rib-sternal rib joint of ribs 4-6, and onto the caudal most end of sternabra 3

(Figure 6). In kogiids, the trajectory of the diaphragm was not notably different from that

of T. truncatus, except for the insertion site; the central tendon of the diaphragm attaches

to hypophyses of lumbar vertebrae 2-5 (Appendix A). Thus, in all models, the caudal

thoracic volume (vertebral ribs 5 - lumbar 3 for T. truncatus and lumbar 5 for kogiids)

was modeled as a slanted right cone (Figure 6, Figure 7).

For both T. truncatus and kogiids, the left thoracic wall was removed during

dissection to obtain lateral images of the interior thorax and position of the diaphragm

muscle. Digital, scaled images were taken to note the position of the lungs, diaphragm,

35 and lateral shape of the thorax. These images were uploaded into EasyCAD and used as references for the geometric models in both T. truncatus and Kogia spp. Each of the geometric shapes used in the models simplify the true shape of the thoracic cavity, and, thus, may yield over- or under-estimates of the volume.

The whole cross-sectioned specimens were used to estimate the error of each thoracic cavity volume model. Images from each cross-sectioned specimen were imported into EasyCAD and scaled. The appropriate lengths and radiuses were measured and then used as inputs into each model to calculate thoracic cavity volume. For each cross-sectioned specimen, the percent difference between the measured thoracic cavity volume (L) (see “Lung Size: Whole Body Cross-Sections” above) and the modeled thoracic cavity volume (L) was calculated. The model that most closely estimated the real measured volume from the cross-sections was considered to be the best model for that species. This model was then used to estimate the thoracic cavity volume changes that occurred between the maximal cranially expanded and caudally collapsed postures for each isolated thorax (n = 3 for T. truncatus and n = 5 for kogiids).

RESULTS

A primary objective of this study was to compare lung size of the shallow diving coastal T. truncatus with that of the deeper diving kogiids. Lung size was measured using three different methods. For a large number of individuals, lung mass was recorded, for a smaller subset of individuals excised lung volume was recorded, and for one individual each of T. truncatus and K. sima lung volume, derived from whole body cross-sections, was computed.

Lung Mass

The slopes of the lines that describe the allometric relationship between lung mass and total body mass for T. truncatus and Kogia spp. across all age classes and body conditions (total data set) were not significantly different from each other and neither was distinguishable from 1 (Table 4, Figure 8). However, their y-intercepts did differ significantly (F = 224.15, P < 0.0001, d.f. = 128). Thus, although their lungs grow at a similar rate, for any given body mass the kogiid lung mass is approximately one-half that of a T. truncatus. Removing emaciated specimens from the data set did not change these relationships. The slopes were similar and neither was distinguishable from 1, and the y- intercepts were significantly different between species (F = 144.06, P < 0.0001, d.f. =

101; Table 4). Because the inclusion of the emaciated animals did not appear to bias the relationship between lung mass and total body mass, the total data set was utilized to investigate how the lung’s contribution to total body mass varied across animals of different body lengths (Figure 9). The slopes of the lines that describe the relationship between the lung mass: total body mass ratio and total length for T. truncatus and kogiids were similar and neither was distinguishable from zero. However, the y-intercepts did differ significantly between species (T. truncatus = 0.0299, Kogia spp. = 0.0133; F=

117.01, P < 0.0001, d.f. = 128). Thus, across all body lengths kogiid lungs contribute approximately one-half as much to total body mass as do those of T. truncatus (Figure 9).

A subset of data for each species that included only sub-adults and adults was used to compare relative lung size of T. truncatus and kogiids to that predicted for adult terrestrial mammals (y = 0.0113x0.986 ± 0.012, n > 100, r2 = 0.922, b ± 0.012; Brody 1945,

Stahl 1967, Lasiewski and Calder 1971, reviewed in Calder 1984 and Peters 1993). For

Table 4. Summary of allometric relationships between organ mass and total body mass. y = axb, where y = organ mass (kg) and x = total body mass (kg). (n = sample size, a = y- intercept, b = slope, C.I. = confidence interval, D-P = Delphinidae-Phocoenidae, and K- Z-P = Kogiidae-Ziphiidae-Physeteridae)

n a b 95% C.I. Lung Mass: Total Data Set T. truncatus 111 0.032 0.973 0.932 - 1.014 Kogia spp. 18 0.014

Non-emaciated T. truncatus 90 0.032 0.975 0.927 - 1.022 Kogia spp. 12 0.014

Sub-adults and Adults T. truncatus 84 0.028 1.004 0.921 - 1.087 Kogia spp. 17 0.012

Family Level Comparisons Delphinidae 256 0.034 0.944 0.905 - 0.984 Kogiidae 19 0.017

Delphinidae 256 0.033 0.949 0.425 - 1.474 Phocoenidae 15 0.016 1.177 0.916 - 1.438

Kogiidae 19 0.031 0.826 0.732 - 0.919 Ziphiidae 8 0.041 Physeteridae 10 0.047

D-P 271 0.033 0.951 0.852 - 1.049 K-Z-P 37 0.022 0.900 0.858 - 0.941

Heart Mass: T. truncatus 38 0.013 0.857 0.794 – 0.919 Kogia spp. 19 0.010

Liver Mass: T. truncatus 104 0.012 1.054 1.004 – 1.104 Kogia spp. 17 0.002 1.235 1.030 – 1.440

Log Lung Mass (kg)

Log Total Body Mass (kg)

Figure 8. Log lung mass (kg) vs. log total body mass (kg) for all age classes and body conditions of T. truncatus and Kogia spp. The slopes of these lines are similar, however, the y-intercepts differ significantly (see Table 4).

Lung Mass / Total Body Mass (kg/kg) Lung Mass / Total Body

Total Body Length (cm)

Figure 9. Ratio of lung mass (kg) vs. total body mass (kg) against total body length (cm) across all age classes and body conditions of T. truncatus and Kogia spp. The slopes of these lines are similar, however, the y-intercepts differ significantly.

this subset of individuals, the slopes of the lines for T. truncatus and Kogia spp. were

similar and neither was distinguishable from 1 (Table 4). As was observed for the total

data set, though, the y-intercepts did differ significantly (F = 213.59, P < 0.0001, d.f. =

100). The allometric equation for kogiids was similar to that of terrestrial mammals, and both predicted lung masses that were approximately half that of a similarly-sized T. truncatus (Figure 10).

To broaden the phylogenetic scope of these comparisons, allometric relationships

between lung mass and total body mass were generated for a variety of odontocete

families. Representatives of the Family Delphinidae (n = 256, Table 1, Appendix A,

Appendix B) were first compared to the Family Kogiidae, which included the sample

from this study and one more individual from a previously published report (n = 19). The

slopes of the lines for the Family Delphinidae and Kogiidae were similar and both were

slightly less than 1 (Table 4). The y-intercepts were significantly different between families (F = 115.56, P < 0.0001, d.f. = 274; Figure 11). Thus, members of the Family

Kogiidae possess lungs that are approximately half the mass of those of a similarly-sized delphinid.

To further broaden the comparative phylogenetic scope, representatives of the

Families Phocoenidae, Ziphiidae, and Physeteridae (Table 1, Appendix B) were also included. For the families Phocoenidae and Delphinidae, the slopes were not significantly different from each other (P = 0.0905) and neither was distinguishable from 1. Given the relatively low P-value, though, the ANCOVA analysis included the slope interaction effect and, thus, both slope values were reported in Table 4. The y-intercepts were also not significantly different (F = 2.32, P = 0.1291, d.f. = 270; Table 4). Because neither

T. truncatus 1.2 Kogia spp. 1.1 Terrestrial Mammals 1.0

0.9

0.8

0.7

0.6 0.5

0.4

0.3

0.2

0.1

0.0

-0.1 Log Lung Mass (kg) -0.2

-0.3

-0.4

1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Log Total Body Mass (kg)

Figure 10. Log lung mass (kg) vs. log total body mass (kg) for sub-adults and adults of T. truncatus and Kogia spp. compared to adult terrestrial mammals (y = 0.0113x0.986, b ± 0.012; Brody 1945). The slopes of the lines for T. truncatus and kogiids are not significantly different, however, the y-intercepts differed significantly.

Log Lung Mass (kg)

Log Total Body Mass (kg)

Figure 11. Log lung mass (kg) vs. log total body mass (kg) for Families Delphinidae and Kogiidae. The slopes of these lines were similar, however, the y-intercepts differed significantly.

slope nor y-intercept were significantly different between families, the data were pooled

together to form the group Delphinidae-Phocoenidae (D-P). A similar comparison was

under taken for the Families Kogiidae, Ziphiidae, and Physeteridae. The slopes of the

lines for these three families were similar, and just under 1. The y-intercepts were also

similar (F = 2.92, P = 0.0678, d.f. = 36; Table 4). Thus, data from the three families were

pooled together to form the group Kogiidae-Ziphiidae-Physeteridae (K-Z-P).

The slopes of the lines that describe the relationship between lung mass and total

body mass for the D-P and K-Z-P groups were not significantly different from each other

(P = 0.0817) (Table 4). Given the relatively low P-value, though, the ANCOVA analysis

included the slope interaction effect and, thus, both slope values were reported in Table 4.

The y-intercepts, though, did differ significantly between these two groups (F = 5.05, P =

0.0253, d.f. = 308; Figure 12). Thus, species within the K-P-Z group possess lungs that

are approximately half the mass of those of similarly-sized species within the D-P group.

Excised Lung Volume

The volume of excised lungs in both an un-inflated (n = 5 for T. truncatus and n =

4 for Kogia spp.) and inflated (n = 4 for T. truncatus and n = 3 for Kogia spp.) condition

were measured by the water displacement method. The total lung capacity (TLC) and

minimum air volume (MAV) were calculated using a method modified from Kooyman

and Sinnett (1979) (Table 5, Table 6).

Overall, lung volumes of kogiids were small relative to those of T. truncatus.

When lung volumes were standardized to total body mass (TBM), the un-inflated lung volume, MAV, and TLC of T. truncatus were approximately five times larger than those

Table 5. Lung volume measurements for kogiids and T. truncatus. The un-inflated, and inflated lung with 1.0L of air, lung volumes were measured during the inflation experiments. The minimum air volume (MAV) and total lung capacity (TLC) were calculated using methods of Kooyman and Sinnett (1979). MAV was conservatively assumed to be 17% of TLC* (which ranged from 0-17% in Phocoena phocoena; Kooyman and Sinnett 1979). MAV and TLC for VAQS 20071081 could not be calculated because MAV was less than zero. (TBM = total body mass)

Lung Vol. Lung Lung Volume Field ID Species TBM (kg) Inflated with MAV (L) TLC (L)* Mass (kg) Un-inflated (L) 1.0L Air (L)

WAM 637 K. sima 134.0 1.515 2.63 4.94 1.11 6.55 VAQS 20071081 K. breviceps 234.5 2.987 2.67 4.31 0.00 ---- MLC 003 K. breviceps 386.2 3.621 4.16 NE 0.54 3.17 WAM 644 K. breviceps 392.0 3.654 5.01 6.98 1.35 7.95

WAM 647 T. truncatus 153.0 5.064 7.61 9.34 2.54 14.95 PBN 003 T. truncatus 173.0 6.065 7.98 10.10 1.91 11.25 WAM 633 T. truncatus 180.0 6.970 11.20 13.01 4.23 24.88 BCB 004 T. truncatus NE 3.951 5.96 3.92 2.00 11.79 RJM 003 T. truncatus NE 1.904 3.87 NE 1.96 11.54

Table 6. Mean lung air volume (± S.E.) measures for T. truncatus and Kogia spp. compared to P. phocoena (n = 4) (*data from Kooyman and Sinnett 1979). (TBM = total body mass, MAV = minimum air volume, TLC = total lung capacity)

Lung Un- Volume inflated / MAV / TBM TLC / TBM Species TBM (kg) Un- MAV (L) TLC (L) TBM (L/kg) (L/kg) inflated (L/kg) (L)

Kogia spp. 286.7 ± 62.6 3.62 ± 0.58 0.01 ± 0.002 1.002 ± 0.241 0.003 ± 0.002 5.892 ± 1.420 0.021 ± 0.012

T. truncatus 168.7 ± 8.1 8.93 ± 1.14 0.05 ± 0.005 2.894 ± 0.692 0.017 ± 0.004 17.025 ± 4.069 0.101 ± 0.021

P. phocoena* 35.0 ± 3.4 ------0.383 ± 0.104 0.011 ± 0.003 3.325 ± 0.250 0.095 ± 0.012

Log Lung Mass (kg)

Log Total Body Mass (kg)

Figure 12. Log lung mass (kg) vs. log total body mass (kg) for species pooled in Families Delphinidae and Phocoenidae (D-P) and Kogiidae, Physeteridae, Ziphiidae (K-P-Z). The slopes of these lines were similar, however, the y-intercepts differed significantly. The line for terrestrial mammals was generated using the equation from Brody (1945) (y = 0.0113x0.986, b ± 0.012).

volume measures of kogiids (Table 6). The MAV and TLC of T. truncatus were more

similar to, though larger than, those of P. phocoena (reported in Kooyman and Sinnett

1979; Figure 13). The TLC volumes calculated for T. truncatus and Kogia spp. were

compared to those that would be predicted based upon existing allometric relationships for marine mammals (Kooyman 1973, Kooyman and Sinnett 1979) and terrestrial mammals (Tenney and Remmers 1963, Stahl 1967) (Table 7). Calculated TLC values for

T. truncatus were similar to those predicted by existing allometric relationships, while all calculated TLC values for the kogiids were smaller by a factor of at least 20-50%.

In situ Lung Volume Calculated from Whole Body Cross-Sections

The volume of the in situ lung was computed from whole body cross-sections of a single T. truncatus and K. sima (Table 8). This volume and those of other structures

within the entire musculoskeletal thorax are also reported as a percent of the entire

thoracic volume. The entire thorax comprised 14% of the total post-cranial body volume

in T. truncatus (7.9L absolute) and 15% of the total post-cranial body volume in K. sima

(9.6L absolute).

Both the lungs and pleural cavity of the K. sima were each half the volume of

those structures in the T. truncatus (Table 8). The cross-sections also revealed that the lungs of K. sima were positioned strictly dorsally throughout the entire thoracic cavity, while in T. truncatus the lungs extended ventro-laterally and encompassed the heart

(Figure 14, Figure 15). The dorsal position of the lungs had been previously noted in a fetal K. breviceps (Kernan and Schulte 1918). The tissue along the ventro-lateral margins

Table 7. A comparison of estimated total lung capacity (TLC), based upon measurements from this study, and those predicted from a variety of existing allometric relationships based upon total body mass (TBM). Marine mammal regression line: VL (L) = 0.135 X (kg) 0.92 1.02 (Kooyman 1973, Kooyman and Sinnett 1979), terrestrial mammal regression lines: VL (mL) = 56.7 X(kg) (Tenney and Remmers 1.06 1963) and VL (mL) = 53.5 X(kg) (Stahl 1967).

Tenney and Remmers Calculated Marine Mammal Stahl Terrestrial Field ID Species TBM (kg) Terrestrial TLC (L) TLC (L) TLC (L) TLC (L)

WAM 637 K. sima 134.0 6.55 12.2 8.4 9.6

MLC 003 K. breviceps 386.2 3.17 32.4 24.7 29.5

WAM 644 K. breviceps 392.0 7.95 32.8 25.0 30.0

WAM 647 T. truncatus 153.0 14.95 13.8 9.6 11.1

PBN 003 T. truncatus 173.0 11.25 15.5 10.9 12.6

WAM 633 T. truncatus 180.0 24.88 16.0 11.3 13.2

Table 8. Absolute (L) and relative (%) volumes of organs, vascular tissues and specific cavities within the entire musculoskeletal thorax calculated from whole body cross-sections of a single K. sima and T. truncatus.

% of Thoracic Structure Volume (L) Region K. sima T. truncatus K. sima T. truncatus Entire musculoskeletal thorax 9.58 7.94 100.0 100.0 pleural cavity 2.14 4.27 22.3 53.8 lungs 1.43 2.91 15.0 36.7 thoracic arterial rete 0.86 0.39 8.9 4.9 heart 0.85 0.81 8.9 10.2 intra-thoracic abdominal cavity 5.81 2.01 60.6 25.4 liver 2.38 0.69 24.9 8.7

Figure 13. Log of calculated total lung capacity (TLC, in liters) vs. log of total body mass (kg) for T. truncatus and Kogia spp. A terrestrial (Tenney and Remmers 1963, Stahl 1967) and marine mammal line (Kooyman 1973) has been added for references. Published values for Phocoena phocoena are from Kooyman and Sinnett (1979).

A B

R R

L L R R

L L

Figure 14. Whole body cross-sections at the level of the heart in a (A) T. truncatus and (B) K. sima. L = lung, H = heart, R = thoracic arterial rete. The lateral extensions of the thoracic arterial rete is K. sima is denoted by the white outline. (black scale bar = 5cm)

Figure 15. In (A) T. truncatus the lungs overlap the heart ventro-laterally. In (B) Kogia spp. the lungs are located strictly dorsal to the heart. The blue thick link traversing from the sternum to the caudal thoracic and lumbar vertebrae represents the dome of the diaphragm. (Image of T. truncatus from Rommel)

of the lungs in this young T. truncatus had not yet fully matured to its inflated condition,

which is a typical characteristic for a juvenile of this species (pers. obs.).

In K. sima, the thoracic arterial rete was also more extensively distributed

throughout the pleural cavity than it was in T. truncatus. In K. sima this rete extends

ventrally, draping over the thoracic inlet, and caudo-laterally along the thoracic wall to

the level of vertebral ribs 3-4. Cranially, this rete completely surrounds both lungs

(Figure 16). The thoracic arterial rete of K. sima was twice the volume of that of T.

truncatus.

Heart volumes were similar between species. The intra-thoracic abdominal cavity

and liver volumes were 2-3 times larger in K. sima than in T. truncatus.

Mobility of Isolated Thoraxes

Results from the manipulations of isolated thoraxes (depicted in lateral and frontal

perspectives in Figure 17) do not support the hypothesis that the thorax of a deeper diving

kogiid is more mobile than that of a shallow diving T. truncatus. There were no

significant differences between the kogiids and T. truncatus in the change in thoracic height, width, and circumference (measured between ribs 1-5), or inlet height measured between the maximally expanded and collapsed postures (Table 9, Figure 17, Figure 2).

Note, though, that there was large inter-individual variability in these measurements.

Only the change in inlet width measured between the two postures differed significantly

across species (t = 4.74, d.f. = 5, P = 0.005; Table 9); T. truncatus underwent a larger

change than Kogia spp.

Table 9. The mean (± S.D.) percent difference between the maximal cranially expanded to caudally collapsed postures (averaged across ribs 1-5) for thoracic dimensions measured for each species (* denotes significant differeces).

Measurement T. truncatus Kogia spp.

Avg. Height (%) 25.8 ± 2.9 25.9 ± 8.1

Inlet Height (%) 32.8 ± 4.1 37.2 ± 12.2

Avg. Width* (%) 9.5 ± 2.3 11.8 ± 6.3

Inlet Width (%) 17.8 ± 1.0 9.1 ± 3.0

Avg. Circum. (%) 21.3 ± 11.8 15.6 ± 6.6

Thoracic Vol. (%) 30.5 ± 25.9 20.6 ± 12.5

A B

L L

Figure 16. Whole body cross-sections at the level of the inlet to the thorax in a (A) T. truncatus and (B) K. sima. In K. sima the thoracic arterial rete completely encompasses both left and right lungs. No homologous vascular tissue is observed in T. truncatus. L = lung, white outline = lung, white arrows = thoracic arterial rete (black scale bar = 5cm)

A C

B D

Figure 17. The range of thoracic mobility in Kogia spp. The body outline in each of the lateral views (C-D) is fixed between positions. (A) and (C) depict the cranially most expanded thoracic posture, while (B) and (D) depict the caudally most collapsed thoracic posture. Note the lack of a first sternal rib (compare to T. truncatus in Figure 2).

The significant difference in the thoracic inlet width measurement is interpretable

given the morphological differences between T. truncatus and Kogia spp. at the first rib.

In T. truncatus, vertebral rib 1 articulates with a relatively long bony sternal rib (74% of

vertebral rib length). These ribs articulate at a highly mobile joint, which flares laterally as the thorax is manipulated from a caudally collapsed to a cranially expanded position

(Figure 2; Cotten et al. 2008). In contrast, in kogiids, vertebral rib 1 forms an immobile articulation with a short (11% of vertebral rib length) sternal cartilage. Thus, kogiids lack a mobile joint between the first vertebral and sternal ribs, which appears to limit lateral flaring at the inlet in the cranially expanded posture (Figure 17A). In kogiids, sternal ribs

2-5 are also cartilaginous, but unlike sternal rib 1, they form highly mobile joints with their respective vertebral ribs. Thus, except for sternal rib 1, the cartilaginous sternal ribs of kogiids form mobile joints with their respective vertebral ribs, in a manner similar to

that of T. truncatus, and unlike that of terrestrial mammals. Kogiids also lack a costal arch, which is typical in terrestrial mammals.

Thoracic Volume Models

Changes in the volume of the cranial thorax, between the cranially expanded and caudally collapsed postures, were estimated using geometric models. Each model was tested for its ability to estimate thoracic volume from known computed values derived from the whole body cross-sections. Of the four geometric models investigated, Model 1

(frustum of right circular cone in series with a slanted complete cone) appeared to best estimate the thoracic cavity volume of the cross-sectioned specimens (Table 10). This model, though, provides only gross estimates of thoracic cavity volume with errors up to

Table 10. Cranial thoracic cavity volume (L) calculated from whole body cross-sections and calculated via four geometric models.

"No ID" Tt VAQS 20081002Ks % % Volume (L) Volume (L) difference difference Measured 5.92 3.78

Models

1 6.87 14 4.26 11

2 7.29 19 4.60 18

3 13.19 55 4.54 17

4 13.89 57 4.65 19

13.8 and 11.3% for T. truncatus and K. sima, respectively (Table 10). Estimates of kogiid thoracic volume were relatively insensitive to the geometric shape chosen. In contrast, the cardioid models poorly predicted the volume of the T. truncatus thorax.

Model 1 was applied to each specimen to estimate changes in thoracic cavity volume that occurred during manipulation experiments (Table 11). On average, thoracic cavity volumes decreased, from the maximally expanded to the maximally collapsed posture, by 30.5% and 20.6% in T. truncatus and Kogia spp., respectively. These values were not significantly different from each other (t = 0.683, d.f. = 5, P = 0.525).

Gross observations, individual measurements and thoracic cavity volume estimates all demonstrate that there exists large intra-specific variability in thoracic mobility. These data also suggest that the thorax of a deep diving kogiid is not more mobile than that of a shallower diving T. truncatus. In kogiids, the permitted change in lateral width of the thoracic inlet appears to be constrained, relative to T. truncatus, by the morphology of the first vertebral-sternal rib pair.

DISCUSSION

This study tested the hypothesis that the deep diving kogiids would possess either smaller lungs and/or a more flexible thorax than the shallow diving T. truncatus. The kogiid lungs were one-half the mass, and between 20-50% of the volume of the lungs of a similarly-sized T. truncatus. There was only one difference in thoracic mobility between these two groups - the change in width of the kogiid thoracic inlet appears to be constrained relative to T. truncatus. The kogiid thorax also contained a larger volume of

Table 11. Cranial thoracic cavity volumes (L), calculated using geometric Model 1, for cranially most expanded and caudally most collapsed postures. The percent change between postures and the mean for each species is also reported.

Cranial Mean Field ID Species Caudal (L) %Δ (L) %Δ WAM 633 T. truncatus 27.08 23.33 13.8 30.5

PBN 003 T. truncatus 29.11 24.04 17.4

BRF 090 T. truncatus 13.94 5.53 60.4

WAM 637 K. breviceps 14.66 11.06 24.6 20.6

WAM 634 K. breviceps 28.22 26.61 5.7

VAQS 20071081 K. breviceps 40.48 26.20 35.3 VAQS 20081003 K. sima 25.09 20.88 16.8

arterial retial tissue than the dolphin thorax. These observations are discussed within both a phylogenetic and functional context below.

Interestingly, kogiid lung mass, relative to total body mass, is similar to that predicted for terrestrial mammals. In a broader phylogenetic comparison, kogiids, physeterids, and ziphiids all possess relative lung masses similar to those of terrestrial mammals. When relative lung mass is mapped onto an existing phylogeny for cetaceans

(Messenger and McGuire 1998, Price et al. 2005), and values for mysticetes and

monodonts are included, a phylogenetic pattern in relative lung mass is observed (Figure

18). Mysticetes, physeterids, ziphiids, and kogiids all possess relative lung masses that

are similar to those predicted for terrestrial mammals. That is, lung size in these cetaceans

is a plesiomorphic character. And across these groups relative lung size is not uniformly

associated with deep or prolonged diving – kogiids, physeterids and ziphiids are deep

divers, but mysticetes are not.

To investigate the relationship between lung mass and diving without regard to

phylogenetic relationships, indices of diving ability (e.g. Williams et al. 2008) were

developed based upon information available in the literature (Table 12). Those species

with dive depth and duration data were coded as either a (1) short duration shallow diver,

(2) short duration deep diver (i.e. “deep sprinters” sensu Soto et al. 2008), or (3) long

duration deep diver. A species was defined as a shallow diver if its commonly reported

dive depth was under 100m (see Figure 1, inflection point on the depth-volume curve).

Common, rather than maximum, dive depths were used because all species, even

typically shallow divers, have been recorded or trained to dive to depths greater than

100m. Short dive duration was defined as a common dive duration under 25min. While,

Table 12. Common diving capacity measures for a variety of odontocetes for which a corresponding lung mass and total body mass ratio was available. (* = common depth or duration did not exist for that species and instead maximum record was used)

Dive Species Family Duration Dive Depth (m) Method Citation (min.) Evans 1971, Ridgway and Harrison D. delphis Delphinidae 5* 260* time depth recorder 1986 1.26 - S. attenuata Delphinidae 22.1 - 24.0 time depth recorder Scott and Chivers 2009 1.68 satellite-linked T. truncatus Delphinidae 1 < 20 Mate et al. 1995, Ridgway et al. 1986 recorder P. phocoena Phocoenidae 1 14-40 recorder Westgate et al. 1995 satellite-linked Heide-Jorgensen et al. 1998, Martin D. leucas Monodontidae 12 to 15 400-600 recorder and Smith 1999 Kogia spp. Kogiidae 12* > 400 recorder transponder Scott et al. 2001

Jaquet et al. 2000, Norris and Harvey ST=sonar 1972, Papastavrou et al. 1989, Watkins P.macrocephalus Physeteridae 40-50O 400-600ST transponder, et al. 1985, Panigada et al. 1999, O=observation Scholander 1940

H. ampullatus Ziphiidae 40 800 recorder Hooker and Baird 1999

D=Digital TAG, T= M.densitrostris Ziphiidae 48-68* 1251*D - 1408*T Baird et al 2004, Tyack et al. 2006 time depth recorder

Family Lung Mass:TBM (%)Diving Capacity

Delphinidae 2.7% Phocoenidae Shallow, Short & Deep, Short 3.0% Shallow, Short Monodontidae 2.6% Deep, Short 1.4% Ziphiidae Deep, Long Terrestrial 1.3% 1.0% Kogiidae Deep, ? Mammals 0.9% Physeteridae Deep, Long

0.9% Mysticetes Shallow, Short

Figure 18. The ratio of lung mass vs. total body mass is mapped upon a phylogeny for cetaceans (Messenger and McGuire 1998, Price et al. 2005). The ratio for terrestrial mammals is derived from the allometric equation of Brody (1945). A shallow dive was defined as a common dive depth < 100m, while a short dive duration was defined as a common dive duration that was < 25min.

these definitions are somewhat arbitrary, they do offer broadly comparative bins into which cetacean species could be placed. The slopes of the lines that describe the allometric relationship between lung mass and total body mass for these three defined groups of divers were not significantly different from each other (Table 13). However, their y-intercepts did differ significantly (F = 62.16, P < 0.0001, d.f. = 176; Table 13).

Deep, prolonged divers possessed significantly smaller lungs than did deep, short divers,

which in turn possessed significantly smaller lungs than did shallow, short divers (Figure

19).

Species were also coded based upon a physiological metric associated with diving

– the myoglobin concentration of locomotor muscle (Table 14). Species were divided

into two bins, dependent upon whether their myoglobin concentrations were below or

above 4g/100g wet weight muscle. In this case, the slopes of the lines that describe the

allometric relationship between lung mass and total body mass for these two defined

groups were significantly different from each other (F = 18.19, P < 0.0001, d.f. = 190;

Table 13). Species with high myoglobin concentrations [Feresa attenuata (a delphinid),

Hyperoodon ampullatus, Kogia spp., and Physeter macrocephalus] possessed smaller lungs. Most species with relatively large lungs possessed a low myoglobin concentration

(Delphinus delphis, T. truncatus, Stenella attenuata, Delphinapterus leucas). The exceptions were Stenella coeruleoalba and Phocoena phocoena, which possessed both high myoglobin concentrations and relatively large lungs (Figure 20). Thus, within the odontocetes, species with relatively small lungs tend to be long-duration, deep divers with relatively high myoglobin concentrations. High myoglobin concentrations can, though, also be found in some large-lunged delphinids and phocoenids.

Table 13. Summary of allometric relationships between lung mass and total body mass for diving behavior and myoglobin content. Shallow dive depth was defined as a common dive depth under 100m. Short dive duration was defined as a common dive duration under 25min. Myoglobin content was defined as below or above 4g/100g wet weight muscle. y = axb, where y = lung mass (kg) and x = total body mass (kg). (n = sample size, a = y-intercept, b = slope, S.E. = standard error)

n a b S.E. Diving Behavior Shallow, Short Duration 126 0.220 0.986 0.023 Deep, Short Duration 37 0.175 Deep, Long Duration 14 0.136

Myoglobin Low Myoglobin 126 0.221 0.984 0.035 High Myoglobin 65 0.253 0.833 0.018

Table 14. Myoglobin values for a variety of odontocetes for which a corresponding lung mass and total body mass were available.

Myoglobin (g /100 g Species Family Citation wet muscle) Dolar et al. 1999, D. delphis Delphinidae 3.4 - 3.55 Noren et al. 2000 F. attenuata Delphinidae 5.7 Dolar et al. 1999

S. attenuata Delphinidae 2.5 Dolar et al. 1999

S. coeruleoalba Delphinidae 5.78 Noren et al. 2000 Dolar et al. 1999, T. truncatus Delphinidae 2.5 to 3.5 Noren et al. 2000 Dolar et al. 1999, P. phocoena Phocoenidae 4.03 - 4.1 Noren et al. 2000 D. leucas Monodontidae 3.44 Noren et al. 2000 Kogia spp. Kogiidae 4.33 Noren et al. 2000 P.macrocephalus Physeteridae 5.7 Dolar et al. 1999

H. ampullatus Ziphiidae 6.3 Dolar et al. 1999

0 Log Lung Mass (kg)

-1 Short, Shallow Divers Short, Deep Divers Long, Deep Divers -2 012345 Log Total Body Mass (kg)

Figure 19. Log lung mass (kg) vs. log total body mass (kg) of species within five families (Kogiidae, Physeteridae, Ziphiidae, Delphinidae and Phocoenidae). Each species was coded as either a short duration shallow diver, short duration deep diver, or long duration deep diver (Table 12). The slopes of all three of these lines are similar, however, the y-intercepts differ significantly (see Table 13).

0 Log Lung Mass (kg) Mass Lung Log

-1 Low Myoglobin (< 4g/100g wet muscle) High Myoglobin (> 4g/100g wet muscle)

-2 012345 Log Total Body Mass (kg)

Figure 20. Log lung mass (kg) vs. log total body mass (kg) of species within five families (Kogiidae, Physeteridae, Ziphiidae, Delphinidae and Phocoenidae). Each species was coded according to its species-specific myoglobin level (Table 14). The slopes of these lines are significantly different (see Table 13).

Although these analyses were undertaken without the explicit use of phylogenetic information, it is clear that these characters co-occur within the closely related families of

Physeteridae, Ziphiidae, and Kogiidae. Species within these families also achieve large body sizes, which has been positively correlated to dive depth and duration within the odontocetes (Schreer and Kovacs 1997).

The mapping of relative lung size onto cetacean phylogeny presented in Figure 18 suggests that the lungs of the deep diving kogiids, physeterids, and ziphiids are not specialized, relative to the putative ancestral condition. In contrast, it does appear that the relatively large lung mass observed in delphinids and phocoenids is a shared, derived

character in these families. Most of these species tend to be short-duration shallow divers,

although there are some short-duration deep divers within Delphinidae. In diving

odontocetes, the lungs function as an oxygen store for respiratory gas exchange, a

dynamic buoyancy regulation organ, and as a reservoir for air required for echolocation

(i.e. pneumatic excitation of the phonic lips; Cranford et al. 1996). What are the

functional consequences for a shallow diving species of possessing relatively large lungs?

It is unlikely that a large lung is required to provide air sufficient to support

echolocation at shallow depths. Deep diving ziphiids and physeterids, with their

relatively small lungs, can echolocate at great depths (800-1,000m) (Baird et al. 2004,

Miller et al. 2004, Tyack et al. 2006, Watwood et al. 2006) when the air within the

respiratory system is compressed to only 1-2% of its volume at the surface.

The buoyant function of a large lung in shallow diving marine mammals has been

considered by Taylor (1994). He hypothesized that lung size would vary between species

that control their position within the water column hydrostatically versus

70 hydrodynamically. The shallow diving manatee, for example, has a large lung within a very dense body (Domning and deBuffrenil 1991). This body composition permits the manatee to hydrostatically control its position within the water column. Its lungs undergo large absolute changes in volume (and therefore density) at shallow depths, and coupled with its dense body, permit the manatee to achieve negative buoyancy at depths of less than 10m.

Taylor (1994) hypothesized that marine mammals, such as odontocete cetaceans, which hydrodynamically control their position within the water column, should possess small lungs and low density bodies. A small lung (and therefore air volume) would reduce the work required to overcome buoyancy during a dive, and decrease the depth at which the swimming animal would become neutrally buoyant. This pattern is observed in the deep diving kogiid, but not in the shallow diving T. truncatus. The dolphin’s relatively large lung likely increases the mechanical work required to dive (see

Stephenson et al. 1989). It also likely causes the dolphin to experience large changes in buoyancy at shallow depths, although the functional consequences of this effect in an animal that controls its position within the water column hydrodynamically are not known.

It seems likely that the large lung size of the shallow diving T. truncatus permits it to function as an important oxygen store for respiratory gas exchange during a shallow dive. Using existing data on mass specific metabolic rates and total lung capacity (TLC)

(Williams et al. 1992, Irving et al. 1941, Ridgway et al. 1969, Kooyman 1973, Kooyman and Sinnett 1979), one can calculate the relative contributions of lung oxygen stores to maintaining metabolic function during different activities for an adult bottlenose dolphin

(200kg) (Table 15). For a resting bottlenose dolphin, experiencing a prolonged (1min)

apnea, the lungs can provide 169-285% of the oxygen required to maintain its metabolic

rate. For a similarly-sized kogiid, assuming it has a metabolic rate similar to that of a

bottlenose dolphin, the lung can provide only 35-60% of the oxygen required. If a

bottlenose dolphin swam on a 1min breath-hold at a typical swimming speed (2.1m/s,

Williams et al. 1992), the lungs could provide 138-231% of the oxygen required.

Assuming metabolic similarity, the kogiid lung could provide only 29-49% of the oxygen

required during a 1min apneic swim. Thus, the lungs of T. truncatus can provide much of

the oxygen required during rest and routine swimming (even on a prolonged 1min breath- hold), whereas kogiid lungs cannot. Bottlenose dolphins do not routinely swim on an extended breath-hold; their respiratory rate is 2-3breaths/min. Bottlenose dolphins are therefore replenishing their lung oxygen stores at a much higher rate than the one used in these “back of the envelope” calculations.

Kooyman (1973) and Scholander (1940) have categorized cetaceans as either

“fast” or “slow” breathers. A “fast breather” usually surfaces on the move and takes a single breath each time that they pass rapidly through the air-water interface. A “slow breather” must stay at the surface after a dive and ventilate a number of times to replenish oxygen stores. By this definition, T. truncatus is a “fast breather” and kogiids are “slow breathers”. During a ventilation cycle, T. truncatus can routinely exchange 75%, and maximally up to 90%, of its total lung volume in a third of a second at the surface (Irving et al. 1941, Ridgway et al. 1969). In contrast, kogiids have been observed to come to the surface after a dive and log for multiple ventilations (recorded in one individual for up to

11min)(Allen 1941, Yamada 1953, Nagorsen 1985, Caldwell and Caldwell 1989, Scott et

Table 15. The estimated contribution of lung oxygen stores to meeting metabolic costs of resting and swimming in T. truncatus and kogiids. Calculations were based on a 200kg* adult animal with a resting metabolic rate of 6.53mL O2 / kg•min (Williams et al. 2001) and a swimming (at 2m/s) metabolic rate of 8.07mL O2 / kg•min (Williams et al. 1992). Under both conditions, the animal’s metabolic costs were calculated for a 1min apnea. The kogiid lung volumes were estimated at 2.1% of the values for T. truncatus (see Table 6).

O2 Amount of O2 Available Air Required Provided TLC (L)* in Lungs for Condition by Lungs (L)** (L) (%) Rest 1min apnea T. truncatus 10-17.7 2.2-3.7 1.3 169-285% Kogia spp. 2.2-3.7 0.46-0.78 1.3 35-60%

2.1m/s swim for 1min apnea

T. truncatus 10-17.7 2.2-3.7 1.6 138-231%

Kogia spp. 2.2-3.7 0.46-0.78 1.6 29-49%

* Ridgway et al. (1969) states that a 200kg carcass possessed a 10-11L lung volume. In contrast, from an allometric equation of lung volume to total body mass in Kooyman (1973) and Kooyman and Sinnett (1979), a 200kg animal should have a lung volume of 17.7L. Both these values were used to bracket the calculations. ** accounts for 21% oxygen in air

al. 2001, Baird 2004). Their high ventilation rate, short ventilation times and

extraordinarily large tidal volumes suggest that bottlenose dolphins require frequent

replenishment of their lung oxygen stores. Thus, it seems likely that the large lung of the

shallow diving bottlenose dolphin functions primarily to support metabolic demands of a

relatively active lifestyle.

Large heart size has also been observed in animals that are capable of high

endurance activity (Crile and Quiring 1940a, Brody 1945, Tenney and Remmers 1963,

Ridgway and Johnston 1966, Bryden 1972). In the young cross-sectioned specimens, the

ratios of heart volume to total post-cranial body volume were very similar between T.

truncatus and K. sima. However, when heart mass to total body mass was compared

across these two species (n = 38 for T. truncatus and n = 18 for Kogia spp.), T. truncatus

possessed a heart mass significantly larger than that of similarly-sized Kogia spp. (Figure

21). Although the slopes of the lines that describe these allometric relationships were

similar, the y-intercepts did differ significantly (F = 28.51, P < 0.0001, d.f. = 57). When

compared to adult terrestrial mammals T. truncatus possessed a relative heart mass similar to that of the thoroughbred race horse, Equipoise, while kogiids possessed a relative heart mass more similar to that of an ox (Table 16; Howell 1930, Crile and

Quiring 1940a, Ridgway and Johnston 1966). Thus, T. truncatus, a shallow diving, “fast”

breathing cetacean possesses significantly larger lungs and heart than a deep diving,

“slow” breathing kogiid.

This study also investigated whether the musculoskeletal thorax of the deep

diving kogiid was more mobile than that of the shallow diving bottlenose dolphin.

Overall, there were no differences in thoracic mobility across species and both species

Table 16. Heart mass (kg) vs. total body mass (kg) ratios for a variety of cetaceans. An allometric equation of terrestrial mammals was used to predict heart mass for each cetacean when total body mass was known (y = 0.006 x0.98, n = 568, r2 = 0.98; Stahl 1967). (* = data represents the mean value for the sample of that species; NR = not reported)

TBM HM / Stahl Species n Citation (kg) TBM prediction Megaptera novaeangliae 3 39311* 1.50%* 0.49% Bryden 1972, Quiring 1943, Kenyon 1961 Balaenoptera borealis NR NR 0.40% --- Omura 1950 Balaenoptera musculus NR NR 0.50% --- Crile and Quiring 1940a, Nishiwaki 1950 Balaenoptera physalus NR NR 0.70% --- Nishiwaki 1950 Eschrichtiidae robustus NR NR 0.50% --- Omura et al. 1969 Physeter macrocephalus NR NR 0.30% --- Crile and Quiring 1940a, Omura 1950 Physeter macrocephalus 1 39,000 0.32% 0.49% Bryden 1972, Quiring 1943, Kenyon 1961 Ziphius cavirostris 1 2952 0.52% 0.51% Bryden 1972, Quiring 1943, Kenyon 1961 Kogia spp. 19 195* 0.48%* 0.54% UNCW database Phocoena phocoena NR NR 0.80% --- Slijper 1958 Phocoenoides dalli 4 NR 1.31%* --- Ridgway and Johnston 1966 Lagenorhynchus obliquidens 5 NR 0.85%* --- Ridgway and Johnston 1966 Delphinapterus leucas NR NR 0.60% --- Omura 1950 Delphinapterus leucas NR 521.18 0.61% 0.53% Crile and Quiring 1940a Tursiops truncatus 38 202.3* 0.61%* 0.54% UNCW database Tursiops truncatus 4 NR 0.54%* --- Ridgway and Johnston 1966 Tursiops truncatus NR NR 1.00% --- Slijper 1958 Thoroughbred race horse, "Equipoise" 1 521.5 0.85% 0.53% Crile and Quiring 1940a Ox 71 536* 0.35%* 0.53% Howell 1930, Crile and Quiring 1940a

0.6 Kogia spp. 0.4 Terrestrial Mammals Tursiops truncatus

0.2

0.0

-0.2

Log Heart Mass (kg) -0.4

-0.6

-0.8 1.41.61.82.02.22.42.62.8 Log Total Body Mass (kg)

Figure 21. Log heart mass (kg) vs. log total body mass (kg) for T. truncatus and Kogia spp. compared to adult terrestrial mammals (y = 0.006 x0.98, n = 568, r2 = 0.98; Stahl 1967). The slopes of these lines are similar; however, the y-intercepts differ significantly (see Table 4). Data obtained from UNCW database.

could achieve a 20-30% change in thoracic cavity volume. However, due to the

differences in the ecology of these cetaceans, this change in thoracic cavity volume may

be accommodating different features of their lifestyle. T. truncatus is a shallow diver and,

thus, routinely experiences rapid and large changes in thoracic air volume. The specialized morphological features of the T. truncatus thorax may also function to permit

rapid changes in thoracic volume that may be required during their explosive ventilatory

event (Cotten et al. 2008).

Although the excised kogiid thorax could change shape similar to that of T.

truncatus, on a dive thoracic collapse may be limited. Kogiids possess small lungs that

will undergo absolutely smaller changes in volume during a dive. Within the thorax,

kogiids also possess more extensive vascular structures that may dynamically increase their volume during a dive. The dorsal arterial rete in K. sima was twice the volume of that in T. truncatus. Interestingly, there may be another structure, bounded by the musculoskeletal thorax, which could function as a blood reservoir during a dive. For

example, in adult harbor seals, the hepatic sinus is approximately the size of the liver and

can store at least 1L of blood when completely filled (Harrison and Tomlinson 1956).

The pericardial venous plexus is fed in part by the hepatic sinus of the liver. The liver of

K. sima was 1.5 times the volume of that of T. truncatus (Table 4, Figure 22). When the relationship between liver mass and total body mass was investigated between these groups (n = 17 for T. truncatus and n = 104 Kogia spp.) the slope of the line for kogiids was significantly higher than for T. truncatus (F = 5.35, P = 0.0225, d.f. = 120). When compared to a variety of other cetaceans, kogiids possessed the largest relative liver mass

(Table 17). The kogiid liver was twice the mass of that predicted for a similarly sized

Table 17. Liver mass (kg) vs. total body mass (kg) ratios for a variety of cetaceans. An allometric equation of terrestrial mammals was used to predict liver mass for each cetacean when total body mass was known (y = 0.033 x0.87, n = 175, r2 = 0.984; Brody 1945). (* = data represents the mean value for the sample of that species; NR = not reported)

TBM LM / Brody Species n Citation (kg) TBM prediction Balaenoptera borealis NR NR 1.30% --- Bryden 1972 Balenoptera musculus NR NR 1.20% --- Bryden 1972 Balenoptera physalus NR NR 1.00% --- Bryden 1972 Eschrichtius robustus NR NR 0.90% --- Bryden 1972 Megaptera novaeangliae 3 39311* 1.20%* 0.83% Bryden 1972 Physeter macrocephalus 1 39000 1.07% 0.83% Bryden 1972 Physeter macrocephalus NR NR 1.60% --- Bryden 1972 Ziphius cavirostris 1 2952 0.88% 1.17% Bryden 1972 Kogia spp. 17 180* 3.60%* 1.68% UNCW database Phocoena phocoena 28 37* 3.19%* --- Slijper 1958 Phocoena phocoena NR NR 3.20% --- Bryden 1972 Tursiops truncatus NR NR 2.20% --- Bryden 1972 Tursiops truncatus 104 117.7* 2.40%* 1.78% UNCW database Delphinapterus leucas NR NR 1.50% --- Bryden 1972

4.5 Kogia spp. Terrestrial Mammals 4.0 T. truncatus

3.5

3.0

2.5 Log LiverLog Mass (g)

2.0

1.5 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 Log Total Body Mass (g)

Figure 22. Log liver mass (kg) vs. log total body mass (kg) for T. truncatus and Kogia spp. compared to adult terrestrial mammals (y = 0.033 x0.87, n = 175, r2 = 0.984; Brody 1945). The slopes of these lines were significantly different (see Table 4). Data obtained from UNCW database.

79 terrestrial mammal. A larger liver may be able to supply more blood to the pericardial venous plexus within the thorax at depth.

A future study is needed to examine the thorax and lungs of a shallow and deep diving cetacean under controlled compression. The morphology and the connections of multiple vascular structures to other structures has yet to be well-described in a deep diving cetacean, and how blood is circulated through these structures at depth is unknown.

CONCLUSION

This study provides evidence to support the hypothesis that the lungs of deep diving kogiids are significantly smaller, by both mass and volume, than those of the shallow diving T. truncatus. Calculations based upon mass specific metabolic rates and total lung capacities suggest that the shallow diving, “fast” breathing T. truncatus may be using its large lung as an oxygen store to meet its metabolic demands. Similar calculations for kogiids, assuming metabolic similarity, suggest that their lungs are too small to provide the oxygen required for even a one minute resting apnea. Overall, the mobility of the isolated thorax of T. truncatus and kogiids was similar. Thoracic mobility in T. truncatus may accommodate the routine large changes in thoracic air volume this species experiences during shallow diving, and may function to permit rapid changes in thoracic volume required during explosive ventilation. In contrast, the small lung of the deep diving, “slow” breathing kogiids likely reduces the absolute change in thoracic volume that occurs during a dive and may contribute to negative buoyancy at shallow depths. Kogiids also possess extensive thoracic vasculature and a large liver, which may

80 also function to limit thoracic collapse. A phylogenetic analysis suggests that the small lung size in deep diving odontocetes is a plesiomorphic character rather than a specialization for diving. The large lung size of delphinids and phocoenids appears to be a derived condition that likely permits it to function as an oxygen store for respiratory gas exchange in these active, relatively shallow divers.

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APPENDIX

Appendix A. Specimens used in this study either stranded or were incidentally killed in fishing operations. All specimens used in this study were in fresh to moderate condition (Smithsonian Institute Code 1 through 3; Geraci and Lounsbury 2005). Each specimen was placed into a life history category as defined by Caldwell and Caldwell (1989), Mead and Potter (1990), Struntz et al. (2004), and Dunkin et al. (2005). (N/E = not examined, CBD = could not be determined, NR = not recorded, BC = body condition, NE = not emaciated, E = emaciated)

Total Total Life Field Identification Body Species Length History Code BC Number Mass (cm) Category (kg) WAM 635 f,h,i K. breviceps 116.5 34.6 Neonate 2 NE ASF 029 f,h,i K. breviceps 124.0 N/E Sub-adult 1 NE PEM 1516/99 f,h,i K. sima 152.5 61.8 Sub-adult NR E VAQS 20081002 d K. sima 160.0 77.0 Sub-adult 2 NE SAM 35799 f,h,i K. sima 178.0 104.5 Sub-adult NR E WAM 637 a,c,e,f,g,h,i K. breviceps 198.0 134.0 Sub-adult 1 E DAP 032 f,h,i K. sima 207.0 137.0 Sub-adult 1 E KLC 025 g K. sima 213.0 N/E Sub-adult 2 NE VAQS 20081003 a,c,f K. sima 218.4 161.5 Sub-adult 1 NE CLP 001 a,b,c,f,h,i K. breviceps 225.0 158.0 Adult 1 E PEM 1516/97 f,h,i K. sima 235.0 209.1 Adult NR NE WAM 634 a,c,f,h,i K. breviceps 237.0 219.0 Adult 1 E NMNH 504221 e K. sima 237.0 NR Adult NR NR VAQS 20071081 K. breviceps 258.0 234.5 Adult 3 E a,c,f,g,h,i MLC 003 b,f,g,h K. breviceps 262.0 386.2 Adult 1 NE BRF 092 f,h,i K. breviceps 267.0 316.6 Adult 1 NE KMS 427 f,h,i K. breviceps 267.0 363.6 Adult 1 NE KMS 429 f,h,i K. breviceps 283.0 371.8 Adult 2 NE KMS 373 f,h K. breviceps 292.0 400.0 Adult 1 NE DAP 033 h K. breviceps 302.0 N/E Adult 1 E WAM 644 b,f,h,i K. breviceps 307.0 392.0 Adult 1 NE NMNH 504737 e,f K. breviceps 328.0 465.0 Adult 1 NE VMSM 971062 h K. breviceps 133.0 40.0 Neonate 1 NE

95 Appendix A. (cont'd)... Total Total Life Field Identification Body Species Length History Code BC Number Mass (cm) Category (kg) PEM 1518/85 i K. sima 189.0 129.5 Sub-adult NR NR PEM 1519/59 i K. sima 189.0 113.2 Sub-adult NR NR PEM 1516/51 h,i K. sima 136.0 47.3 Sub-adult NR NR No Tag f T. truncatus 101.5 13.9 Neonate 2 NE VMSM 951022 f T. truncatus 104.0 13.8 Neonate 2 E VMSM 961035 f,i T. truncatus 105.0 14.4 Neonate 2 NE EMM 010 f,i T. truncatus 106.0 14.4 Neonate 2 NE VMSM 20001020 f,i T. truncatus 106.5 14.4 Neonate 2 NE CALO 99-19 f,i T. truncatus 108.0 16.5 Neonate 2 NE NC 98-079 f,i T. truncatus 108.0 14.7 Neonate 2 NE CALO 99-13 f,i T. truncatus 109.5 14.7 Neonate 2 NE VMSM 20011080 f,i T. truncatus 110.0 15.5 Neonate 2 E VMSM 20021042 f,i T. truncatus 111.0 19.8 Neonate 2 NE MMB 003 f,i T. truncatus 113.0 15.6 Neonate 2 E VGT 073 f,i T. truncatus 113.6 16.5 Neonate 2 NE WAM 550 f,i T. truncatus 114.5 19.8 Neonate 1 NE VMSM 19991086 f,i T. truncatus 115.0 22.7 Neonate 2 NE VMSM 931046 f,i T. truncatus 115.0 20.4 Neonate 2 NE WAM 584 f T. truncatus 119.0 N/E Neonate 2 NE CALO 96-23 f,i T. truncatus 119.2 17.6 Neonate 2 E VMSM 20001031 f,i T. truncatus 127.0 29.2 Neonate 2 NE VMSM 20011087 f,i T. truncatus 129.7 30.7 Neonate 2 NE NC 98-097 f,i T. truncatus 131.5 23.8 Neonate 2 NE VMSM 971049 f,i T. truncatus 138.0 39.0 Neonate 2 NE VMSM 20031082 f,i T. truncatus 142.0 41.8 Neonate 2 NE VMSM 971045 f,i T. truncatus 144.0 43.3 Neonate 2 NE ASF 042 f,i T. truncatus 146.0 46.4 Neonate 2 NE VMSM 20021089 f,i T. truncatus 148.0 49.4 Neonate 2 NE VMSM 971053 i T. truncatus 108.0 18.6 Neonate 2 NE VMSM 961028 i T. truncatus 110.5 20.5 Sub-adult 2 NE

96 Appendix A. (cont'd)... Total Total Life Field Identification Body Species Length History Code BC Number Mass (cm) Category (kg) WAM 569 f,i T. truncatus 150.0 56.0 Neonate 2 NE VMSM 971060 f,i T. truncatus 153.3 48.1 Neonate 2 NE NEFC 00101 571382 f,i T. truncatus 157.6 53.8 Sub-adult 2 NE VMSM 921021 f,i T. truncatus 158.0 61.8 Sub-adult 2 NE NEFC 00102 571383 f,i T. truncatus 158.3 58.7 Sub-adult 2 NE NEFC 00103 571385 f,i T. truncatus 159.1 55.8 Sub-adult 2 NE ASF 001 f,i T. truncatus 160.5 47.4 Sub-adult 2 NE VMSM 931028 f,i T. truncatus 163.0 54.4 Sub-adult 2 NE 93-MM-AO-TT-07 f,i T. truncatus 164.5 63.3 Sub-adult 3 NE DAP 034 f,i T. truncatus 169.0 61.3 Sub-adult 2 NE "No ID" d T. truncatus 170.9 62.5 Sub-adult 1 NE KMT 091 f,i T. truncatus 172.0 70.6 Sub-adult 3 NE WAM 607 f,i T. truncatus 173.0 74.5 Sub-adult 2 NE VAQS 20061008 f,i T. truncatus 175.1 90.0 Sub-adult 3 NE VMSM 951037 f,i T. truncatus 177.0 64.8 Sub-adult 2 NE BRF 090 a,c T. truncatus 182.0 79.0 Sub-adult 3 NE NCAFFTT 053104 f,i T. truncatus 187.0 93.0 Sub-adult 3 NE RJM 003 g T. truncatus 188.0 N/E Sub-adult 2 CBD PTM 117 f,i T. truncatus 189.0 73.6 Sub-adult 2 E VAQS 20051118 f T. truncatus 189.0 90.2 Sub-adult 1 NE VMSM 20031043 f,i T. truncatus 190.5 94.8 Sub-adult 2 NE VMSM 19961061 f,i T. truncatus 191.0 107.2 Sub-adult 2 NE WAM 627 f,h,i T. truncatus 194.0 93.0 Sub-adult 2 NE SDZ 001 f,i T. truncatus 195.0 84.5 Sub-adult 2 NE VMSM 2004 1079 f,i T. truncatus 195.0 98.0 Sub-adult 2 NE VAQS 20051086 f,h,i T. truncatus 195.4 93.0 Sub-adult 2 NE VGT 176 f,i T. truncatus 196.0 84.0 Sub-adult 2 NE WAM 609 f,h,i T. truncatus 197.5 110.0 Adult 2 NE VMSM 20041024 i T. truncatus 190.0 51.2 Sub-adult 2 E WJW 007 i T. truncatus 273.0 294.8 Adult 1 NE

97 Appendix A. (cont'd)... Total Total Life Field Identification Body Species Length History Code BC Number Mass (cm) Category (kg) DAP 031 f,i T. truncatus 201.0 92.0 Sub-adult 2 NE MMSC 92-11571496f,o,i T. truncatus 202.2 75.2 Sub-adult 3 E NEFSC 03976 f T. truncatus 203.0 128.6 Sub-adult 2 NE VMSM 2004 1042 f,i T. truncatus 203.6 95.7 Sub-adult 3 NE KMT 013 f,i T. truncatus 204.0 98.2 Sub-adult 2 NE VMSM 20031104 f,i T. truncatus 204.0 114.0 Sub-adult 2 NE VMSM 20001049 f,i T. truncatus 207.0 114.0 Sub-adult 1 NE NEFC 016 571381 f,i T. truncatus 208.5 114.3 Sub-adult 2 NE VGT 087 f,i T. truncatus 209.8 122.7 Sub-adult 2 NE KMT 099 f,i T. truncatus 212.0 114.0 Sub-adult 2 NE PTM 047 f,i T. truncatus 212.0 124.6 Sub-adult 2 NE VMSM 941001 f,i T. truncatus 212.0 83.0 Sub-adult 2 E VGT 155 f,i T. truncatus 216.0 181.8 Sub-adult 2 NE NEFSC 5451 f,i T. truncatus 218.0 158.0 Sub-adult 2 NE KMT 100 f T. truncatus 219.0 153.6 Sub-adult 2 E RJM 004 g T. truncatus 221.0 N/E Sub-adult 3 NE WAM 591 f,i T. truncatus 222.8 114.0 Sub-adult 2 E KR 001 (571521) f,i T. truncatus 223.0 149.0 Sub-adult 2 NE KMT 023 f,i T. truncatus 223.5 150.4 Sub-adult 2 NE PTM 007 f,i T. truncatus 225.0 162.6 Adult 2 E VMSM 2004 1027 f,i T. truncatus 225.0 141.4 Sub-adult 2 NE WAM 647 f,g T. truncatus 225.0 153 Sub-adult 2 NE BCB 004 g T. truncatus 228.0 N/E Sub-adult 2 NE NEFSC 5332 f,i T. truncatus 228.0 163.0 Sub-adult 1 NE f,o,i KMT 051 T. truncatus 229.0 96.0 Sub-adult 2 E VAQS 20071081 h T. truncatus 275.0 234.5 Adult 3 E CJH 003 f,i T. truncatus 229.5 138.0 Adult 2 NE WAM 553 f,i T. truncatus 232.0 160.0 Sub-adult 2 NE

98 Appendix A. (cont'd)... Total Total Life Field Identification Body Species Length History Code BC Number Mass (cm) Category (kg) VMSM 2004 1040 f,i T. truncatus 234.5 139.0 Sub-adult 2 NE MMB 002 f T. truncatus 235.0 167.0 Sub-adult 2 NE PTM 074 f,h,i T. truncatus 237.0 197.2 Adult 2 NE WAM 579 f,h,i T. truncatus 237.0 184.0 Adult 2 E MML 0412 (FB119) f,h,i T. truncatus 238.0 168.0 Adult 2 CBD HOF 007 f,h,i T. truncatus 238.8 184.0 Adult 1 NE VMSM 951045 f,h,i T. truncatus 239.0 126.4 Adult 2 E WAM 559 f,h,i T. truncatus 239.0 166.0 Adult 1 NE VMSM 951023 f,h,i T. truncatus 240.0 232.8 Adult 2 NE WAM 573 f,h,i T. truncatus 241.5 200.0 Adult 2 NE WAM 633 a,c,f,g,h,i T. truncatus 244.0 180.0 Adult 1 NE WAM 560 f,h,i T. truncatus 245.0 193.0 Adult 1 NE PBN 003 a,c,f,g,h,i T. truncatus 246.0 173.0 Adult 2 E WAM 545 f,h,i T. truncatus 246.0 238.0 Adult 2 NE WAM 628 f,h,i T. truncatus 246.0 213.0 Adult 2 NE VGT 168 f,h,i T. truncatus 247.0 164.6 Adult 2 E VMSM 951039 f,h,i T. truncatus 249.0 190.0 Adult 2 NE WAM 642 f,h,i T. truncatus 250.0 226.0 Adult 2 NE NEFSC 01408 f,o,h,i T. truncatus 252.0 236.0 Adult 2 NE VGT 049 f T. truncatus 253.0 254.0 Adult 2 NE WAM 533 f,o,h T. truncatus 261.0 170.0 Adult 2 E BRF 164 f,o,h,i T. truncatus 262.0 216.5 Adult 1 NE WAM 535 f,h,i T. truncatus 264.0 297.0 Adult 2 NE REL 014 f,h,i T. truncatus 265.0 260.4 Adult 2 NE VMSM 921006 f,h,i T. truncatus 265.0 210.0 Adult 2 NE VMSM 941101 f,o,h,i T. truncatus 265.0 263.1 Adult 2 NE

Appendix A. (cont'd)... Total Total Life Field Identification Body Species Length History Code BC Number Mass (cm) Category (kg) VMSM 951035 f,h,i T. truncatus 267.0 199.0 Adult 2 NE AJW 001 f,o,h,i T. truncatus 274.0 231.0 Adult 1 NE BRF 061 f,h,i T. truncatus 275.0 257.0 Adult 2 NE WAM 631 f,o,h,i T. truncatus 275.0 209.0 Adult 2 NE WAM 639 f,h,i T. truncatus 276.0 290.0 Adult 3 NE MML 0111 f,h,i T. truncatus 277.0 170.2 Adult NR E VMSM 951051 f,h,i T. truncatus 277.0 196.0 Adult 2 E KLC 020 f,o,i T. truncatus 282.0 228.0 Adult 3 E DAP 027 f,o,h T. truncatus 283.0 233.0 Adult 2 E 571687 f,i T. truncatus 300.5 312.0 Adult 2 E a Gross description of thorax b Gross description of mycology in Kogia c Thorax manipulations d Cross-section anatomy and volume e Skeleton drawing f Lung mass: Total Body Mass g Lung volume h Heart mass i Liver mass o Offshore morphotype based on morphological characteristics

100 Appendix B. Cetacean species, investigated by VAQS or UNCW, used to compare lung mass to total body mass across a broader phylogenetic sample. All specimens used in this study were in fresh to moderate condition (Smithsonian Institute Code 1 through 3; Geraci and Lounsbury 2005). Each specimen was placed into a life history category as defined by stranding records. (NE = not examined, NR = not recorded, CBD = could not be determined)

Field Total Length Total Body Life History Identification Species Family Code Body Condition (cm) Mass (kg) Category Number EgNEFL 0704 Eubalaena glacialis Balaenida 401.0 749.0 Sub-adult 3 CBD KLC 022 Eubalaena glacialis Balaenida 495.0 1586.0 Neonate 1 not emaciated CTH 001 Balaenoptera acutorostrata Balaenopteridae 284.0 211.4 Sub-adult 1 emaciated VMSM 20031051 Delphinus delphis Delphinidae 183.0 66.8 Sub-adult 1 not emaciated VMSM 20041024 Delphinus delphis Delphinidae 190.0 51.2 Sub-adult 2 emaciated VAQS 20081012 Delphinus delphis Delphinidae 199.0 70.0 Sub-adult 1 emaciated VAQS 20081009 Delphinus delphis Delphinidae 200.0 88.0 Adult 1 not emaciated VAQS 20081013 Delphinus delphis Delphinidae 200.5 86.0 Adult 1 not emaciated VMSM 20031099 Delphinus delphis Delphinidae 202.0 86.0 Adult 1 not emaciated VMSM 20031090 Delphinus delphis Delphinidae 207.0 74.4 Adult 2 emaciated VMSM 19981025 Delphinus delphis Delphinidae 215.0 100.8 Adult 1 not emaciated VAQS 20051007 Delphinus delphis Delphinidae 222.0 130.0 Adult 2 not emaciated VMSM 20011030 Delphinus delphis Delphinidae 223.0 112.7 Adult 1 not emaciated VMSM 20011045 Delphinus delphis Delphinidae 232.0 114.4 Adult 2 not emaciated VMSM 20041020 Delphinus delphis Delphinidae 232.0 132.0 Adult 1 not emaciated GNL 080 Feresa attenuata Delphinidae 205.0 122.4 Adult 1 not emaciated SC 0735 Feresa attenuata Delphinidae 207.0 128.0 Adult 1 not emaciated VMSM 20031052 Globicephala melas Delphinidae 201.0 103.0 Sub-adult 1 not emaciated VMSM 951002 Grampus griseus Delphinidae 173.0 57.2 Neonate 2 emaciated VMSM 19981045 Grampus griseus Delphinidae 277.0 197.6 Adult 1 NR VAQS 20051082 Grampus griseus Delphinidae 277.2 301.4 Adult 1 NR

101 Appendix B. (cont'd)...

Total Field Identification Total Body Life History Species Family Length Code Body Condition Number Mass (kg) Category (cm)

DMB 008 Lagenorhynchus acutus Delphinidae 164.0 55.0 Sub-adult 1 not emaciated LRD 003 Lagenorhynchus acutus Delphinidae 165.0 46.4 Neonate 1 NR VAQS 20081066 Peponocephala electra Delphinidae 247.8 157.0 Adult 2 NR VAQS 20081069 Peponocephala electra Delphinidae 249.4 166.5 Adult 1 not emaciated VMSM 941095 Stenella attenuata Delphinidae 174.4 64.7 Sub-adult 2 NR WAM 602 Stenella clymene Delphinidae 202.0 92.0 Adult 1 not emaciated VMSM 971064 Stenella coeruleoalba Delphinidae 135.5 28.8 Sub-adult 2 NR SGB 127 Stenella coeruleoalba Delphinidae 152.8 41.0 Sub-adult 2 not emaciated SGB 126 Stenella coeruleoalba Delphinidae 172.5 63.0 Sub-adult 1 not emaciated VAQS 20051117 Stenella coeruleoalba Delphinidae 181.8 64.0 Sub-adult 2 NR VMSM 20031005 Stenella coeruleoalba Delphinidae 184.5 76.0 Sub-adult 1 not emaciated WAM 622 Stenella coeruleoalba Delphinidae 219.0 116.0 Adult 2 not emaciated WAM 619 Stenella coeruleoalba Delphinidae 222.0 120.0 Adult 2 not emaciated WAM 615 Stenella coeruleoalba Delphinidae 223.0 120.0 Adult 2 not emaciated WAM 612 Stenella coeruleoalba Delphinidae 225.0 120.5 Adult 2 not emaciated WAM 614 Stenella coeruleoalba Delphinidae 225.5 118.0 Adult 2 not emaciated WAM 618 Stenella coeruleoalba Delphinidae 226.0 129.5 Adult 2 not emaciated WAM 623 Stenella coeruleoalba Delphinidae 229.5 128.5 Adult 2 not emaciated WAM 621 Stenella coeruleoalba Delphinidae 230.0 134.0 Adult 2 not emaciated WAM 620 Stenella coeruleoalba Delphinidae 233.5 146.0 Adult 2 not emaciated WAM 613 Stenella coeruleoalba Delphinidae 235.0 123.0 Adult 2 not emaciated WAM 645 Stenella frontalis Delphinidae 177.0 59.0 Adult 2 not emaciated SGB 114 Stenella sp. Delphinidae NR 47.8 NR 2 NR

102

Appendix B. (cont'd)...

Total Field Identification Total Body Life History Species Family Length Code Body Condition Number Mass (kg) Category (cm)

VMSM 971002 Phocoena phocoena Phocoenidae 109.5 20.4 Sub-adult 2 NR MLC 001 Phocoena phocoena Phocoenidae 113.0 20.0 Sub-adult 1 not emaciated VMSM 20011013 Phocoena phocoena Phocoenidae 113.0 22.8 Sub-adult 2 not emaciated VMSM 971010 Phocoena phocoena Phocoenidae 119.0 22.3 Sub-adult 2 NR DMB 001 Phocoena phocoena Phocoenidae 133.0 28.2 Adult 1 emaciated VMSM 977013 Phocoena phocoena Phocoenidae 170.5 21.6 Adult 2 NR WAM 593 Mesoplodon densitrostris Ziphiidae 423.0 940.3 Adult 2 not emaciated VMSM 20021056 Mesoplodon europeus Ziphiidae 204.0 75.5 Sub-adult 1 not emaciated VMSM 931036 Mesoplodon europeus Ziphiidae 214.6 86.2 Neonate 1 NR NMNH 571568 Mesoplodon europeus Ziphiidae 422.0 760.0 no sperm 2 NR VMSM 20031098 Mesoplodon mirus Ziphiidae 391.0 618.2 Sub-adult 3 emaciated

103 Appendix C. Description of measurements taken during thoracic mobility manipulations.

In the maximally expanded and collapsed postures the following measurements were taken:

(1) length of the thorax from the cranial margin of vertebral rib 1 to the caudal margin of

the last vertebral rib, at mid-shaft (“1” on datasheet);

(2) length of cranial vertebral ribs only, from the cranial margin of vertebral rib 1 to the

caudal margin of last sternally connected vertebral rib (for T. truncatus vertebral rib 6

and for kogiids vertebral rib 4), at mid-shaft (“2” on datasheet);

(3) length of the sternal ribs from the cranial margin of sternal rib 1, at its articulation

with the sternum, to the caudal margin of the terminal sternal rib (for T. truncatus

vertebral rib 8 and for kogiids vertebral rib 11 or 12), at mid-shaft (“3” on datasheet);

(4) length of the thorax from the cranial margin of sternal rib 1, at its articulation

with the sternum, to the caudal margin of the last vertebral rib, at mid-shaft (“4” on

datasheet);

(5) length of the distance between the cranial margin of the manubrium, at the midline,

to the ventro-cranial margin of cervical vertebra 1, at the midline (“5” on datasheet);

(6) two measures of thoracic inlet height:

(a) perpendicular distance between the ventral surface of the vertebral body and

the dorsal surface of the sternal midline (“a” on datasheet);

(b) straight-line measurement between the ventral surface of the angle of

vertebral rib 1 and dorsal surface of the manubrium wing (“b” on datasheet);

(7) external thoracic height, taken at the level of each rib, as the perpendicular distance

from the dorsal most aspect of the rib (either dorsal rib angle or articulation of the

104 vertebral rib with the transverse process, whichever is highest) to the ventral

articulation with the sternum (ventral most aspect of the sternum or for the caudal ribs

their ventral most tip) (“Lat. Height” on datasheet);

(8) thoracic inlet width as the perpendicular distance between the medial surfaces of the

first vertebral-sternal rib joints in T. truncatus (“c” on datasheet) and between the

medial surfaces of the first vertebral rib, at mid-shaft, in kogiids (“d” on datasheet);

(9) external thoracic width, taken at the level of each rib as the perpendicular distance

between the widest position between vertebral ribs (usually mid-shaft) (“Ext. Width”

on datasheet);

(10) thoracic vertebral width as the distance from right to left transverse processes at

each vertebra, which is added to the circumference measurements (“Vert. Width” on

datasheet).

(11) thoracic circumference was measured at the level of each sternal rib between the

left and right vertebra-vertebral rib joints; the circumferences were measured for

the cranial vertebral ribs only, i.e. those that have associated sternal ribs

(“Circumference” on datasheet).

(12) thoracic wall thicknesses was measured at the level of vertebral rib 1 and the

last sternally connected vertebral rib (for T. truncatus vertebral rib 6 and for kogiids

vertebral rib 4), at three circumferential positions: (a) angle of rib, (b) mid-shaft and

105

Appendix D. Datasheet used to define measurements of the thorax in a cranially expanded and caudally collapsed position. Each dimension was measured (cm) three times and then the mean was calculated. Measurements include, lateral lengths (1), (2), (3), (4), (5); widths at each vertebral rib; lateral height at each vertebral rib; lateral vertebra width at each vertebra; circumference at each vertebral rib that has an associated sternal rib (i.e. for T. truncatus at each vertebral rib for vertebral ribs 1-6); inlet dimensions (a), (b), (c), (d); and thoracic wall thicknesses at six different locations (not shown here).

THORAX MOBILITY

Field ID: ______Species: ______TL: _____ TBM: ______

Position: suspended OR supine Muscle Present: ______

cranial OR caudal Spring Load (kg): ______

Fishing Line Position: ______

106 Appendix E. The equations of all four models used to estimate thoracic cavity volume

are listed here.

The first model to estimate thoracic cavity volume used a frustum of a right

circular cone in series with a slanted right circular cone (see Figure 6A; Sandifer and

Moshos 1996):

(1) frustum of a right circular cone,

V ⅓ π l r2 Rr R2,

where l = length between vertebral ribs 1-5, r = radius of a circle at the cross-section between vertebral ribs 1-2 (derived from the circumference measurement taken between ribs 1-2), and R = radius of a circle at the cross-section between vertebral ribs 5-6

(derived from the circumference measurement taken between ribs 5-6).

(2) slanted right circular cone,

V ⅓ π cos θ L R2,

where L = length between vertebral rib 5 and lumbar vertebra 3, R= radius of circle at the cross-section between ribs 5-6 (derived from the circumference measurement taken between vertebral ribs 5-6) and θ = the angle of approach of the diaphragm to lumbar vertebra 3 (Sandifer and Moshos 1996).

The second model included a right circular cylinder for the cranial thorax in

series with the same slanted right circular cone used above for the caudal thorax (see

above for slanted cone equation; Figure 6B; Sandifer and Moshos 1996):

(1) right circular cylinder,

107 V π r2 l ,

where r = average radius of circle at the cross-section between ribs 1-2 (derived from the

circumference measurements taken between vertebral ribs 1-6), and l = length between

vertebral ribs 1-5.

A third model to estimate thoracic cavity volume included a frustum of a right

cardioid cone in series with a slanted right cardioid cone (Figure 6C):

(1) frustum of a right cardioid cone,

V 1/2 l π r2 rR R2,

where a = radius of cardioid at the cross-section between vertebral ribs 1-2 (derived from

the lateral width measurements taken between vertebral ribs 1-2), A = radius of cardioid

at the cross-section between vertebral ribs 5-6 (derived from lateral width measurements

taken at vertebral ribs 5-6), and l = length between vertebral ribs 1-5.

(2) slanted right cardioid cone,

V 1/2 π cos θ L R2, where L = length between vertebral rib 5 and lumbar vertebra 3, A= radius of cardioid at the cross-section between ribs 5-6 (derived from the lateral width measurement taken between vertebral ribs 5-6) and θ = the angle of approach of the diaphragm to lumbar vertebra 3 (Sandifer and Moshos 1996).

The fourth and final model used to estimate thoracic cavity volume utilized a right cardioid cylinder, for the cranial thorax, in series with the slanted right cardioid

108 cone for the caudal thorax. The following equation was used for the cardioid shaped cylinder (see equation in model 3 for slanted cone) (Figure 6D):

(1) right cardioid cylinder,

V 3/2 π r2 l, where a = average radius of cardioid at the cross-section between vertebral ribs 1-6

(derived from the average of lateral width measurements taken between vertebral ribs 1-

6), and l = length between vertebral ribs 1-5.

109