POSTCRANIAL SKELETAL PNEUMATICITY, BONE STUCTURE, AND

FORAGING STYLE IN TWO CLADES OF NEOGNATH BIRDS

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A thesis

Presented to

The Honors Tutorial College

Ohio University

______

In Partial Fulfillment

of the Requirements for Graduation

from the Honors Tutorial College

with the degree of

Bachelors of Science in Biological Sciences

______

by

Sarah C. Gutzwiller

June 2010

This thesis has been approved by

The Honors Tutorial College and the Department of Biological Sciences

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Dr. Patrick O’Connor Associate Professor, Biomedical Sciences Thesis Advisor

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Dr. Soichi Tanda Honors Tutorial College, Director of Studies Biological Sciences

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Jeremy Webster Dean, Honors Tutorial College

Acknowledgements

I would like to thank the Ohio University Honors Tutorial College for

providing countless opportunities and gracious support throughout my undergraduate

career. A special thanks to my Director of Studies, Dr. Tanda, and my Thesis

Advisor, Dr. O’Connor, for their direction and guidance.

Thank you to the Carnegie Museum of Natural History, the Ohio University

Vertebrate Collections, the Ohio University microCT, and the Ohio University Office of the Vice Provost for Research for providing specimens, equipment, and funding.

I also wish to thank Ryan Ridgely, Anne Su, and other colleagues and friends for helping me throughout my project.

TABLE OF CONTENTS

List of Figures ii List of Tables iii List of Abbreviations iii Abstract 1 Introduction 2 Rationale of the Current Study 11 Goals of the Current Study 23 Materials and Methods 24 Hypotheses and Predictions 29 Results 30 Discussion 44 Conclusions 50 Literature Cited 52 Appendices 57

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LIST OF FIGURES

Fig. 1 Phylogenetic hypothesis of extant birds 3 Fig. 2 Pulmonary latex injection detailing the avian air sac system. 6 Fig. 3 Pulmonary injection of the Blackheaded gull (Larus 7 ridibundus). Fig. 4 Pneumatic bones from Marabou stork (CM 118743). A, 8 cervical vertebra; B, humerus; PF, pneumatic foramen. Fig. 5 Example of anatomical distribution of pneumaticity in extant 10 birds. Fig. 6 The Common Murre (Uria aalge). 16 Fig. 7 The Western Gull (Larus occidentalis). 17 Fig. 8 The Great Skua (Catharacta skua). 18 Fig. 9 The Double-crested Cormorant (Phalacrocorax auritus). 21 Fig. 10 The Anhinga (Anhinga anhinga). 22 Fig. 11 The Brown Pelican (Pelecanus occidentalis). 23 Fig. 12 MicroCT scanner housed at Ohio University. 26 Fig. 13 Cross-sectional μCT slice of a middle cervical vertebra from 27 a Great Skua (Catharacta skua). Fig. 14 Average Pneumaticity Index (PI) values for two dedicated 31 diving specialists (murre and puffin) and two non-diving specialists (gull and skua). Fig. 15 The log transformed average species body mass regressed 33 upon the arcsine transformed average Pneumaticity Index for each charadriiform species. Fig. 16 Phylogenetic hypothesis of Charadriiformes with relative 35 pneumaticity Fig. 17 Box and whisker plots of cervical trabecular bone volume 37 fractions for each charadriiform species. Fig. 18 Box and whisker plots of thoracic trabecular bone volume 38 fractions for each charadriiform species. Fig. 19 Box and whisker plots of cortical bone thickness for each 39 charadriiform species. Fig. 20 Average Pneumaticity Index (PI) values for two dedicated 40 diving specialists (anhinga and cormorant) and non-diving specialist (pelican). Fig. 21 Box and whisker plots of cervical trabecular bone volume 41 fractions for each pelecaniform species. Fig. 22 Box and whisker plots of thoracic trabecular bone volume 42 fractions for each pelecaniform species. Fig. 23 Box and whisker plots of cortical bone thickness for each 43 pelecaniform species.

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LIST OF TABLES

Table 1 Focal species 24

LIST OF ABBREVIATIONS

ABD abdominal air sac AU anatomical unit AXD axillary diverticulum BV/TV trabecular bone volume fraction CA caudal vertebrae CAC caudal cervical vertebrae CAT caudal thoracic vertebrae CAUDTH caudal thoracic air sac Cb.T cortical bone thickness CERV cervical air sac CL clavicular air sac CoV coefficient of variance CRC cranial cervical vertebrae CRT cranial thoracic vertebrae CRTH cranial thoracic air sac IMDIV intermuscular diverticula MC middle cervical vertebrae PF pneumatic foramen PI pneumaticity index SS synsacral vertebrae TR trachea μCT micro-computed tomography VOI volume of interest

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ABSTRACT

Extant birds represent the only living sauropsid group in which pulmonary air

sacs aerate the postcranial skeleton. The degree of variability in birds is notable,

ranging from taxa that are completely apneumatic to those characterized by air

within most of the postcranial skeleton. Although numerous factors (e.g., body size)

have been linked with ‘relative’ pneumaticity, comparative studies examining this

system remain limited. This project sought to (1) examine whole-body patterns of

pneumaticity in select charadriiform and pelecaniform birds, (2) evaluate relationships

among relative pneumaticity, body size and locomotor specializations (e.g. diving,

soaring) and (3) examine if/how bone structure is altered in pneumatic versus

apneuamtic vertebrae.

Species-specific pneumaticity profiles were used to examine relative

pneumaticity. Results suggest that the largest flying birds exhibit a higher degree of

pneumaticity relative to smaller birds (e.g. larids). In contrast, skeletal

pneumaticity has been independently lost in multiple lineages of diving specialists.

Such reductions in skeletal pneumaticity likely result in decreased buoyancy in birds

specialized for dive foraging. Conversely, aerating the postcranial skeleton offers a

mechanism that allows volumetric increases in bone without concomitant increases

in body mass. Thus, the potential to differentially pneumatize the postcranial

skeleton may have played a role in relaxing constraints on body size evolution and/or

habitat exploitation during the course of avian evolution. Patterns of pneumaticity and

bone structure within charadriiforms and pelecaniforms are also discussed.

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INTRODUCTION

Living birds include a vast diversity of approximately 10,000 species that occupy 22 orders (Fig. 1; Perrins, 2009). They have exploited an array of niches from the terrestrial seed-eating finches of the Galapagos to the aquatic fish-eating penguins of the Antarctic. They utilize a variety of social systems, breeding behaviors, and locomotor styles (Perrins, 2009; Simons, 2010). Paralleling this diversity in lifestyle, birds are diverse in morphology. They can range greatly in size and body mass (Dunning, 1993). For example, the Vervain Hummingbird

(Mellisuga minima) weighs an average of 2.4 grams, whereas the ostrich (Struthio camelus) averages 83,500 grams. Whether in size or in shape, the morphology often reflects the function (Perrins, 2009). For example, the shape of a bird’s beak may reflect its diet. A Hawfinch has a stout, thick beak optimal for cracking open hard-shelled seeds, while a Sword-billed hummingbird has a long, thin beak ideal for collecting nectar from passionflowers. Thus a distinct relationship exists between form (morphology) and function (lifestyle).

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Figure 1. Phylogenetic hypothesis of extant birds. Taken from O’Connor (2009)

Form and Function

When studying the relationship between form and function, birds make an

excellent study group. Such diversity in lifestyle and morphology allows

researchers to examine form-function relationships and to be able to apply these

insights to the study of the ecology, conservation, and evolution of birds.

Researchers can use the understanding of form-function relationships in three important ways: (1) they can apply the knowledge of the anatomy and physiology of one species to another species that is less understood; (2) they may be able to examine the morphology of an extinct species and hypothesize the function and

Gutzwiller Senior Thesis—3

lifestyle of that animal; (3) they can examine mechanisms of evolution that have resulted in the vast diversity of living animals around us today.

Foraging Specializations and Evolutionary Adaptation

An aspect of lifestyle that varies among neognath birds is foraging mode or style. Foraging style is a significant aspect of an organism’s ecology. Not only does it determine how and what an organism consumes, it represents a trade-off in organismal energetics (Maurer, 1996). For optimal pay-off, the energy gained through foraging must be greater than the energy used to forage. Due to the impact of this energetic balance on the survival and fitness of the organism, foraging style is highly susceptible to natural selection. Adaptive foraging styles can reflect the environmental pressures and evolutionary constraints that are acting upon the organism. Understanding foraging style can reveal what factors have led to the diversification of birds.

Some birds are specialized dive foragers, whereas others are nondiving forms, specialized in other means of foraging, such as in-flight capture and/or shoreline feeding. Specialization in a certain foraging strategy can be reflected in the morphology of the bird. For example, foot-propelled diving specialists have elongated and more posteriorly-positioned hind limbs (Raikow, 1970; Johnsgard,

1987; McCracken, et al. 1999). This adaptation aids in the pursuit of prey under water. Likewise, birds specialized in soaring (extended flight with minimal flapping) have evolved long forearms to aid in this flight style. It has been

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hypothesized that other anatomical systems have similarly been modified in relation to foraging style. For example, some birds exhibit postcranial skeletal pneumaticity, the invasion of the vertebral column and limbs by the air sacs connected to the lung and the subsequent creation air-filled cavities in these bones

(Hogg, 1984b; O’Connor, 2004). It has been hypothesized that this morphological feature is an evolutionary adaptation to reduce mass and make locomotion (flight in particular) more energetically efficient (O’Connor, 2004; 2009). Likewise, it is thought that a lack of postcranial pneumaticity observed in some birds is an adaption for specialized dive foragers, possibly to counteract buoyancy.

The Avian Air Sac System

The development of postcranial pneumaticity is possible due to the unique avian respiratory system, one that consists of a lung ventilated by nine compliant air sacs (Fig. 2). The avian lung is a rigid structure that does not greatly change in volume during respiration. The rigidity of the lung leads to small terminal respiratory units and thus a large respiratory surface area (Duncker, 1972; Maina, 2006). A series of thin-walled air sacs grow from the surface of the lung during embryonic development (Locy and Larcell, 1916). As an embryo, a bird has five pairs of air sacs

(King, 1966; McLelland, 1989). However, in most birds one of the pairs fuses during development leaving a single (clavicular) and four paired (abdominal, caudal thoracic, cervical, and cranial thoracic) air sacs (Christiansen, 2000; Dunker, 2004). This air sac system is considered a low-pressure system (Dunker, 2004). The air sacs are thin-

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walled and highly compliant and serve to facilitate the movement of air to and from the functional surface of the lung during ventilation. Movement of air throughout the system results from localized pressure gradients created by volumetric changes related to deformation of the body wall (Fedde, 1998). The physical separation of the lung and the air sacs allows a continuous and unidirectional movement of air across the respiratory surface of the lung (Dunker, 1971; Maina, 2006). It has been hypothesized that the specialized locomotion of flight allows for this unique respiratory setup, because the muscles used to power the wing and leg movements are separate from the thoracic muscles used in respiration (Dunker, 1971). The high compliance of the air sacs allows them to spread throughout the body, making postcranial pneumaticity possible.

Figure 2. Pulmonary latex injection detailing the avian air sac system. ABD, abdominal air sac; CAUDTH, caudal thoracic air sac; CERV, cervical air sac; CRTH, cranial thoracic air sac; CL, clavicular air sac; TR, trachea. Taken from O’Connor (2004). Gutzwiller Senior Thesis—6

Development of Pneumaticity

The development of postcranial pneumaticity has been documented in some species and has been shown to occur after hatching (Hogg, 1984b). Pneumatization of the postcranial skeleton occurs when intraosseous pneumatic diverticula, epithelium- lined outgrowths of the air sacs, invade the bone (Witmer, 1990; O’Connor, 2004).

During embryonic development, diverticula penetrate the bone through pneumatic foramina or osseous sutures in the cortical bone (Bremer, 1940). The diverticula then displace the bone marrow as they extend through the medullary cavity, leaving the bone air-filled (Fig. 3; Schepelmann, 1990). The invasion of some bones by the diverticula during the pneumatization process is well documented. However, less is known about the mechanisms driving its development (see O’Connor, 2009).

Figure 3. Pulmonary injection of the Blackheaded gull (Larus ridibundus). ABD, abdominal air sac; AXD, axillary diverticulum; CAUDTH, caudal thoracic air sac; IMDIV, intermuscular diverticula. Taken from O’Connor (2004).

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Characteristics of Pneumatic Bone

Pneumatic bones share several characteristics that distinguish them from apneumatic bones (Müller, 1908; O’Connor, 2006). In pneumatic bones, bone marrow is replaced by epithelium-lined outgrowths of the air sac. Because air is lighter than bone marrow, pneumatic bones have a lower specific gravity. The lack of bone marrow also results in the bones being less oily and lighter in color. Pneumatic bones are less vascularized and have the presence of a pneumatic foramen (Fig. 4), the site of entrance of the pneumatic diverticulum into the bone. In some cases, the diverticulum will not completely penetrate a bone, but instead leave an osteological marker in the form of a depression, called a pneumatic fossa, on the surface of the apneumatic bone

(Witmer, 1990).

A B PF

Figure 4. Pneumatic bones from Marabou stork (CM 118743). A, cervical vertebra; B, humerus; PF, pneumatic foramen.

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Anatomical Distribution of Pneumaticity

Certain bones are more likely to be pneumatized by specific air sacs according to their location within the body (Fig. 5; Müller, 1908; McLelland, 1989; Evans,

1996). For example, the cervical air sacs tend to pneumatize the cervical and thoracic vertebrae and their associated ribs. Thoracic vertebrae may also be pneumatized directly from lung diverticula. The clavicular air sac pneumatizes the humerus, sternum, sternal ribs and pectoral girdle. The abdominal air sacs pneumatize the posterior thoraic vertebrae, the pelvis, and the pelvic limbs. The thoracic air sacs do not tend to pneumatize any portion of the postcranial skeleton, excluding some species of budgerigar (Evans, 1996) and storks (P. O’Connor, pers.com) where the cranial thoracic air sacs have been observed to pneumatize sternal ribs. Both osteological and pulmonary injection studies have shown that distal limb elements are rarely pneumatized (see Hunter, 1774; Owen, 1835; Crisp, 1857; King, 1956; Bellairs and

Jenkin, 1960; Hogg, 1984b; McLelland, 1989; O’Connor, 2009). A level of variation in pneumaticity within species and asymmetry within individuals can occur (Hogg,

1804a).

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Figure 5. Example of anatomical distribution of pneumaticity in extant birds. Gray shading denotes a pneumatized bone. Vertebrae: CA, caudal; CAC, caudal cervical; CAT, caudal thoracic; CRC, cranial cervical; CRT, cranial thoracic; MC, middle cervical; SS, synsacral. Taken from O’Connor (2004).

Biomechanical Implications

Aspects of biomechanics have been shown to be altered in pneumatic bone.

The location of the pneumatic foramen on a particular bone tends to be similar across species, suggesting that the diverticulum enters at a site that will cause the least amount of structural and mechanical harm to the bone (Müller, 1908).

However, the biomechanics of the bone are influenced by the invasion of air sac diverticula. Cortical thickness and the presence of marrow may be relevant for bone strength and stiffness (Kafka, 1983; Cubo and Casinos, 1999). Pneumatic

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bones tend to have a lower cortical bone thickness, bending strength, and flexural

Young’s modulus when compared to apneumatic bones (Cubo and Casinos, 1999;

Fajardo et al., 2007). These results suggest that the breaking moment of apneumatic bones should be 2-2.5 times higher than pneumatic bones (Cubo and

Casinos, 1999). The prevalence of pneumatic bones in certain species of birds even though they exhibit a decrease in biomechanical strength indicates that there is indeed some selective advantage for pneumatic bones, such as aiding in flight by decreasing body mass.

RATIONALE OF THE CURRENT STUDY

The unique respiratory system observed in birds, that includes a rigid lung connected to compliant air sacs, has exceptional potential for exploring potential evolutionary adaptations. The diverticula that extend from the air sacs can penetrate and change the morphology of surrounding bone. In some birds, postcranial skeletal pneumaticity may reduce body mass and alter energy expenditure, making some forms of locomotion, such as flight, more energetically efficient. The loss of pneumaticity observed in diving birds may be an evolutionary adaptation to counteract buoyancy, allowing for more energetic diving bouts.

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Previous Studies

Anseriform birds (ducks and relatives) represent the only group in which

detailed studies of relative pneumaticity have been conducted to date (O’Connor,

2004; Fajardo et al., 2007). O’Connor (2004) examined postcranial pneumaticity with

regards to body mass and locomotion. He observed a suggested relationship that

postcranial skeletal pneumaticity increases as body mass increases, perhaps reflecting

a mechanism to decouple typical mass-volume relationships and make locomotion

more energetically efficient. He hypothesized that pneumaticity allows for an increase

in the volume of a bird without the concomitant increase in energetically-costly mass

(see O’Connor, 2009 for additional discussion on this topic). He also observed more

pronounced patterns. Soaring birds were highly pneumatic, perhaps as an adaptation

to reduce body mass and allow for more energetically efficient flight. Also, in three

independently derived occasions, diving specialists were observed to have

significantly reduced postcranial pneumaticity relative to their non-diving clade-mates.

O’Connor hypothesized that this lack of pneumaticity evolved to reduce buoyancy, making diving more energetically efficient. He concluded that, at least in anseriforms, locomotor patterns such as extended diving can restrict the extent of postcranial pneumaticity.

Apart from a basic understanding of which bones are air-filled, much less is known about the actual structure (e.g., relative density of trabecular bone or thickness of cortical bone) of pneumatic and apneumatic bone. An understanding

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of the structure of pneumatic and apneumatic bone will allow researchers to better examine the evolution of pneumaticity.

Fajardo et al. (2007) aimed to better understand pneumatic and apneumatic bone structure in anseriforms. With the use of micro-computed tomography (μCT, an X-ray imaging technique), they examined the third thoracic vertebra of two species, the pneumatic wood duck (Aix sponsa) and the apneumatic ruddy duck

(Oxyura jamaicensis). They created three volumes of interest, including the trabecular bone, the cortical bone, and the whole centrum. Results indicated that the pneumatic vertebrae have a significantly lower bone volume to total volume fraction of the whole centrum. However, trabecular bone volume fraction is similar in both species. Contrary to previous thoughts (although ones based not on explicit, quantitative studies), these results suggest that pneumatization is not associated with a decrease in trabecular bone. Instead they attributed the decreased total bone volume fraction to the significantly thinner cortical bone observed in pneumatic bone. With conclusions similar to O’Connor (2004), they hypothesized that thinner cortical bone in A. sponsa, a generalized dabbling duck, may reflect an adaptation to reduce bone mass and allow more energetically efficient flight.

Similarly, thicker cortical bone in the apneumatic bones may reflect the locomotory specialization on O. jamaicensis, a dedicated diving forager. It may act to reduce the amount of low-density bone marrow and increase skeletal mass, as a means of reaching neutral buoyancy. This preliminary study demonstrates the

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application of μCT techniques to exploring bone structure in pneumatic and apneumatic vertebrae.

Previous work has focused on anseriform birds. In this study, I aimed to explore if similar patterns are observed independently in other neognath clades and if the pneumaticity profiles identified in anseriform birds represent patterns in birds more generally. Specifically, I examined the relationships between pneumaticity, bone structure, and foraging style in two orders of living birds, Charadriiformes and Pelecaniformes. Within charadriiforms and pelecaniforms, the species are united by several characteristics, yet are also diverse in many aspects of their morphology and lifestyle.

Introduction to Charadriiformes

Charadriiformes includes 350 species of shorebirds and allies, including gulls, sandpipers, and plovers (Ericson et al., 2003). Though they share some morphological characteristics, the phylogenetic resolution within charadriiforms has been more successfully documented using molecular data (sequencing and

DNA-DNA hybridization) rather than morphological data. This order is very diverse in social mating systems, ecology, and life history (Thomas et al., 2004).

Some charadriiforms specialize in dive foraging, while others specialize in various flight styles for foraging.

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Highlighted charadriiform families:

Within Charadriiformes, two families exhibit this dichotomy in foraging styles (Bull and Farrand, 2005). In the family Alcidae (including murres and puffins), birds forage by diving. In the family Stercorariini (including skuas), the birds are non-diving specialists and forage by other means. Some families, including Laridae, exhibit intermediate foraging specializations, with splash diving and shoreline foraging being most common.

Family Alcidae: Puffins and Murres (Fig. 6)

The thick-billed murre (Uria lomvia) breeds in colonies in open water areas in ice-covered polar oceans (Falk et al., 2002). This bird uses pursuit diving to forage for marine invertebrates and fish. They exhibit plasticity in their diving behavior depending upon food availability, sometimes diving at shallower depths

(2-10 m), sometimes deeper (>70 m). One murre has been recorded reaching 210 m in depth (Perrins, 2009). The length of the dive, which correlates with the depth of the dive, averages 55 seconds (Croll et al., 1992).

The charadriiforms specialized in dive foraging display a host of adaptations related to this behavior (Gaston, 1998). Puffins and murres exhibit a fusiform body with posteriorly-positioned hind limbs, a characteristic also seen in other foot-propelled divers. They possess covert feathers that are stiffer due to higher levels of connective tissue, resulting in the forearm being functional as a paddle. The hypapophyses of the last cervical vertebra are greatly developed and these birds have a greater number of thoracic vertebrae. This allows for a longer

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body and greater flexibility during swimming. The humerus is quite robust, oval

shaped in cross-section, and the articular surface is larger, perhaps in response to

the higher force applied to the bone during swimming. In order to perform wing-

propelled diving, auks bend their wing to decrease surface area and allow

swimming to be more energetically efficient. They also have a high blood volume

and high myoglobin content to increase oxygen storage during their long dives that

may exceed 48 seconds (at which point aerobic processes transition to anaerobic).

These adaptations for swimming may also influence other locomotion styles. Due

the small wings specialized for swimming, flight in auks is dependent on constant

flapping.

Figure 6. The Common Murre (Uria aalge). www.cvlbirding.co.uk

Family Laridae: Gulls (Fig. 7)

Gulls utilize a variety of foraging strategies, including splash diving and

shoreline foraging (Knopf, 2000; Bull and Farrand, 2005; Perrins, 2009). While

gulls are distributed throughout the world, most are located within the northern

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hemisphere. They tend to be colonial breeders, nesting on islands, beaches, and

inland lakes and rivers. They forage by scavenging and eating a number of aquatic

animals, including clams and other shellfish. Gull locomotion can vary from

gliding and soaring to flap gliding. Like the alcids, larid morphology also varies

widely, reflecting their variety of foraging and locomotory specializations (Perrins,

2009). They range in size from the Little gull (Larus minutes, ~118g) to the Great

black-headed gull (Larus ridibundus, 1659g; Dunning, 1993). Likewise, other

aspects of their morphology, such as bill shape, vary from species to species.

Figure 7. The Western Gull (Larus occidentalis). www.birdsasart.com

Family Stercorariini: Skuas (Fig. 8)

The great skua (Catharacta skua) is specialized in soaring flight (Bull and

Farrand, 2005). They have a great diversity in diet and a large geographic range

(Knopf, 2000; Votier, 2003; Perrins, 2009). The great skua feeds on a variety of

fish, shrimp, rodents, and the eggs and young of other seabirds. They also have

been observed to kill herons, geese, and hares. Whereas skuas have a wide

geographic range in both hemispheres, great skuas span throughout Norway,

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Svalbard, and northern Russia. Great skuas are the largest of the northern skuas and have a barrel-shaped body with a short tail. They tend to fly at intermediate speeds (15 m/s) with steady slow wing beats, occasionally gliding (Alerstam et al.,

2007). With less angled wings than the southern skuas, Catharacta pursue their prey with active flight, chasing them to the surface of the water. Although most of

Catharacta are short-distance migrants, great skuas perform moderately long migrations during the breeding season (Olson and Larson, 1997).

Figure 8. The Great Skua (Catharacta skua). www.surfbirds.com

Introduction to Pelecaniformes

Pelecaniformes comprise 55 living species in six families of aquatic birds, including pelicans, cormorants, anhingas and darters, boobies and gannets, frigate birds, and tropicbirds (Hedges and Sibley, 1994). Whereas several molecular studies suggest that pelecaniforms may not be monophyletic, the group is united by a host of morphological features (Johnsgard, 1993; Donoghue, 2004). All pelecaniforms have a totipalmate foot, which is one that has all four toes webbed

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together. The tongue and the hyoid structure are highly reduced, which is thought to be important for a thermoregulatory behavior called “gular fluttering”, the opening of the mouth for evaporative cooling. Other traits that are widespread within pelecaniforms include the lack of a brood patch, and the presence of a gular pouch and an uropygial gland.

Along with the morphological traits that unite pelecaniforms, some of the birds share ecological and life history traits (Johnsgard, 1993; Enstipp et al., 2005).

Cormorants, darters, and pelicans are united by their general diet. All feed largely on fish or other aquatic vertebrates and invertebrates. This diet geographically limits these birds to waters that are rich in these food sources and often promotes colonial breeding at food sites. Excluding tropicbirds, pelecaniforms incubate their eggs by use of their foot webs since brood patches are lacking. This method of incubation limits clutch size to one or two eggs, limiting the reproductive potential of pelecaniforms. The young are highly altricial when hatched and require a prolonged nesting period and a great deal of parental care, so monogamous mating is typical within pelecaniforms.

Similar to charadriiforms, pelecaniforms also have a large degree of diversity in morphology and ecological traits, including locomotor and foraging strategies (Johnsgard, 1993; Brewer and Hertel, 2003; Ryan, 2007). Three of the six families are highly pelagic (living near the ocean) and spend much of the nonbreeding season away from land. For example, the family Sulidae (gannets and boobies) is found primarily in temperate and tropical regions. Whereas all species

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of pelecaniforms have characteristically limited locomotion on land that consists mainly of waddling and hopping, they vary greatly in their airborne and water locomotion. Some groups specialize in a variety of diving foraging strategies, including both shallow (surface) or plunge diving (as deep as 100 m). For example, the family Phaethontidae includes the highly pelagic tropicbird (Bull and

Farrand, 2005). These birds forage for fish and squid by deep-plunge diving from the air. Within pelecaniforms, other groups specialize in aerial pursuit of prey.

The family Fregatidae includes the great frigatebird (Brewer and Hertel, 2007).

Frigatebirds directly capture their prey from the water’s surface or perform aerial pursuit of flying fish. These birds are capable of sustained flight for long periods of time.

Highlighted pelecaniform families:

Three families of interest within Pelecaniformes are Phalacrocoracidae

(cormorants), Anhingidae (anhingas and darters) and Pelecanidae (pelicans).

Phalacrocoracids and anhingids are subsurface diving specialists, whereas pelecanids are non-diving foragers.

Family Phalacrocoracidae: Cormorants (Fig. 9)

Cormorants are foot-propelled diving specialists (Johnsgard, 1993; White,

2008). During dives lasting from 10-60 seconds, cormorants often dive to depths of

6 to 30 meters from a floating position on the surface of the water. Under the water, cormorants are foot-propelled swimmers, holding wings close to their sides

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and using their tail and feet at rudders. Anatomical specializations related to these

abilities in cormorants include a highly developed patella consisting of a true

sesamoid patellar portion and a portion of the cnemial crest of the tibio-tarsus. The

patella is large, suggesting specific interactions with leg musculature that most

likely aid in foot-propelled diving.

Figure 9. The Double-crested Cormorant (Phalacrocorax auritus). www.urbanhawks.blogs.com

Family Anhingidae: Anhingas (Fig. 10)

Anhingas are diving specialists that typically enter the water by dropping

down from a perch (Johnsgard, 1993). As opposed to the deep dives seen in

cormorants, they tend to dive only a few meters deep. They are also foot-propelled swimmers, but they keep their wings partially open for steering purposes. Dives may last up to 60 seconds. Anhingas forage by moving slowly or sitting motionless in the water, then spearing a fish with their bill. The femur of the

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anhinga is longer than that of the cormorant and is bowed in shape, perhaps reflecting their tree-climbing abilities.

Figure 10. The Anhinga (Anhinga anhinga). www.marioncountyaudubon.com

Family Pelecanidae: Pelicans (Fig. 11)

Pelicans forage at the surface of the water and are not specialized for deep diving, although some species are specialized in splash diving (Johnsgard, 1993;

Brewer and Hertel, 2007). Note: although the brown pelican is known to splash dive, it does not pursue prey beneath the surface of the water (Richardson, 1939).

Soaring is well documented in pelicans. Within the air, pelicans tend to fly with their neck retracted in order to support their heavy bill. Pelicans have

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characteristically long forearms with an ulna that is longer than the radius, which

may be an important adaptation to their flight mode.

Figure 11. The Brown Pelican (Pelecanus occidentalis). www.richard-seaman.com

GOALS OF THE CURRENT STUDY

My study expands upon the concepts and methods explored by O’Connor

(2004, 2009) and Fajardo et al. (2007). With the foundation of anseriform

research, I focused my I study on charadriiform and pelecaniform birds. The goals

of my study occupied two levels. I examined postcranial pneumaticity at the level

of the whole skeleton and at the level of bone structure. At the whole skeletal

level, I contributed to further developing postcranial skeletal pneumaticity profiles for four charadriiform and three pelecaniform species (Table 1). I examined how relative pneumaticity relates to body mass, phylogeny, and foraging specialization.

At the bone structure level, I explored the use of μCT techniques combined with

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additional methods in determining the effects of pneumaticity and lifestyle on bone structure.

MATERIALS and METHODS

Specimens were provided by the Ohio University Vertebrate Collections

(OUVC) and the Carnegie Museum of Natural History (CM).

Table 1. Focal species (body mass data from Dunning, 1993) Order Species Common Name Average BM Foraging Specialization Charad. Uria aalge Common murre 993g Dedicated diver Fratercula Atlantic puffin 381g Dedicated diver arctica Larus Western gull 1011g Generalized flier occidentalis Catharacta Great skua 446g Soaring specialist skua Pelec. Anhinga Anhinga 1235g Dedicated diver anhinga Plalacrocorax Double-crested 1674g Dedicated diver auritus cormorant Pelecanus Brown pelican 3438g Soaring specialist occidentalis

Whole skeleton postcranial pneumaticity profile

Following the methods used in O’Connor (2004, 2009), a whole skeleton postcranial pneumaticity profile was completed for each charadriiform and pelecaniform specimen. Twelve discrete anatomical units (AUs) of the postcranial skeleton were examined (Appendix I).

Individual AUs were scored for the presence or absence of pneumaticity, by determining the presence or absence of pneumatic foramina. A pneumaticity index (PI) was created by totaling the number of pneumatic anatomical units and then dividing this total by 12. Gutzwiller Senior Thesis—24

Pneumaticity Index = # pneumatic AUs total # AUs

The use of the PI allows for the quantification of the extent of pneumaticity

and a comparison of the degree of relative pneumaticity among several species that

may have different vertebral counts. The data collected for selected charadriiform

specimens was combined with a larger charadriiform dataset collected by Dr.

O’Connor. In order to examine the relationship between body mass and relative

pneumaticity in charadriiforms, average body masses were taken from Dunning

(1993).

For a historical perspective, relative pneumaticity was mapped on a

charadriiform phylogeny using Mesquite® (Maddison and Maddison, 2007).

Phylogenetic hypotheses for charadriiforms were taken from Livezey and Zusi

(2007).

MicroCT scanning

To examine bone structure in targeted charadriiform and pelecaniform species, selected bones were identified as candidates for micro-computed

tomography scanning. Specifically, a middle cervical vertebra and a free thoracic

vertebra were scanned using a GE eXplore Locus micro-computed tomography

(µCT) scanner housed at Ohio University (Fig. 12). All scans were acquired at an

x-ray tube voltage of 80 kV, a current of 450µA, 1200 views, 400 ms exposure

time, 360 degree scan technique, 2x2 detector bin mode, and an effective pixel size

Gutzwiller Senior Thesis—25

of 0.045mm. Three-dimensional reconstructions were acquired and imported into

an image rendering program called Amira®.

Figure 12. MicroCT scanner housed at Ohio University. www.oucom.ohiou.edu/ou-microct/

Trabecular bone volume to total volume fraction

Trabecular bone volume fraction (BV/TV) = trabecular bone volume total bone volume

To determine the trabecular bone volume fraction (BV/TV), thoracic and

middle cervical vertebrae were examined (Fig. 13). A spherical volume of interest

(VOI) was created for each vertebra using the wrap function within Amira. In

order to account for differences in size and shape among species, the sphere was

created at the cranial end of the vertebra and had a maximum diameter as large as

the trabecular bone volume would allow. Cortical bone was excluded, so the edge

of each sphere hit the boundary of cortical and trabecular bone. This VOI was then

Gutzwiller Senior Thesis—26

exported as a 16 bit 2D tiff image sequence into ImageJ®, where it was converted

to an 8 bit image sequence. This image sequence was imported into a program

called Quant3D® (see Ketcham and Ryan, 2004). An additional user-entered

spherical VOI was created with identical dimensions and overlaid on the sphere

exported from Amira. Using an iterative threshold (see Fajardo et al., 2007),

BV/TV was determined for selected vertebra.

Figure 13. Cross-sectional μCT slice of a middle cervical vertebra from a Great Skua (Catharacta skua). A, cortical bone; B, trabecular bone.

Cortical bone thickness

Within Amira, mean cortical bone thickness (Cb.T) was measured for the

same middle cervical vertebrae for which BV/TV calculations were performed (Fig

13). I created a cross-section located at the most posterior extent of the cranial zygapophyses. On this cross-section, three cortical bone thickness measurements were acquired: ventral, medial, and lateral sides of the centrum. These three measurements were averaged to obtain the mean cortical bone thickness. Mean Gutzwiller Senior Thesis—27

cortical bone thickness was then standardized by dividing by species mean body

mass estimates (Dunning, 1993).

Statistical approaches

Species-specific pneumaticity profiles were regressed on body mass (in

charadriiforms only) using the protocol developed in O’Connor (2004). Body mass

was log10 transformed and pneumaticity profiles were arcsine transformed. Since

pneumaticity was quantified as an index and not an exact measurement of air present

in the bone, the regression was used to identify directionality of trends between

pneumaticity and body mass, not to analyze slope of the trend line. R2 of the trend

line was determined using Microsoft Excel®. Spearman’s rho was used to determine statistical significance of the correlation in SPSS®.

Species mean values and coefficients of variation for the two bone parameters

(BV/TV and Cb.T) were calculated. In SPSS, Kruskal-Wallis (K-independent

samples) and Mann-Whitney tests (pairwise, 2-independent samples) were used to

determine significant differences among the species in the bone structure parameters.

For statistical purposes, one missing data point (in the pelican thoracic trabecular bone

volume fraction) was substituted with the mean.

Gutzwiller Senior Thesis—28

HYPOTHESES and PREDICTIONS

My overarching hypotheses address postcranial pneumaticity at two levels: species comparisons of relative pneumaticity in the whole skeleton and species comparisons of bone structure.

Whole skeleton pneumaticity comparisons:

Using the species-specific pneumaticity indices, I was able to examine the relationships between pneumaticity and body mass (ahistorical) within charadriiforms. I also examined the distribution of relative pneumaticity across charadriiforms. For both charadriiforms and pelecaniforms, I was able to use PI to explore the relationship between relative pneumaticity and foraging specialization.

Hypotheses:

(A) H1: Pneumaticity index (PI) increases with increasing body mass among

species examined, as an adaptation to decouple the mass/volume relationship.

(B) H1: Pneumaticity index (PI) decreases with increasing diving behavior among

species examined, as an adaptation to decrease buoyancy.

(C) H1: Pneumaticity index (PI) increases with increasing soaring behavior among

species examined, as an adaptation to decrease mass and allow for more

energetically efficient flight.

Gutzwiller Senior Thesis—29

Bone structure comparisons:

Using trabecular bone volume fractions (BV/TV) and cortical bone thickness

(Cb.T), I was able to examine the differences in vertebral bone structure between diving specialists (with apneumatic bones) and nondiving specialists (with pneumatic bones).

Hypotheses:

(D) H1: Trabecular bone volume fraction (BV/TV) is lower in nondiving birds, as

an adaptation to further decrease mass.

(E) H1: Cortical bone thickness (Cb.T) is lower in nondiving birds, as an

adaptation to further decrease mass.

RESULTS

Charadriiforms:

Relative whole skeletal pneumaticity

Very little intraspecific variability in relative pneumaticity (PI) was observed for charadriiforms (Fig. 14). For example, neither of the dedicated divers (puffin and murre) pneumatized any portion of their postcranial skeleton (mean PI = 0). The generalized flier (gull) pneumatized 5 of 12 AU's, namely all of the cervical and thoracic vertebrae (mean PI = 0.42). The soaring specialist (skua) pneumatized 6 of

12 AU's, the cervical and thoracic vertebrae and the humerus (mean PI = 0.50).

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Figure 14. Average Pneumaticity Index (PI) values for two dedicated diving specialists (murre and puffin) and two non-diving specialists (gull and skua). Note: where there was no variability within species, no error bars are shown.

Relative pneumaticity and body mass

When my PI data was integrated with a larger charadriiform data set and the arcsine transformed PI was regressed on the log BM, four patterns emerged (Fig. 15).

(1) All of the dedicated diving specialists (Alcidae, including puffins and murres) had completely apneumatic postcranial skeletons (PI = 0). (2) Small-bodied (23-215g) generalized waders (e.g., Scolopacidae, Sternidae) had PI values that ranged narrowly between 0.1 to 0.2 and pneumatized only the vertebrae at the cervicothoracic junction.

(3) The highest PI values were observed in the static soaring skuas (Stercorariidae). In these first three cases, there was no relationship between relative pneumaticity and body size, as evidenced by slope values for each group that were not significantly

Gutzwiller Senior Thesis—31

different from 0. And (4), it was only within the larids (gulls) that a significant positive relationship was observed between PI and body mass (R2=0.53, p≤.001). As body mass increased, the extent of pneumaticity increased. Specifically, smaller- bodied larids (e.g. Franklin’s gull, Larus pipixcan) pneumatized only a small section of the vertebral column whereas larger forms (e.g. Herring gull, Larus argentatus) pneumatized all of the vertebral column excluding the caudal vertebrae.

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skua Gutzwiller Senior Thesis Senior Gutzwiller Gutzwiller Senior Thesis Senior Gutzwiller — — 3 34 3

Figure 15. The log transformed average species body mass regressed upon the arcsine transformed average Pneumaticity Index for each charadriiform species. Each point represents a species; each color represents a charadriiform family. There is a positive relationship between body mass and pneumaticity in Laridae (gulls, green).Gutzwiller Diving specialists Senior Thesis (red)— are1 completely apneumatic. The generalized waders (orange and blue) have intermediate levels of pneumaticity. The soaring specialists (pink) are highly pneumatic.

Postcranial pneumaticity and phylogeny

When relative pneumaticity was mapped onto the charadriiform phylogeny, the most common pneumaticity pattern observed among all charadriiforms involved vertebrae at the cervicothoracic junction (Fig. 16). Deviations from this pattern were observed in alcids, stercorariids, rynchopids, and larids. In Stercorariidae (skuas) and

Rynchopidae (skimmers), pneumaticity is extended throughout the postcranial axial and proximal appendicular skeleton. Larids have variable postcranial axial pneumaticity and postcranial pneumaticity is completely absent in alcids.

Thus, whereas the majority of charadriiforms were static in their pattern of pneumaticity, five relatively closely related families (Alcidae, Stercorariidae,

Rynchopidae, Sternidae and Laridae) were dynamic, showing several patterns of pneumaticity. The static portion of the phylogeny may have shared a phylogenetic constraint, the inability of a phenotype to evolve under the pressure of selection

(Derrickson and Ricklefs, 1988). Phylogenetic constraints are underlain by genetic and developmental limitations that lead to a particular morphology (McKitrick, 1993).

The ancestral morphology may have restricted pneumaticity to one pattern. Likewise, the dynamic portion of the phylogeny may have shared a common ancestor that lacked this phylogenetic constraint or possessed a novel characteristic allowing for the diversification in pneumaticity.

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Figure 16. Phylogenetic hypothesis of Charadriiformes with relative pneumaticity mapped. Blue, cervicothoracic junction only; Green, variable postcranial axial; White, postcranial axial and proximal appendicular; Black, absent. Red dash, diversification event. Outgroup based on Livesey and Zusi (2007).

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Bone Structure

A large degree of intraspecific variability was observed in charadriiform trabecular bone volume fraction in both cervical and thoracic vertebrae. Among all species examined, BV/TV ranged from a fraction of 0.11 to 0.81 for cervical vertebrae

(Fig. 17). In this, gulls exhibited the greatest amount of variability with a range in cervical BV/TV of 0.11 to 0.68 (CoV=0.44; Appendix IV). Among all species examined, BV/TV ranged from 0.16 to 0.66 for thoracic vertebrae (Fig. 18). Thoracic vertebrae were slightly less variable, with the greatest range being seen in skuas (0.16 to 0.37; CoV=0.41). Overall, no significant pattern between BV/TV and species (or foraging specialization) can be observed. Likewise, average cortical bone thickness is highly variable within charadriiforms (Fig. 19; Appendix V). Interestingly, both a non-diver and a diver exhibit relatively thinner cortical bone (gull and murre) and a non-diver and a diver exhibit relatively thicker cortical bone (skua and puffin).

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Figure 17. Box and whisker plots of cervical trabecular bone volume fractions for each charadriiform species. The top of the box denotes the 75th percentile; the bottom of the box denotes the 25th percentile. The horizontal line in the box represents the 50th percentile. The extent of the whiskers denotes the range, excluding outliers (asterisk).

Gutzwiller Senior Thesis—37

Figure 18. Box and whisker plots of thoracic trabecular bone volume fractions for each charadriiform species. The top of the box denotes the 75th percentile; the bottom of the box denotes the 25th percentile. The horizontal line in the box represents the 50th percentile. The extent of the whiskers denotes the range, excluding outliers (asterisk/dot).

Gutzwiller Senior Thesis—38

Figure 19. Box and whisker plots of cortical bone thickness for each charadriiform species. The top of the box denotes the 75th percentile; the bottom of the box denotes the 25th percentile. The horizontal line in the box represents the 50th percentile. The extent of the whiskers denotes the range.

Pelecaniforms:

Relative whole skeleton pneumaticity:

Similar to charadriiforms, there is very little intraspecific variability in PI for

pelecaniforms (Fig. 20). The anhinga, a dedicated diver, has a completely apneumatic

(mean PI = 0) postcranial skeleton. The cormorant, another dedicated diver, variably

pneumatizes one AU, the humerus or the caudal cervical vertebrae (mean PI = 0.09).

The pelican, a soaring specialist, exhibits extensive pneumaticity (mean PI = 0.92).

The only AU not pneumatized in the pelican is the femur. Gutzwiller Senior Thesis—39

Figure 20. Average Pneumaticity Index (PI) values for two dedicated diving specialists (anhinga and cormorant) and non-diving specialist (pelican). Note: where there was no variability within species, no error bars are shown.

Bone structure:

Similar to charadriiforms, some pelecaniform species show a large degree of

variability in cervical trabecular bone volume fraction (Fig. 21; Appendix IV).

Similarly, there is no significant pattern between species (and foraging strategies) and

BT/TV for cervical vertebrae. In contrast, a fairly strong signal emerges when

examining thoracic vertebrae. The pelican has significantly lower thoracic BV/TV

when compared to the cormorant and anhinga (p≤0.05; Fig. 22). A similar pattern is observed for cortical bone thickness, whereby the pelicans exhibited significantly thinner cortical bone for their body mass (p≤0.05; Fig. 23).

Gutzwiller Senior Thesis—40

Figure 21. Box and whisker plots of cervical trabecular bone volume fractions for each pelecaniform species. The top of the box denotes the 75th percentile; the bottom of the box denotes the 25th percentile. The horizontal line in the box represents the 50th percentile. The extent of the whiskers denotes the range.

Gutzwiller Senior Thesis—41

Figure 22. Box and whisker plots of thoracic trabecular bone volume fractions for each pelecaniform species. The top of the box denotes the 75th percentile; the bottom of the box denotes the 25th percentile. The horizontal line in the box represents the 50th percentile. The extent of the whiskers denotes the range.

Gutzwiller Senior Thesis—42

Figure 23. Box and whisker plots of cortical bone thickness for each pelecaniform species. The top of the box denotes the 75th percentile; the bottom of the box denotes the 25th percentile. The horizontal line in the box represents the 50th percentile. The extent of the whiskers denotes the range.

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DISCUSSION

Researching form-function relationships can allow for a greater understanding of the evolutionary pressures that have led to the vast diversification of birds that we see on the planet today. Postcranial pneumaticity is just one aspect of avian morphology that appears to have responded to natural selection. Ahistorical and historical (phylogenetic) perspectives of what pressures may have influenced the evolution of pneumaticity and how bone structure is affected are discussed below.

Postcranial pneumaticity and body size

When examining the entire dataset (Appendix III) of charadriiforms, several patterns emerge. In one clade, the gulls, there is a positive relationship between body mass and the extent of pneumaticity. This means that as interspecific body mass increases, the extent of postcranial pneumaticity also increases. This supports my hypothesis that pneumaticity index (PI) increases with increasing body mass among species examined and supports observations on other neognath clades (e.g., some anseriforms; O’Connor, 2004). This pattern may reflect an evolutionary adaptation by which skeletal pneumatization works to decouple mass/volume relationships

(O’Connor, 2009). Increasing body size has costs and benefits. Having a larger body size may allow a bird greater protection from predators, a wider home range/foraging radius and a wider size range of prey (Peters, 1983; Lindstedt, 1986; Neal, 2004).

However, this increase in body volume is often paralleled by a concomitant increase in body mass. A large body mass means increased energy expenditure during locomotion. Postcranial skeletal pneumaticity may play a role in decoupling this

Gutzwiller Senior Thesis—44

mass/volume relationship, allowing a bird to increase body size without increasing body mass to the same extent.

The correlation is significant among larids, but is not observed in the other clades examined. Laridae is a diverse clade that spans a wide range of body masses.

The positive relationship between body mass and pneumaticity may have not been observed in other groups because in some cases (skuas) the specific species examined do not span a wide enough size range for the pattern to be evident. Perhaps if additional species were acquired and scored for postcranial pneumaticity, similar patterns would be seen in other charadriiform groups. In other cases (e.g., small- bodied waders), it may be the case that since most of the clade is relatively small- bodied that the selective pressure to reduce skeletal density (and thus, whole body density) was not engaged. On the other hand, the lack of this pattern in other groups may suggest that other factors have influenced the evolution of postcranial pneumaticity.

Postcranial pneumaticity and foraging specialization

One factor that may influence the extent of postcranial pneumaticity is foraging specialization. I observed two distinct patterns when examining postcranial pneumaticity in charadriiforms. Birds that specialize in soaring, including skuas, have evolved extensive postcranial pneumaticity that has filled at least half of the postcranial elements examined with air. Birds that specialize in dive foraging, including murres and puffins, have evolved a completely apneumatic postcranial skeleton. These patterns support my hypotheses that pneumaticity index (PI)

Gutzwiller Senior Thesis—45

decreases with increasing diving behavior and increases with increasing soaring behavior. Thus the patterns between pneumaticity and foraging style observed in anseriforms (O’Connor 2004, 2009) are also seen in charadriiforms.

The evolution of flight has allowed birds numerous ecological benefits. They can occupy niches unavailable to land animals and use a wider variety of foraging strategies. However, flight is an energetically costly form of locomotion (Norberg,

1996). The greater the mass of the bird, the more energy expended during flight. The data discussed in this report further support the hypothesis that flight may play a role in the development of pneumaticity. Postcranial pneumaticity may reduce mass to make flight more energetically efficient. This hypothesis is also supported by the pelecaniform data. Large-bodied flying birds (such as the pelican) seem to have altered both behavioral and morphological characteristics (O’Connor, 2009). The pelican utilizes static soaring and is hyperpneumatic, pneumatizing 11 of 12 elements, excluding only the femur.

The observation of significantly reduced or a complete lack of pneumaticity in the postcranial skeleton of dedicated divers also supports the general hypothesis of a relationship between dive foraging and skeletal apneumaticity (O’Connor 2004, 2009).

In both diving charadriiforms (murres and puffins) and diving pelecaniforms

(cormorants and anhingas), pneumaticity was lacking or only found in one anatomical unit. Locomotion underwater requires a great deal of energy. Air in the skeleton would cause increased buoyancy and increased energy expenditure during diving.

Thus, the reduction in postcranial pneumaticity may be an adaptation to reduce

Gutzwiller Senior Thesis—46

buoyancy and make diving more energetically efficient. These results suggest that differing foraging specializations have played a role in the evolution of postcranial skeletal pneumaticity.

Postcranial pneumaticity and phylogeny

The common pattern of pneumaticity (located at the cervicothoracic junction) was observed in the majority of charadriiform families (Fig. 16). In fact, only four families deviate from this pattern: Alcidae, Stercorariidae, Rynchopidae, and Laridae.

Interestingly, these four families lie within a group of five relatively closely related families within the charadriiform phylogeny. However, they do not deviate from the common pattern of pneumaticity in the same manner. Larids have evolved increased pneumaticity that is variable along the axial skeleton. Stercorariidae and Rynchopidae have increased the extent of pneumaticity even further to include most of the postcranial axial and proximal appendicular skeleton. Alcids, on the other hand completely lack postcranial pneumaticity. These results are quite interesting because it seems that somewhere in the evolutionary history of these clades a diversification in the extent of pneumaticity occurred (red dash in phylogeny; Fig. 16). This may suggest that, although these four families do not all share the same pattern of pneumaticity, they share a common ancestor that evolved the “ability” to deviate from the common pattern. This “ability” may have come in the form of a morphological restraint that was lifted. From that point on, birds would have been able to adapt their postcranial pneumaticity according to different environmental pressures. Although it would be hard to say what ancestral adaptation occurred, the patterns observed in

Gutzwiller Senior Thesis—47

relative charadriiform pneumaticity suggest that phylogenetic constraints may have played a role in the evolution of this morphological trait.

Postcranial pneumaticity and bone structure

Past studies have hypothesized that birds that specialize in flight to forage have decreased energy expenditure by evolving decreased bone mass (trabecular bone volume and cortical bone thickness; e.g. some anseriformes; Fajardo et al., 2007).

Likewise, researchers hypothesized that dedicated diving birds have evolved increased trabecular bone volume and cortical bone thickness to increase mass and displace low- density bone marrow. Increased density in diving birds would allow them to be more neutrally buoyant and increase the energetic efficiency of diving. The charadriiform dataset examined in this project do not support these hypotheses. Charadriiforms exhibit a large degree of intraspecific variability in both trabecular bone volume fraction and cortical bone thickness (see CoV’s in Appendix IV and V). The two dedicated diving birds (murres and puffins) do not have significantly different bone structure parameters than the flight specialists (gull and skua). These results suggest that the specific bone structure parameters that I examined are not greatly different in pneumatic versus apneumatic bone. Other factors may influence bone structure to a greater extent than foraging style and pneumaticity. For example, the size, age, sex, and health at the time of death of the individuals included in this study may account for the observed intraspecific and interspecific variability. Although there is no significant difference in trabecular bone volume and cortical bone thickness between charadriiform pneumatic and apneumatic bone, it is important to recall that there is

Gutzwiller Senior Thesis—48

one major difference. The non-bone spaces in pneumatic bone are filled with air, while the same spaces in apneumatic bone are marrow-filled.

In the pelecaniform cervical trabecular bone volume fraction, an intraspecific variability similar to that seen in charadriiforms is observed. In contrast, distinct patterns were observed in thoracic trabecular bone volume fraction and cervical cortical bone thickness. The pelican, a soaring specialist, has a significantly lower thoracic trabecular bone volume fraction and cortical bone thickness than the dedicated divers, the cormorant and anhinga. This may support my initial hypotheses that bone structure can be influenced by the foraging strategy (and presence or absence of vertebral pneumaticity) of a bird.

However, other factors could also have importance. The cervical and thoracic trabecular bone volume fractions (BV/TV) of the pelican and anhinga are good examples. Cervical and thoracic BV/TV of the anhinga are not significantly different from each other. Both values are around 0.51. But in the pelican, thoracic BT/TV is significantly lower than cervical BV/TV (0.14 and 0.50 respectively; p≤0.05). This may suggest that the location of the vertebra (cervical or thoracic) influences the bone structure. The degree of this influence may depend on the species. Although pelicans are specialized in flight and are expected to have low trabecular bone volume, they have a characteristically large and heavy bill. The pressure applied to base of the neck by this large bill may require them to have more trabecular bone in the cervical vertebrae than the thoracic vertebrae for structural support. The anhinga, conversely,

Gutzwiller Senior Thesis—49

may not have differential pressures on different vertebrae, so the cervical and thoracic

trabecular bone volume fractions are similar.

My data suggest that, although there is some support that bone structure can be

influenced by the presence or absence of air in the bones of dedicated diving and

flying foraging specialists, other aspects the individual’s and species biology may

influence this bone structure to a greater extent.

CONCLUSIONS

After examining relative postcranial skeletal pneumaticity through both

historical and ahistorical perspectives at the level of the whole skeleton, I conclude

that several factors, including morphological pressures (mass/volume relationships)

and ecological pressures (foraging style) may have influenced the evolution of this

morphological trait in charadriiforms, an ecologically diverse order of neognath birds.

Some of these ecological hypotheses are also supported within pelecaniforms.

However, it is also necessary to keep a historical (phylogenetic) perspective.

Ancestral morphology may have restricted the evolution of postcranial pneumaticity in some families and the lack of this limitation may have allowed for diversity in

pneumaticity in other families. It seems that the evolution of postcranial pneumaticity

has been a dynamic process, in some cases constrained by phylogeny and in other

cases shaped by environmental pressures for birds to change in size and to occupy new

niches (aerial and aquatic). The same morphological and ecological factors that may

Gutzwiller Senior Thesis—50

have influenced the evolution of pneumaticity could have also influenced other aspects of a skeletal morphology, including bone structure.

Future studies

Future studies may be able to further tease apart the many factors that influence pneumaticity. By increasing sample size and including more species, researchers may be able observe a greater degree of clarity in the relationships of body mass and bone structure with pneumaticity. More specifically, with increased sample size, phylogenetic comparative approaches (for example, Phylogenetically-

Independent Contrasts) may be employed to further refine the statistical framework for examining these relationships. It would also be interesting to explore the phylogenetic history of pneumaticity in other clades of birds where a large degree of pneumatic variability exists (pelecaniforms, for example) to see what patterns emerge. Similarly, the degree to which phylogenetic constraints may have influenced charadriiforms may be examined in other orders of neognath birds. Finally, research on the ontogeny of pneumaticity within an individual could allow for a greater understanding of the process and what factors may influence it.

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Richardson F. 1939. Functional aspects of the pneumatic system of the California brown pelican. Condor 41:13–17. Ryan P. 2007. Diving in shallow water: the foraging ecology of darters (Aves: Anhingidae). J Avian Biol 38:507-514. Schepelmann K. 1990. Erythropoietic bone marrow in the pigeon: development of its distribution and volume during growth and pneumatization of bones. J Morphol 203:21–34. Simons E. 2010. Forelimb skeletal morphology and flight mode evolution in pelecaniform birds. Zoology 113:39-46. Thomas G. et al. 2004. A supertree approach to shorebird phylogeny. BMC Evolutionary Biology 4: doi:10.1186/1471-2148-4-28. White C. 2008. Pedestrian locomotion energetics and gait characteristics of a diving bird, the great cormorant, Phalacrocorax carbo. J Comp Physiol B 178:745–754. Witmer LM. 1990. The craniofacial air sac system of Mesozoic birds (Aves). Zool J Linn Soc 100:327–378. Votier S. et al. 2003. Assessing the diet of great skuas, Catharacta skua, using five different techniques. Polar Biol (2003) 26: 20–26.

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APPENDICES

Appendix I Abbreviations of Anatomical Units Used for Pneumaticity Index H, Humerus D, Diving F, Femur G, Non-diver CRC, Cranial Cervical Vertebrae PI, Pneumaticity Index MC, Middle Cervical Vertebrae CAC, Caudal Cervical Vertebrae Pneumatic denoted by (+) CRT, Cranial Thoracic Vertebrae Apneumatic denoted by (-) CAT, Caudal Thoracic Vertebrae CA, Caudal Vertebrae TT, Tibiotarsus/Fibula/Tarsometatarsus TP, Tarsus/Pes AB, Antibrachium CM, Carpus/Manus

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Appendix II: Pneumatic Profiles for Focal Charadriiform and Pelecaniform Species Focal Charadriiform Pneumatic Profiles Species ID Foraging H F CRC MC CAC CRT CAT CA TT TP AB CM PI Uria aalge CM S-14348 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-3958 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-11577 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-399 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Gutzwiller Senior Thesis Senior Gutzwiller Uria aalge CM S-11929 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-8250 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-15183 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-11725 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-14180 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Uria aalge CM S-11930 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Fratercula arctica CM S-11726 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 —

58 Fratercula arctica CM S-398 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00

Fratercula arctica CM S-11794 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Catharacta skua CM S-10116 G (+) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.50 Catharacta skua CM S-11606 G (+) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.50 Larus occidentalis CM S-11756 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-11758 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-9877 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-12321 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-11054 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-11776 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42

Larus occidentalis CM S-13890 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-11757 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-18428 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42 Larus occidentalis CM S-11105 G (-) (-) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) 0.42

Focal Pelecaniform Pneumatic Profiles Species ID Foraging H F CRC MC CAC CRT CAT CA TT TP AB CM PI Anhinga anhinga CM S-13826 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00

Anhinga anhinga CM S-13811 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00

Gutzwiller Senior Thesis Senior Gutzwiller Anhinga anhinga CM S-14362 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00

Anhinga anhinga CM S-14313 D (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.00 Phalacrocorax auritus OUVC 10479 D (+) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.08 Phalacrocorax auritus OUVC 9771 D (+) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.08 Phalacrocorax auritus OUVC 10234 D (+) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.08 Phalacrocorax auritus OUVC 10233 D (-) (-) (-) (-) (+) (-) (-) (-) (-) (-) (-) (-) 0.08 Phalacrocorax auritus OUVC 10291 D (+) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 0.08 —

59 Phalacrocorax auritus OUVC 9772 D (-) (-) (-) (-) (+) (-) (-) (-) (-) (-) (-) (-) 0.08

Phalacrocorax auritus OUVC 10241 D (-) (-) (-) (-) (+) (-) (-) (-) (-) (-) (-) (-) 0.08 Phalacrocorax auritus OUVC 10505 D (+) (-) (-) (-) (+) (-) (-) (-) (-) (-) (-) (-) 0.17 Phalacrocorax auritus OUVC 10482 D (-) (-) (-) (-) (+) (-) (-) (-) (-) (-) (-) (-) 0.08 Pelecanus occidentalis OUVC 10433 G (+) (-) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) 0.92

Pelecanus occidentalis OUVC 10484 G (+) (-) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) 0.92

Appendix III: charadriiform pneumaticity profiles

Order Taxon n CRC MC CAC CRT CAT CA HU FM TT TP AB CM logBM arcsinPI LS Charadriiformes RYNCHOPIDAE Rynchops niger 4 + + + + + - + - - - - - 2.47 0.52 G

STERCORARIIDAE Gutzwiller Senior Thesis Senior Gutzwiller Catharacta skua 2 + + + + + - + - - - - - 2.65 0.52 G Stercorarius pomarinus 2 + + + + + - + - - - - - 2.84 0.52 G Stercorarius parasiticus 1 + + + + + - + - - - - - 2.66 0.52 G Stercorarius longicaudus 3 + + + + + - + - - - - - 2.47 0.52 G

ALCIDAE Alca torda* 7 ------2.86 0.00 D —

60 Aethia cristatella 4 ------2.42 0.00 D

Aethia pusilla 6 ------1.92 0.00 D Alle alle 1 ------2.21 0.00 D Uria aalge* 15 ------3.00 0.00 D Uria lomvia 2 ------2.98 0.00 D Fratercula arctica* 7 ------2.58 0.00 D Fratercula cirrhata 4 ------2.89 0.00 D Cephus columba 6 ------2.69 0.00 D Cephus grylle 3 ------2.61 0.00 D Synthliborhamphus antiquus 1 ------2.31 0.00 D

Ptychoramphus aleuticus 2 ------2.27 0.00 D Cyclorrhynchus psittacula 1 ------2.41 0.00 D Bradyrhampus marmoratus 2 ------2.35 0.00 D Cerorhinca moncerata 4 ------2.72 0.00 D

JACANIDAE Jacana spinosa 4 - - + + ------1.97 0.17 G Actophilornis africanus 1 - - + + ------2.28 0.17 G

ROSTRATULIDAE

Gutzwiller Senior Thesis Senior Gutzwiller Rostratula semicollaris 2 ? ? ? ? ? ? ------1.89 G

SCOLOPACIDAE Scolopax minor 5 - - + + ------2.29 0.17 G Scolopax rusticola* 1 - + + + ------2.49 0.25 G Gallinago gallinago 4 - - + + ------2.09 0.17 G Limosa fedoa 1 - - + + ------2.57 0.17 G —

61 Numenius americana 2 - + + + ------2.78 0.25 G

Bartramia longicauda 3 - - + + ------2.17 0.17 G Tringa totanus 1 - - + + ------2.11 0.17 G Tringa solitaria 3 - - + + ------1.68 0.17 G Tringa flavipes 8 - - + + ------1.91 0.17 G Tringa melanoleuca 8 - - + + ------2.23 0.17 G Actitis macularius 8 - - + + ------1.60 0.17 G Heteroscelus incanus 1 - - + + ------2.04 0.17 G Catoptrophorus semipalmatus 2 - - + + ------2.33 0.17 G

Arenaria interpres 6 - - + + ------2.05 0.17 G Arenaria melanocephala 3 - - + + ------2.07 0.17 G Limnodromus scolpaceus 8 - - + + ------2.01 0.17 G Limnodromus griseus 3 - - + + ------2.05 0.17 G Aphriza virgata 4 - - + + ------2.27 0.17 G Calidris alba 4 - - + + ------1.76 0.17 G Calidris canutus 4 - - + + ------2.13 0.17 G Calidris pusilla 4 - - + + ------1.49 0.17 G Calidris minutilla 4 - - + + ------1.36 0.17 G Calidris mauri 4 - - + + ------1.36 0.17 G

Gutzwiller Senior Thesis Senior Gutzwiller Calidris alpina 2 - - + + ------1.73 0.17 G Calidris bairdii 2 - - + + ------1.60 0.17 G Calidris fuscicollis 2 - - + + ------1.54 0.17 G Calidris maritima 2 - - + + ------1.91 0.17 G Calidris himantopus 2 - - + + ------1.75 0.17 G Calidris melanotos 2 - - + + ------1.94 0.17 G Tringites subruficollis 3 - - + + ------1.78 0.17 G —

62 Steganopus (Phalaropus) tricolor 3 - - + + ------1.75 0.17 G

Phalaropus lobatus 6 - - + + ------1.52 0.17 G Phalaropus fulicaria 4 - - + + ------1.73 0.17 G

THINOCORIDAE Thinocorus rumicivorus 1 - - + ------1.64 0.08 G

BURHINIDAE Burhinus bistriatus 4 - - + + ------2.90 0.17 G

HAEMATOPODIDAE Haematopus ostralegus 2 - +/- + + ------2.72 0.25 G

RECURVIROSTRIDAE Recurvirostra avocetta 1 - + + + ------2.49 0.25 G Recurvirostra americana 13 - +/- + + ------2.50 0.25 G Himantopus mexicanus 9 - +/- + + ------2.22 0.25 G

Gutzwiller Senior Thesis Senior Gutzwiller GLAREOLIDAE Pluvianus aegyptius 1 - - + + ------1.91 0.17 G Rhinoptilus chalcopterus 1 - - + + ------2.18 0.17 G Rhinoptilus africanus 1 - - + + ------1.92 0.17 G

CHARADRIIDAE Pluvialis squatarola 1 - - + + ------2.34 0.17 G —

63 Charadrius semipalmatus 2 - - - +/------1.67 0.08 G

Charadrius wilsonia 1 - - - +/------1.74 0.08 G Charadrius alexandrinus 1 - - - +/------1.61 0.08 G Charadrius collaris 2 - - - +/------1.45 0.08 G Charadrius vociferus 4 - - - +/------1.98 0.08 G Vanellus vanellus* 2 ------2.34 0.00 G Vanellus spinosus 2 ------2.18 0.00 G Vanellus chilensis 2 ------2.51 0.00 G

LARIDAE Larus philadelphia 4 - - + + ------2.33 0.17 G Larus novaehollandiae 2 +/- + + + + ------2.51 0.43 G Larus ridibundus* 13 - - + + ------2.45 0.17 G Larus atricilla 8 - - + + + ------2.51 0.25 G Larus pipixcan 2 - - + + ------2.45 0.17 G Larus melanocephalus 1 - - + + ------2.41 0.17 G Larus heermani 4 +/- + + + + ------2.70 0.43 G Larus canus* 5 - + + + + ------2.61 0.34 G Larus delawarensis 6 + + + + + ------2.71 0.43 G Gutzwiller Senior Thesis Senior Gutzwiller Larus occidentalis 11 + + + + + ------3.00 0.43 G Larus dominicanus 2 + + + + + ------2.95 0.43 G Larus marinus* 5 + + + + + ------3.22 0.43 G Larus argentatus* 17 + + + + + ------3.06 0.43 G Larus californicus 6 + + + + + ------2.78 0.43 G Larus glaucescens 8 + + + + + ------3.00 0.43 G Rissa brevirostra 1 + + + + ------2.59 0.34 G —

64 Xema sabini 2 + + + + ------2.28 0.34 G

STERNIDAE Sterna caspia 2 - - + + ------2.82 0.17 G Sterna maxima 2 - - + + ------2.63 0.17 G Sterna sandvicensis 2 - - + + ------2.32 0.17 G Sterna elegans 2 - - + + ------2.41 0.17 G Sterna dougallii 2 - +/- + + ------2.04 0.25 G Sterna paradisaea 2 - +/- + + ------2.04 0.25 G

Sterna hirundo* 4 - +/- + + ------2.08 0.25 G Sterna vittata 2 - - + + ------2.15 0.17 G Sterna forsteri 2 - - + + ------2.20 0.17 G Sterna antillarum 3 - - + + ------1.63 0.17 G Sterna anaethetus 1 - - + + ------1.98 0.17 G Sterna fuscata 2 - - + + ------2.26 0.17 G Sterna aleutica 2 - - + + ------2.08 0.17 G Larosterna inca 3 - - + + ------2.26 0.17 G Anous stolidus 1 - - + + ------2.30 0.17 G

Gutzwiller Senior Thesi Senior Gutzwiller Gygis alba 1 - - + + ------2.05 0.17 G Chlidonias hybridus 1 - - + + ------1.94 0.17 G Phaetusa simplex 1 - - + + ------2.37 0.17 G

s —

65

Appendix IV Trabecular bone volume fraction species means and coefficients of variance Order Species Element Mean BVTV COV Charadriiformes Skua CV 0.24 0.01 TV 0.26 0.41 Gull CV 0.30 0.44 TV 0.33 0.16 Puffin CV 0.25 0.18 TV 0.25 0.22 Murre CV 0.39 0.28 TV 0.51 0.14 Pelecaniformes Cormorant CV 0.42 0.02 TV 0.36 0.12 Pelican CV 0.50 0.14 TV 0.14 0.20 Anhinga CV 0.52 0.07 TV 0.51 0.06

Appendix V Adjusted cortical bone thickness species means and coefficients of variance Order Species Element Mean Adj. Cb.T COV Charadriiformes Skua CV 7.10E-05 0.05 Gull CV 3.38E-05 0.13 Puffin CV 7.87E-05 0.22 Murre CV 3.36E-05 0.13 Pelecaniformes Cormorant CV 2.14E-05 0.13 Pelican CV 6.30E-06 0.18 Anhinga CV 2.97E-05 0.20

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