TUPAIID MASTICATORY ANATOMY AND THE

APPLICATION OF EXTANT ANALOGS TO RECONSTRUCTING

PLESIADAPIFORM JAW ADDUCTORS

by

Heather L. Kristjanson

A dissertation submitted to Johns Hopkins University in conformity with the

requirements for the degree of Doctor of Philosophy

Baltimore, Maryland

June 2019

© 2019 Heather L. Kristjanson

All Rights Reserved i

v

Abstract

The plesiadapiforms, archaic stem primates first appearing in the Paleocene

66-63 Ma), are integral to investigating the ecological context for primate origins emphasizing diet as a key factor. Fossil evidence comprised almost entirely of jaw fragments has led to dental analyses dominating most dietary research. Yet despite a tremendous diversity in jaw morphologies, no analyses have focused on quantifying the functional morphology of non-dental parts of the plesiadapiform masticatory apparatus, resulting in a chronic deficit in the study of mandibular morphology and muscles of mastication. Non-dental parts of the masticatory apparatus refine dental-based dietary inferences. This project aims to reconstruct plesiadapiform muscles of mastication using both a tupaiid (treeshrews: Order

Scandentia) and strepsirrhine (lemurs, lorises and galagos: Order Primates) model.

These two taxa form an extant phylogenetic bracket around plesiadapiforms and will serve as extant analogs during reconstruction of their masticatory muscles.

Jaw adductor muscles of tupaiid treeshrews were dissected and their architecture described. Muscle mass and fiber architecture were measured and used to calculate the physiological cross-sectional area (PCSA). Results indicate that tupaiid jaw adductor muscle mass, fiber length, temporalis muscle PCSA, and masseter muscle PCSA scale positively and allometrically to jaw length, while medial pterygoid PCSA scales isometrically. Diffusible Iodine-based Contrast-enhanced

ii

MicroCT scans of two tupaiid specimens show promise as an alternative method to traditional dissection.

PCSA in tupaiids was found to correlate to mandibular muscle insertion area, which was then combined with strepsirrhine data to infer jaw adductor dimensions in a sample of plesiadapiform primates. Reconstructing PCSA in this sample suggests niche partitioning in insectivorous species, dental convergence in frugivorous species and specialization in folivorous species. From the reconstructed

PCSA, bite force was estimated along the tooth row for plesiadapiforms and compared to estimates in treeshrews and strepsirrhines. In all cases, bite force increased distally along the tooth row and was greatest in specialized extant folivores and weakest in insectivorous plesiadapiforms.

Incorporating data from a variety of sources including dental, mandibular, muscular, and biomechanical metrics provides a more comprehensive picture of the challenges and opportunities proffered by a unique mandibular morphology than can be painted by looking at dental evidence alone.

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Thesis Committee Members

Jonathan Perry, Ph.D. (dissertation advisor)

Associate Professor, Center for Functional Anatomy and Evolution

Johns Hopkins University School of Medicine

Siobhán Cooke, Ph.D.

Assistant Professor, Center for Functional Anatomy and Evolution

Johns Hopkins University School of Medicine

Sharlene Santana, Ph.D.

Associate Professor, Department of Biology

Curator of Mammals, Burke Museum

University of Washington

Eric Sargis, Ph.D.

Professor, Department of Anthropology

Curator of Mammals and Vertebrate Paleontology, Yale Peabody Museum of Natural

History

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Acknowledgements

I thank the American Museum of Natural History and the Staatliches

Museum Fur Naturkunde (R. Ziegler) for access to plesiadapiforms in their care. I thank the United States National Museum of Natural History-Smithsonian

Institution (D. Lunde), Yale University (G. Aronsen and E. Sargis), and the Max

Planck Florida Institute for Neuroscience for access to treeshrew specimens. I thank the Shared Materials Instrumentation Facility at Duke University (D. Boyer, J.

Gladman) and the Gignac Lab at Oklahoma State University (P. Gignac) for the use of their facilities.

I thank the American Museum of Natural History, American Association of

Anatomists, and Society for Vertebrate Paleontology for funding.

I thank the members of my committee, Drs. Siobhán Cooke, Sharlene Santana, and Eric Sargis, and Drs. Mary Silcox, Doug Boyer, Paul Gignac, and Chris Kirk for helpful advice. I would also like to thank Arlene Daniel, current and former faculty members Kenneth Rose, David Weishampel, Valerie DeLeon, Chris Ruff, Elizabeth St.

Clair, Janine Chalk, and Terry Mitchell, as well as former and current students from the Center for Functional Anatomy and Evolution for their friendship, expertise, and advice over the years. A special thank you to my fieldwork companions Heather

Ahrens, Stephanie Canington, Tony Harper, Katrina Jones, Kristen Prufrock, Nicky

Squyres, and Kaya Zelazny for the good times and memories.

v

I would especially like to thank my advisor, Dr. Jonathan Perry, for all of his advice and friendship over the years; it was a lot of fun bonding over our shared pop-culture references and love of all things purple.

To my family, who supported me on my journey in the pursuit of knowledge, it was your love, encouragement, and stability that made this possible. In particular,

I thank my dad for instilling in me the curiosity to embark upon this path and my mom for the perseverance to see it through.

These acknowledgements would not be complete without thanking my friend and fellow student, Rachel, for her confidence in me, her support both as a colleague and friend, and always providing an excuse to drink tea. Whether it was a midnight dissection or midnight wedding flower arranging, you were always there for me.

Finally, I thank my husband, Steve, for being an invaluable source of encouragement and support; it seems fitting that it was anatomy that brought us together and I am very grateful to always have you by my side.

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Contents

Abstract ...... ii

Acknowledgements ...... v

List of Tables ...... xiii

List of Figures ...... xv

Chapter 1: Introduction ...... 19

1.1 Primate origins and the case for diet ...... 19

1.2 Introduction to plesiadapiforms ...... 22

1.2.1 Overview ...... 22

1.2.2 Previous dietary research on plesiadapiforms ...... 24

1.3 Relationships between primate morphology and diet ...... 27

1.3.1 Categorizing diet ...... 28

1.3.2 Muscles of Mastication ...... 30

1.3.2.1 Applications of iodine staining for reconstructive work ...... 31

1.3.2 The primate mandible...... 32

1.3.2.1 Corpus ...... 33

1.3.2.2 Symphysis ...... 35

1.4 Non-dietary factors influencing primate mandibular morphology ...... 36

1.4.1 Food material properties ...... 37

1.4.2 Sexual dimorphism and size ...... 38

1.5 Reconstructing diet in the primate fossil record ...... 40

vii

1.5.1 Establishing modern analogs for plesiadapiform by constructing an extant phylogenetic bracket ...... 42

1.5.1.1 Plesiadapiform reconstruction ...... 44

1.5.2 Introduction to tupaiids ...... 45

1.5.2.1 Systematics and ecology ...... 45

1.5.2.2 Tupaiid suitability as an extant analog ...... 47

1.6 Goals and hypotheses ...... 49

1.6.1 Goals and Hypotheses: Chapter Two ...... 49

1.6.2 Goals and Hypotheses: Chapter Three ...... 49

1.6.3 Goals and Hypotheses: Chapter Four ...... 50

1.6.4 Goals and Hypotheses: Chapter Five ...... 50

1.6.5 Goals and Hypotheses: Chapter Six ...... 51

1.7 Significance of this study ...... 51

Chapter 2: An Overview of Tupaiid Masticatory Musculature ...... 53

2.1 Introduction ...... 53

2.2 Materials ...... 56

2.3 Methods ...... 57

2.3.1 Removal of the masseter muscle group ...... 59

2.3.2 Removal of the temporalis muscle group ...... 61

2.3.3 Removal of the masseter muscle group ...... 63

2.4 Results ...... 66

2.4.1. Superficial Masseter ...... 66

2.4.2. Deep Masseter ...... 71

viii

2.4.3. Zygomatico-mandibularis ...... 74

2.4.4. Zygomatic Temporalis ...... 77

2.4.5. Superficial Temporalis ...... 80

2.4.6. Deep Temporalis ...... 82

2.4.7. Medial Pterygoid ...... 86

2.5 Discussion ...... 88

2.5.1 Comparisons to previous treeshrew dissections ...... 88

2.5.2. Comparisons to C. Volans ...... 95

2.5.3. Comparisons to strepsirrhine primates ...... 97

2.6 Conclusions ...... 100

Chapter 3: Applications of iodine staining to tupaiid masticatory musculature ...... 104

3.1 Introduction ...... 104

3.2 Materials ...... 107

3.3 Methods ...... 110

3.3.1. Diffusible Iodine-based Contrast-Enhanced Computed Tomography ..... 110

3.3.2. MicroCT scanning ...... 112

3.4 Results ...... 112

3.5 Discussion ...... 117

3.5.1 Dissection and muscle architecture ...... 117

3.5.2 Muscle volume ...... 119

3.5.3 Future Work ...... 121

3.6 Conclusions ...... 123

ix

Chapter 4: Tupaiid Jaw Adductor Properties and their Suitability for Plesiadapiform

Dietary Reconstruction ...... 125

4.1 Introduction ...... 125

4.2 Materials ...... 128

4.3 Methods ...... 131

4.3.1 Muscle mass measurements ...... 131

4.3.2 Fiber length measurements ...... 131

4.3.3 Calculating Physiological Cross-sectional Area ...... 133

4.3.4 Scaling Calculations...... 133

4.4 Results ...... 135

4.4.1 Measurements ...... 135

4.4.2 Scaling ...... 135

4.5 Discussion ...... 148

4.5.1 Muscle Mass ...... 148

4.5.2 Fiber Length ...... 151

4.5.3 PCSA ...... 154

4.5.4 Implications for Diet ...... 158

4.5.5 Caveats ...... 165

4.6 Conclusions ...... 167

Chapter 5: Reconstructing Jaw Adductor Muscles in Plesiadapiformes and the

Implications for Dietary Interpretations ...... 170

5.1 Introduction ...... 170

x

5.2 Materials ...... 176

5.3 Methods ...... 179

5.3.1 Insertion Area Measurements ...... 179

5.3.2 Osteological Proxy Validations ...... 183

5.3.3 Calculating Physiologic Cross-sectional Area ...... 184

5.4 Results ...... 185

5.4.1 Osteological Proxy Validations ...... 185

5.4.2 Plesiadapiform PCSA reconstruction ...... 202

5.5 Discussion ...... 204

5.5.1 PCSA ...... 204

5.5.2 Implications for Plesiadapiform Diet ...... 215

5.5.2.1 Omnivory in Paromomyids ...... 216

5.5.2.2 Frugivorous Convergence ...... 217

5.5.2.3 Insectivorous Niche Partitioning ...... 218

5.5.2.4 Plesiadapid Evolution ...... 220

5.5.2.5 Caveats ...... 221

5.6 Conclusions ...... 224

Chapter 6: Biomechanically modelling the plesiadapiform mandible and estimating bite force ...... 227

6.1 Introduction ...... 227

6.2 Materials ...... 231

6.3 Methods ...... 231

6.3.1. PCSA ...... 232 xi

6.3.2. Calculating Muscle Group Orientation ...... 232

6.3.3. Calculating the Jaw Adductor Resultant ...... 234

6.3.4. Measuring the Lever Arm ...... 235

6.3.5. Measuring the magnitude of the moment arm of the jaw adductor resultant ...... 236

6.3.6. Calculating the moment arm of bite force ...... 237

6.3.7. Calculating Bite Force ...... 237

6.4 Results ...... 238

6.5 Discussion ...... 250

6.5.1 Bite Force ...... 250

6.5.2 Implications for plesiadapiform diet ...... 255

6.5.2.1 Frugivory in the fossil record ...... 258

6.5.2.2 Exudate and nectar feeding in the fossil record ...... 260

6.5.3 Fallback foods and the importance of food mechanical properties ...... 262

6.5.4 Limitations of the model and future work ...... 264

6.6 Conclusions ...... 266

Chapter 7: Conclusions ...... 269

7.1 Summary ...... 269

7.1.2 Tupaiid musculature ...... 269

7.1.3 DiceCT ...... 270

7.1.4 Osteological proxy correlations ...... 271

7.1.5 Reconstruction of plesiadapiform PCSA ...... 272

7.1.6 Bite force estimations ...... 274

xii

7.2. Future research directions ...... 275

Works Cited ...... 277

xiii

List of Tables

Table 2.1: Specimens dissected ...... 58

Table 2.2 Muscle equivalencies1 ...... 91

Table 3.1: Parameters for staining and CT scanning ...... 110

Table 3.2: Comparison of muscle volume ...... 118

Table 4.1: Specimens in this study ...... 131

Table 4.2: Muscle mass measurements (g) ...... 137

Table 4.3: Fiber length measurements (cm) ...... 138

Table 4.4: Physiological Cross-sectional Area (cm2) ...... 139

Table 4.5: Regression statistics for variables against jaw length cubed ...... 140

Table 4.6: Tupaiid dietary categories and PCSA muscle percentages ...... 163

Table 4.7: Strepsirrhine dietary categories and PCSA muscle percentages ...... 164

Table 5.1: Plesiadapiform specimens ...... 179

Table 5.2: Extant specimen muscle insertion areas ...... 187

Table 5.3: Statistics for RMA regressions onto jaw length squared ...... 192

Table 5.4: Statistics for OLS regressions onto jaw length ...... 196

Table 5.5: Statistics for RMA regressions of PCSA onto insertion area ...... 200

Table 5.6: Reconstructed plesidapiform muscle insertion areas (cm²) and PCSA

(cm²) ...... 204

Table 5.7: Plesiadapiform jaw adductor PCSA percentages ...... 209

Table 6.1 Measurements and calculations for estimating bite force in tupaiids ...... 240

xiv

Table 6.2: Measurements and calculations for estimating bite force in strepsirrhines

...... 241

Table 6.3: Measurements and calculations for estimating bite force in plesiadapiforms ...... 242

Table 6.4: Load arm measurements and estimations of bite force in tupaiids ...... 243

Table 6.5: Load arm measurements and estimations of bite force in strepsirrhines

...... 244

Table 6.6: Load arm measurements and estimations of bite force in plesiadapiforms

...... 245

Table 6.7: Regression statistics for bite force estimations ...... 250

xv

List of Figures

Figure 2.1: Exposed muscles of mastication ...... 59

Figure 2.2: Revealing deep masseter tendon ...... 61

Figure 2.3: Origins of deep temporalis muscle ...... 65

Figure 2.4: Deep temporalis insertion ...... 66

Figure 2.5: Removal of hemimandible ...... 67

Figure 2.6: Superficial masseter ...... 69

Figure 2.7: Ridge for insertion of superficial masseter ...... 70

Figure 2.8: Strutting around malar foramen ...... 71

Figure 2.9: Deep masseter tendon ...... 73

Figure 2.10: Insertion for deep masseter ...... 74

Figure 2.11: Zygomatico-mandibularis aponeurosis ...... 76

Figure 2.12: Zygomatico-mandibularis muscle fibers ...... 77

Figure 2.13: Large zygomatic temporalis ...... 80

Figure 2.14: Tendon of insertion for the zygomatic temporalis ...... 81

Figure 2.15: Superficial temporalis overlying the deep temporalis ...... 82

Figure 2.16: Deep temporalis aponeurosis ...... 84

Figure 2.17: Temporal tendon on deep temporalis ...... 85

Figure 2.18: Temporal tendon as seen after removal of the muscle ...... 86

Figure 2.19: Medial pterygoid muscle after removal ...... 88

Figure 2.20: Aponeurosis arrangement of the temporalis muscle group ...... 94

xvi

Figure 3.1: Tendon of the Temporalis muscle ...... 114

Figure 3.2: Fiber direction in the masseter muscle group ...... 115

Figure 3.3: Fiber direction in the masseter muscle group ...... 116

Figure 3.4: 3D modeling muscles of mastication ...... 123

Figure 4.1: Muscle Chemical Dissection ...... 133

Figure 4.2: RMA regression of body mass (g) upon jaw length cubed (cm3)...... 142

Figure 4.3: RMA regression of jaw adductor muscle mass (g) upon jaw length cubed

(cm3) ...... 144

Figure 4.4: RMA regression of fiber length (cm) upon jaw length (cm) ...... 145

Figure 4.5: RMA regression of masseter PCSA (cm2) upon jaw length squared (cm2)

...... 147

Figure 4.6: RMA regression of temporalis PCSA (cm2) upon jaw length squared (cm2)

...... 148

Figure 4.7: RMA regression of medial pterygoid PCSA (cm2) upon jaw length squared (cm2) ...... 149

Figure 4.8: Comparing tupaiid and strepsirrhine jaw adductor muscle mass ...... 151

Figure 4.9: Comparing tupaiid and strepsirrhine jaw adductor fiber length ...... 154

Figure 4.10: Comparing tupaiid and strepsirrhine masseter PCSA ...... 156

Figure 4.11: Comparing tupaiid and strepsirrhine temporalis PCSA ...... 157

Figure 4.12: Comparing tupaiid and strepsirrhine medial pterygoid PCSA ...... 158

Figure 4.13: Comparison of jaw adductor muscle group percentages by taxon ...... 161

xvii

Figure 5.1: Measurements for calculating temporalis insertion area ...... 181

Figure 5.2: Measurement for calculating masseter insertion area ...... 182

Figure 5.3: Measurement for calculating medial pterygoid insertion area ...... 182

Figure 5.4: RMA regression of MIA (cm2) upon jaw length squared (cm2) ...... 188

Figure 5.5: RMA regression of TIA (cm2) upon jaw length squared (cm2) ...... 189

Figure 5.6: RMA regression of MPIA (cm2) upon jaw length squared (cm2) ...... 190

Figure 5.7: OLS regressions of insertion areas and PCSA for calculating residuals . 194

Figure 5.8: RMA regressions of PCSA residuals onto insertion area residuals ...... 195

Figure 5.9: OLS regression of masseter PCSA (cm2) upon masseter insertion area

(cm2) for plesiadapiform formula ...... 197

Figure 5.10: OLS regression of temporalis PCSA (cm2) upon temporalis insertion area (cm2) for plesiadapiform formula ...... 198

Figure 5.11: OLS regression of medial pterygoid PCSA (cm2) upon medial pterygoid insertion area (cm2) for plesiadapiform formula ...... 199

Figure 5.12: Comparing masseter PCSA in tupaiids, strepsirrhines and plesiadapiforms ...... 206

Figure 5.13: Comparing temporalis PCSA in tupaiids, strepsirrhines andplesiadapiforms ...... 207

Figure 5.14: Comparing medial pterygoid PCSA in tupaiids, strepsirrhines and plesiadapiforms ...... 208

xviii

Figure 5.15: Comparing jaw adductor PCSA in tupaiids, strepsirrhines, and plesiadapiforms ...... 210

Figure 5.16: Comparing masseter PCSA by dietary category ...... 212

Figure 5.17: Comparing temporalis PCSA by dietary category ...... 213

Figure 5.18: Comparing medial pterygoid PCSA by dietary category ...... 214

Figure 6.1: Calculating the orientation of the temporalis muscle group ...... 235

Figure 6.2: Lever and load arm measurements and placing the jaw adductor resultant ...... 237

Figure 6.3: RMA regression of tupaiid bite force at three points along the tooth row

...... 247

Figure 6.4: RMA regression of strepsirrhine bite force at three points along the tooth row ...... 248

Figure 6.5: RMA regression of plesiadapiform bite force at three points along the tooth row ...... 249

Figure 6.6: Allometry of bite force estimations at the mesial premolar ...... 253

Figure 6.7: Allometry of bite force estimations at the first molar ...... 254

Figure 6.8: Allometry of bite force estimations at the third molar ...... 255

Figure 6.9: Bite force estimations at the mesial premolar ...... 257

Figure 6.10: Bite force estimations at the first molar ...... 258

Figure 6.11: Bite force estimations at the third molar ...... 259

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CHAPTER 1. Introduction

Chapter 1: Introduction

1.1 Primate origins and the case for diet

One of the most hotly-debated topics in primate paleontology is the context for primate origins, where arguments focus on activity patterns and dietary habits of the earliest primates. There are three main sceniarios that dominate arguments explaining the evolution of key primate traits within an ecological scenario: the

Arboreal Hypothesis (Smith, 1912; Wood Jones, 1916) stresses that key primate traits arose as adaptations for an arboreal lifestyle. The Visual Predation Hypothesis

(Cartmill, 1972, 1974a, 1974b) challenges the arboreal hypothesis and suggests the traits evolved in the context for nocturnal visual predation on insects. Finally, the

Angiosperm Coevolution h=Hypothesis (Sussman, 1991; Sussman and Raven, 1978) counters that it is more likely those traits evolved for preying on fruit in the terminal branches of angiosperms.

Szalay (1968a) postulated that diet was a driving force behind primate evolution whereby insectivorous forms moved into a previously unoccupied adaptive zone by becoming increasingly reliant on fruit and leaves as a dietary resource. Others have highlighted specific aspects, e.g., locomotion in terminal branches (Bloch and Boyer, 2002; Schmitt and Lemelin, 2002), locomotion in the form of grasp-leaping (Dagosto, 2007; Szalay and Dagosto, 1980), or suggested a combined scenario (Rasmussen, 1990). Silcox and colleagues (2007) added another

20

CHAPTER 1. Introduction level of complexity by demonstrating that these discussions are inherently flawed because these traits evolved at different times. Regardless, it is clear from the fossil evidence that diet plays a crucial role in explaining primate origins.

Thought by many researchers to be the most likely candidates for the ancestors of living primates (E.g., Bloch et al., 2016; Chester et al., 2015; Silcox et al.,

2010), Plesiadapiforms, stem archaic primates, lie at the heart of primate origins.

Dentally, they share some traits with living primates and possess a tremendous diversity of jaw morphologies, suggesting a variety of approaches to food processing

(Bloch et al., 2007; Silcox and Williamson, 2012). With the established relationship of diet to body size (e.g., Bloch and Boyer, 2007; Kay, 1975) and habitat (e.g.,

Lehmann, 2004) in primates, investigating this diversity is integral to studying plesiadapiform niche occupation and ultimately their role in the origin of primates.

Most plesiadapiform fossil evidence comprises isolated teeth and jaws (Rose,

1981; Szalay, 1968a), with dental analyses overwhelmingly providing the basis for most research into plesiadapiform dietary patterns (e.g., Biknevicius, 1986; Kay and

Cartmill, 1977; López-Torres et al., 2018; Rose and Bown, 1982; Szalay, 1969). In contrast, despite providing more accurate dietary reconstructions than alternative, dental-only methods, non-dental quantification of their masticatory apparatus has been rare (Perry, 2001).

Reconstructing the diet of plesiadapiforms has been an enduring topic of study (Biknevicius, 1986; Gingerich, 1976; Rose, 1975; Rosenberger, 2013; Szalay,

21

CHAPTER 1. Introduction

1968a, 1969; Ungar, 2002), yet, this research has largely focused on dental adaptations, often overlooking the mandible and muscles of mastication. To date,

“no one has attempted a comprehensive comparison of masticatory apparatus form, beyond the teeth, or jaw-muscle activity patterns between primates and closely related non-primates” (Vinyard et al., 2007, pg 194).

Given their importance for primate origins, a more full understanding of the plesiadapiform feeding apparatus is needed. Non-dental parts of the masticatory apparatus refine dental-based inferences, providing information unobtainable from teeth, such as gape adaptations and the forces available to break food.

Therefore, this dissertation will focus on dietary adaptations of a specific group of fossil primates in order to contribute to a complete picture of the ecological context for primate origins. The study of plesiadapiforms is significant to understanding the broad topic of primate origins because of their close relationship and morphological similarities to true primates (Bloch et al., 2007; Silcox et al.,

2007). The major goal of this dissertation is to reconstruct plesidapiform masticatory soft tissue parameters by employing tupaiid and strepsirrhine species as extant analog models.

22

CHAPTER 1. Introduction

1.2 Introduction to plesiadapiforms

1.2.1 Overview

Plesiadapiforms are an extinct group of mammals recovered from North

America, Asia and Europe (Silcox, 2007; Silcox and Gunnell, 2008), represented by

11 families and over 140 species (Silcox et al., 2017). They existed from the

Cretaceous into the Eocene, with those from the western United States showing an especial diversity (Silcox et al., 2015) that peaked 65-51 Ma where they are a large component of faunal remains (Rose, 1981).

Plesiadapiforms have remained an enigma for some time, in that they present a problematic mosaic of primitive and derived features (Beard, 1990,

1993a; Gingerich, 1976; Szalay and Delson, 1979) and their place among the primates is still disputed. For example, the carpolestid Carpolestes simpsoni retains a primitive groove for the passage of the internal carotid artery, but also possessed a nail on the hallux, a derived primate trait (Bloch and Silcox, 2006; Sargis, 2002d;

Sargis et al., 2007).

The presence of diagnostically euprimate traits has been used to argue for the inclusion of plesiadapiforms within (MacPhee et al., 1983) or exclusion from

(e.g., Cartmill, 1974a) Primates. For example, the presence of a petrosal bulla in

Plesiadapis, Carpolestes, and Dryomomys, as discussed by Szalay (1987), Bloch and

Silcox (2006) andBlech et al. (2016), is a characteristically euprimate trait. So too is the opposable hallux with a nail found in Carpolestes by Bloch and Boyer (2002), 23

CHAPTER 1. Introduction

Szalay also presented a suite of postcranial evidence to argue for inclusion of plesiadapiforms in Primates (Szalay, 1975; Szalay and Decker, 1974; Szalay et al.,

1975).

Despite similar molar morphology to early euprimates (true primates), including a broad talonid basin and similar cusp patterns, most plesiadapiforms show specialized dental adaptations precluding them from being direct ancestors to primates (Bloch and Boyer, 2002). Based on a suite of postcranial similarities and cranial features, including the presence of an entotympanic bulla, some authors argue that at least some plesiadapiforms are more closely related to colugos (Order:

Dermoptera) than primates (Beard, 1990, 1993a, 1993b, Kay et al., 1992, 1990;

Meredith et al., 2011; Ni et al., 2013, 2016). In addition to a close relationship with lagomorphs, Meredith et al. 2011 also found evidence for a Scandentia + Glires

(Lagomorpha + Rodentia) clade.

Others argue that plesiadapiforms, while not directly ancestral to extant primates, are still more closely related to extant primates than to any other modern mammals (Bloch et al., 2016, 2007; Chester et al., 2015; Silcox, 2001, 2008; Silcox et al., 2010). For example, in their work, Silcox and colleagues (2007) supported a

Scandentia + Dermoptera clade, a finding supported by Bloch et al. (2007) and O’Leary et al. (2013). They assert plesiadapiforms should be considered to be stem primates, arguing for a sister group pairing between euprimates and a paraphyletic

Plesiadapiformes.

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CHAPTER 1. Introduction

1.2.2 Previous dietary research on plesiadapiforms

The functional significance of teeth has been well studied in the primate literature (Butler, 2007; Gingerich et al., 1982; Kay, 1975; Kay and Williams, 1994;

Lucas, 1982, 2004; Mills, 1963; Plavcan, 1993; Szalay and Delson, 1979; Ungar,

1998). Not only are teeth one of the primary tools by which food is broken down, but also their morphology reflects dietary adaptations. For this reason, and the ubiquity of teeth in the fossil record, teeth are an integral part of mammal paleontological research.

Recent work analyzing teeth has focused on quantifying the surface of the tooth in order to investigate diet. Dental topographic analysis, as these methods are known, are thought to be more sensitive for inferring diet (Ungar and M’Kirera,

2003). A study by Bunn (2011) of Dirichlet normal energy (DNE) and relief index

(RFI) found overwhelming support for these metrics to differentiatie diet in primates. Notably, Prufrock et al. (2016) then used three different measures of topographic analysis, DNE, RFI and orientation patch count rotated (OPCR), to investigate dietary niche overlap between plesiadapoid primates and early rodents.

Because most of the plesiadapiform fossil remains are dentary fragments and isolated teeth, much of the previous research on plesidapiforms concerns their diet, and often behavioral inferences that can be made from their ecology. Previous research on the teeth of plesiadapiforms points to many (e.g., Palaechthonidae), as being predominantly insectivorous, piercing chitinous exoskeletons with sharp

25

CHAPTER 1. Introduction teeth (Kay and Cartmill, 1977; Kay and Hiiemae, 1974). However, insectivory is not the inference for all plesiadapiforms, as many have specializations suggested as adaptations for fruit and leaf eating (Szalay, 1968a).

Purgatorius, the oldest plesiadapiform, from the Puercan North American

Land Mammal Age (NALMA) has dental features that place it as a possible ancestor of both plesiadapiforms and euprimates (Chester et al., 2015; Silcox et al., 2017).

Smaller purgatoriids were likely insectivorous and supplemented their diet with fruit (Scott et al., 2016). Larger-bodied forms exhibited greater shearing potential, which, combined with a greater body mass, has been suggested as adaptations for folivory. (Fox, 1991). The relatively primitive Palaechthonidae (Torrejonian -

Tiffanian NALMA) were also hypothesized to be primarily insectivorous, perhaps supplementing with frugivory, based on high crowned molars (Gunnell, 1989; Kay and Cartmill, 1977).

The small body sizes of micromomyids, (middle Tiffanian-early Wasatchian

NALMA), picromomyids (Wasatchian NALMA), picrodontids ((Torrejonian -

Tiffanian NALMA), and Toliapinids (Ypresian European Land Mammal Age) having body masses within the range of 10-37g (Chester and Bloch, 2013; Silcox et al.,

2017), suggest a diet comprised chiefly of insects. Micromomyids have bunodont teeth and an enlarged fourth premolar, indicating they may have had an increased reliance on fruit (Szalay, 1974). Toliapinids exhibit low crowned molars and bunodont teeth, with wear facets that perhaps favor a diet of fruit and nectar

26

CHAPTER 1. Introduction

(Hooker et al., 1999). Picromomyids and picrodontids are very similar in also having low crowned molars (Silcox et al., 2002) but have a further specialization of one enlarged tooth, the fourth premolar in picromomyids and the first molar in picrodontids. This may have been an adaptation for eating fruit and nectar (Rose and Bown, 1996).

Paromomyids (Torrejonian – Chadronian NALMA) show some similarities to these groups, also having low-crowned molars with additional specialized premolariform lower premolars. Representatives from these families have also been suggested to have been exudate feeders (Boyer and Bloch, 2008).

Microsyopids (Tiffanian - Chadronian NALMA) and plesiadapids (Torrejonian

- Ypresian ELMA) were two groups that were both long lived and diverse enough that their range of body sizes suggests several dietary adaptations. Smaller species of microsyopids were similar in size to micromomyids and therefore also hypothesized to be insectivorous (Kay and Cartmill, 1977). Gunnell’s (1989) assessment of tooth shape and wear facets led him to hypothesize that medium sized microsyopid species were probably frugivorous and large bodied species omnivorous. Plesiadapids show similar trends, where the older and smaller species were likely insectivorous (Kay and Cartmill, 1977), while the later species were large and had molars adapted for shearing (Gingerich, 1976) that have been suggested as an adaptation for specialized folivory (Boyer et al., 2012a, 2010).

27

CHAPTER 1. Introduction

Members of the Carpolestidae (Torrejonian - Chadronian NALMA) and

Saxonellidae (Tiffanian NALMA, Thanetian ELMA) were also hypothesized to have adaptations for frugivory and hard object feeding. Carpolestids and saxonellids display a further specialization, plagiaulacoid dentition, in which the lower fourth premolars in carpolestids (Simpson, 1933) and lower third premolars in saxonellids

(Fox, 1991) are blade-like, and thought to have aided in processing nuts, seeds, invertebrates, and fruit (Biknevicius, 1986).

There have been few studies that look to model the plesiadapiform masticatory apparatus (Perry, 2001), likely due to the paucity of plesiadapiform crania. Rather, cranial material is usually investigated from an evolutionary standpoint investigating brain anatomy or other anatomical characteristics. The relationships of plesiadapiform mandibular morphologies to diet are also not well studied, again due to the fact that complete dentaries are uncommon. Even so, studies that have discussed the mandible focus entirely on superficial morphology and what that says about diet and dietary behaviors.

1.3 Relationships between primate morphology and diet

Beyond teeth, several studies have linked the morphology of the primate mandible to the stress and strain associated with food processing (Daegling and

McGraw, 2009; Daegling and Scott Mcgraw, 2001; Dumont et al., 2011; Hylander,

1984, 1985; Hylander and Johnson, 1993; Hylander et al., 1998, 2000; Ravosa, 1991;

Williams et al., 2008). Assuming stress and strain would have acted on the 28

CHAPTER 1. Introduction plesiadapiform mandible in the same way it does on extant primates (Kay and

Covert, 1984), studying the diets, muscular anatomy, and mandibular morphology in extant primates should give clues as to the dietary proclivities of fossil forms.

1.3.1 Categorizing diet

Diet is an important factor in studying primates because of the relationship with many other aspects of primate ecology, including foraging habitat, feeding behavior, and activity patterns. Primate dietary categories are often classified as insectivorous, folivorous, frugivorous, or omnivorous, the latter usually when vertebrates are eaten as well, for ease of comparing diets of different primate groups (Ganzhorn, 1999). These categories are, however, far too wide to accurately describe feeding behaviors of most primates. Additional categories exist, including gummivory (exudate feeding), gramnivory (seed feeding) and nectarivory (nectar feeding), but these are far less common and often considered subsets of frugivory

(Lambert et al., 2004).

Diet categories are defined differently by different authors, usually as a certain percentage of food either observed to be ingested or as stomach contents

(Kurland and Gaulin, 1987). Body size is often used as a metric as well, for example using Kay’s threshold (500g). Kay’s Threshold separates insectivorous and folivorous primates, having similar dental specializations, based on body size: larger primates can not meet their nutritional requirements from insects alone. especially larger species that fall above of 500g (Kay, 1984). There is also the argument to be 29

CHAPTER 1. Introduction made to include fallback foods, eaten during times of hardship or unavailability of regular resources, into these categories as well (Marshall and Wrangham, 2007;

Rosenberger, 2013).

Some species certainly do obtain most of their food within one category, such as the folivorous colobine monkeys (Chivers and Hladik, 1980), or the frugivorous red howler monkey Alouatta seniculus (Julliot, 1996), or even from one species of plant, such as Hapalemur, consuming between 72-95% of its diet from giant bamboo

(Tan, 1999). But for most primates, specialization is rare and can even increase a primate species’ likelihood of extinction (Kamilar and Paciulli, 2008). Most species rely on foods that are readily available (Overdorff, 1993; Overdorff et al., 1997) and capable of processing, which usually means consuming food from more than one category.

Thus, many primates cannot easily be assigned to one dietary category, like the folivore-frugivore Japanese macaque, Macaca fuscata yakui. (Hill, 1997).

Unfortunately, these relatively more descriptive terms such as “folivore-frugivore” are arguably found more often amongst bird literature than primate literature (E.g., carnivore/ insectivore/ pisicivore, Zakaria et al., 2009).

The difficulty in defining dietary categories in extant primates, unfortunately, also highlights the difficulties in inferring diet from fossil primates. Most plesiadapiforms are categorized based on a combination of tooth morphology and body size (i.e., Kay’s Threshold, Kay, 1975; Kay and Covert, 1984), with some

30

CHAPTER 1. Introduction extreme specializations garnering more detailed investigations. Combining these dental analyses with inferences on muscle architecture and mandibular morphology may help clarify these currently broad categorizations.

1.3.2 Muscles of Mastication

The muscles of mastication provide the main forces acting on the mandible.

The jaw adductors act to close the jaw during mastication and are divided into three groups: the temporalis muscle group acts to elevate and retract the mandible, having forces that act in a posterior and superior direction; the masseter muscle group is an elevator of the jaw with fibers running in an antero-superior to infero- posterior direction. Laterally and postero-inferiorly coursing fibers in the medial pterygoid allow it to elevate the mandible and create medial excursion.

Prior work looking at the masticatory muscles and chewing biomechanics in strepsirrhine primates highlights their usefulness for enriching dietary inferences in primates (e.g., Hylander et al., 1998; Perry, Hartstone-Rose, and Logan, 2011; Perry,

Hartstone-Rose, and Wall, 2011; Ravosa, 2000; Christopher J. Vinyard et al., 2007).

In particular, strepsirrhine chewing muscle groups are known to scale differently based on dietary differences (Perry et al., 2011a). In general, folivorous and insectivorous strepsirrhines have great chewing muscle cross-sectional area for their body size compared to frugivorous and gummivorous ones. By contrast, chewing muscle fiber length is relatively greater in frugivores. Presumably, this is related to ingesting their foods at larger gapes (Perry and Hartstone-Rose, 2010). 31

CHAPTER 1. Introduction

Anton (1999) found a correlation between jaw adductor muscle architecture and mandibular bone morphology in macaques. Specifically, muscle size and cross- sectional area primarily correlated with body size. Taylor and Vinyard have conducted a number of studies on the relationship between muscle fiber architecture, mandibular morphology, and diet in anthropoid primates. For example, their work on callitrichid monkeys found that the tree-gouging species have relatively longer masseter and temporalis fibers which they attribute to the production of wide jaw gapes (Taylor and Vinyard, 2008). Their study on capuchins indicated large muscle masses and PCSA as a function of a diet heavy of large tough foods (Taylor and Vinyard, 2009). Further work revealed differences in PCSA between anthropoids and hominoids, scaling close to isometry relative to body size in the former, but likely with positive allometry in in the latter (Taylor and Vinyard,

2013).

1.3.2.1 Applications of iodine staining for reconstructive work

A new trend in comparative anatomy, commonly referred to as DiceCT

(Diffusable Iodine-based Contrast-Enhanced Computed Tomography), has the potential to revolutionize the methodology of researchers studying the muscle fiber architecture (Gignac et al., 2016). This promising new method involves immersing specimens in an iodine solution, usually Lugol’s iodine (I2KI) followed by CT scanning (e.g., Cox and Jeffery, 2011; Metscher, 2009) in order to give structures of interest a higher x-ray attenuation. 32

CHAPTER 1. Introduction

Despite the small number of mammals represented in the literature, pioneering fiber muscle architecture research on muscles of mastication using

DiceCT has been performed on squirrels, rats, and guinea pigs (Cox and Jeffery,

2011) as well as on mice (Baverstock et al., 2013), the naked mole-rat (Cox and

Faulkes, 2014) and bats (Santana, 2018). Collectively, this work highlights the usefulness of DiceCT to clarify dissection inconsistencies, obtain accurate fiber and volume measurements, and simplify direct comparisons between species and specimens.

One of the most obvious advantages to adopting this method is the ability to spare rare museum specimens from destructive sampling because the staining technique is reversible. This allows the specimens to be returned de-stained for future work. The second immediate advantage is scientific reproducibility, whereby more than one researcher is able to study the same specimen. Therefore, the ephemeral nature of dissected specimens can be avoided and further research can be conducted on the same specimen multiple times for various purposes.

In this dissertation, two tupaiid specimens will serve as an exciting test case for the method; tupaiids have never been subjected to iodine staining.

1.3.2 The primate mandible

Many studies have been undertaken to investigate how the chewing musculature impacts mandibular morphology of primates. These studies have taken a range of approaches, including examining the effects of diet (E.g., Anapol and Lee, 33

CHAPTER 1. Introduction

1994; Hogg et al., 2011), phylogeny (eg., Meloro et al., 2015; Ravosa et al., 2000), ontogeny (eg., Vinyard and Ravosa, 1998), and allometry (eg., Bouvier, 1986;

Ravosa, 1991, 2000). The chewing musculature in primates has also been studied as a way of interpreting diet in fossil forms (Ravosa, 1996) using a range of methods, such as biomechanical modelling (eg., (Hylander et al., 1992; Rapoff et al., 2008), in vivo bone strain analyses (eg., Daegling, 1993; Hylander, 1979), 3D analyses (eg.,

Burrows and Smith, 2005), and Finite Element Analysis (eg., Korioth et al., 1992;

Ross et al., 2005; Toro-Ibacache and O’Higgins, 2016).

1.3.2.1 Corpus

During mastication, the corpus of the mandible undergoes different types of strain and stress depending on its position and actions during the cycle of chewing.

As described by Hylander (1984), the lateral pterygoid muscles cause medial transverse bending to the mandibular corpus during jaw opening. During the power stroke of mastication, the corpus then experiences lateral forces from the jaw adductor muscles, causing lateral transverse bending. The mandible is also subject to reactionary forces in the temporomandibular joint and teeth. These stresses and strains work to deform the mandible in both the primary direction of force, as well as perpendicular to it (van Eijden, 2000).

Folivorous primates in particular have been argued to have a deeper mandible as a consequence of increased forces on the mandible (Bouvier, 1986;

Hylander, 1979b; Ravosa, 1996). Although this pattern is seen in folivorous 34

CHAPTER 1. Introduction catarrhines and strepsirrhines, when investigated further the most folivorous strepsirrhines do not have relatively deeper corpora than closer relatives (Ravosa,

1991). This pattern is also not true for platyrrhines, where it is the hard-object feeders that exhibit the deepest mandibular corpora (Bouvier, 1986). Consequently, were researchers to classify deep mandibles as adaptations to folivory, this would only be correct for some phylogenetic groups.

Studies of exudate feeding primates have garnered attention for a rare feeding style amongst primates. Behavioral studies indicate that tree-gouging marmosets, including Callithrix and Cebuella, rely on large jaw gapes to gouge trees and stimulate exudate flow, but do not appear to generate large bite forces (Hogg et al., 2011; Vinyard and Ryan, 2006). A study of Otolemur crassicaudatus, the brown greater galago (Burrows and Smith, 2005) also found evidence for a long mandible, suggesting this might be an adaptation for harvesting gum.

These patterns in the corpus can be seen in humans as well. For example a study by von Cramon-Taubadel (2011) looking at a global sample of agricultural and hunter gatherer subsistence populations demonstrates that the mandible significantly reflects subsistence strategy, where the hunter-gatherers having longer and narrower mandibles than agriculturalists who exhibit shorter mandibles. The authors confirm that it is the masticatory stress driving the overwhelming number of these differences and not genetic differences.

35

CHAPTER 1. Introduction

1.3.2.2 Symphysis

The primate symphysis is another aspect of the primate masticatory apparatus that has received considerable attention. Many studies have sought to explain the functional relationships and evolutionary significance of the symphysis through biomechanical experiments and modelling (Beecher, 1977, 1979, 1983;

Bucinell et al., 2010; Greaves, 1988; Hylander, 1977, 1979b, 1984, 1985; Hylander and Johnson, 1993; Hylander et al., 1987, 1998; Ravosa, 1991; Ravosa and Hogue,

2004; Ravosa and Hylander, 1994; Scapino, 1965, 1981).

Like the mandibular corpus, the symphysis deforms with respect to the chewing cycle: during jaw opening it undergoes tensile stress labially and compressive stress lingually (Hylander, 1984). During the power stroke, the forces are reversed with the added effect of dorsoventral shear, where one side is pulled dorsally and the other ventrally due to the presence of food on the contralateral side.

Experimental studies have shown that the evolution of symphyseal fusion was likely an adaption to reduce stress in the mandible. By strengthening the joint between the two sides of the mandible, forces produced by the balancing side could be recruited with greater efficiency to aid the muscles on the working side (Bouvier and Hylander, 1984; Hylander, 1979b, 1985; Hylander and Johnson, 1993; Hylander et al., 1998, 2000, 2011, 2005; Ravosa and Hogue, 2004; Ravosa and Hylander,

1994; Vinyard et al., 2007, 2005).

36

CHAPTER 1. Introduction

The degree of stress therefore plays a role in the symphyseal morphology.

Several studies have examined the symphysis from the point of view of presence/absence. Their results show that primates relying on tougher foods or foods that require more chewing have a higher likelihood of symphyseal fusion

(Scott et al., 2012). However, not many studies have examined the morphology of the symphysis itself (Beecher, 1979; Daegling and Jungers, 2000).

Signals of diet found in the symphysis (Beecher, 1983; Ravosa, 1991) and corpus (Bouvier, 1986; Taylor, 2006) of the mandible clearly point to a relationship to dietary adaptations. Rather than using diet as a categorical variable for mandibular shape, it would be more precise to use mandibular loading regimes to categorize primate mandibles (Ross et al., 2012).

1.4 Non-dietary factors influencing primate mandibular morphology

Morphological variation may not be as thorough a signal of diet as often hypothesized. Not only are there external factors influencing stress and strain, but behavioral factors as well. For example, tool usage is one instance which might mitigate the effects of dietary signal, as found by Wright and colleagues (2009). In studying fallback foods on skeletal morphological variation in Cebus monkeys, their results indicated moderate mandibular robustness, despite a diet incorporating tough foods, due to preprocessing their foods with tools.

37

CHAPTER 1. Introduction

Although plesiadapiforms were unlikely to be using tools to process their food, there are certainly other factors that would have affected their food processing, including food processing habits, such as frequency of muscle engagement during chewing. Further considerations include whether or not dietary habits are affected by other factors such as age and sexual dimorphism. Not only are these considerations for the plesiadapiforms, but considerations that must be taken into account when conducting research using extant analogs for reconstructive purposes; habits and biologic factors affecting the study sample will almost unavoidable be transferred and applied to discussions about fossil forms.

1.4.1 Food material properties

In discussing stresses on the mandible, material properties of specific foods also need to be taken into account. For example, tougher foods, like leaves and seeds, should produce a greater amount of stress and strain on the mandible, requirig a deeper corpus, due either to their toughness or because they might simply require more chewing to process (Lucas et al., 2000). The importance of food material properties has only begun to be appreciated in recent years and thus far there is limited research on the subject.

A comprehensive study of food mechanical properties was undertaken by

Williams and colleagues (2005) and Lucas and colleagues (2012) looking at a range of food, mostly fruit. Yamashita measured food mechanical properties in Lemur catta and Propithecus verreauxi (2003) as well as in three species of Hapalemur 38

CHAPTER 1. Introduction

(2009). Lambert measured hardness in foods associated with cercopithecine monkeys (2004), Kinzey and Norconk measured hardness in foods related to two platyrrhines (1990), and Wright and colleagues (2008) measured toughness in leaves eaten by colobine monkeys. All studies found that species eating tougher foods had more robust mandibles. However, these studies were small in scale and do not include food associated with plesiadapiform diets that are heavy in insects.

1.4.2 Sexual dimorphism and size

Many studies with a substantial sample size do not specify whether specimens are male or female, thus making it hard to draw conclusions as to whether dietary correlations are truly the result of diet, or influenced by sexual dimorphism (e.g., Anapol et al., 2008; Taylor, 2006). This compromises the quality of the data because sex differences in primates are known to exist when specifically evaluated. In many anthropoid primates, males are often much larger than the females (Clutton-Brock and Harvey, 2009; Crook, 1972; Leutenegger and Kelly,

1977; Plavcan and van Schaik, 1997; Smith and Jungers, 1997). For example

Daegling and Jungers (2000), in analyzing the shape of the symphysis in great apes, revealed that within species, size differences between males and females were pronounced in gorillas, although not in chimpanzees or orangutans.

Although purely morphometric differences caused by sex are probably a reflection of diet correlating with body size, this does have consequences for making inferences about the fossil record. For example, Terhune et al. (2015) found 39

CHAPTER 1. Introduction evidence that male Macaca fascicularis have experienced selective adaptation for longer jaws, possibly to facilitate longer canine teeth. However, longer jaws are also an adaptation for specific types of feeding in primates, most notably nectivory

(Dumont, 1997). This could confound results investigating fossil forms where sexual dimorphism is confused for gape adaptations and vice versa.

Generally speaking, there are usually not suffiecient remains of fossil primates to confidently determine the sex of an individual let alone measure sexual dimorphism. There are exceptions, however, for example, a large sample of the

Miocene hominoid Lufengpithecus lufengensis enabled a comprehensive study determining extreme sexual dimorphism (Kelley and Qinghua X., 1991). In looking at euprimates, Kristalka et al. (1990) found evidence of sexual dumorphism in canine size in a species of Notharctus, and Gingerich (1981) asserts that previous work declaring new species of the euprimate Adapis was in reality misidentifying males and females as different species. And through analogy with living primates,

Fleagle and colleagues (1980) declared they found evidence of sexual dimorphism in early fossil catarrhines from the Oligocene Fayum Depression in Egypt.

Given the extant sample that is the focus of this study, there is not enough research to determine whether treeshrews exhibit sexual dimorphism and researchers have evidence supporting conflicting arguments. In her study of

Bornean treeshrews, Emmons (2000) found an absence of sexual dimorphism across six species of treeshrew, finding males and females “vitually identical in size”

40

CHAPTER 1. Introduction

(p. 17). However, work by Tsang and Collins (1985) assert male specimens of

Tupaia belangeri are approximately 20% larger than females. This was corroborated by Sargis and colleagues (2018) in their study of island dwarfism among treeshrews, who found T. glis males to be considerably larger than females,

Research into strepsirrhines suggests that they are among the least sexually dimorphic primates (Lindenfors and Tullberg, 1998; Plavcan, 2001; Weckerly,

1998). Additionally, research by Plavcan (2000) investigating the sexual dimorphism and behavior in primates did not support a relationship between the two and cautioned against using the presence of sexual dimorphism to infer behavior. Any differences found in diet between specimens of the same species but differe sexes are not likely to be based on sex alone, or possibly at all, but rather sex- correlated size differences.

1.5 Reconstructing diet in the primate fossil record

As established, diet is an important part of the ecological niches of primates, yet to the frustration of many paleontologists, it cannot be observed for fossils, merely inferred (Ungar, 2002). Therefore, the only recourse is to evaluate morphology found in extant primates and relate it to certain behaviors, then apply the same logic to fossil forms to infer behavior. This is all assuming that a given trait has remained unchanged in its function. A popular application, the comparative method, has formed the basis of much of what is known about fossil primates. For example, Gingerich and colleagues (1982) evaluated allometric scaling of the molar 41

CHAPTER 1. Introduction crown area to body mass in a substantial sample of extant primates in order to produce a regression for estimating body mass in fossil primates from dental remains. This regression line is still commonly used (Silcox et al., 2009).

There are several samples from the literature specifically using extant primates to infer diet in fossil primates. For example, Ungar and Kay (1995) reconstructed the diets of European Miocene hominoids and pliopithecids through analyzing molar shear and crest development and compared their results with an extant sample of hominoids, concluding that dietary adaptations included hard- object feeding, soft-object frugivory, and folivory. Similar methods were used by

Strait (2001) to reconstruct diet in fossil omomyid primates by comparing them to strepsirrhines and tarsiers, inferring adaptations for frugivory.

Perry (2008) reconstructed the muscles of mastication in a sample of

European adapid primates using strepsirrhine primates as extant analogs. He inferred a folivorous diet of resistant foods in Adapis and Leptadapis due to both great PCSA, leverage and bite force similar to extant folivorous strepsirrhines.

Dumont et al., 2011 also used the same strepsirrhine PCSA data combined with platyrrhine electromyography (EMG) data for reconstructing bite forces in fossil lemurs Archaeolemur and Hadropithecus. From their findings, they support the hypothesis that Archaeolemur was a partially hard object feeder, but reject that diet for Hadropithecus, instead arguing for a diet based on bulbs and succulent leaves.

42

CHAPTER 1. Introduction

As argued by Ross et al., (2012), diet is best studied through the loading regimes of the mandible. Since the greatest source of forces on the mandible are the muscles of mastication, reconstructing muscles of mastication in fossil plesiadapiforms is necessary to analyze their force output. In order to estimate these muscle parameters, correlations between muscle dimensions and the mandible must first be established for an extant sample for use as a model (Kay and

Cartmill, 1977). Patterns can be assessed in extinct primates once two conditions are met: first, the modern sample must be a suitable analog, possessing the traits of interest in the fossil sample (Lauder, 1995). Second, reliable correlations between the trait and its function must be verified.

1.5.1 Establishing modern analogs for plesiadapiform by constructing an extant phylogenetic bracket

Bryant and Russell (1992) posit that the most appropriate comparison group for studying a trait is the extant sister group of the taxon in question. When considering soft anatomy and behavior in fossil taxa, extant groups must be examined because they are the only option for obtaining precise and accurate information (Witmer, 1995). However, relying on but a single comparison group creates the assumption that the trait of interest was present in the common ancestor as well. To avoid making a priori predictions about traits in plesiadapiforms, two major groups of extant taxa will be used to create an extant phylogenetic bracket and make comparisons to ensure solid inferences: treeshrews 43

CHAPTER 1. Introduction

(Scandentia: Tupaiidae) and lemurs, lorises, galagos, and pottos (Primates:

Strepsirrhini).

One reason I focus on treeshrews is their close relationship to, and thus frequent usage as, an outgroup for Primates (e.g., Matsui et al., 2009; Perelman et al.,

2011). While molecular studies (Janečka et al., 2007; Nie et al., 2008) have disagreed as to whether colugos are more closely related to primates than are treeshrews, the most recent evidence (Mason, 2016) supports a sister group relationship between colugos and primates. However, colugos are autapomorphic in a number of ways that preclude them from serving as extant analogs for reconstructive purposes. One such autapomorphy is the extremely long limbs proportions an adaptation for gliding (Byrnes et al., 2011). These proportions, and gliding as a locomotory style, are not found in primates. Furthermore, their comb-like dentition is unique among mammals (Aimi and Inagaki, 1988) and suggests they process food unlike any other mammal. Finally, due to their scarcity, colugos are extrememly difficult to obtain for dissection.

Scandentian supraordinal relationships, while often under debate, have nonetheless often suggested a close relationship to euprimates (Kay et al., 1992;

Wible and Covert, 1987), with others suggesting them as more closely related to colugos (Bussche and Hoofer, 2004; Liu et al., 2001; Murphy et al., 2001; Sargis,

2004), or lagomorphs (e.g., (Arnason et al., 2002; Graur et al., 1996; Lin et al., 2002).

Although the living sister group of Primates is not fully settled (Godinot, 2007),

44

CHAPTER 1. Introduction within this paper Scandentia will be considered the sister taxon for a Primates +

Plesiadapiform clade as per Silcox et al. (2007). Bracketing plesiadapiform analyses between the two chosen living taxa will make a strong argument for reasonable dietary inferences.

1.5.1.1 Plesiadapiform reconstruction

Confidently reconstructing the soft tissue of the plesiadapiform specimens requires both the treeshrews and strepsirrhines to possess the same soft tissue structures and associated osteological features. Previous work in strepsirrhines

(Perry, 2008; Perry et al., 2011b) has already established muscle presence, muscle architecture and muscle attachment patterns, as well as the bony ridges for muscular attachment. In order to confirm this in treeshrews however, treeshrew masticatory anatomy must be investigated first-hand by way of gross dissections.

As part of this dissertation, osteological proxies for estimating soft tissue dimensions (muscle size, cross-sectional area, and fiber length) in plesiadapiforms will be assessed using tupaiid and strepsirrhine data. Proxies are already known for muscle size and cross-sectional area in strepsirrhines (Perry, 2008; Perry et al.,

2015), although none yet exist for fiber length. The best proxies for muscle cross- sectional area are the areas of muscle origin and insertion. The best proxies for muscle mass are the product of these attachment sites and distances representing individual muscle length. To satisfy these conditions, osteological proxies known from strepsirrhines will be verified on the extant tupaiid sample.

45

CHAPTER 1. Introduction

1.5.2 Introduction to tupaiids

1.5.2.1 Systematics and ecology

Tupaiids, commonly known as treeshrews, are small, omivorous mammals from South East Asia. Superficially, they resemble squirrels, and the name Tupaia itself comes from the Malay word for squirrel, tupai (Emmons, 2000). They range in size from ~40-241g (Sargis, 2002a; Table 4)and their pellage ranges from grey- brown (T. belangeri, T. glis) to brown (Tupaia everetti) to almost black (T. montana) to red (T. nicobarica, T. tana).

Belonging to their own order (Mammalia: Scandentia) the treeshrews are comprised of two families: the Tupaiidae consists of 3 recognized genera: Tupaia,

Anathana, and Dendrogale, now that Urogale has been synonymized with Tupaia

(Hawkins, 2018; Kennerly, 2019; Roberts et al., 2011). The single species Ptilocercus lowii in the family Ptilocercidae (Hawkins, 2018). Originally placed by Wagner

(1855) within Insectivora alongside true shrews, they became grouped with colugos

(Mammalia: Dermoptera), Primates, and Chiroptera when Gregory (1910) proposed the superorder Archonta, including elephant shrews. They were then moved to

Primates by Carlsson (1922) where they remained for several decades before van

Valen (1965) removed them and Butler (1972) placed them into their own Order,

Scandentia.

Treeshrews have a less derived dentition than do extant primates, having dilambdodont molars and a dental formula of 2.1.3.3/3.1.3.3. Upper incisors and 46

CHAPTER 1. Introduction canines are cylindrical and very alike in appearance, with large diastema between each. Similarly, the lower incisors and canines are also alike in size and appearance, being more spatulate than the uppers. The lower incisors in particular are procumbent and create a clasping mechanism with the upper incisors; however, neither the incisors nor canines from the upper and lower mandibles occlude with each other. The premolars are small but increase in size distally along the tooth row.

Both upper and lower premolars have one cusp, with the exception of the fourth lower premolar which has two cusps. The molars, in contrast to the premolars, decrease in size when moving distally along the tooth row. Each upper molar has three cusps and the lower molars display tall trigonids with high protoconids in relation to the lower talonid basin. The dilambdodont dentition of tupaiids is adapted for shearing and crushing (Butler, 1980).

Treeshrews have been a popular laboratory study animal in the clinical sciences for decades, especially with genome sequencing indicating that the treeshrew nervous, immune, and metabolic systems are very close to those of humans (Fan et al., 2013). Yet for all of these laboratory studies, few field studies on treeshrews have been conducted (Chorazyna and Kurup, 1975; D’Souza and Martin,

1974; Emmons, 2000; Kawamichi and Kawamichi, 1979; Langham, 1982; Oomen and Shanker, 2008). In her field study of Bornean treeshrews, Emmons (2000) followed six treeshrew species in Sabah, Borneo. After analyzing scat, she found the more terrestrial species (T. gracilis, T. longipes, T. montana, and T. tana) to eat a diet

47

CHAPTER 1. Introduction composed largely of ants, crickets, beetles, termites, spiders, and fruit. The more arboreal species (T. minor and P. lowii) ate more fruit and moths. Such as composition makes their insectivorous diet similar to that inferred for early eutherians (Fish, 1983), and probably broadly similar to those inferred for at least some plesiadapiforms (Kay and Hiiemae, 1974).

1.5.2.2 Tupaiid suitability as an extant analog

Tupaiids have often been used as an outgroup for Primates when studying primate intraordinal relationships (Sargis, 2004; Silcox et al., 2007) and as a model for plesiadapiform paleobiology, including ancestral primate size (Gebo, 2004) and postcranial functional morphology (Bloch and Boyer, 2002, 2007).

Molecular (Olson et al., 2005; Roberts et al., 2011) and morphological studies

(e.g., Bloch and Boyer, 2002; Murphy et al., 2001; Sargis, 2004) have established that the sister taxon to tupaiids, Ptilocercus lowii (Family Ptilocercidae) would make an excellent analog to plesiadapiforms in being plesiomorphic and having similar postcrania (E.g., Sargis, 2002d, 2002c, 2002b, 2002e, 2004; Sargis et al., 2007).

Szalay and Dogasto (1988) and Sargis et al. (2007) note the grasping similarities between the plesiadapiform Plesiadapis and P. lowii, while Bloch and Boyer (2007) and Bloch et al. (2007)note additional grasping similarities to paromomyid and micromomyid plesiadapiforms. They compare the micromomyid Dryomomys szalayi to P. lowii and suggest that the earliest primates were morphologically very similar to P. lowii, probably filling a similar ecological role. 48

CHAPTER 1. Introduction

With regard to dietary inferences, many researchers have noted that the shared anatomical similarities between tupaiids and plesiadapiforms make tupaiids excellent functional models for reconstructing reliable masticatory muscles and calculating realistic jaw biomechanics (e.g., Butler, 1980; Fish, 1983; Le Gros Clark,

1959; Vinyard et al., 2007). These similarities include the posteriorly jutting angular process of the mandible, as well as primitive traits like an overall small size, an unfused symphysis, and tribosphenic molars.

Previous work has utilized tupaiids as extant analogs for plesiadapiforms in a functional context (e.g., Sargis, 2001; Tattersall, 1984) and specifically regarding chewing in primitive primates (e.g., Butler, 1980; Fish and Mendel, 1982; Hiiemae and Kay, 1973; Jablonski, 1986; Kay and Hiiemae, 1974). Work by Vinyard et al.

(2005) further argued for treeshrews as an acceptable model for early primates in that treeshrews display similar enough feeding behaviors to early primates to make inferences about fossil forms. They analyzed the firing patterns of different masticatory muscles during chewing in order to quantify and compare muscle recruitment patterns. They concluded that, like primates with an unfused symphysis, treeshrews rely more on their working-side muscles of mastication than their balancing-side muscles. The work presented here is novel in using tupaiids for reconstructive purposes.

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CHAPTER 1. Introduction

1.6 Goals and hypotheses

The goals of this dissertation involve understanding the relationship between muscles of mastication and diet in treeshrews in order to apply the same logic to fossil specimens of the plesiadapiforms. Hypotheses are primarily concerned with the differences between treeshrews and strepsirrhines, due to extensive prior work on the suitability of the latter as an extant analog for adapid primates. Further hypotheses address the inferences that can be made about fossil plesidapiform specimens through the reconstruction of extant masticatory musculature to the fossil specimens.

1.6.1 Goals and Hypotheses: Chapter Two

Chapter Two concerns the first goal of the dissertation: to quantify muscle attachment sites and aspects of muscle anatomy in treeshrews. Gross dissection will allow for a detailed description of the muscles as they are removed for further processing.

1.6.2 Goals and Hypotheses: Chapter Three

Chapter three has the same goals as Chapter two, but uses a different method. This second method is a new technique called Diffusible Iodine Contrast

Enhanced Computed Tomography (DiceCT) where its accuracy is compared to gross dissection is compared and its effectiveness as a replacement for gross dissection is

50

CHAPTER 1. Introduction evaluated. Hypotheses are concerned with differences between muscle dimensions as measured by the two methods.

1.6.3 Goals and Hypotheses: Chapter Four

The second, third, and fourth goals of this dissertation are to quantify muscle mass, fiber length, and physiological cross-section area (PCSA) of the muscles of mastication in several species of treeshrew. To address Goal Two, the muscles of mastication are weighed upon removal from the specimens. To address Goal 3, the muscles undergo a chemical dissection process in order to measure the individual muscle fibers of each chewing muscle. To address goal four, muscle fiber length and mass are used to calculate PCSA. Hypotheses in this chapter are strictly based on previous work using the same methodology as conducted on strepsirrhine primates.

1.6.4 Goals and Hypotheses: Chapter Five

The fifth goal of this dissertation is to statistically verify osteological correlations in treeshrews while goal six is to reconstruct the muscles of mastication in plesiadapiforms. To address Goal Five, osteological correlations previously verified in strepsirrhines will be applied to treeshrews and evaluated. To address

Goal Six, osteological measurements will be taken from plesiadapiform fossil specimens and input into treeshrew and strepsirrhine muscle parameter models from which plesiadapiform PCSA will be estimated.

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CHAPTER 1. Introduction

1.6.5 Goals and Hypotheses: Chapter Six

The seventh and final goal of this dissertation is to test hypotheses about plesidapiform diet using the reconstructed muscles of mastication. To address Goal seven, observable data from both treeshrews and strepsirrhine as well as the reconstructed data for plesiadapiforms will then be used to model bite force at points along the tooth row. Following these results, I will test hypotheses surrounding the relationship between muscle group activity and diet between plesiadpaiform species as well as strepsirrhine primates and treeshrews.

1.7 Significance of this study

This dissertation will be the first to examine and evaluate dietary patterns within plesiadapiform primates by going beyond dental analyses to reconstruct the soft tissue structures of the masticatory apparatus. It will test hypotheses of interest to the greater scientific community, such as dietary patterns among major plesiadapiform clades, and morphological similarities between sister taxa and outgroups. Testing these hypotheses will contribute to interdisciplinary areas of anthropology and mammal paleontology, including dietary niche occupation,

Paleogene ecology and early primate community structure.

It will also establish a database of tupaiid masticatory muscle fiber architecture and anatomical measurements. As the most commonly used outgroup to studying primate phylogeny, these tupaiids are indispensable to anthropologists.

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CHAPTER 1. Introduction

Notably, examining muscle fiber architecture through enhanced contrast imaging will provide a non-destructive alternative to traditional dissection, leaving rare museum specimens intact. By incorporating DiceCT techniques into this project, I will help pave the way for applications of iodine staining far beyond the limits of this dissertation.

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CHAPTER 2. MASTICATORY ANATOMY

Chapter 2: An Overview of Tupaiid Masticatory

Musculature

2.1 Introduction

Living taxa are often examined for information on the current function of a specific trait also found in a fossil specimen because it is thought that a trait functioned similarly in the past as it does today. Here, I consider the mastication in early primates, specifically plesiadapiforms, using treeshrews (Order: Scandentia) as extant analogs for reconstructing the muscles of mastication. Other researchers have utilized living analogs to reconstruct certain aspects of the masticatory system across Mammalia. For example Wroe (2008) modelled cranial stress and masticatory biomechanics in the extinct marsupial lion, Thylacoleo carnifex, using the extant Panthera leo; (Maglio, 1973) reconstructed the skull musculature of the extinct mammoth, Mammuthus, using the extant elephants Loxodonta and Elephas;

Perry (2008) estimated jaw adductor resultants in adapids using extant strepsirrhines as a model.

Many researchers have noted anatomical similarities between plesiadapiforms and the primitive treeshrews and have inferred that plesiadapiforms occupied similar ecological niches to those of treeshrews (Bloch and Boyer, 2007; Hiiemae and Kay, 1972; Le Gros Clark, 1959). Treeshrews present

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CHAPTER 2. MASTICATORY ANATOMY a plesiomorphic condition with specific similarities to early primates including an unfused mandibular symphysis, small body size, and little-modified tribosphenic molars. Together, these traits make treeshrews excellent functional models for early placental mammals (Butler, 1980; Fish, 1983; Gingerich, 1976; Kay and Hiiemae,

1974; Le Gros Clark, 1959; Vinyard et al., 2007).

Despite their name, treeshrews are not shrews, and many of them do not live in trees (Hawkins, 2018). They are small-bodied and omnivorous mammals endemic to the Indomalayan region, Borneo, and the Philippines (Emmons, 2000).

They range in size from 40 to 350g.

Unfortunately, there is a limited amount of previous work conducted on the muscles of mastication in treeshrews. The first researcher to address this was Le

Gros Clark (1924), who conducted a skeletal muscle dissection on a specimen of

Tupaia minor. In that paper, he briefly described each muscle and included diagrams of the temporalis muscle and of muscle insertions on the lateral mandibular ramus.

He later published the results of a whole-body dissection of the pen-tailed treeshrew Ptilocercus lowii that included a description of the chewing muscles and a diagram detailing innervation (Le Gros Clark, 1926). Although these dissections served as the basis for treeshrew anatomy for decades, several aspects of masticatory anatomy were omitted. These include the large orbital head of the medial pterygoid muscle and the zygomatico-mandibularis muscle.

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In 1982, Fish and Mendel studied the complexities of mandibular movement in the common treeshrew Tupaia glis, concluding that the large range of motion in the jaw is primitive for placental mammals and that it might have been key to the success of early placental mammals (Fish and Mendel, 1982). Later, Fish (1983) expanded on the notion that the condition of the masticatory system in treeshrews is primitive for placentals and argued for the use of treeshrews as a model to describe mastication in early placental mammals. The basis for this notion was largely their tribosphenic molars and omnivorous behavior. He undertook a more comprehensive study of T. glis, describing the cranium, its joints, dentition, and muscles of mastication (Fish, 1983). He described the chewing muscles in greater detail than did Le Gros Clark and addressed the specific functions of each muscle during mastication as well as the difficulty in distinguishing some of the muscle layers.

However, these previous studies were not sufficiently detailed to use as a guide for reconstructing muscles of fossil forms for this dissertation. Not only did these studies conflict with regards to gross anatomy, they did not touch on many aspects of the finer details of masticatory anatomy such as fiber architecture.

Furthermore, they did not describe the bony attachments of muscles in sufficient detail required for mapping muscles onto fossil mandibles. Additionally, these studies were restricted to only three species of treeshrew: T. glis, T. minor, and P.

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CHAPTER 2. MASTICATORY ANATOMY lowii. This is especially important as unique aspects of masticatory anatomy were revealed in several other species.

Given the studies on both treeshrew (Fish and Mendel, 1982; Vinyard et al.,

2007, 2005) and primate chewing systems (Dechow and Carlson, 1983; Vinyard and

Taylor, 2010; Vinyard et al., 2008; Wall et al., 2006), treeshrews will help fill a void in knowledge left by a lack of research on plesiadapiform chewing systems. By dissecting the muscles of mastication in several species of treeshrew, this study will serve as a guide for future reconstructions of the same muscles in plesiadapiforms.

2.2 Materials

Cadaveric specimens of adult tupaiids were obtained for dissection (Table

2.1). Specimens obtained from a study colony at the Max Planck Florida Institute were bred at the facility and held in captivity until death, then frozen until dissection. Specimens obtained from the United States National Museum of Natural

History-Smithsonian Institution include wild caught specimens, specimens bred in captivity at the National Zoo (Washington, D.C.), and wild caught specimens subsequently held in captivity at the National Zoo. Each of these specimens was preserved and stored in fluid. Additional specimens obtained from Eric Sargis at

Yale University were frozen until dissection. More than one individual per species, where possible, was dissected in order to account for individual variation and age.

Ptilocercus is an endangered species with few known specimens ever collected, and thus Ptilocercus cadaveric material was unavailable for this study. 57

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2.3 Methods

The muscles of mastication are located on the lateral and medial aspects of the mandible as well as lateral and ventral portions of the cranium. To begin dissecting these muscles, the skin and fascia on the head must be retracted (Figure

2.1). Using a number 15 scalpel, an incision is made along the sagittal plane of the cranium from the anterior tip of the snout caudally to the nuchal line. Another incision is made from the anterior of the mandible, inferiorly and posteriorly to the posterior-most aspect of the jaw where it meets the throat. Careful skinning of the head reveals the muscles encased within their deep fascia.

Table 2.1: Specimens dissected Species Specimen Side Dissected Preservation JHUTS2* Right Frozen Tupaia belangeri 734BEF2* Right Frozen 734BABA* Left Frozen USNM 497016 Left 10% Formalin Tupaia glis USNM 399582 Right 10% Formalin Tupaia minor YBL 9011 Right 10% Formalin USNM 296647 Left 10% Formalin Tupaia montana USNM 292532 Right 10% Formalin Tupaia nicobarica USNM 111783 Left 10% Formalin USNM 580476 Left 10% Formalin Tupaia tana USNM 546344 Right 10% Formalin YBL 9010 Left 10% Formalin

Abbreviations: JHU - Johns Hopkins University USNM – Unites States National Museum of Natural History YBL – Yale Biological Anthropology Laboratory

*Obtained from the Max Planck Florida Institute

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CHAPTER 2. MASTICATORY ANATOMY

The muscles are removed in a specific order, beginning with the masseter group, followed by the temporalis group, and finally the pterygoid group. The muscles are layered upon one another and removing them individually minimizes damage. Muscle nomenclature follows Turnbull (1970).

Figure 2.1: Exposed muscles of mastication

The skin has been removed on one half of the head of a specimen of T. belangeri (734BABA) to expose the muscles to be dissected.

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2.3.1 Removal of the masseter muscle group

Beginning with the masseter group, the superficial masseter muscle is removed from the lateral surface of the mandible. It is removed in an inferior to superior direction. A small portion of the muscle is attached at the inferior and caudal edge of the mandible, wrapped around the angular process to attach to the medial side of the mandible. This medial insertion is located inferiorly to the insertion point of the medial pterygoid muscle. Once this portion has been detached, the scalpel is used to gently push up the superficial masseter to reveal the white tendon of the deep masseter, which has its insertion along the inferior and caudal portion of the mandibular ramus. Once this tendon has been identified, the superficial masseter is peeled back to its origin on the lateral and inferior portions of the zygomatic processes of the frontal and temporal bones (Figure 2.2). A sharp cut removes it from the bone of the zygomatic arch.

The deep masseter is not as large as the superficial masseter, and in appearance it is triangular and medio-laterally thin, pressed between the extremely large superficial masseter and zygomatico-mandibularis. It inserts just superiorly to the inferior edge of the mandible in a U-shaped hollow. The deep masseter is removed in a superior to inferior direction. Another scalpel cut severs the deep masseter from its attachments to the inferior border of the zygomatic bone; the tendon of the zygomatico-mandibularis is visible deep to the cut. The underlying zygomatico-mandibularis muscle is separated from it by a thin fascial sheet.

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Figure 2.2: Revealing deep masseter tendon

Peeling back the superficial masseter to reveal the tendon of the deep masseter (734BABA; T. belangeri)

The zygomatico-mandibularis is thicker than the deep masseter at its point of origin where it fills the space between the medial edge of the zygomatic bone and the lateral edge of the mandibular ramus. The superior attachment lies along the medial and inferior edge of the zygomatic bone. This muscle does not extend as far inferiorly as the deep masseter, but rather fills the masseteric fossa of the ramus of the mandible. The zygomatico-mandibularis is removed in an inferior to superior direction. This muscle is carefully scraped from its attachment along the masseteric 61

CHAPTER 2. MASTICATORY ANATOMY fossa. Beneath it, the thin tendon of the zygomatic temporalis muscle is visible along the anterior edge of the coronoid process.

2.3.2 Removal of the temporalis muscle group

Once the masseter group is removed, the temporalis group (superficial, deep, and zygomatic temporalis) is dissected. The zygomatic temporalis is a curved muscle whose origin lies between the temporal bone of the cranium and the zygomatic bone, arising from the dorsal and medial surfaces of the posterior part of the zygomatic arch. From there, the muscle stretches down between the coronoid process of the mandible and zygomatic arch. It is compartmentalized with the other temporalis muscles beneath a thin fascial sheet that separates it from the masseter muscles.

The origin of the muscle is thick and oval shaped, with the long axis of the oval parallel to the long axis of the skull. Most of the muscle is hidden from view by the zygomatic bone, but it does protrude slightly above the zygomatic arch. The muscle inserts on the lateral surface of the coronoid process, down to the superior edge of the masseteric fossa where it abuts the zygomatico-mandibularis. To remove it, the muscle is separated from the other temporalis muscles on the cranium, and the tendon detached from the anterior edge of the lateral surface of the coronoid process. The tendon is separated from the zygomatico-mandibularis by a thin fascial sheet. This tendon is of variable size and its length depends on the species. 62

CHAPTER 2. MASTICATORY ANATOMY

The superficial temporalis muscle is removed from superior and rostral to inferior and caudal. The superficial temporalis muscle has its origins immediately caudal to the superior orbital process and medially along the sagittal crest. The belly of the muscle sits in the furrow caudal to the orbit and superficial to a thin sheet of fascia of the deep temporalis muscle (i.e., the temporal tendon). The muscle ends as a fascial sheet covering the deep temporalis muscle caudally. This fascial sheet (part of the temporal fascia) is usually quite extensive and often larger in area than the belly of the muscle. Caudally, the temporal fascia is attached to the nuchal line along with the deep temporalis muscle. It is removed by cutting the site of origin and slowly peeling it from the deep temporalis, slicing its attachment to the sagittal crest while doing so. It is then cut from the nuchal line. The superficial temporalis has its inferior point of origin as a common tendon with the deep temporalis that inserts on the medial and superior aspects of the coronoid process and is removed with a slicing action against the points of attachment on the coronoid process.

Deep to the superficial temporalis is the deep temporalis, which originates on the lateral portion of the cranium and occupies the temporal fossa (Figure 2.3). It is usually the largest muscle of mastication, but in some specimens the superficial masseter is the largest. The deep temporalis is removed from superior and caudal to inferior and rostral. The muscle is cut from its attachments along the sagittal crest and nuchal line, then slowly and gently scraped from the cranium. The deep temporalis muscle is fan shaped, and has its insertion in the form of a thick tendon

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CHAPTER 2. MASTICATORY ANATOMY attached to the supero-medial side of the coronoid process and the superior most point of the medial aspect of the coronoid process (Figure 2.4). To remove the tendon, the scalpel is inserted between the cranium and zygomatic bone to access the coronoid process.

2.3.3 Removal of the masseter muscle group

Last to be removed is the medial pterygoid muscle. It has two heads (orbital head and pterygoid head) and to access it requires the removal of the ipsilateral hemimandible (Figure 2.5). The scalpel is used to break the mandible at the symphysis and slice away the tissue and muscles of the floor of the mouth. The mandible is slowly retracted caudally while the scalpel blade is scraped along the medial aspect of the ramus to free the medial pterygoid from its insertion on the medial pterygoid fossa of the mandible. Retraction of the jaw exposes the insertion surface of the medial pterygoid muscle. The muscle can then be removed from its attachments of origin. First, the muscle is scraped from the bone immediately inferior to the eyeball, on the caudal aspect of the maxillary bone. It is also carefully cut from its attachment between the medial and lateral pterygoid plates (scaphoid fossa). The medial pterygoid is large and irregularly shaped, but somewhat like a cylindrical prism.

After its removal, each muscle is weighed, and then preserved with a 10% buffered solution of formalin to await fiber architecture analyses (see Chapter 3).

During preservation, the anatomical position of each muscle is maintained. 64

CHAPTER 2. MASTICATORY ANATOMY

Figure 2.3: Origins of deep temporalis muscle

The deep temporalis of specimen 734BABA (T. belangeri) attached to the nuchal line (denoted by arrows).

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Figure 2.4: Deep temporalis insertion

The temporal tendon (arrows) inserting onto the tip of the coronoid process of the mandible (734BABA; T. belangeri). Unseen is the attachment of the tendon along the medial aspect of the process.

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Figure 2.5: Removal of hemimandible

The left hemimandible of specimen USNM 497016 (T. glis) retracted to access the medial pterygoid muscle (which was detached from its insertion on the hemimandible before retraction).

2.4 Results

2.4.1. Superficial Masseter

The superficial masseter (SM) is the largest muscle in the masseter group and frequently the largest muscle of mastication with regard to mass. It can compose up to 43% of chewing musculature, as in specimen USNM 111783 (T. nicobarica). The largest recorded superficial masseter dissected in this study was from a specimen of the common treeshrew T. glis (USNM 399582), which had a mass of 0.876g. However, it can be quite small as well, as seen in a specimen of the 67

CHAPTER 2. MASTICATORY ANATOMY lesser treeshrew T. minor (YBL 9011), which has both the absolutely smallest superficial masseter (0.118g) among the individuals dissected as well as the smallest as a percentage of the total weight of the muscles of mastication (26%). In specimens where the superficial masseter is not the largest, that rank is held by the deep temporalis muscle.

The SM has varying degrees of visible aponeurosis, depending on the individual, with the most developed muscles also having the largest tendons. In the aforementioned specimens USNM 111783 and USNM 399582, the tendon covers the entirety of the lateral surface of the muscle. In contrast, the smaller individuals

USNM 296647 (T. montana) and YBL 9011 had no obvious aponeurosis; the aponeurosis was likely present, but thin to the point of transparency (Figure 2.6). In all species, the inferior attachments of the SM are on the angle of the mandible. On the lateral aspect of the mandible, the muscle is attached along the angle, to its posterior projection, inferior and posterior to the inferior attachment of the tendon of the deep masseter muscle. On the medial aspect of the mandible, it is attached in the same fashion along the inferior edge of the jaw inferior to the inferior attachment of the medial pterygoid muscle.

It is interesting to note that the SM must wrap around the inferior and posterior aspect of the mandible in order to attach itself to the medial aspect of the angle. Although the muscle wraps around the inferior border of the mandible, anterior to the deep masseter insertion, it does not in fact insert itself to the inferior

68

CHAPTER 2. MASTICATORY ANATOMY border. This results in the superficial masseter being curved in the sagittal plane, with the inferior part of the muscle turning medially. The differences in attachments to the angle are reflected in the bony morphology of the angle itself. For example in specimen USNM 111783, the medial aspect of the angle has a pronounced ridge for the SM insertion (Figure 2.7), more so than in the other specimens.

Figure 2.6: Superficial masseter

Superficial masseter of specimen YBL 9011 (T. minor) showing very little aponeurosis on its superficial surface.

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Figure 2.7: Ridge for insertion of superficial masseter

Medial view of left hemimandible of specimen USNM 111783 (T. nicobarica) displaying a ridge (arrows) for the insertion of the superficial masseter, which must wrap around the inferior aspect of the mandible to reach its insertion area.

The superior attachment of the superficial masseter muscle is the inferior and lateral aspect of the zygomatic process. Anteriorly, on the zygomatic process of the maxillary bone, this begins approximately at the anterior edge of the upper second molar, inferior to the bone contributing to the bony orbit. The posterior border of the origin area is at the point where the zygomatic process of the temporal bone begins to rapidly expand.

The superior attachment of the SM also covers the large malar foramen, which contains the zygomaticofacial nerve and adipose tissue in life. The superior border of the foramen is an attachment site for the superficial masseter and the 70

CHAPTER 2. MASTICATORY ANATOMY inferior border of the foramen is an attachment point for the deep masseter. The foramen has a triangular shape with sharp ridges for borders; these might act as struts to distribute forces from chewing. This kind of strutting is especially notable on specimen USNM 580476 (T. tana) (Figure 2.8). The size of the foramen varies between individuals.

Figure 2.8: Strutting around malar foramen

Left zygomatic arch on specimen USNM 546344 (T. tana) illustrating strutting around the large malar foramen (arrows)

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The fibers of the SM run in an antero-superior to infero-posterior direction and work to elevate the mandible. By wrapping itself around the inferior portion of the mandible, the muscle can increase its surface area to increase its mass, thus increasing the force it can produce.

2.4.2. Deep Masseter

The deep masseter (DM) is not as large as the superficial masseter, and does not show as much size variation. Unlike the superficial masseter, which can enlarge into the cheek, the DM is constrained in size between the SM and zygomatico- mandibularis, presenting as a sheet between the two. Like the SM, it has varying degrees of visible tendon depending on the individual. For example, a thick white tendon covers most of the lateral surface of the DM in both specimens of T. tana, but only about half of the surface in T. montana (USNM 497016) (Figure 2.9)

The inferior attachment of the DM is the V-shaped depression within the lateral aspect of the mandibular angle. This V accommodates the thick tendon of the

DM and varies in size. The edges are frequently ridged in larger specimens such as T. glis (USNM 399582) (Figure 2.10).

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Figure 2.9: Deep masseter tendon

Specimen USNM 497016 (T. glis) with the superficial masseter removed to see the white tendon of the deep masseter underneath.

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Figure 2.10: Insertion for deep masseter

A pronounced V-shaped ridge on the lateral surface of the mandible (arrows) for the insertion of the large deep masseter present on specimen USNM 399582 (T. glis).

As with the SM, the anterior edge of the DM’s superior attachment is on the zygomatic process of the maxillary bone and its posterior attachment terminates where the zygomatic process of the temporal bone abruptly expands. Unlike the SM, however, the DM has its attachment along the inferior border of the zygomatic arch, including the section of bone contributing to one edge of the malar foramen. As mentioned previously, this attachment, in conjunction with that of the SM, may be determining the somewhat triangular shape of the foramen.

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Although the fibers of the DM also run in an antero-superior to infero- posterior direction like the SM, the fibers are angled far more vertically. There is also greater variation in their orientation relative to the SM due to the fan shape of the muscle, created by the common tendon.

2.4.3. Zygomatico-mandibularis

The smallest muscle within the masseter group, the zygomatico- mandibularis muscle (ZM) sits within the mandibular fossa on the lateral side of the mandible. Situated medial to the deep masseter and lateral to the mandible, the ZM is limited in size due to spatial constraints. As with the other masseter muscles, the appearance of the tendon (i.e., mote white) is dependent on the size of the individual and its muscles. Here, it is only visible on specimens T. nicobarica (USNM 111783) and T. glis (USNM 399582) and as seen in (Figure 2.11), presents as a white sheet covering the majority of the lateral edge of the muscle, abutting the underside of the deep masseter muscle.

The attachment area of the ZM is more extensive than that of either the deep or superficial masseter and lies against the masseteric fossa. This muscle is attached to most of the ramus of the mandible, with bony ridges for attachment running vertically along the anterior and posterior borders of the ramus. Superiorly, the muscle is attached to the ramus beginning at the approximate height of the zygomatic arch. The inferior attachment runs the antero-posterior length of the ramus inferiorly to the inferior border of the fossa. This fossa does not extend to the 75

CHAPTER 2. MASTICATORY ANATOMY edge of the ramus, however, due to the inferior attachment and wrapping of the superficial and deep masseters.

Figure 2.11: Zygomatico-mandibularis aponeurosis

An aponeurosis is visible on the lateral surface of the zygomatico-mandibularis in specimen USNM 111783 (T. nicobarica). This specimen has relatively large muscles and visibly defined aponeuroses.

Similar to the superficial and deep masseter muscles, the superior attachment of the ZM is along the zygomatic process. Its attachment is along the medial aspect of the inferior border of the zygomatic, and is not as extensive as

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CHAPTER 2. MASTICATORY ANATOMY those for the other two parts of the masseter complex. The anterior and posterior edges are the bony edges of the zygomatic arch, thus confining its size.

Similar to the DM, the fibers of the ZM run in an antero-superior to infero- posterior direction. However, they are positioned far more vertically and vary between specimens. For example, specimen USNM 580476 (T. tana) has particularly vertical fibers (Figure 2.12).

Figure 2.12: Zygomatico-mandibularis muscle fibers

The muscle fibers of the zygomatico-mandibularis in specimen USNM 580476 (T. tana) are relatively more vertical than the same fibers in other specimens. They are also more vertically positioned than the fibers of the overlying deep and superficial masseter muscles, which run more antero-posteriorly. Arrow denotes the nerve to masseter.

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2.4.4. Zygomatic Temporalis

The zygomatic temporalis (ZT) is the smallest muscle in the temporalis group. It is a crescent-shaped muscle and lies superficial to the anterior-most portion of the deep temporalis muscle. The posterior head of this muscle is bulbous, arching and tapering into a tendon (that itself blends with the tendon of insertion for the superficial temporalis) on the lateral surface of the ramus of the mandible.

This bulbous head is initially hidden from view, deep to the fascia that also covers the deep temporalis. It is separated from the deep temporalis by another, thinner, layer of fascia.

This muscle is not distinctly different in size between species, likely due to the bony constraints enveloping it. Larger ZT muscles are found in specimens with overall larger jaw adductor muscles, such as T. glis (USNM 399582), where it has expanded superiorly into the space anterior to the deep temporalis (Figure 2.13).

The length of the tendon varies as well, presenting as longer and thinner relative to the muscle belly when the muscle belly itself is small.

The origin of this muscle lies medial to the attachment of the SM muscle, on the superior aspect of the zygomatic arch at its posterior most point. Here, it sits in a large groove from which it projects anteriorly, continuing anteriorly between the anterior process of the deep temporalis muscle medially and the zygomatic bone laterally.

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As this muscle arches superiorly and laterally over the coronoid process, short fibers insert onto the lateral aspect of the coronoid process just below the tip, inferior to the attachment of the deep temporalis muscle. The remaining fibers continue anteriorly and inferiorly, ultimately converging as a common tendon of insertion on the anterior edge of the mandibular ramus at the junction where it meets the body of the mandible (Figure 2.14).

The majority of the posterior fibers and the anterior fibers course anteriorly and then inferiorly to insert on the ramus. However, the short posterior fibers that insert onto the lateral aspect of the coronoid process course mainly medially. Given its fiber orientation, this small muscle provides a limited posterior directed force on the ramus.

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Figure 2.13: Large zygomatic temporalis

Right side of specimen USNM 399582 (T. glis) illustrating a large zygomatic temporalis with a muscle belly protruding superiorly over the zygomatic arch (arrow).

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Figure 2.14: Tendon of insertion for the zygomatic temporalis

Arrow illustrating the tendon of insertion for the zygomatic temporalis for specimen USNM 580476 (T. tana). The tendon inserts along the lateral anterior border of the coronoid process

2.4.5. Superficial Temporalis

This triangular muscle lies in the anterior third of the temporal fossa. It is separated from the subjacent deep temporalis muscle by a layer of fascia. The superficial temporalis muscle (ST) varies in shape between specimens, primarily in how far it extends posteriorly. For example in T. tana (USNM 546344) (Figure 2.15), it projects posteriorly to lie superficially upon the anterior portion of the deep

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CHAPTER 2. MASTICATORY ANATOMY temporalis. In specimens with well-developed muscles, such as specimen USNM

546344, there is a concavity within the temporal fossa for the superficial temporalis, creating a clear boundary between the superficial and deep temporalis muscles.

The superior attachment of the fleshy part of this muscle is within the temporal fossa. However, fascia connects the muscle to the anterior edge of the fossa and sagittal crest; the fascia can be particularly thick and strong in some specimens. The inferior attachment of the ST is a common tendon on the tip and medial aspect of the coronoid process, as well as inferiorly along the anterior edge.

This tendon is shared with the other parts of the temporalis muscle.

Figure 2.15: Superficial temporalis overlying the deep temporalis

The superficial temporalis muscle thins out posteriorly, where it sometimes overlies the deep temporalis, as seen here in specimen USNM 546344 (T. tana).

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2.4.6. Deep Temporalis

A fan shaped muscle, the deep temporalis (DT) is frequently the largest muscle of mastication. Although the mass of the DT averages at approximately one- third of chewing muscle mass for the treeshrew sample, it can comprise as much as

41.5%, as seen in a specimen of T. nicobarica (USNM 111783). The largest recorded

DT muscle (1.15 g) was dissected from the 224 g T. nicobarica as well, and comprised 0.5% of its body mass. As with the ST, the body of the muscle lies within the temporal fossa, and occupies most of the fossa on the temporal bone. Anteriorly, it is separated from the ST by a thin sheet of fascia.

Along with the ST, this muscle initially presents deep to a thick fascial sheet.

Once the sheet is removed, the white aponeurosis of the muscle is visible, and is present on every specimen. It is more developed in certain specimens, including T. nicobarica (USNM 111783) (Figure 2.16). At the anterior edge of the muscle, along the edge of the ST muscle belly, the tendon of the DT is visible as it projects anteriorly and inferiorly to insert on the mandible (Figure 2.17).

The superior attachment of the DT is approximately the posterior two thirds of the temporal fossa. Like the ST, the DT usually has thick fascia connecting the muscle to the sagittal crest and the superior edge of the nuchal line, down to the zygomatic arch. The inferior point of attachment of the DT muscle is a common tendon shared with the ST muscle (Figure 2.18). This tendon inserts on the medial and superior aspects of the coronoid process. A larger proportion of the tendon

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CHAPTER 2. MASTICATORY ANATOMY accommodates fibers of the DT than of the SM. This tendon is exceptionally strong and can be difficult to remove from the bone during dissection.

Figure 2.16: Deep temporalis aponeurosis

The aponeurosis of the deep temporalis muscle in specimen USNM 111783 (T. nicobarica) is particularly thick and covers the majority of the lateral side of the muscle.

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Figure 2.17: Temporal tendon on deep temporalis

The temporal tendon (arrow)of specimen USNM 546344 (T. nicobarica). The tendon inserts on the tip of the coronoid process and continues inferiorly along its medial aspect. Fibers from the superficial temporalis course anteriorly and infero-laterally from the sagittal crest to insert in the common tendon. This gives the muscle a superior and posterior direction of force.

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Figure 2.18: Temporal tendon as seen after removal of the muscle

Right deep temporalis muscle from specimen USNM 399582 (T. glis).

From their origin on the posterior aspect of the cranium, the fibers of the DT course in different directions depending on their point of origin. Those from the nuchal line course slightly superiorly in order to join the common tendon, while those from the sagittal crest course slightly inferiorly. Once part of the tendon, these fibers course in an antero-inferior direction. On the whole, however, these fibers probably exert a posterior as well as superiorly directed force. You can differentiate the muscle from the overlying ST muscle due to the difference in fiber orientation.

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2.4.7. Medial Pterygoid

The medial pterygoid (MP) is a large muscle occupying most of the infratemporal fossa. Long and ovoid in shape, it is arguably the most complex muscle of mastication (Figure 2.19). Having both a pterygoid head and orbital head of origin, it is further complicated by having several internal tendons and more than one fiber direction.

One head of origin lies in the scaphoid fossa, between the medial and lateral pterygoid plates. This head is located medial to the body of the lateral pterygoid muscle, which has its origin on the lateral pterygoid plate. The second head of origin is large and originates on the floor of the orbit, on the superior aspect of the palatine bone, medial to the part of the maxilla containing the last molar. From these two heads, the fibers course laterally and postero-inferiorly to converge and attach to the angle of the mandible.

Fibers from the pterygoid head course a great deal more laterally than those from the orbital head, which are longer and course more posteriorly than they do laterally. I have omitted internal dissection of the MP muscle so as avoid damaging the fibers which are arranged in a complex geometry; this enables me to include the medial pterygoid muscle in the chemical dissections and follows procedures in

Perry et al. (2011b). Further comments on the internal structure of the MP can be found in Chapter 4.

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Figure 2.19: Medial pterygoid muscle after removal

Lateral view of the right medial pterygoid muscle from specimen USNM 399582 (T. glis).The insertion area is bounded by the mandibular condyle superiorly and the angle of the mandible inferiorly, including the medial pterygoid ridge. On its inferior attachment point, the medial pterygoid abuts the medial attachment point of the Superficial Masseter. In specimens with an inferiorly coursing sheet of Deep Temporalis, this sheet will attach at a point immediately anterior to the Medial Pterygoid.

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2.5 Discussion

Specimens representing closely related taxa will be discussed to examine the similarities they may share with treeshrews. These include dissections of a

Phillipine colugo (Cynocephalus volans) (Diogo, 2009) and strepsirrhine primates

(Perry, 2008). Comparisons will not be made to members of the Glires (rodents and lagomorphs) due to the unique nature of their masticatory musculature (Cox et al.,

2012).

2.5.1 Comparisons to previous treeshrew dissections

Previous studies of the muscles of mastication in specimens of treeshrews agree with the above observations generally, although they do not agree entirely.

Major points of disagreement include the proper separation of layers of masseter muscle, the appropriate grouping for the zygomatic temporalis muscle, and the number of heads of the temporalis that are identified.

Studies of the superficial and deep masseter muscles and the zygomatico- mandibularis have been approached differently by different researchers. The resulting disagreements arise from interpreting or missing the blending of fibers between the superficial masseter and deep masseter, between the deep masseter and zygomatic temporalis, and, to an extent, the relative amount of fiber insertions from the zygomatic temporalis onto the medial aspect of the zygomatic arch.

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In his dissection of the pen-tailed treeshrew P. lowii (1926), Le Gros Clark states that he has little difficulty identifying a superficial, intermediate, and deep layer of the masseter, which correspond to the superficial masseter, deep masseter, and zygomatico-mandibularis, respectively, presented in this study. Le Gros Clark describes the tendinous nature of the anterior border of the superficial masseter as well as its horizontal fiber direction, but does not describe any wrapping of the muscle around the angle of the mandible. He also describes the fiber blending of the superficial and intermediate layer of masseter (here the deep masseter).

Fish later conducted a thorough dissection of a specimen of T. glis (1983).

That study agrees with many of the findings presented here; however, the muscle he refers to as the superficial masseter is what I believe to be the superficial and deep masseter muscles dissected together (refer to Table 1.2 for muscle equivalencies).

He notes the “The orientation of the deeper fibers of superficial masseter differs from that of the more superficial ones.” (p23); that is, they run more vertically than the superficial fibers. This is in an indication that superficial masseter and deep masseter, with differing fiber orientations, were removed simultaneously.

He further describes the lack of any apparent fascial plane to distinguish between potential layers possessing different fiber orientations. Although this indicates he was prepared to find two distinct layers, I believe the reason he did not find a fascial separation is due to removing the layers in a superior to inferior direction and therefore immediately encountering the anteriorly blending fibers of

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CHAPTER 2. MASTICATORY ANATOMY the superficial and deep masseter muscles. If the superficial masseter is removed from inferior to superior, then the bright white external aponeurosis of the deep masseter, thinner dorsally than ventrally, is easily seen.

Table 2.2 Muscle equivalencies1

Muscle Synonym Superficial masseter (Includes the deep masseter Superficial masseter with this layer) (Diogo, 2009; Fish, 1983 and Le Gros Clark, 1924) Superficial masseter Deep masseter (Diogo, 2009; Fish, 1983; Le Gros Clark, 1924) Intermediate masseter (Le Gros Clark, 1926) Zygomatico- Deep masseter (Diogo, 2009; Fish, 1983; mandibularis Le Gros Clark, 1924; Le Gros Clark, 1926) Zygomaticomandibularis (Diogo, 2009; Fish, 1983) Zygomatic temporalis Zygomatic head of the temporalis (Le Gros Clark, 1924; Le Gros Clark, 1926) Superficial temporalis Temporalis (Diogo, 2009) Temporalis (Diogo, 2009) Deep temporalis Deep temporalis, zygomatic temporalis, intraorbital temporalis (Fish, 1983) Pterygoideus medialis (Diogo, 2009) Medial pterygoid Pterygoideus internus ( Le Gros Clark, 1924; Le Gros Clark, 1926)

1Equivalencies are limited to authors describing Scandentian muscles of mastication and do not include comparisons to primate or other mammalian muscle nomenclature.

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As described during the methods, the dissection of the superficial masseter from the deep masseter must always be performed in an inferior to superior direction. With the anterior fibers of the superficial and deep masseter muscles blending, it is important to dissect them from each other beginning at the angle of the mandible so as to not dissect the external aponeurosis of the deep masseter away with the superficial masseter.

Further evidence that Fish dissected the superficial and deep masseter muscles away together is indicated by how he describes what he is calling the deep masseter, and what I call the zygomatico-mandibularis: namely, the origin along the medial and inferior aspect of the zygomatic arch as well as the almost vertical fiber orientation.

The muscle I refer to as the zygomatic temporalis muscle, which Fish calls the zygomatico-mandibularis and Le Gros Clark calls the zygomatic head of the temporalis is accurately described by both authors. The discrepancy here is to which muscle group it has been assigned. Le Gros Clark groups it with the temporalis group as a third head of the temporalis muscle while Fish groups it with the masseter muscles. This is due to the nature of the muscle, having similarities to both the temporalis and masseter muscle groups.

I have categorized the zygomatic temporalis with the temporalis group, finding more similarities between this muscle and the temporalis group. Like the deep temporalis, this muscle has an area of origin superior to the zygomatic arch

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CHAPTER 2. MASTICATORY ANATOMY and forms a tendon to insert on the coronoid process. This makes it both anatomically and functionally similar to the temporalis group, having a posterior direction of pull. Although short fibers interdigitate with those of the zygomatico- mandibularis on the lateral aspect of the coronoid process, I see no evidence that the fibers of the zygomatic temporalis blends with the deepest fibers of the zygomatico- mandibularis, as Fish argues.

Gaspard (1973) avoids grouping the zygomatico-mandibularis (and zygomatic temporalis) with either the masseter or the temporalis by referring to both muscles as part of the “transitional layers” (“faisceaux de transition”). In galagos, both the zygomatico-mandibularis and the zygomatic temporalis are innervated, at least in part, by the nerve to masseter (Cordell, 1991).

Fish also argues for a distinct zygomatic head of the temporalis muscle, which is not supported by either Le Gros Clark or this study. The muscle to which

Fish refers is incorporated here and by Le Gros Clark as part of the deep temporalis muscle. As observed during dissections for this study, some specimens display a section of deep temporalis not covered with aponeurosis, and slightly bulging laterally, located posterior to the zygomatic temporalis (Figure 2.20). It inserts into the common temporalis tendon. When identifiable, it was not removed as its own muscle due to the lack of any fascial plane between it and the deep temporalis. It was found more frequently in specimens with a pronounced aponeurosis of the deep temporalis, but a pronounced aponeurosis did not guarantee its presence.

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One feature noted by Fish but not by Le Gros Clark, is the presence of what

Fish refers to as the intraorbital temporalis. He describes this as a thin slip of muscle originating on the posterior rim of the orbit, and passing directly inferolaterally to insert on the mylohyoid line, with a few fibers inserting along with the common tendon of the temporalis muscles. This muscle was observed in three specimens dissected for this study: T. tana, T. glis, and T. montana.

Figure 2.20: Aponeurosis arrangement of the temporalis muscle group

The aponeurosis of the deep temporalis is frequently much thicker than the superficial temporalis muscle, as seen in specimen USNM 399582 (T. glis). There is frequently a portion of the deep temporalis not covered in aponeurosis (arrow), which previous work has labelled a zygomatic of head of the deep temporalis (Fish, 1983).

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Although Fish describes this muscle as originating on the posterior side of the orbit, I believe these slips of muscle to be continuations of the deep (and possibly superficial) temporalis muscle, as Fish also speculates. This is upheld by the fact that during dissection, this muscle presents as a sheet with all of the fibers running directly inferolaterally. It is first observed upon removal of the hemi- mandible to dissect away the medial pterygoid muscle. If the fibers had originated on a point at the posterior aspect of the orbit, as Fish’s diagram illustrates and the orbit’s anatomy requires, they would instead fan so as to attach along the mylohoid line. This evidence supports the theory that this muscle is a continuation of the deep temporalis that did not combine with the common temporalis tendon, instead continuing inferiorly.

Interestingly, this slip of muscle is found in specimens that have relatively large zygomatico-mandibularis muscles. It could be that this additional growth of temporalis works to counteract the zygomatico-mandibularis to keep the jaw stable around an antero-posterior axis. With the zygomatico-mandibularis pulling laterally, this extra excursion of deep temporalis muscle would help rotate the jaw slightly medially around the long axis of the mandible.

This muscle does not interfere with the action of the medial pterygoid muscle, located immediately posterior and medial to this sheet. The medial pterygoid is an overall point of consensus between previous workers and this study, with some differences. Le Gros Clark describes this muscle as large and separable

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CHAPTER 2. MASTICATORY ANATOMY into two equal halves by a central tendon, but makes no mention of the large orbital head found in other specimens. Fish, meanwhile, describes this muscle as complex but makes note of only one head (intra-orbital) in passing.

2.5.2. Comparisons to C. Volans

A dissection of C. volans by Diogo (2009) reveals that the colugo has many similarities in its musculature to the treeshrews. Like Fish (1983), Diogo describes the masseter as comprising three layers, corresponding to the combined superficial and deep masseter, zygomatico-mandibularis, and zygomatic temporalis. The differences he describes include the narrower superior attachment of the superficial masseter. It does not extend the length of the zygomatic arch and as such, results in a smaller muscle than those found in the treeshrews. According to Diogo’s illustrations, it does not completely obscure either the mandibular ramus nor the deep masseter as it does in tree shews. He also does not describe the superficial masseter as wrapping around the angle of the jaw. The angle of the jaw in colugos does not have an elongate posterior projection the way it does in treeshrews, rather colugos possess a large ramus with a flared inferior border for the inferior attachment of the superficial masseter. Diogo does not specify where upon the mandible the muscle inserts.

Additionally, Diogo describes the zygomatic temporalis as grouping with the masseter because of the extent of the interdigitating fibers between it and the deepest layer of the masseter. However, he also concedes that it “is deeply mixed 96

CHAPTER 2. MASTICATORY ANATOMY with the medial fibers of the deep bundle, as well as with the lateral fibers of the temporalis” (p17). Unlike the treeshrews, whose short fibers of the zygomatic temporalis are attached sparsely to the lateral aspect of the coronoid process, in colugos the majority of its fibers are attached to the coronoid process; Diogo makes no mention of a tendon of attachment on the anterior border of the mandibular ramus, as found in treeshrews (and strepsirrhines).

Finally, Diogo claims that he found no clear divisions within the temporalis muscle. Although the attachment points were described as homologous to the treeshrews, he finds no facial plane to separate any layers. Furthermore, while he states the inferior attachment for the temporalis to be the medial and dorsal surfaces of the coronoid process, he makes no mention of a tendon of insertion, as the treeshrews have. However, in my experience with treeshrews, the superficial temporalis muscle must be removed prior to removal of the deep temporalis or the two muscles will be removed together. Therefore, divisions of the temporalis

(superficial and deep) may have been present in the colugos dissected by Diogo, but the temporal tendon forming the border between them might have been obscured due to dissection techniques. An additional possibility is that the specimens dissected had very thin superficial temporalis muscles with little muscle definition and consisting mainly of connective tissue.

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2.5.3. Comparisons to strepsirrhine primates

Several researchers have studied the chewing musculature of strepsirrhines for their similarities to early primates. The most comprehensive study was undertaken by Perry (2008), who studied them to make dietary inferences about adapid euprimates. His study encompasses 25 species of lemurs, lorises, and galagos, for which he dissected the same muscles as described above in the methods.

Like in treeshrews, in strepsirrhines the superficial masseter muscle is frequently the second largest jaw adductor after deep temporalis. Unlike in treeshrews, in strepsirrhines there are specimens where the superficial masseter is smaller than both the deep and superficial temporalis muscles. The most obvious difference between the superficial masseter in treeshrews and strepsirrhines is the posterior attachment of the muscle. In treeshrews, the muscle wraps around the angle of the jaw to insert on the medial edge, abutting the medial pterygoid muscle.

In strepsirrhines, the superficial masseter muscle attaches along the inferior edge of the lateral side of the jaw, leaving the inferior border of the jaw bare and not in contact with the medial pterygoid.

An interesting observation made by Perry (2008) but not seen in these dissections, is the existence of what he refers to as the anterior zygomatic tubercle

(or point, if it is less prominent), at the anterior end of the zygomatic arch for the attachment of the muscle. In treeshrews there is an attachment scar running along

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CHAPTER 2. MASTICATORY ANATOMY the inferior surface of the arch, but there is no obvious tubercle on any of the specimens; this location might be the site of a concentration of masticatory forces.

The deep masseter as described by Perry (2008; 2011b) conforms to the deep masseter muscles dissected here. One difference is the degree to which fascia covers the deep masseter muscle, and divides it from the zygomatico-mandibularis beneath, to which fibers are attached. From his dissections, most specimens present similar fascia between them. However, the tupaiid specimens varied greatly in this respect. Some specimens presented fascia that was thin and transparent, that caused difficulty separating layers, while other specimens presented with much thicker fascia.

The zygomatico-mandibularis as described by Perry differs from those seen in treeshrews mainly in the fascial thickness. Where he describes the fascia in strepsirrhines as occurring relatively posteriorly, the tupaiid specimens present a pattern in which the fascial thickness follows the entire zygomatic arch and runs more superiorly to inferiorly.

The contentious zygomatic temporalis, not fitting neatly into either the masseter or temporalis group, is markedly larger as seen in strepsirrhines than in treeshrews. In strepsirrhines, the muscle belly is wider both medio-laterially and antero-posteriorly and constitutes a greater percentage of the overall muscle; the muscle belly is visible in its lateral descent past the zygomatic arch and has only a short visible tendon at its insertion point. In each of the treeshrew specimens, on

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CHAPTER 2. MASTICATORY ANATOMY the other hand, this muscle has a relatively longer tendon of insertion that attaches along the entire anterior border of the coronoid process of the mandible. There is also no evidence of a tubercle of insertion as seen in some strepsirrhine specimens.

This is likely due to the tapering of the tendon as it inserts along the mandible rather than the robust tendon seen in the strepsirrhines. The muscle belly is restricted to its position lateral to the superficial temporalis and medial to the zygomatic arch.

One clear difference between the superficial temporalis of treeshrews and that of strepsirrhine primates dissected by Perry (2008) is its posterior range. In some larger species of strepsirrhine, including Varecia and Propithecus, the superficial temporalis almost completely overlies the deep temporalis. Yet in treeshrews and some smaller strepsirrhines, like Eulemur, the muscle is restricted to the anterior part of the temporal fossa, with its posterior border lying well anterior to the external auditory meatus. Like in strepsirrhines, there is frequently a ridge of bone delineating its posterior border in treeshrews. In strepsirrhine specimens dissected by Perry, however, the muscular fibers frequently overlie the deep temporalis muscle, sometimes making it difficult to dissect from the deeper muscle.

The deep temporalis is overall very similar between treeshrews and strepsirrhines. Both groups share a fascial layer that extends posteriorly over the deep temporalis, attaching to the sagittal crest. However, in some strepsirrhines, the

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CHAPTER 2. MASTICATORY ANATOMY postero-dorsal extent of this tendon is reduced and it never makes it (visibly, at least) as far as the sagittal crest; in such specimens, this temporal tendon is concealed between the superficial temporalis and deep temporalis. Additionally, the deep temporalis was not observed by Perry (2008) to have any inferiorly descending fibers attaching to the medial aspect of the jaw, only to the medial side of the coronoid process, as observed in three specimens of treeshrews dissected for this study.

Another muscle comparable between the two groups is the medial pterygoid.

Like the strepsirrhines, the medial pterygoid in treeshrews has a larger pterygoid head than orbital head. One notable difference is that the muscle is more anteroposteriorly oriented in treeshrews than strepsirrhines, possibly as a function of their longer jaw.

2.6 Conclusions

The muscles of mastication dissected as part of this study illustrate a great deal of uniformity amongst specimens. The most notable differences between corresponding muscles were predominantly size, but also degree of aponeurosis development and presence of the additional muscle “sheet” of deep temporalis that forms the muscle’s medial-most component. It should be noted that although P. lowii is considered to be the most primitive treeshrew (Sargis, 2004) the dissection performed by Le Gros Clark did not introduce any unique anatomical findings to distinguish it from the tupaiid dissections performed here. 101

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A major point of contention in dissection reports concerns the number of layers into which the masseter muscle of treeshrews is divided. For example, while both Le Gros Clark (1924) and Fish (1983) divided the muscle itself into two groups by dissecting the superficial and deep masseter together as one layer and the zygomatico-mandibularis as the second, Fish included a third layer comprised of the zygomatic temporalis. Thus, although Fish and I have reported a total of three layers from our dissections, the layers to which I refer are contained within the masseter muscle and his layers include the masseter as well as a muscle from another group.

Another problematic muscle is the zygomatic temporalis. Previous researchers have been conflicted as to whether this muscle should be associated with the masseter or temporalis group and each has his/her own reasons for categorizing this muscle as part of either group, with Fish arguing the zygomatic temporalis blends with the deepest fibers of the zygomatico-mandibularis and Le

Gros Clark arguing it is a third head of the temporalis muscle. Here, I did not categorize the muscle with either group as I consider it to act more as a transition muscle between the groups than to align with either group wholly. Cordell (1991) describes this muscle in galagos and notes that it is innervated by a branch of the nerve to masseter; nevertheless, she considers the muscle to be a part of the temporalis complex and Perry (2008) follows that categorization for strepsirrhines as a group.

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When compared to closely related taxa, the treeshrew muscles of mastication are remarkably similar to those of both colugos and strepsirrhine primates. Like treeshrews, colugos have the same muscles present in the masseter group, although with differing areas of attachment. More research needs to be conducted on the temporalis and pterygoid groups of muscle before any further comparisons can be made.

Comparisons to strepsirrhine primates highlight the similarity in structure and arrangement of the chewing musculature while differences are focused in relative size of muscles, the different degree of fascia present, and the differing forms of insertion. For example, the masseter muscles in strepsirrhines overall have the same points of origin and are similar in relative size to each other and the other muscles of mastication. However, the muscles more frequently had thick fascial coverings, which the treeshrews on average lacked, and the superficial masseter muscle did not share the same “wrap around” inferior insertion onto the angle of the jaw that the treeshrews presented.

The zygomatic temporalis muscle was the greatest point of difference between the two groups: small with a long, thin tendon in treeshrews, yet large and with a short, robust tendon in strepsirrhines. This muscle was robust enough in strepsirrhines that a tubercle accommodates the tendon on the body of the jaw which is not present in treeshrews.

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The fewest differences between treeshrews and strepsirrhines were found in the superficial and deep temporalis muscles and in the medial pterygoid. The superficial temporalis differed largely in how far posteriorly the muscle body extended, and the deep temporalis differed primarily in the greater degree of aponeurosis displayed in strepsirrhines. The medial pterygoid muscle was fairly consistent between the two groups, although the relative size of the two heads differed within strepsirrhines. It is important to note that the internal structure of the medial pterygoid was not examined in detail here nor was it described systematically by Perry; therefore, differences between treeshrews and strepsirrhines in internal structure of the medial pterygoid might exist.

Further work on treeshrew muscles of mastication would provide additional insight into their chewing systems and contribute to a greater understanding of how these remarkable mammals process food. In particular, additional detailed dissections of colugos and dissections of additional genera of treeshrews (especially

Ptilocercus) would shed light on variation within the orders of Euarchonta.

Currently, however, alongside strepsirrhines, they stand as promising model for reconstructing the masticatory anatomy in plesiadapiforms.

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CHAPTER 3. DiCeCT

Chapter 3: Applications of iodine staining to tupaiid

masticatory musculature

3.1 Introduction

Computed Tomography (CT) as a form of non-invasive imaging has proven to be extremely valuable to the field of comparative biology. In producing digital renderings of mineralized tissues, anatomical structures can be studied despite, for example, being embedded deep within other tissues (Gignac and Kley, 2014) or within hard matrix (Garwood et al., 2016). These tissues retain their original orientations and relationships, providing clear advantages to studies spanning ontogeny, phylogeny, and biomechanics.

Mineralized tissues have great attenuation because of an increased capacity for blocking x-rays. Soft tissues on the other hand have lower attenuation and are not so easily differentiated by x-ray (Metscher, 2009). Thus, a burgeoning technique within the realm of CT scanning involves staining fixed specimens with a solution of iodine, followed by CT scanning. This technique is commonly referred to as

Diffusible Iodine-based Contrast-Enhanced Computed Tomography (DiceCT). The iodine provides excellent 3D visualization of soft tissue by giving it a higher x-ray attenuation.

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DiceCT has become very popular in the last decade and used to investigate soft tissue morphology in a range of vertebrates, including primates (Burrows et al.,

2019), small mammals like mice (Badea et al., 2008; Baverstock et al., 2013), naked mole rats (Cox and Faulkes, 2014), squirrels (Cox and Jeffery, 2011), bats (Hedrick et al., 2018; Santana, 2018; Yohe et al., 2018), birds (Lautenschlager et al., 2014; Li et al., 2016; Tahara and Larsson, 2013), arthropods (Akkari et al., 2018), as well as mineralized structures like mollusk shells (Sasaki et al., 2018) and teeth (Alstrup et al., 2017; Nasrullah et al., 2018) in relation to soft tissues.

In their work on archosaurs, Gignac and Kley (2014) found that lipids and skeletal muscle had the highest attenuation values of the soft tissue stained with iodine, followed by glandular and myelinated peripheral nervous system tissues, and non‐myelinated tissues of both the central and peripheral nervous systems. The resulting images enable clear differentiation of cranial structures including arteries, nerves, the brain, and muscles of mastication.

Due to the relativelyhigh level of x-ray attenuation found in skeletal muscle compared to other soft tissues, digital isolation of muscles is now possible with

DiceCT, including muscles of mastication as well as their individual muscle fascicles.

It is especially well suited for visualizing the medial and lateral pterygoid muscles; located internally, they are often difficult to dissect in small mammals. This method is therefore of great use for investigating bite force mechanics because the common method of approximating muscle force, Physiological Cross-sectionial Area (PCSA),

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CHAPTER 3. DiCeCT involves measuring both muscle volume and muscle fascicle length. These variables are usually measured through traditional gross dissection.

A recent experiment conducted by Dickinson et al (2018) tested the reliability of this method as an alternative to traditional dissection, specifically with regards to obtaining accurate volume and muscle fascicle length measurements in primate chewing musculature. Using a specimen of Macaca fascicularis (the crab eating macaque) as its own control, the muscles of mastication on one side of the mandible were subjected to DiceCT and the muscles of the other side to traditional gross dissection. Their results indicate that muscle volume and fascicle length were found to correspond closely between the two methods, with the temporalis muscle group showing the greatest variation.

Fascicles were the subject of another intriguing study using the superficial masseter of the dog (Kupczik et al., 2015). Here the authors sought to use pattern recognition to create a vector field of fascicle orientations for use in creating more accurate PCSA calculations. They found the model to faithfully represent the complexity of fascicle pinnation. In a study of Noctilionoid bats, Santana (2018) compared traditional dissection with DiceCT scans to investigate jaw adductor muscle architecture, including compartmentalization and PCSA scaling. She found a strong correlation between dissection-based and DiceCT-based PCSA estimates, with DiceCT generating greater estimates.

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Similar to Santana’s study, the research presented here will employ tupaiid specimens as a test case to demonstrate that traditional and digital dissection are comparable methods. Specimens will be stained using a potassium iodine solution and subsequently microCT scanned, followed by digital segmentation and measurement of the muscles of mastication. Once scanned, specimens will be de- stained and manually dissected to compare digital versus manual dissection-based fiber architecture measurements.

I predict that, like the findings presented by Dickinson et al. (2018), both methods will return equivalent measurements for muscle volume and fiber length.

Such a finding will then demonstrate the use of DiceCT for studying rare specimens, in that they can be stained, scanned, and returned to museums undamaged. The effects would be far-reaching, and allow for a dramatic increase in sample size for research limited by specimen availability.

3.2 Materials

Two treeshrew specimens were included in this study, namely one specimen representing one of the largest treeshrews (YBL 9010, T. tana) and one representing one of the smallest (YBL 9011, T. minor) treeshrew. Not only are are they different in size, but also in substrate use and dietary preferences (Emmons, 2000). These specimens were loaned for destructive sampling from the Yale Biology Laboratory.

Both specimens were frozen from death and preserved in 10% buffered formalin in

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CHAPTER 3. DiCeCT preparation for staining, as per published protocols (Gignac et al., 2016). Specimens can be found in Table 3.1.

All staining preparation took place in the laboratory of Dr. Paul Gignac of The

Department of Anatomy and Cell Biology in the Health Sciences Center at the

University of Oklahoma, Tulsa. All micro-CT scanning took place in the Shared

Materials Instrumentation Facility at Duke University using a Nikon XTH 225 ST

Micro-CT scanner. Digital segmenting and analyses were performed using Avizo 8.0 at Johns Hopkins University.

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Table 3.1: Parameters for staining and CT scanning

Specimen Taxon Staining Days in Isometric X-ray Tube X-ray Tube Concentration Solution Voxel Size (µm) Voltage (kv) Amperage (µA) CHAPTER 3. DiCeCT

110 YBL 9011 T. minor 3% I KI 14 26.5 100 300 2

YBL 9010 14 100 300 T. tana 3% I2KI 44.5

Abbreviations: YBL – Yale Biological Anthropology Laboratory

CHAPTER 3. DiCeCT

3.3 Methods

3.3.1. Diffusible Iodine-based Contrast-Enhanced Computed Tomography

Several contrast enhancing agents are commonly used to increase soft tissue attenuation, including osmium tetroxide, which binds preferentially to lipids, as well as phosphomolybdic acid and phosphotungstic acid, which bind preferentially to protein and collagen (Kiernan, 2008). Here, I use Iodine-potassium iodide (Lugol’s iodine; I2KI). I2KI is both cost effective and has an affinity for a wide range of tissues, enabling more comprehensive 3D visualizations.

The amount of time a specimen needs to be perfused in order to adequately absorb enough iodine is proportional to its size and the accessibility of the muscles to the perfusion solution. Here, the muscles of most concern for staining were the masseter muscles, as they are the muscles with the greates cross-sectional area being studied. However, because both specimens had been necropsied, exposing the tissue in the neck, the superficial masseter muscles were directly accessible to the iodine solution. Both specimens were stained in a 3% aqueous solution of I2KI (1% w/v of I2 and 2% w/v of KI) for 14 days, a period of time chosen following Cox and

Faulkes’ (2014) method for staining the masticatory muscles of a naked mole-rat

(Heterocephalus glaber), and Dr. Gignac’s personal experience with staining timelines.

The I2KI solutions were prepared using I2 and KI compounds measured using a digital scale accurate to the thousandth of a gram. The percentage of each solution 111

CHAPTER 3. DiCeCT

(3% and 5%) was verified using titration composed of 1% sodium thiosulfate

(Na₂S₂O₃xH₂O). Staining parameters can be found in Table 3.1.

Specimens were prepared for staining by first being fixed in a 10% buffered formalin solution. After ensuring full preservation, they could now be moved to containers in which they would stay during the staining process. Glass jars were used for their non-porous nature and ability to be sealed. They were wrapped with aluminum foil so as to not allow any light into the jars, due to the highly photosensitive nature of iodine.

Testing for perfusion completeness usually requires either iterative scans of the same specimen (e.g., Santana, 2018), or successive bisections of identically perfused specimens. Because an appropriate CT scanner was unavailable on-siteand the specimens were too precious to bisect, neither scanning nor sectioning were possible for this study. Therefore, full perfusion was estimated based on the work of

Cox and Faulkes (2014) and was verified during subsequent CT scanning.

After the specimens had been scanned, the iodine in the specimens was neutralized using a 1% solution of sodium thiosulfate, a naturally clear substance in opposition to the brown color of the iodine solution. The specimens were allowed to sit in the solution until the iodine was thoroughly neutralized, identifiable when the brown of the iodine was replaced entirely with the clarity of the sodium thiosulfate.

Depending on the size of the specimen, this may require the sodium thiosulfate to be replaced until it runs clear; in this case the specimens were small enough that this

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CHAPTER 3. DiCeCT was not required and neutralization was complete in approximately 10 minutes.

Afterwards, the specimens were returned to their formalin solution, which has the additional benefit of leaching the iodine from the tissue.

3.3.2. MicroCT scanning

Specimens were removed from their iodine solutions and patted dry with paper towels to absorb excess iodine so as to not have pooling iodine affecting the image. Specimens were then placed in standard polyethylene bags and situated into the scanner such that the resolution of their heads was maximized. The minimum resolution required was used for each specimen, meaning a resolution of 44.5 µm for T. tana and a resolution of 26.5µm for T. minor. Both specimens were scanned with a voltage of 150 kV and an amperage of 300µA. Scanning parameters can be found in Table 3.1.

After scanning, 3D reconstructions of the muscles of mastication were created using the labelling tools and segmentation function in Avizo 8.0.

3.4 Results

Muscles groups were easily identified based upon muscle origins and insertions as well as muscle fiber direction. Larger bundles of fascicles contained within connective tissue compartments were identified and used to delineate separate muscles layers (e.g., superficial versus deep masseter). Muscle relationships were verified afterward by gross dissection.

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Many aspects of the masticatory system were clearly visible during the image segmentation, for example, the tendon of the temporalis muscle (Figure 3.1).

Figure 3.1: Tendon of the Temporalis muscle

Transverse view of muscle fascicles in T. tana illustrating the clearly defined tendon of the deep temporalis muscle (DT) as well as blending of muscle fascicles from zygomatic temporalis (ZT) and zygomatico-mandibularis (ZM). Other muscles labeled include the deep masseter (DM), superficial masseter (SM) and medial pterygoid (MP).

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Due to muscle fiber blending, there were difficulties isolating muscles.

Despite not finding many blending fibers in the dissections performed in Chapter

Two (section 2.4.4), the zygomatic temporalis muscle proved to be the most difficult muscle to isolate due to fiber blending. Figure 3.2 illustrates muscle fascicle bundles of the zygomatic temporalis and zygomatico-mandibularis in T. tana. Inferior fibers of the zygomatic temporalis blended with the superior fibers muscle of the masseter muscle group (seen in Figure 3.1). The latter belong to the zygomatico- mandibularis, the deepest member of the masseter group.

Figure 3.2: Fiber direction in the masseter muscle group

Coronal view of muscle fascicles in T. tana. The blending of muscle fascicles from zygomatic temporalis (ZT) and zygomatico-mandibularis (ZM) is clearly visible.

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Furthermore, blending also meant that not every muscle of mastication could be individually isolated; the masseter group had to be segmented as one unit, as seen highlighted in pink in Figure 3.3.

Figure 3.3: Fiber direction in the masseter muscle group

Sagittal section through the masticatory muscles of T. minor illustrating the differing fiber directions of the superficial and deep masseter muscles. Other muscles labeled include the superficial temporalis (ST), zygomatic temporalis (ZT), deep masseter (DM), superficial masseter (SM) and medial pterygoid (MP).

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Careful evaluation of the muscle fibers did reveal a difference between glycolytic versus oxidative muscle fibers. Muscles with more glycolytic fibers (the superficial masseter) appeared brighter and more white from binding more iodine than did muscles with more oxidative fibers (the medial pterygoid), which appeared darker. This supports the same findings as reported by Gignac and Kley (2014).

Fascicles were clearest in T. minor, but only in the masseter muscle group and only for a select few fibers. Further manipulation of the data would have been required to take additional measurements, such as resampling the data along an axis parallel to the direction of muscle fibers of interest. Therefore, sampling only a few fibers from one muscle from one specimen to compare to fibers measured under the microscope using traditional dissection was not considered statistically reliable.

Isolating the muscles digitally provided an alternative measure of muscle volume as compared to traditional dissection. After each muscle group was digitally segmented, volume calculations were performed in Avizo, as reported in Figure 3.2, and compared to volume measurements of the same muscles collected during gross dissection. Muscle volume was calculated for the gross dissections by dividing muscle mass by the density constant of muscle, 1.056g/cm3 (Murphy and Beardsley,

1974). These differences range from 15% in the zygomatic temporalis of T. tana to

66% in the deep temporalis of T. minor. Overall, apparent muscle volume is greater when using traditional dissection than digital dissection, with the exception of the zygomatic temporalis from T. minor, which appeared larger in digital dissection.

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Table 3.2: Comparison of muscle volume

Vol. (cm3) Vol. (cm3) Specimen Taxon Muscle % Diff. Dissection CT Masseter Group 0.176 0.119 32 Zygomatic Temporalis 0.02 0.016 20 YBL 9011 T. minor Superficial Temporalis 0.027 0.022 17 Deep Temporalis 0.132 0.045 66 Medial Pterygoid 0.059 0.039 34 Masseter Group 0.436 0.343 21 Zygomatic Temporalis 0.054 0.062 -15 YBL 9010 T. tana Superficial Temporalis 0.049 0.029 41 Deep Temporalis 0.149 0.115 23 Medial Pterygoid 0.099 0.062 38

3.5 Discussion

3.5.1 Dissection and muscle architecture

With regards to my first prediction, that muscle fascicles would present with similar lengths to measurements taken from traditional dissection, this was neither supported nor refuted. Fascicles, primarily due to their bending between planes, were not measurable in these specimens due to the resolutions of the scans.

Fascicles are generally visible between 30-35µm (P. Gignac, personal communication) and both specimens have resolutions falling outside of this window.

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Muscle architecture from the MicroCT scans was consistent with that found during the gross dissection of these specimens as well as gross dissections of other tupaiid specimens (see Chapter Two, Discussion). Differences in architecture encountered between the two methods are attributed to the delicate nature of the tissue, possibly leading to human error in dissecting, and the layered nature of the masticatory complex.

In practice, this primarily manifests as small pieces of tissue being left behind during removal, usually where a ligament attaches to bone. For example, the deep temporalis tendon may not be removed in its entirely due to its thick and strong attachment to the medial and superior aspect of the coronoid process. It is also possible that small pieces of muscle are removed with the wrong muscle where muscles are layered, for example, accidentally removing a portion of the deep masseter with the superficial masseter if removed in the wrong order the tendon of the deep masseter (refer to Chapter Two, section 2.3.1).

Traditional dissection can therefore cause issues for measuring aspects of a muscle, especially muscle volume. For example, removing a piece of one muscle with another could significantly alter its mass. Although probably not an issue for a large specimen with muscles of mastication of a great size, this could impact the accuracy of the muscle volume of a small specimen such as a treeshrew.

These issues alone do not make an argument for employing the time sensitive and expensive DiceCT to avoid these potential errors. However, in some

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CHAPTER 3. DiCeCT cases, digitally segmenting muscles of mastication can provide advantages over gross dissection. For example, the preferential uptake of the iodine means fatty tissue is not dissected with the muscle and incorporated into measurements as might happen with traditional dissection. Also, more detail can be observed using this method, including placement of blood vessels and nerves. High scanning resolution also allows researchers to observe and measure small differences unavailable to the naked eye and compare these differences with more accuracy.

3.5.2 Muscle volume

Measuring muscle volume is perhaps one of the largest current debates in using iodine staining and MicroCT scanning for measuring and calculating muscle parameters. This is because of its potential use in addressing the issue of shrinkage incurred during preservation. One the one hand, traditional dissection may cause small portions of muscle to be left behind or misattributed, as aforementioned, but presumably causes no (specimen fresh) or limited (specimen frozen or preserved) tissue shrinkage from loss of water.

On the other hand, segmenting scanned material allows for a greater degree of accuracy with regards to muscle completeness, orientation, and two-dimensional measuring, for example, digitally isolated muscles omit the facial layers surrounding and permeating each muscle fiber bundle; this fascia represents a large portion of the volume of a traditionally dissected muscle. However, the process of fixing and staining muscle tissue causes shrinkage. As reported by Gignac and Kley (2014) and

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Vickerton et al. (2013), longer exposure of soft tissue to the iodine solution can cause greater shrinkage to occur, and this might be exacerbated by the concentration of the solution as well as the surface area of the tissue absorbing the solution.

Differences in muscle volume as measured by traditional dissection versus digital dissection for the specimens in this study can be found in Table 3.2. The range of differences is great (15%-66%) and does not appear to follow any specific pattern. That being said, these differences uphold the aforementioned problem of shrinkage due to staining, whereby each muscle, or muscle group, exhibits greater volume when measured using traditional dissection methods than digital dissection methods, with one exception.

The outlier, a zygomatic temporalis muscle, may display a smaller volume when measured traditionally due to the previously mentioned issues of dissecting delicate tissue: this is a small muscle almost completely hidden behind the coronoid process of the mandible when viewed laterally, and is therefore difficult to dissect.

Furthermore, this muscle belonged to a specimen of T. minor, one of the smallest treeshrews, meaning this muscle was very small in absolute terms, and makes any error in dissecting it away from the zygomatico- mandibularis disproportionate to its size.

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Ultimately I believe a combination of gross and digital dissection is best, a sentiment also expressed by Dickinson et al. (2018) and illustrated in Santana’s

(2018) work. Here, she employed DiceCT to measure the volume and create 3D visualizations of the muscles of mastication in noctilionoid bats. Unfortunately, due to the small physical size of the specimens, resulting images were too low in resolution to calculate the lengths of muscle fibers. This therefore required the author to perform traditional dissections to collect muscle fiber measurements rather than using DiceCT to calculate PCSA.

3.5.3 Future Work

The advantages to DiceCT and digital dissection include digitally isolating muscles and preserving their orientation; accurate muscle orientations are important for calculating muscle force and thereby estimating gape, bite force, and stress and strain. Estimating these variables open up many exciting possibilities for comparative and developmental biologists to investigate soft tissue anatomy through the advent of DiceCT. However, ongoing issues with shrinkage will need to be addressed more thoroughly in order to provide faithful results. For example, in this study, muscle PCSA, a measure of a muscle’s force, was correlated to muscle mass meaning that any substantial levels of tissue shrinkage could drastically alter bite force estimates.

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Future research would focus on building a wholly 3-Dimensional model of a treeshrew skull. For example, CT scanning the cranium and mandible before introducing the specimen to the iodine stain would enable a digital skull base onto which I could map the muscles of mastication (Figure 3.4). By studying the forces acting on the skull, these data could then be applied to fossil primate primates as an analog for studying early primate chewing systems.

Figure 3.4: 3D modeling muscles of mastication

Muscles of mastication segmented from T. minor superimposed onto a 3D rendering of a skull of T. belangeri. Superficial temporalis (ST), deep temporalis (DT), zygomatic- temporalis (ZT) and the superficial masseter group (SM)

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3.6 Conclusions

These results indicate that using DiceCT to digitally isolate the muscles of mastication provide more accurate measures of muscle tissue volume as compared to traditional dissection, while also preserving their orientation and position relative to bone. While muscle fascicles are not measureable using these scans, they may be measurable if resolution could be increased or more time was taken to resample each muscle to orient fibers in a plane.

If using DiceCT for reconstructive purposes, the difference in volume between digital and traditional dissection must be taken into account. Digital segmentation should still be paired with traditional dissection for isolating small, interdigitated muscles like the masseter muscles and the zygomatic temporalis. In addition, care needs to be taken when segmenting specific muscles due to the lack of clear fascial separation in digital space.

There are advantages and disadvantages to both traditional and digital dissection, as I have outlined here, and I suggest future researchers apply these methods based on careful consideration of their research questions. For example, research focused on muscle architecture might benefit more from applications of iodine staining and MicroCt scanning specimens, where the high level of detail will be advantageous. Meanwhile research with a need for accurate muscle mass might benefit from manually dissecting fresh or frozen tissue.

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Another advantage of this method is the immediate and permanent availability of scan data for research purposes, especially from afar; digital muscles are not subject to degradation as preserved muscles are. In the same vein, this also means that the same muscles are available to be studied by multiple researchers simultaneously. This will allow reproducibility studies to be performed, impossible before with traditionally dissected specimens.

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Chapter 4: CORRELATIONS AND PCSA

Chapter 4: Tupaiid Jaw Adductor Properties and their

Suitability for Plesiadapiform Dietary Reconstruction

4.1 Introduction

Body size (Gould, 1975; Schlichting and Pigliucci, 1998)and food properties

(Daegling, 1992; Hylander, 1979a) account for much of the variation in the hard tissue morphology of the mammalian masticatory system. While the mandible is involved with behaviors that go beyond mastication, including breathing and vocalizing (Smith, 1984), it is going to be the influence of body size and food properties on the soft tissue anatomy that will be discussed here, which remains relatively understudied in many species, including treeshrews (Order: Scandentia).

Treeshrews have been argued to make excellent analogs for both early primates (Szalay and Drawhorn, 1980; Vinyard et al., 2007) and plesiadapiforms

(Bloch et al., 2007) for their small size, plesiomorphic characteristics, and omnivorous diet (e.g., Tattersall, 1984). All treeshrews are omnivorous and consume a variety of fruit, leaves, insects, sap, earthworms, small arthropods

(Emmons, 2000; Oomen and Shanker, 2008). I begin my investigation into plesiadapiform dietary patterns by first studying the muscles of mastication in treeshrews.

The jaw adductor muscles act upon the mandible to process food primarily through elevation, retraction, and lateral and medial rotation. While all three muscle 126

Chapter 4: CORRELATIONS AND PCSA groups (temporalis, masseter, medial pterygoid) work to provide balance and force during mastication, three muscles in particular have the largest impact on mandibular shape due to their function and size (Hiiemae, 1967). The superficial temporalis works as a stabilizer of the jaw, due to its almost vertically inserting fibers that align with the mandibular condyle, here assumed to be the fulcrum of the masticatory apparatus (Anapol and Lee, 1994; Perry, 2018). It is particularly strong during protrusion of the jaw and during a larger gape, such as when incising (Taylor and Vinyard, 2009). The superficial masseter muscle is a powerful elevator of the jaw and performs optimally when molar chewing (Hylander et al., 1992). Due to the lateral rotation the masseter muscle imparts onto the mandible, the medial pterygoid works to counteract this pull by medially rotating the jaw about the long axis.

Several researchers have studied how chewing muscle variation can lead to inferences for diet. In his decade-long study of strepsirrhine primates, Perry has investigated the muscles of mastication from a dietary vantage point, finding correlations between strepsirrhines dietary categories and jaw adductor dimensions, including muscle mass, muscle fascicle length, and cross-sectional area

(Perry, 2008; Perry et al., 2016, 2011a, 2011b). His work shows that insectivorous and frugivorous strepsirrhines have a larger ratio of temporalis muscle mass to masseter muscle mass, while the opposite is true for more folivorous strepsirrhines.

Anapol and Lee (1994) found the same pattern of temporalis to masseter mass ratio

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Chapter 4: CORRELATIONS AND PCSA in platyrrhine primates, where the ratio of masseter muscle was larger in folivorous and frugivorous species (e.g., Ateles, Alouatta) with the temporalis greater in the relatively more omnivorous Cebus, eating fauna and seeds. Looking deeper, a recent survey of masticatory muscle architecture by Hartstone-Rose and colleagues (2018) across all primates reveals that muscle fiber architecture correlates with diet, whereby species eating softer food like fruit have longer fibers that would accommodate an increased gape.

My work expands on this research by studying the muscles of mastication in treeshrews for use as extant models for studying plesiadapiforms. In particular, I focus on the Physiological Cross-section of the muscles of mastication, which should yield a measurement proportional to the maximum force a muscle can generate

(Powell et al., 1984; Weijs and Hillen, 1985).

The muscle dimensions and scaling relationships found in strepsirrhine primates will be used to test hypotheses about muscle dimensions and muscle scaling relationships in treeshrews. The primary interactions I test involve muscle scaling. Specifically, I predict that like strepsirrhines (see table 4.12 in Perry, 2008;

Cachel, 1979), tupaiids will demonstrate isometry in jaw adductor mass relative to jaw length cubed, fiber length in relation to jaw length cubed, and physiological cross-sectional area (PCSA) relative to jaw length cubed. Isometry is predicted for these variables due to their relationships to body size. In addition to its probable relationship to overall body size, fiber length should also increase proportionally to

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Chapter 4: CORRELATIONS AND PCSA jaw length as a function of gape (Perry, 2008). PCSA, itself a function of the two aforementioned variables, would therefore also exhibit isometry, perhaps for multiple reasons. Departures from simple isometric relationships might reflect situations where gape itself does not track body size (E.g., allometry in bite size).

Concerning relative muscle sizes, I make three predictions regarding the three muscle groups: the masseter, temporalis, and medial pterygoid. All predictions are based on the work of Cachel (1979) and supported by Perry (2008). First, I hypothesize that due to the anterior forces required for incisive biting, the temporalis muscle group will be larger in the insectivorous strepsirrhines and treeshrews relative to all other strepsirrhines. Secondly, I hypothesize that the masseter group will be larger in cross section in the more folivorous strepsirrhines relative to all other strepsirrhines and treeshrews. Third, I hypothesize that the relative cross-sectional area of the medial pterygoid will be greater in folivorous and insectivorous strepsirrhines and treeshrews relative to other strepsirrhines.

Potential uses of these metrics include study of both extant and fossil species dietary behaviors, including assigning major dietary categories and estimating bite force in treeshrews and fossil primates.

4.2 Materials

To begin analyzing the muscles of mastication, I examined the jaw adductor muscles of six species of treeshrew, represented by ten individuals (Table 4.1). As a comparative sample, data from 13 species of strepsirrhines were included from the 129

Chapter 4: CORRELATIONS AND PCSA literature (see Table 1 in Perry et al., 2011b). Specimens were either preserved in formalin or frozen since death.

No preference was given to dissecting based on sex, as treeshrews have been found to be extremely sexually monomorphic (Emmons, 2000). While sexual dimorphism does have an impact on jaw morphology in primates, several studies

(Lindenfors and Tullberg, 1998; Plavcan, 2001; Weckerly, 1998) have confirmed that strepsirrhines are among the least sexually dimorphic mammals, showing no significant degree of sexual size dimorphism Sexual dimorphism among strepsirrhines is the lowest among primates, with some researchers willing to label them as sexually monomorphic (Kappeler, 1991).

Seven adductor muscles were included in this study: superficial and deep masseter, zygomatico-mandibularis, zygomatic temporalis, superficial and deep temporalis, and medial pterygoid. The muscles are from both the left and right sides of an individual; no preference was made for either right or left. When only one side of the head was intact, that was the side that was chosen for dissection; when both sides of the head were intact, the side chosen was that which brought left and right side representation into greatest equilibrium.

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Table 4.1: Specimens in this study Specimen Taxon Jaw Length (cm) Body mass (g)a JHUTS2* Tupaia belangeri 3.48 171 734BEF2* Tupaia belangeri 3.32 124 734BABA* Tupaia belangeri 3.12 103 USNM 497016 Tupaia glis 3.488 88 YBL 9011 Tupaia minor 2.436 60 USNM 292532 Tupaia montana 3.295 119 USNM 296647 Tupaia montana 3.35 107 USNM 111783 Tupaia nicobarica 3.811 224 USNM 546344 Tupaia tana 4.306 231 USNM 580476 Tupaia tana 4.226 228 AMNH 170501 Avahi laniger 3.445 1178 DUPC 5800m Eulemur collaris 6.445 2300 DUPC 6394f Eulemur macaca flavifrons 5.95 2120 DUPC 6631m Eulemur rubriventer 6.05 1940 DUPC 1322f Hapalemur griseus 4.48 1030 BAA C2f Lemur catta 5.935 2207 AMNH 170790 Lepilemur leucopus 3.339 742 DUPC 889f Microcebus murinus 2.075 63 DUPC 874f Microcebus murinus 2.035 58 BAA C6m Nycticebus coucang 3.84 679 DUPC 1731m Otolemur crassicaudatus 5.29 1400 AMNH 200640 Perodicticus potto 4.32 1100 DUPC 6110f Propithecus coquereli 6 3700 DUPC 6560m Propithecus coquereli 5.255 2780 DUPC 6769m Varecia rubra 7.51 3650 AMNH 201395 Varecia rubra 7.46 3865

Abbreviations: AMNH – American Museum of Natural History BAA – Biological Anthropology & Anatomy, Duke University DUPC – Duke University Primate Center (now Duke Lemur Center) JHU - Johns Hopkins University USNM – Unites States National Museum of Natural History YBL – Yale Biological Anthropology Laboratory

*Obtained from the Max Planck Florida Institute a Body mass was measured as the weight of the specimen before dissection

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4.3 Methods

4.3.1 Muscle mass measurements

Muscle mass was measured as the wet weight, in grams, of a muscle immediately after removal, using a digital scale accurate to 0.001g.

Chemically dissecting the muscles of mastication is necessary for measuring muscle fibers used in dietary calculations. To begin this process, each muscle is placed in a petri dish with 10% sulfuric acid and cooked in an oven at 60-80° C for between 45 mins to 3 hours, depending on the size and density of connective tissue present in the muscle. All muscles are checked periodically to ensure there is no overcooking. Once cooked, the sulfuric acid is removed, the muscles flushed with distilled water, and then the muscles are placed in a 10% buffered formalin solution again until ready for measuring. Throughout this process, the muscles remain in a single petri dish and every effort is made to maintain anatomical position.

4.3.2 Fiber length measurements

Fibers are measured following a protocol used by Rayne and Crawford

(1972), and modified by Perry and Wall (2008). To measure the fascicle lengths in a muscle, a dissection microscope is equipped with a reticle and a dissection needle and tweezers are used to gently tease apart fascicles and align them with the reticle

(Figure 4.1). As in previous studies of chewing muscle fiber architecture (Hartstone-

Rose et al., 2012; Perry et al., 2011b; Perry and Wall, 2008), fibers were found to run

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Chapter 4: CORRELATIONS AND PCSA the entire length of their fascicle; therefore, here fascicle length and fiber length are treated as equivalent.

Fibers are sampled from throughout each muscle, moving from one side of the muscle (e.g., anterior) to the other (e.g., posterior) in order to capture variation across each muscle. A minimum of 10-15 fibers are recorded for every muscle, although up to 30 is not uncommon.

Figure 4.1: Muscle Chemical Dissection

A. Zygomatic temporalis after cooking and after measuring fascicles. B. Deep temporalis after cooking and measuring fascicles. Scale bar is 5mm.

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4.3.3 Calculating Physiological Cross-sectional Area

Physiologic Cross sectional Area (PCSA) is a representation of the strength of a muscle at a given cross section (Weijs and Hillen, 1985). It is calculated as muscle mass divided by fascicle length, and corrected by a density constant of muscle:

Muscle Mass (g) PCSA (cm²) = avg. Fiber Length (cm) x 1.0564 (g/cm³)

Here, muscle mass is measured as the wet weight, in grams, of a muscle immediately after removal, using a digital scale accurate to 0.001g. 1.0564g/cm³ is the density constant of muscle that was used here (Murphy and Beardsley, 1974).

PCSA is reconstructed for an individual muscle following the formula above. For calculating a given specimen’s total PCSA, I used the sum of the PCSA for each of the individual muscles. This only approximates total muscle force as some components of the muscle vectors are subtractive, not additive.

4.3.4 Scaling Calculations

Due to the overwhelming representation of cranial remains, particularly mandibular remains, in plesiadapiform fossil collections, jaw length has been chosen for use as a body size metric in the analyses within this chapter rather than body mass. While body mass would be the preferable metric (Gingerich et al., 1982), body mass is not directly measureable for the vast majority of specimens, given the

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Chapter 4: CORRELATIONS AND PCSA osteological remains that have survived. However, because jaw length is not only a function of overall size, but also a function of gape and leverage (Smith, 1984; Taylor and Vinyard, 2008), as a variable it may be found unsuitable as a proxy for overall size in scaling analyses. Gape is at least partly a function of food size in strepsirrhines (Fricano and Perry, 2019) while jaw length differs between strepsirrhines having different diets but similar body masses (Perry, 2008). For the research conducted here, however, since food size is not thought to vary systematically in treeshrews (Emmons, 2000), I am confident in the use of jaw length as a size proxy in this group.

Because previous work has shown that muscle dimensions scale differently when using body mass versus jaw length (Perry, 2008), and tupaiids have relatively longer jaws than strepsirrhines, the ratio of jaw length cubed / body mass in tupaiids will be calculated and compared to that in strepsirrhines. Body mass is measured by weighing the specimen before dissections. An isometric relationship and strong correlation between these two variables would provide additional support for the use of jaw length as a body mass proxy in the extant and fossil samples.

In assuming error for all measurements, Reduced Major Axis regressions

(Smith, 2009)will be used to assess relationships between independent variables in this study. All statistics were performed using the R statistical package (R Core

Team, 2013).

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4.4 Results

4.4.1 Measurements

All figures are presented such that y-axis data and x-axis data conform to the same logarithmic space. Results and tables are therefore presented with this in mind.

Tables 4.2 – 4.4 report the measurements taken and calculations made from tupaiid jaw adductors. Table 4.2 reports the muscle mass (in grams) of each muscle dissected from tupaiid cadavers as well as the total summed mass of these muscles.

Table 4.3 reports both the mean fiber length for each of the adductor muscles as well as the total average of individual muscle averages. Table 4.4 reports the tupaiid physiological cross-sectional area calculated from the measurements listed in Table

4.2 and Table 4.3. Strepsirrhine data used for comparisons were taken from the literature (see Tables 4.6, 4.7, and 4.4 in Perry, 2008).

4.4.2 Scaling

Jaw length as a measure of body size, and for use as a scaling variable, is dependent on an isometric relationship with a known body size metric. Here, body mass is used as an independent determinant of body size to validate the use of jaw length in further calculations for this study. Jaw length measurements were cubed and log

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Table 4.2: Muscle mass measurements (g)

Specimen Taxon SMa DM ZM ZT ST DT MP SUM JHUTS2 T. belangeri 0.74 0.48 0.08 0.04 0.20 0.74 0.12 2.40 734BEF2 T. belangeri 0.28 0.06 0.07 0.04 0.07 0.31 0.15 0.97 734BABA T. belangeri 0.22 0.05 0.06 0.07 0.04 0.21 0.08 0.72

USNM 497016 T. glis 0.14 0.06 0.02 0.02 0.05 0.17 0.08 0.55 YBL 9011 T. minor 0.12 0.04 0.03 0.02 0.03 0.14 0.06 0.44 USNM 292532 T. montana 0.18 0.05 0.03 0.04 0.05 0.21 0.08 0.65 USNM 296647 T. montana 0.08 0.13 0.04 0.03 0.05 0.17 0.08 0.57 Chapter 4: CORRELATIONS AND PCSA USNM 111783 T. nicobarica 0.75 0.25 0.10 0.15 0.23 1.15 0.16 2.77

137 USNM 546344 T. tana 0.38 0.11 0.03 0.07 0.06 0.31 0.19 1.13

USNM 580476 T. tana 0.43 0.14 0.06 0.07 0.09 0.36 0.14 1.28

aAbbreviations: SM = superficial masseter; DM = deep masseter; ZM = zygomatico-mandibularis; ZT = zygomatic temporalis; ST – superficial masseter; DT = deep temporalis; MP = medial pterygoid

Table 4.3: Fiber length measurements (cm)

Specimen Taxon SM DM ZM ZT ST DT MP Mean FL JHUTS2 T. belangeri 0.85 0.62 0.60 0.41 0.68 0.79 0.52 0.638

734BEF2 T. belangeri 0.84 0.60 0.51 0.50 0.59 0.56 0.64 0.606 734BABA T. belangeri 0.62 0.59 0.73 0.64 0.59 0.68 0.71 0.650 USNM 497016 T. glis 0.85 0.70 0.56 0.53 0.63 1.07 0.63 0.711 Chapter 4: CORRELATIONS AND PCSA YBL 9011 T. minor 0.57 0.49 0.36 0.45 0.46 0.57 0.36 0.466

138 USNM 292532 T. montana 0.62 0.64 0.51 0.64 0.60 0.73 0.64 0.625 USNM 296647 T. montana 0.74 0.70 0.57 0.57 0.62 0.83 0.63 0.667

USNM 111783 T. nicobarica 1.39 0.91 0.67 0.80 0.86 1.57 0.71 0.984 USNM 546344 T. tana 1.25 0.68 0.46 0.61 0.91 1.04 0.86 0.830 USNM 580476 T. tana 1.30 0.79 0.65 0.68 0.71 0.72 0.67 0.787

Table 4.4: Physiological Cross-sectional Area (cm2)

Mass. Temp. MP Specimen Taxon SM DM ZM ZT ST DT MP PCSA PCSA PCSA

JHUTS2 T. belangeri 0.821 0.727 0.134 0.082 0.280 0.887 0.222 1.681 1.248 0.222 734BEF2 T. belangeri 0.313 0.094 0.138 0.077 0.104 0.516 0.214 0.546 0.698 0.214 734BABA T. belangeri 0.341 0.079 0.074 0.105 0.063 0.289 0.100 0.494 0.457 0.100 USNM 497016 T. glis 0.161 0.082 0.034 0.037 0.077 0.149 0.121 0.277 0.263 0.121 Chapter 4: CORRELATIONS AND PCSA YBL 9011 T. minor 0.195 0.074 0.080 0.044 0.058 0.230 0.163 0.349 0.332 0.163 139 USNM 292532 T. montana 0.272 0.080 0.060 0.065 0.085 0.270 0.116 0.412 0.420 0.116

USNM 296647 T. montana 0.100 0.170 0.065 0.043 0.082 0.193 0.113 0.335 0.318 0.113 USNM 111783 T. nicobarica 0.509 0.259 0.137 0.173 0.249 0.696 0.218 0.905 1.117 0.218 USNM 546344 T. tana 0.285 0.147 0.056 0.105 0.058 0.278 0.208 0.488 0.441 0.208 USNM 580476 T. tana 0.309 0.167 0.085 0.100 0.114 0.480 0.192 0.561 0.695 0.192

Table 4.5: Regression statistics for variables against jaw length cubed y- Allometric RMA H0e Taxon Variablea Slope LCLb UCLc RSE(%)d R² intercept Trend p-value p-value Body Mass 0.98 0.678 1.406 0.564 13.75 Isometric 0.86 0.001 0.880 Total Muscle Mass 1.29 0.694 2.390 -2.097 22.32 Positive 0.35 0.008 0.397 Mean Fiber Length 1.25 0.841 1.862 -0.838 6.46 Positive 0.75 <0.001 0.237 Tupaiid Masseter PCSA 1.64 0.798 3.334 -2.042 21.79 Positive 0.08 0.416 0.170 Temporalis PCSA 1.63 0.813 3.289 -2.034 18.03 Positive 0.12 0.316 0.157 Medial Pterygoid PCSA 0.99 0.492 1.963 -1.860 9.41 Isometric 0.15 0.278 0.957

Body Mass 1.06 0.935 1.453 0.940 17.14 Isometric 0.90 <0.001 0.5111 Total Muscle Mass 0.87 0.733 1.027 -1.046 16.47 Negative 0.91 <0.001 0.093 Mean Fiber Length 0.93 0.732 1.175 -0.737 7.90 Isometric 0.83 <0.001 0.509 Chapter 4: CORRELATIONS AND PCSA Strepsirrhine Masseter PCSA 0.94 0.691 1.274 -0.902 15.87 Isometric 0.71 <0.001 0.668

140 Temporalis PCSA 0.97 0.746 1.257 -0.851 17.14 Isometric 0.79 <0.001 0.799

Medial Pterygoid PCSA 1.02 0.729 1.439 -0.902 17.08 Isometric 0.63 <0.001 0.885

a All variables were converted to log space for calculations

b Lower Confidence Limit (95%)

c Upper Confidence Limit (95%)

d Relative Standard Error: deviation of the estimate from the actual population. Calculated as (SE/Estimate) x 100

e H0: Test of homogeneity against a slope of 1 (isometry)

Chapter 4: CORRELATIONS AND PCSA transformed before being regressed onto log transformed body mass measurements, the results of this regression can be found in (Table 4.5).

The RMA regression of body mass onto jaw length cubed (Figure 4.2) illustrates an isometric relationship (b=0.98, p<0.05, where b=slope) between body mass and jaw length. The sample size of eight is small, with a moderate coefficient of determination at 0.86. Two specimens could not be included in the regression due to the removal of their thoraco-abdominal organs during necropsy thus resulting in an underestimated and unreliable body mass.

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Figure 4.2: RMA regression of body mass (g) upon jaw length cubed (cm3)

Although body mass is perhaps the ideal measure of size when one has access to soft tissues, it is not practical for osteological samples. The isometric relationship and strong correlation (R2=0.86) found here between body mass and jaw length permits the use of jaw length as a body size variable in this study. This further allows me to compare fossil specimens to my extant sample of tupaiids and strepsirrhines using a uniform body size metric.

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Figure 4.3 illustrates an RMA regression of log-transformed tupaiid jaw adductor mass regressed onto log transformed jaw length cubed, where the regression indicates a positively allometric relationship (b=1.29) between jaw adductor mass and jaw length. Furthermore, the relationship is statistically significant (p-value = 0.008), meaning the mass of a given treeshrew’s jaw adductor muscles is correlated with the length of its jaw. The results of this regression can be found in Table 4.5.

The coefficient of determination is low (R²=0.35), and partly an artifact of the relatively higher muscle mass reported for three specimens (JHUTS2, YBL9011, and

USNM 111783; T. belangeri, T. minor, and T. nicobarica). Like muscle mass, fiber length was also investigated without breaking it down into individual muscles.

Figure 4.4 illustrates an RMA regression of log-transformed fiber length regressed onto log-transformed jaw length, where a positively allometric relationship is recovered between the two variables (b=1.25). The strength of the correlation between fiber length and jaw length is moderate in treeshrews (R²=0.75). This relationship is statistically significant (p-value=0.00), indicating that jaw adductor fiber length is directly correlated to jaw length. The results of this regression can be found in Table 4.5.

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Figure 4.3: RMA regression of jaw adductor muscle mass (g) upon jaw length cubed (cm3)

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Figure 4.4: RMA regression of fiber length (cm) upon jaw length (cm)

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An ANCOVA testing the relationship between the slope of total fiber length regressed onto jaw length and the slope of total muscle mass regressed against jaw length showed no statistical difference in fiber length between each of the adductor muscles (p-value>0.05).

Turning to the results of the physiological cross sectional area RMA regression, results have now been divided according to muscle group. All three muscle groups present statistically significant (p-value<0.05) correlations when

PCSA is regressed upon jaw length squared. Temporalis PCSA and masseter PCSA display positive allometry (b=1.63, 1.64; Figure 4.5 and Figure 4.6) while the medial pterygoid displays isometry (b=0.94; Figure 4.7). The results of this regression can be found in Table 4.5.

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Figure 4.5: RMA regression of masseter PCSA (cm2) upon jaw length squared (cm2)

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Figure 4.6: RMA regression of temporalis PCSA (cm2) upon jaw length squared (cm2)

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Figure 4.7: RMA regression of medial pterygoid PCSA (cm2) upon jaw length squared (cm2)

4.5 Discussion

4.5.1 Muscle Mass

Unlike the predictions made earlier concerning tupaiid jaw adductor mass, the tupaiid sample shows a pattern of positive allometry in the muscle mass variable

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Chapter 4: CORRELATIONS AND PCSA when regressed onto jaw length in an RMA regression. This is in opposition to strepsirrhine primates, which exhibit negative allometry when comparing muscle mass to body size (refer to Table 4.5). However, a 95% CI for both of these regressions includes isometry.

In comparing the regressions between the two samples (Figure 4.8), the tupaiids have a lower jaw adductor mass for a given jaw length. These data indicate that, on average, strepsirrhines will have larger muscles of mastication, by weight, than treeshrews at a given body size. This is not an artifact of jaw length in treeshrews given the isometry between jaw length and body mass in this group.

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Figure 4.8: Comparing tupaiid and strepsirrhine jaw adductor muscle mass

Interestingly, this highlights the interdependence of jaw length and muscle mass in the tupaiid chewing system. Also found by Perry (2008) in his work on strepsirrhine jaw adductor mass scaling, Tupaiids can optimize their chewing system (E.g., PCSA) by increasing the size of their muscles as the length of their jaw increases, compensating for the loss of leverage that comes with having bite points at a greater distance from the center of rotation. Here, specimens of T. belangeri and

T. nicobarica have an increased jaw adductor muscles size, respectively; see Figure

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4.3, to the extent that their muscles plot with the much larger bodied strepsirrhines,

T. nicobarica despite having a relatively shorter jaw length. T. minor, while not quite plotting with the strepsirrhines, plots more closely to them than to the tupaiids.

In looking more closely at T. nicobarica, an outlying specimen in terms of both jaw adductor mass and fiber length, we can see that its masticatory system is different when compared to T. tana, a treeshrew of comparable size. For example, at

~224g, T. nicobarica has a jaw approximately 8% shorter than T. tana (~229g) but with an adductor mass 46% larger. This may be related to its specific diet. A field study conducted on T. nicobarica (Oomen and Shanker, 2008), the Nicobar treeshrew, has initial results indicating a pronounced insectivorous diet in relation to other treeshrews. This might explain its larger temporalis adductor muscles required for incisive biting. Despite strong molecular evidence to the contrary

(Roberts et al., 2011), Oomen and Shanker (2008) posited a shared ancestry between T. nicobarica and T. minor, despite their difference in size, because of similarity in appearance, degree of arboreality, and behavior. The shared muscle mass proportions between the two species could indicate at, a minimum, a shared diet and potentially a shared approach to optimizing PCSA.

4.5.2 Fiber Length

Fiber length has interesting connotations for gape adaptations in tupaiids.

Fiber length may be increasing relative to jaw length simply because, like jaw length, it is an adaptation for gape. While prior calculations found no statistical difference in 152

Chapter 4: CORRELATIONS AND PCSA fiber length between each of the adductor muscles (p-value>0.05), this does not mean that there is not a difference in fiber length within each muscle. For example recent research by Perry et al. (2016) found that the anterior portions of the temporalis and masseter muscles in strepsirrhine primates contain longer muscle fibers than the posterior portions, suggesting an adaptation to a larger gape at the anterior aspect of these muscles. This conforms to theoretical expectations of mammalian chewing muscle (Herring and Herring, 1974).

These potential differences in fiber architecture within the muscle therefore influence actual PCSA and have implications for reconstructing PCSA in fossil forms.

Because PCSA is calculated using an average of fiber lengths from throughout the muscle, this creates a sort of average PCSA that presumably masks the effects of longer fibers in the anterior portion of the muscle

Overall, fiber length measurements followed the same pattern as jaw adductor muscle mass when regressed against jaw length in an RMA regression.

Tupaiids presented with positive allometry, unlike my predictions that they would follow strepsirrhines and present with isometry (refer to Table 4.5). Therefore, for treeshrews, larger specimens will have relatively longer muscle fibers relative to their body size than smaller treeshrews, as seen in Figure 4.9.

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Figure 4.9: Comparing tupaiid and strepsirrhine jaw adductor fiber length

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4.5.3 PCSA

PCSA, for the most part, follows the aforementioned relationships between both total jaw adductor muscle mass (refer to Figure 4.3) and fiber length (refer to

Figure 4.4) in relation to jaw length. Strepsirrhine primates exhibit isometry; however, an RMA regression shows an allometric relationship between PCSA and jaw length in the tupaiid masseter and temporalis jaw adductors (Figure 4.10 and

Figure 4.11; Table 4.5). This positively allometric relationship means that treeshrews with longer jaws will have, on average, greater PCSA in these adductors than treeshrews with shorter jaws.

The tupaiids, however, exhibit isometry in their medial pterygoid PCSA in relation to jaw length just as the strepsirrhines do (Figure 4.12). Therefore, treeshrews with greater temporalis and masseter PCSA will not have medial pterygoid muscles increasing at the same proportions. This could potentially lead to unbalanced action of the jaw adductors that would need to be modulated via differential muscle activation or some other mechanism.

There may be a spatial explanation for medial pterygoid isometry; for example, whereas the temporalis and masseter muscles are free to expand laterally, the medial pterygoid is both medially and laterally constrained. Its isometric (i.e., less than positively allometric) relationship to jaw length is likely a reflection of geometric constraints, whereby the medial pterygoid can increase only as the individual increases in size and the jaw can accommodate a larger insertion area.

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Figure 4.10: Comparing tupaiid and strepsirrhine masseter PCSA

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Figure 4.11: Comparing tupaiid and strepsirrhine temporalis PCSA

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Figure 4.12: Comparing tupaiid and strepsirrhine medial pterygoid PCSA

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Within treeshrews, different species seem to optimize PCSA in different ways. For example the PCSA of T. nicobarica and T. tana have followed the pattern described above in the relationship between their adductor muscle mass and jaw length. Namely, that T. tana has increased its jaw length in a predictable fashion to the other treeshrews, whereas T. nicobarica has instead increased its adductor mass and fiber length, giving it a much greater PCSA than T. tana but having a reduced jaw length. Because PCSA is a function of both muscle mass and fiber length, these results are not unexpected.

Strepsirrhines increase the size of their chewing muscles in order to increase their PCSA depending on their food source. Greater PCSA would have advantages such as increasing bite force without needing to decrease jaw length. However, increasing muscle mass is only one way to increase PCSA. Another is to increase the number of the muscle fibers within the chewing muscles.

4.5.4 Implications for Diet

In order to make a proper comparison between tupaiid and strepsirrhine

PCSA,, it must be broken out by dietary group. This is because strepsirrhines have different configurations of their muscles of mastication depending on the dominant food in their diet (Perry, 2008; Perry et al., 2011b). For example, frugivores and folivores place emphasis, respectively, on their temporalis or masseter group preferentially, as seen in the ratio between the two (Cachel, 1979).

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Dietary categories are based on the food groups comprising the majority of the diet of each species. Species that rely on one major food source, for example leaves, are categorized thusly (e.g., folivore). Species that rely on more than one food group for their energy intake reflect this is their designations, for example insectivore-frugivore (e.g., T. belangeri). While several species studied here are frequently considered omnivorous, such as treeshrews and Otolemur (Emmons,

2000), omnivory is not synonymous between groups and was therefore considered too broad a term for the research conducted here.

Figure 4.13 shows a comparison between the percentage of total PCSA each functional muscle group (temporalis, masseter, medial pterygoid) comprises in the tupaiids and strepsirrhines studied. The dots represent tupaiid PCSA and the triangles represent strepsirrhine PCSA. The number of specimens in each dietary group can be found in Table 4.6 and Table 4.7, along with PCSA percentages.

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Figure 4.13: Comparison of jaw adductor muscle group percentages by taxon

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Treeshrews (circles) group with the frugivorous and insectivorous strepsirrhines. They favor the masseter and temporalis muscles almost equally

(43% and 43.3% of total PCSA, respectively), with less reliance on the medial pterygoid muscle (13.7%) like in the more insectivorous strepsirrhines (M. murinus

- 12.9%). Treeshrews, on average, have an equal ratio of masseter PCSA to temporalis PCSA (0.99). One treeshrew (JHUTS2R; T. belangeri) has a ratio significantly greater than one (1.3) which is on par with the ratio seen in folivorous strepsirrhines (e.g., A laniger, P. coquereli), indicating a greater reliance on the masseter muscle than in other treeshrews. This may suggest that this individual had a greater proportion of hard objects in its diet relative to the other treeshrews. The strepsirrhine whose diet most closely resembles that of the treeshrews is the frugivore-insectivore Microcebus murinus.

Generally speaking, the masseter and temporalis each make up over a third, and in some cases closer to half, of total jaw adductor PCSA. Insectivorous and frugivorous strepsirrhines, especially, exhibit larger temporalis muscles relative to masseter muscles than other dietary groups (48.6% for categories including frugivory compared to 42.6% for categories without). This increased reliance on the temporalis group is probably a reflection of the stability this muscle provides during an incisive biting force at large gapes (Shi et al., 2012) due to its vertically oriented muscle vectors (in the coronal plane). The temporalis could assist, for example, with providing a powerful puncturing action for breaking through a hard fruit rind.

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Table 4.6: Tupaiid dietary categories and PCSA muscle percentages Category Mass. Temp. MP Ratioa Specimen Taxon Dietary Category Citatations PCSA% PCSA% PCSA% Mass:Temp JHUTS2 T. belangeri Insectivore-Frugivore Li and Ni, 2016 53.35 39.61 7.04 1.35 734BEF2 T. belangeri Insectivore-Frugivore Li and Ni, 2016 37.48 47.87 14.65 0.78 734BABA T. belangeri Insectivore-Frugivore Li and Ni, 2016 46.98 43.49 9.53 1.08 USNM 497016 T. glis Insectivore-Frugivore Langham, 1982 41.91 39.78 18.31 1.05 YBL 9011 T. minor Frugivore-Insectivore Emmons, 2000 41.32 39.33 19.35 1.05 USNM 292532 T. montana Insectivore-Frugivore Emmons, 2000 43.45 44.31 12.24 0.98

USNM 296647 T. montana Insectivore-Frugivore Emmons, 2000 43.70 41.53 14.77 1.05

Oomen and USNM 111783 T. nicobarica Frugivore-Insectivore 40.41 49.88 9.71 0.81 Chapter 4: CORRELATIONS AND PCSA Shanker, 2008 USNM 546344 T. tana Insectivore-Frugivore Emmons, 2000 42.91 38.77 18.32 1.11

163 USNM 580476 T. tana Insectivore-Frugivore Emmons, 2000 38.76 47.99 13.25 0.81

aCalculated as Masseter/ Temporalis. In bold are ratios greater than 1.

Table 4.7: Strepsirrhine dietary categories and PCSA muscle percentages Category Mass. Temp. MP Ratio Specimen Taxon Dietary Category Citations PCSA% PCSA% PCSA% Mass:Temp AMNH 170501 A. laniger Folivore Harcourt 1991 44.70 32.86 22.44 1.36 DUPC 5800m E. collaris Frugivore-Folivore Donati et al., 2007 37.65 52.54 9.81 0.72 E. macaca Simmen et al., 2007 DUPC 6394f 35.38 49.89 14.73 0.71 flavifrons Frugivore DUPC 6631m E. rubriventer Frugivore-Folivore Overdorff, 1993 36.25 47.67 16.08 0.76 Overdorff et al., DUPC 1322f H. griseus 39.09 44.36 16.55 0.88 Folivore 1997

BAA C2f L. catta Frugivore-Folivore Sussman 1977 34.64 48.22 17.14 0.72 AMNH 170790 L. leucopus Folivore-Frugivore Dröscher et al., 2016 36.34 44.89 18.77 0.81 Frugivore- Martin, 1972 DUPC 889f M. murinus 37.03 49.93 13.04 0.74 Insectivore Chapter 4: CORRELATIONS AND PCSA Frugivore- Martin, 1972

164 DUPC 874f M. murinus 38.97 48.29 12.75 0.81 Insectivore

Gummivore- Wiens et al., 2006 BAA C6m N. coucang 41.09 44.52 14.39 0.92 Frugivore DUPC 1731m O. crassicaudatus Omnivore Harcourt, 1986 34.43 52.32 13.25 0.66 AMNH 200640 P. potto Omnivore Oates, 1984 37.53 46.18 16.28 0.81 Ganzhorn et al., DUPC 6110f P. coquereli 29.65 47.77 22.57 0.62 Folivore 2017 Ganzhorn et al., DUPC 6560m P. coquereli 45.49 32.03 22.49 1.42 Folivore 2017 DUPC 6769m V. rubra Frugivore Rigamoniti, 1993 39.75 49.03 11.22 0.81 AMNH 201395 V. rubra Frugivore Rigamonti, 1993 34.23 51.21 14.56 0.67

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Within strepsirrhines, folivorous species show the greatest reliance on the masseter muscle (39.7%). The more laterally and anteriorly oriented masseter group is beneficial to folivorous strepsirrhines, who use the horizontally angled muscle vector to achieve transverse movement (Hylander, 1979a; Ravosa et al.,

2000). In considering the ratio of masseter PCSA to temporalis PCSA, it is greatest in the folivorous strepsirrhines (1.07), being the only group having a ratio greater than

1. This is greater than either the omnivorous (0.74) or frugivorous strepsirrhines

(ratio=0.77).

Part and parcel to the masseter muscle is the medial pterygoid. Constituting less than one fifth of adductor mass in the majority of specimens, the medial pterygoid muscle is largest in folivorous primates (21% of PCSA), followed by strepsirrhines with a mixed folivorous-frugivorous diet (15.5%). These groups rely on the muscle to assist the masseter muscles in generating transverse chewing movements. An important stabilizer muscle, the medially and anteriorly oriented medial pterygoid muscle vector works to counteract the lateral rotation of the masseter muscle (especially the superficial masseter) in order to prevent the mandible from undue twisting about its long axis (Lieberman and Crompton, 2000).

Therefore, the medial pterygoid is relatively large (though always absolutely small) in strepsirrhines with a diet relatively higher in folviory. The medial pterygoid is smallest in insectivorous treeshrews and strepsirrhines (13.6%) although the importance of the muscle in insectivorous primates is currently unclear. It is 165

Chapter 4: CORRELATIONS AND PCSA possible that, given the largely orthal jaw movements in insectivorous strepsirrhines (Kay and Hiiemae, 1974), the medial pterygoid does not have a different role from that of the masseter and is reduced based on spatial constraints.

4.5.5 Caveats

In this study, the chewing muscles of both preserved and frozen individuals were dissected, which may have implications for the results presented. This is primarily due to the fact that fixed specimens can lose between 10-56% of their mass (Buytaert et al., 2014), thus giving a lower muscle mass measurement compared to fresh specimens. Ideally, one would create a sample of specimens composed of either entirely frozen or entirely preserved specimens in order to reduce error of this kind. However, due to the rarity of suitable specimens, this is not always possible. Additionally, the state of the specimens is not always ideal. For example two specimens had to be excluded from the jaw length validation regression calculation due to the removal of their organs during necropsy, giving an artificially low body mass value.

Additionally, given the role diet plays in masticatory muscle size (Kiliaridis,

1986), it is important to select treeshrew individuals who consumed a diet as close to their natural diet as possible. Ideally, a study like the one conducted here would include wild-caught specimens only. However, this contributes to problems

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Chapter 4: CORRELATIONS AND PCSA associated with having a smaller sample size. Future work would include trying to source additional specimens subsisting off of their natural diet.

Due to the availability of specimens, the results of this study were heavily constrained by the tupaiid sample size. This applies not only to specimen count, but also to the number and breadth of species. I agree with Sargis (2004)that the study of primate systematics is impaired by a lack of variation in treeshrew specimens studied. The work done here, as well as that of others, is guilty of this, having used members of the Tupaiidae exclusively to represent all treeshrews, to the exclusion of specimens representing the other treeshrew genera, (Dendrogale, Anathana, and

Ptilocercus). This lack of variation hinders more comprehensive research, not only with regards to diet and, but physically and behaviorally as well.

This study was constrained to the tupaiid family due to the rarity of specimens from the other treeshrew family. While preserved specimens do exist, for example at the Field Museum, they are scarce, precious, and not readily replaceable.

Therefore destructive sampling, such as the dissections performed here, irrevocably removes an important specimen from the scientific community. My hope is that the

Dice-CT work as outlined in Chapter 3 will help to solve this problem and allow for these rare specimens to be sampled and included in future studies.

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4.6 Conclusions

Treeshrew muscle scaling did not follow the prediction made earlier following the muscle scaling found in strepsirrhine primates. Unlike strepsirrhines, which exhibited isometry (Perry et al., 2011b), when looking at jaw adductor mass relative to body size (jaw length cubed), treeshrews exhibited positive allometry.

However, the low number of specimens and low R² value leave room for interpretation. Additionally, specimens such as the ones of T. nicobarica, are probably throwing this relationship off due to their relatively higher muscle mass.

Poor correlation between muscle mass and body size does not imply a negative result, however. Differences in function between taxa will affect the strength of this relationship. Future research should try to incorporate more individuals, especially larger individuals such as Tupaia everetti (Sargis, 2002a), to investigate further the phenomenon of relatively higher muscle mass.

Like muscle mass, muscle fiber length in relation to jaw length cubed also exhibited positive allometry. By contrast, this relationship in strepsirrhines was one of isometry. In tree shews, this positive allometry may be an adaptation for longer muscle fibers at larger gapes. Treeshrews also showed positive allometry in their

PCSA, but again, the extremely low R² value means this too may be an unreliable conclusion and would benefit from the addition of more specimens. However, PCSA was different for each muscle group, and the treeshrews do in fact exhibit isometry in medial pterygoid PCSA, like the strepsirrhine primates. This may be a reflection of 168

Chapter 4: CORRELATIONS AND PCSA diet, or more likely, a result of physical constraints within the masticatory apparatus.

Overall, tupaiids exhibit similar jaw adductor muscle proportioning as some strepsirrhine primates, primarily grouping with omnivorous and insectivorous strepsirrhine primates. Counter to the predictions made earlier, the temporalis muscle group is not largest in treeshrews or insectivorous strepsirrhines relative to all other strepsirrhines. Rather, it is largest in insectivorous and frugivorous strepsirrhines. Since these strepsirrhines rely more on the temporalis muscle group than do treeshrews, this implies they practice a greater degree of insectivory and anterior incising than treeshrews, which display greater omnivory.

The tupaiid diet most closely resembles that of the strepsirrhine primate M. murinus in comprising primarily insects and fruit. This is especially true for the relatively insectivorous members of the tupaiids (e.g., T. nicobarica).

The predictions made concerning the masseter and medial pterygoid muscles were supported in most aspects. For example, the folivorous strepsirrhine

Propithecus coquereli did have the largest masseter PCSA relative to jaw length (see

Table 6.). While the folivorous strepsirrhines as a whole do not have a greater percentage of masseter PCSA compared to treeshrews, it needs to be taken into account that they also have much larger medial pterygoid muscles than do the treeshrews. Their large medial pterygoid muscles also support the last prediction I made, whereby the relative cross-sectional area of the medial pterygoid will be

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Chapter 4: CORRELATIONS AND PCSA greater in folivorous strepsirrhines, which are pointedly larger in those strepsirrhines categorized as folivorous and as frugivore-folivores. However, the prediction that medial pterygoid muscles would also be larger in insectivorous strepsirrhines was not supported.

Therefore, the research conducted here reinforced the chcarcterization of tupaiids as an omnivorous family with some members slightly more insectivorous.

Thus, tupaiid jaw adductor muscles match the hypothesized pattern of omnivory / insectivory and any predictions about jaw adductor size in plesiadapiforms will be enriched by the use of tupaiid data.

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Plesiadapiformes and the Implications for Dietary

Interpretations

5.1 Introduction

Plesiadapiforms present a wide range of dental characteristics which, combined with their range of body sizes, suggests many different adaptations to feeding behavior (Silcox and Williamson, 2012). Dental evidence suggests that many were specialized for insectivory (Kay and Cartmill, 1977; Kay and Hiiemae, 1974).

However, others present adaptations in both the jaw and teeth that display specializations for practicing frugivory, omnivory, and folivory (Gingerich, 1976;

Rose, 1975; Szalay, 1968a, 1969).

Insectivorous plesiadapiforms include members of the micromomyid and microsyopid families. Evidence for insectivory in micromomyids includes small estimated body mass (estimations between 10-16g with some up to 37g: Silcox et al., 2017)) and high-crowned teeth with tall cusps (Chester and Bloch, 2013; Kay and Cartmill, 1977). Insectivory may have been supplemented with seeds and angiosperm exudates, as Szalay (1974) suggested for the species Tinimomys graybulliensis, due to an expanded lower fourth premolar possessing a well- developed paracristid and cingula.

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Microsyopid plesiadapiforms display similar adaptations for insectivory

(such as in Navajovius kohlhassae). Microsyopids possess a unique lanceolate lower incisor and a caniniform upper incisor with a debated function (Gunnell, 1989).

Insectivory is not the likely inference for all plesiadapiforms, especially larger species that fall above Kay’s threshold of 500g (Kay, 1984). For example, plesiadapid plesiadapiforms have been hypothesized as potentially folivorous based on both body size and dental specializations including high molar crown relief and greater cusp complexity (Boyer et al., 2010; Gingerich, 1976). Omnivorous plesiadapiforms include older members of these families; Nannodectes and

Pronothodectes are earlier occurring members of the Plesiadapidae (Gingerich,

1976) exhibiting less molar crown relief and less cusp complexity (Boyer et al.,

2012b).

Paromomyids have been broadly classified as omnivores, due to their low crowned molars and tall, pointed premolars (Gingerich, 1974; Silcox and Gunnell,

2008). Some well-preserved paromomyid specimens possess a mitten-like upper incisor and display procumbent incisors that have been hypothesized as adaptations for spearing insects (Gunnell, 1989). Some authors have speculated this incisor could have been employed as a gouging mechanism for feeding on tree exudates

(Boyer and Bloch, 2008), though this is still debated (Rosenberger, 2010).

Hypothesized frugivorous plesiadapiforms include members of the picrodontid, carpolestid and saxonellid families. The low crowned, elongate first

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION lower molar of the small bodied pictrodontids may have been an adaptation for accommodating fruit and, therefore, picrodontids have been suggested to have been specialized frugivores (Burger, 2013; Szalay, 1968b). Carpolestids and saxonellids display characteristic plagiaulacoid dentition, in which the lower fourth premolar in carpolestids (Simpson, 1933) and lower third premolar in saxonellids (Fox, 1991) are blade-like, possibly for cracking seeds and fruit (Biknevicius, 1986; Perry, 2001).

The purpose of this study is to look beyond dental evidence and introduce soft tissue analyses, namely reconstructing Physiological Cross-Sectional Area

(PCSA) to contribute to ongoing investigations surrounding diet in plesiadapforms.

By incorporating more evidence into these discussions, I aim to test current hypotheses centering on adaptations for insectivorous sympatric niche partitioning, evolution towards folivory, and convergent evolution towards frugivory.

In this chapter, I build on the work of Chapter 4, characterizing the muscles of mastication in treeshrews, to test whether the dimensions of their muscles of mastication correlate to their mandibular insertion sites independent of body size.

Such a relationship has already been established in strepsirrhine primates, but has yet to be established in treeshrews. Validating this relationship in treeshrews will allow me to reconstruct muscle parameters in plesiadapiforms using both treeshrews and strepsirrhines as extant analogs and test hypotheses about plesiadpiform diet.

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There is little previous work investigating either the methods for reconstructing muscles of mastication or applications in primates. In a notable study, Anton (1999) showed that jaw adductor PCSA in macaques has a low predictability from bony proxies. A recent study by Holliday (2009) investigated osteological correlations in muscles of mastication across a range of dinosaur and archosaur taxa. His results showed a strong correlation for only one muscle, and supports arguments for using extant phylogentic bracketing to reconstruct muscles not entirely supported by bony proxies.

Otherwise, most work has been performed on human cadavers (e.g.,

(Koolstra et al., 1988; Toro-Ibacache and O’Higgins, 2016). For example, a study by

Anton (1994) examined this same relationship in human cadavers as well, also showing a lack of correlation between PCSA and the size of masseter and medial pterygoid bone attachment areas.

In this study, I make the following three predictions based on the results in

Chapter 4. First, I predict that insectivorous and frugivorous plesiadapiforms will present greater temporalis group PCSA relative to the other plesiadapiforms. More specifically, I predict that the temporalis group is largest in cross-sectional area in the inferred insectivorous (Dryomomys, Tinimomys, Navajovius) followed by inferred frugivorous (Carpolestes, Picrodus, Saxonella) plesiadapiforms relative to omnivorous (Nannodectes, Paromomys, and Pronothodectes) and inferred folivorous

(Platychoerops, Plesiadapis) plesiadapiforms, as the temporalis was in

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION strepsirrhines. Second, I predict that the masseter group will be larger in cross section in the more folivorous plesiadapiforms relative to all other plesiadapiforms.

Third, I predict that the relative cross-sectional area of the medial pterygoid will be greater in folivorous plesiadapiforms relative to other plesiadapiforms.

Furthermore, I predict that my results will support general dietary inferences made about plesidapiform diet put forth by previous researchers. For some of the taxa in my study, no direct inferences of diet have been published. For these, I have assigned them a hypothesized diet based on published dietary inferences for plesiadapiform taxa that share key anatomical characteristics with them.

For example, Dryomomys and Tinimomys are both hypothesized to have been insectivorous based on their dental characteristics and small body size (Chester and

Bloch, 2013). Because Dryomomys and Tinimomys are extremely similar in body size and tooth morphology and co-occur in the early Clarkforkian (56.7-56.4Ma) of the

Washakie Basin and the late Clarkforkian (56.1-55.7) of the Clarks Fork Basin

(Chester and Beard, 2012), they are hypothesized to have practiced further niche partitioning. Due to differences in their premolars and jaw morphology, these authors have speculated that Dryomomys was slightly more plesiomorphic. I therefore hypothesize that T. graybulliensis will present with greater temporalis

PCSA than D. szalayi due to greater specialization for insectivory.

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With regards to adaptations for frugivory, it has been hypothesized that the

European Saxonella crepaturae filled a dietary niche similar to the North American

Carpolestes (Russell, 1964). I therefore predict that C. simpsoni and S. crepaturae will display similar overall muscle PCSA due to their similar body size and dental specializations.

Although some authors have hypothesized that paromomyids fed primarily on insects (Godinot, 1984) and others have implicated tree exudates (Beard, 1990;

Boyer and Bloch, 2008), the first quantitative analysis on their dentition was performed by Lopez-Torres and colleagues (2017). These authors did not assign Pa. depressidens to a dietary category, but it was noted to have Dirichlet Normal Energy

(DNE) values closely resembling those of Ignacius fremontensis, which was reconstructed as an omnivore. Therefore, in this chapter, I hypothesize that Pa. depressidens will display PCSA values similar to other omnivorous species (E.g., treeshrews).

Boyer’s recent investigations into the dental adaptations of plesiadapids

(Boyer et al., 2012a, 2010) indicate that later occurring plesiadapids were probably more folivorous than earlier occurring plesiadapids. Based on their larger body size and more complex and higher crowned molars, they show that Plesiadapis cookei and Platychoerops. antiquus likely were more specialized for folivory than older plesiadapids, such as Pronothodectes and Nannodectes. I, therefore, hypothesize that

Pla. antiquus will exhibit greater masseter and medial pterygoid PCSA relative to its

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION hypothesized ancestor, Ple. cookei (Gingerich, 1976), which in turn will present with greater PCSA values relative to Plesiadapis tricuspidens, Pronothodectes gaoi, and

Nannodectes gidleyi.

5.2 Materials

Thirteen mandibles representing seven families of plesiadapiforms were measured (Table 5.1). Specimens were chosen for reconstruction based on their completeness and only specimens from which jaw adductor insertion areas could be measured were chosen. This included scaled photographs from the literature and specimens photographed for this study. While it would have been preferable to include specimens with intriguing mandibular adaptations, such as Chiromyoides, no know specimens exist with a complete enough posterior mandible from which to reconstruct muscle insertion areas.

The sample encompasses the full range of hypothesized plesidapiform body size, with specimens estimated to fall between 37g and 2183g (Boyer, 2009; Silcox et al., 2017). Since plesiadapiforms exhibit a range of body sizes, and probably a range of feeding behaviors and adaptations, it is probable that neither living strepsirrhines nor treeshrews alone have the morphological breadth to encompass the variation found in plesiadapiforms. Therefore, strepsirrhine primate jaw adductor data are included here alongside the treeshrew data in order to provide a broader comparative sample.

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The largest plesiadapiform specimen included in this study is estimated to be around the size of a modern ruffed lemur (Varecia - 3kg), whereas the smallest is estimated to have a body mass similar to the smallest treeshrews, Ptilocercus lowii and Dendrogale murina (45g, Sargis, 2002a, Table 4). This, therefore, means that muscle size predictions for the smallest plesiadapiforms in this study lie beyond the range of extant values used to make the predictions.

This is a problem not uncommon in estimating fossil parameters; for example, subfossil Malagasy lemurs are so large that among primates, their size range falls within that of non-human hominoids rather than extant strepsirrhines.

Jungers (1990) explored this issue by reconstructing body mass in several specimens of subfossil lemur using two regression equations created from extant strepsirrhines as well as non-human humanoids. However, while the size differences between extant strepsirrhines and these extinct lemurs are quite large, that is not the case for the data presented here. Of the thirteen plesiadapiforms included, six fall within the size range of extant strepsirrhines and tupaiids, and all are within ±2 standard deviations of the mean. Therefore, given the close body size measurements within my samples, I expect data trends for plesiadapiforms to continue somewhat uniformly beyond the range of my sample.

A table of extant specimens can be found in Chapter 4 (Table 4.1). Extant specimens include 26 specimens of tupaiid (Family Scandentia, n=10) and strepsirrhine (Primates, n=16) cadavers, either preserved or frozen.

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Table 5.1: Plesiadapiform specimens Est. Jaw Body mass Specimena Taxon Abbreviation Family body length Citations mass Bloch and UM 86273 Carpolestes simpsoni C. simpsoni Carpolestidae 2.223 100 Gingerich (1998) UM 41870 Dryomomys szalayi D. szalayi Micromomyidae 1.494 37 Silcox et al., 2009 USNM 425583 Tinimomys graybulliensis T. graybulliensis Micromomyidae 1.381 38 Silcox et al., 2009 Silcox and AMNH 17390 Navajovius kohlhassae Nav. kohlhassae Microsyopidae 1.571 80 Gunnell, 2007 AMNH 35546/35547 Paromomys depressidens Pa. depressidens Paromomyidae 2.123 90 Conroy, 1987 AMNH 35459/ Picrodus silberlingi Pi. silberlingi Picrodontidae 1.748 40 35453/ 89502 Fleagle, 2013 Gingerich and UM 87990 Plesiadapis cookei Ple. cookei Plesiadapidae 6.869 2200 Gunnell (2005) AMNH 17379 Nannodectes gidleyi Nan. gidleyi Plesiadapidae 4.145 400 Boyer, Chapter 2009 5: PLESIADAPIFORM RECONSTRUCTION St 45822 Plesiadapis tricuspidens Ple. tricuspidens Plesiadapidae 7.794 2656 Silcox et al., 2009

179 St 45825 Plesiadapis tricuspidens Ple. tricuspidens Plesiadapidae 7.029 2656 Silcox et al., 2009 b St 45580 Platychoerops antiquus Pla. antiquus Plesiadapidae 9.519 3582 UALVP 46764 Pronothodectes gaoi Pr. gaoi Plesiadapidae 3.448 777 Silcox et al., 2009 Wa/351 Saxonella crepaturae S. crepaturae Saxonellidae 2.297 82 Rose et al., 2015

Abbreviations: AMNH – American Museum of Natural History St – Staatliches Museum für Naturkunde Stuttgart UM – University of Michigan Wa – Geiseltalmuseum USNM – Unites States National Museum of UALVP – University of Alberta Laboratory for Vertebrate Natural History Paleontology aTwo specimens (Pa. depressidens and Pi. silberlingi) are composites of more than one specimen b Body mass estimated using the generalize primate regression from Gingerich et al., 1982

Chapter 5:PLESIADAPIFORM RECONSTRUCTION

5.3 Methods

Muscle origin and insertion sites have previously been confirmed as proxies for reconstructing jaw adductor cross-sectional area in strepsirrhine primates

(Perry, 2008; Perry et al., 2015), but not in treeshrews. Therefore, confirmation of these proxies in treeshrews is required before work can be undertaken on plesiadapiforms. The method used for reconstructing muscle mass and cross- sectional area follows work done on strepsirrhines (Perry, 2008; Perry et al., 2015).

5.3.1 Insertion Area Measurements

Muscle cross-sectional area can be estimated from muscle origin or insertion area in strepsirrhines, as both the area of origin and insertion area were validated to be appropriate proxies for the temporalis, masseter, and medial pterygoid (Perry et al., 2015). These were also used to estimate jaw muscle dimensions in adapids and subfossil lemurs (Perry, 2018; Perry et al., 2015). Given a fossil sample composed primarily of mandibles, the proxies used here will be limited to the area of insertion.

Insertion area can be captured from a photograph, where the surface of insertion is aligned in parallel to the lens, and is calculated using Fiji software (Schindelin et al.,

2012). I expect minimal difference from a 3-Dimensional area with respect to a photograph because mandibular surfaces are incredibly flat.

Figure 5.1 illustrates the linear measurements used to calculate insertion area for the temporalis muscle group. Modified from Perry et al. (2015), A is the 180

Chapter 5:PLESIADAPIFORM RECONSTRUCTION distance from the dorsal-most point on the coronoid process to the anterior-most point on the ramus where the zygomatic temporalis attaches, marked by the end of a ridge. B is the distance from the dorsal-most point on the coronoid process to the deepest point on the mandibular notch. The product of these two distances approximates the area of attachment for the temporalis muscle on both the medial and lateral aspects of the mandible.

The insertion area of the masseter group is approximated by the masseteric fossa on the lateral side of the ramus (Figure 5.2). The medial pterygoid is approximated by the medial pterygoid fossa on the medial surface of the ramus

(Figure 5.3). Each measurement was taken three times and the average used as the final measurement.

Lateral

Figure 5.1: Measurements for calculating temporalis insertion area

A: Height of the dorsal most aspect of the coronoid process to the deepest part of the mandibular notch B: Distance from the dorsal most point on the coronoid process the end of the tubercle for the zygomatic temporalis tendon.

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Lateral

Figure 5.2: Measurement for calculating masseter insertion area

This area corresponds to the masseteric fossa and angle of the mandible

Medial

Figure 5.3: Measurement for calculating medial pterygoid insertion area

The blue fill represents the depression of the insertion for the medial pterygoid, comprising primarily the angle of the mandible. Inferiorly, the insertion abuts that of the superficial masseter, and superiorly does not exceed the mandibular foramen.

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Measuring insertion area on fossil specimens followed the same protocol as for the extant specimens. Specimens were photographed, or photographs were taken from the literature. Only mostly complete specimens could be included in this study; however, since so few completely intact plesiadapiform mandibular specimens exist, damaged specimens had to be included in order to achieve a reasonable sample size. In cases where a specimen was damaged in an area containing part of an insertion area, this damaged area was either reconstructed using a complete specimen for reference or if the damage was not severe, then the missing portion was filled in freehand.

For specimens where the posterior aspect of the jaw was preserved but broken in the middle of the tooth row, a composite specimen was created, augmenting the original by combining it with the anterior half of a jaw of the same side and species, scaled to the first specimen. The representative paromomyid (Pa. depressidens) and picromomyid specimens (Pi. silberlingi), are composites made using more than one individual. The representative microsyopid specimen (Nav. kohlhassae) is also a composite; however, it is a composite made from parts of the right and left hemimandibles of the same individual and therefore listed under one specimen number.

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5.3.2 Osteological Proxy Validations

Because the use of jaw length affects both proxies and muscle dimensions, the relationship between PCSA and muscle dimension, independent of their relationship with jaw length, need to be assessed in tupaiids.

To do this, the relationship between muscle insertion area and jaw length as well as the relationship between PCSA and jaw length will be assessed using a

Reduced Major Axis (RMA) regression for each muscle group (temporalis, masseter, medial pterygoid). An RMA regression is used in order to take into account the error present in both the x and y variables. The coefficient of determination, R², is used as a first validation because it is the measure of the strength of a relationship.

In parallel, the same relationships between muscle insertion area, PCSA, and jaw length will be assessed using an OLS (Ordinary Least Squares) regression.

Following Perry et al. (2015), OLS regressions will be plotted to assess the relationship between the residuals from the regression of muscle insertion area onto jaw length and the residuals from the regression of PCSA onto jaw length. The residuals of these six regressions will then be plotted against jaw length squared to ascertain whether any further relationships are at play beyond size. Then, the residuals will be plotted against each other and lines of fit will be used to to ascertain whether the osteological proxy of insertion area correlates with PCSA by examining the p-value as a second validation.

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Once independence from jaw length is determined for muscle insertion area and PCSA, an OLS regression of PCSA onto muscle insertion area may then be plotted. In estimating error only for the y variable, this OLS regression line can then be used to predict PCSA from muscle insertion areas in plesiadapiforms. Insertion area as an osteological proxy for PCSA has already been established in strepsirrhines, and so strepsirrhine data will be plotted alongside the results found here for tupaiids to construct the OLS regression line.

5.3.3 Calculating Physiologic Cross-sectional Area

Combining the aforementioned validated tupaiid data with previously independently verified strepsirrhine data (Perry, 2008) will generate a regression line that will be the formula for estimating PCSA in fossil specimens. Including both large strepsirrhines and smaller tupaiids will allow for a range of plesiadapiform body sizes to be faithfully reconstructed.

Individual muscle PCSA cannot be reconstructed in fossil specimens because the borders of the individual muscle layers do not leave faithfully recognizeable markers on bones. Therefore, each of the temporalis and masseter muscle group is reconstructed as a single unit. The medial pterygoid is a muscle composed of four overlapping sheets of fascicles (Gaspard et al., 1973); here it is be reconstructed as a single muscle unit.

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5.4 Results

All figures are presented such that y-axis data and x-axis data conform to the same logarithmic space. Results and tables are therefore presented with this in mind.

5.4.1 Osteological Proxy Validations

Table 5.2 reports the insertion areas measured for each muscle group on the extant sample of tupaiids and strepsirrhines.

Results of RMA regressions of muscle insertion area onto jaw length have been divided by muscle group (Figure 5.4 - Figure 5.6). All three muscle groups show statistical significance in their relationship between jaw length squared and insertion area. The temporalis and masseter insertion areas follow a similar pattern, having statistically significant and similar negatively allometric slopes (temporalis b=0.75, masseter b=0.76, where b is slope). The regression of the medial pterygoid insertion area upon jaw length squared returns an isometric relationship (b=0.94).

The r-squared values are moderate (0.61, 0.79, 0.68). The statistics for these regressions can be found in Table 5.3.

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Table 5.2: Extant specimen muscle insertion areas

Specimen Taxon Jaw Length TIA MIA MPIA JHUTS2 R T. belangeri 3.48 0.802 0.496 0.115 734BEF2 R T. belangeri 3.32 0.715 0.409 0.101 734BABA L T. belangeri 3.12 0.665 0.379 0.065 USNM 497016 T. glis 3.49 0.726 0.392 0.129 YBL 9011 T. minor 2.44 0.523 0.299 0.067 USNM 292532 T. montana 3.30 0.542 0.391 0.085 USNM 296647 T. montana 3.35 0.565 0.435 0.088 USNM 111783 T. nicobarica 3.81 0.969 0.637 0.151 USNM 546344 T. tana 4.31 0.814 0.663 0.149 USNM 580476 T. tana 4.23 1.096 0.531 0.122 AMNH 170501 A. laniger 3.45 2.262 2.823 1.606 DUPC 5800m E. collaris 6.45 3.760 4.041 1.805 DUPC 6394f E. macaca flavifrons 5.95 4.059 2.826 1.429 DUPC 6631m E. rubriventer 6.05 4.588 3.091 1.443 DUPC 1322f H. griseus 4.48 1.982 2.419 1.558 BAA C2f L. catta 5.94 3.095 3.455 1.698 AMNH 170790 L. leucopus 3.34 1.646 1.445 0.716 DUPC 889f M. murinus 2.08 0.465 0.602 0.580 DUPC 874f M. murinus 2.04 0.637 0.428 0.469 BAA C6m N. coucang 3.84 1.920 2.645 0.951 DUPC 1731m O. crassicaudatus 5.29 4.282 4.007 1.713 AMNH 200640 P. potto 4.32 4.052 3.018 1.181 DUPC 6110f P. coquereli 6.00 4.297 5.567 3.091 DUPC 6560m P. coquereli 5.26 3.808 4.851 2.145 DUPC 6769m V. rubra 7.51 5.590 4.201 1.772 AMNH 201395 V. rubra 7.46 6.592 5.162 2.121

TIA=Temporalis Insertion Area, MIA = Masseter Insertion Area, and MPIA = Medial Pterygoid Insertion Area. Refer to Table 4.1 for the full taxon names.

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Figure 5.4: RMA regression of MIA (cm2) upon jaw length squared (cm2)

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Figure 5.5: RMA regression of TIA (cm2) upon jaw length squared (cm2)

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Figure 5.6: RMA regression of MPIA (cm2) upon jaw length squared (cm2)

Referring to the results of the physiological cross sectional area regression from Chapter 4, results have again been divided according to muscle group. Unlike the above regressions of insertion area on jaw length, the PCSA regressions upon jaw length are not statistically significant (p-value>0.05), nor do they have strong correlation (R2= 0.8, 0.12, 0.15, for masseter, temporalis, and medial pterygoid, respectively; refer to Table 5.3). This lack of significance may be due to the small

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION sample size of tupaiids, as PCSA has been show to statistically correlate with negative allometry in platyrrhines (Taylor et al., 2015); close to isometry in anthropoids (Taylor and Vinyard, 2013); encompassing isometry, negative allometry, and positive allometry in strepsirrhines (Perry and Wall, 2008); and suggested to be close to negative isometry in bats (Santana, 2018).

Based on this body of work supporting statistical correlation between masticatory muscle PCSA and body mass in close living relatives to treeshrews and bats, I shall therefore report on PCSA scaling in treeshrews with the hope that future work will increase the sample size to statistical significance. As such, here, temporalis PCSA and masseter PCSA display positive allometry against jaw length squared (b=1.63, 1.64) as opposed to the negative allometry associated with insertion area; the medial pterygoid displays isometry (b=0.99) in both. For plots, refer to Figure 4.5 - Figure 4.7 in Chapter 4; regression statistics can be found in

Table 5.3.

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Table 5.3: Statistics for RMA regressions onto jaw length squared

Allometry Taxon Y Variable Slope LCL UCL y-intercept RSE(%) R² p-value H0 pvalue Trend MIA 0.76 0.54 1.09 -1.17 8.0 Negative 0.79 0.001 0.123 TIA 0.75 0.47 1.22 -0.95 7.9 Negative 0.61 0.007 0.225 MPIA 0.94 0.60 1.46 -2.00 9.2 Isometric 0.68 0.759

0.003 Tupaiid Mass PCSA 1.64 0.80 3.33 -2.04 21.8 Positive 0.08 0.416 0.170 Temp PCSA 1.63 0.81 3.29 -2.03 18.0 Positive 0.12 0.316 0.157 MP PCSA 0.99 0.49 1.96 -1.86 9.4 Isometric 0.15 0.278 Chapter0.957 5: PLESIADAPIFORM RECONSTRUCTION MIA 0.91 0.72 1.15 -0.79 11.8 Isometric 0.83 <0.001 0.391 192 TIA 0.93 0.77 1.11 -0.80 12.9 Isometric 0.90 <0.001 0.376

MPIA 0.63 0.47 0.84 -0.70 10.9 Negative 0.74 <0.001 0.003 Strepsirrhine Mass PCSA 0.94 0.69 1.27 -0.90 15.9 Isometric 0.71 <0.001 0.668 Temp PCSA 0.97 0.75 1.26 -0.85 17.1 Isometric 0.79 <0.001 0.799 MP PCSA 1.02 0.73 1.44 -0.90 17.1 Isometric 0.63 <0.001 0.885

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Insertion area and PCSA were also regressed onto jaw length squared using an OLS regression (Figure 5.7). Residuals from these six regression plotted against jaw length were equally and randomly spaced around the horizontal axis, having low R² values (all <0.05) and high p values (~1) indicating no further relationships; regression statistics can be found in Table 5.4.

The residuals were then plotted against each other (Figure 5.8). Although correlation was low (R2 = 0.33, 0.4, and 0.34 for the masseter, temporalis, and medial pterygoid, respectively), p-values were either significant (0.05, temporalis), or approaching significance (0.08 for both masseter and medial pterygoid). This indicates that solving an OLS regression equation for PCSA against muscle insertion area can yield data for estimating the soft tissue proxy. Correlations are likely to improve with an increase in sample size.

The relationship between PCSA and muscle insertion areas for all muscle groups for both strepsirrhine and tupaiid specimens was then assessed using an

RMA regression. PCSA measurements were log-transformed and regressed onto log- transformed insertion area measurements for each muscle group, as seen in Figure

5.9 - Figure 5.11. The results from the regression analyses can be seen in (Table 5.5).

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Figure 5.7: OLS regressions of insertion areas and PCSA for calculating residuals 194

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Figure 5.8: RMA regressions of PCSA residuals onto insertion area residuals

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Table 5.4: Statistics for OLS regressions onto jaw length

R² p-valuea Taxon Y Variable Slope LCL UCL y-intercept R² p-value Residuals Residuals

MIA 0.68 0.26 0.86 -1.07 0.79 0.001 <0.001 0.998 TIA 0.59 -0.09 0.82 -0.78 0.62 0.007 <0.001 0.977

MPIA 0.77 -0.39 0.98 -1.82 0.69 0.003 <0.001 0.990

Tupaiid Mass PCSA 0.48 -0.55 1.17 -0.79 0.08 0.416 <0.001 1.000 <0.001 Temp PCSA 0.58 -0.82 1.35 -0.90 0.12 0.316 1.000 MP PCSA 0.38 -0.70 0.74 -1.20 0.14 0.278 <0.001 1.000Chapter 5: PLESIADAPIFORM RECONSTRUCTION MIA 0.82 0.65 1.27 -0.68 0.83 <0.001 <0.001 1.000

196 TIA 0.88 0.76 1.14 -0.73 0.90 <0.001 <0.001 1.000 MPIA 0.54 0.40 0.78 -0.58 0.74 <0.001 <0.001 1.000 Strepsirrhine Mass PCSA 0.79 0.58 1.11 -0.70 0.71 <0.001 <0.001 1.000 Temp PCSA 0.86 0.67 1.04 -0.71 0.79 <0.001 <0.001 1.000 MP PCSA 1.34 <0.001 <0.001 1.000 0.81 0.52 -1.12 0.63

aRMA regression of residuals onto jaw length

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Figure 5.9: OLS regression of masseter PCSA (cm2) upon masseter insertion area (cm2) for plesiadapiform formula

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Figure 5.10: OLS regression of temporalis PCSA (cm2) upon temporalis insertion area (cm2) for plesiadapiform formula

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Figure 5.11: OLS regression of medial pterygoid PCSA (cm2) upon medial pterygoid insertion area (cm2) for plesiadapiform formula

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Table 5.5: Statistics for RMA regressions of PCSA onto insertion area

y- ANCOVAa Regression Taxon Muscle Group Slope LCL UCL Allometry R² p-value intercept p-value Masseter 1.35 0.42 2.11 0.13 Positive 0.42 0.060 - Tupaiid Temporalis 2.16 0.59 3.17 0.03 Positive 0.41 0.045 - Medial Pterygoid 1.04 0.48 1.41 0.23 Isometric 0.38 0.380 -

RMA Masseter 1.04 0.36 1.29 -0.09 Isometric 0.78 <0.001 - Strepsirrhine Temporalis 1.05 0.50 1.22 -0.02 Positive 0.78 <0.001 - Medial Pterygoid 1.63 1.15 2.07 -0.25 Positive 0.73 <0.001 Chapter- 5: PLESIADAPIFORM RECONSTRUCTION Masseter 1.08 -0.54 1.85 0.09 Positive 0.26 0.135 *

200 Tupaiid Temporalis 1.39 0.21 2.19 -0.08 Positive 0.41 0.045 * Medial Pterygoid 0.64 1.14 Negative *

0.15 -0.16 0.38 0.058 OLS Masseter 0.91 0.55 1.13 -0.03 Negative 0.78 <0.001 0.415 Strepsirrhine Temporalis 0.92 0.62 1.08 0.04 Negative 0.78 <0.001 0.775 Medial Pterygoid 1.40 0.98 1.96 -0.22 Positive 0.73 <0.001 0.12

aANCOVA was performed for each OLS muscle regression between tupaiids and strepsirrhines, with results reported in the strepsirrhine row

Chapter 5:PLESIADAPIFORM RECONSTRUCTION

The results from these analyses indicate there is a significant relationship between PCSA and muscle insertion area (from the RMA regression) in the tupaiid sample for each muscle group (p-values<0.05). The same analyses performed on strepsirrhine specimens (both from the literature and performed here as a check) also show statistical significance (p-values<0.05). Treeshrews display positive allometry in both their temporalis (b=2.16) and masseter PCSA (b=1.35) regressed onto jaw length but isometry in their medial pterygoid PCSA (b=1.04) regressed onto jaw length. Strepsirrhines present the opposite results, having isometry in their temporalis (b=1.05) and masseter PCSA (b=1.04) regressed onto jaw length and positive allometry in their medial pterygoid (b=1.63) regressed onto jaw length.

In order to use these correlations to predict PCSA in fossil specimens, OLS regressions of strepsirrhine and tupaiid specimens for each muscle group were created and their slopes compared (see Table 5.5). An ANCOVA performed on the tupaiid and strepsirrhine regression lines within each muscle group concludes that there is no significant difference in either the regression slope or y-intercept for the temporalis, masseter, or medial pterygoid muscle groups (p-values=0.42; 0.77,

0.12). The significance of these relationships now allows for the construction of a common OLS regression line for each muscle group from which to predict fossil

PCSA values.

While the aforementioned ANCOVA indicates that the two medial pterygoid regression lines are not statistically significantly different (p-value=0.12), this p-

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION value is very close to the significance level (p=0.05). Therefore, a common regression containing tupaiids and strepsirrhines was not used to predict medial pterygoid PCSA values since doing so would underestimate medial pterygoid size, especially in the very large plesiadapids.

Medial pterygoid PCSA values for fossils were therefore estimated in accordance with their size, using either the individual strepsirrhine or tupaiid OLS regression lines (rather than a common strepsirrhine-tupaiid line as for the temporalis and masseter): those fossils falling within the size range of the strepsirrhine primates (all plesiadapid plesiadapiforms; jaw length > 3.448cm) have their medial pterygoid PCSA values estimated from the strepsirrhine regression line and those falling within the size range of the tupaiid specimens (all non-plesiadapid plesidadpiforms; jaw length < 2.297cm) have their medial pterygoid values estimated from the tupaiid regression line.

The regression formulae are as follows, with all data in log space:

Log Temporalis PCSA = 1.115 × Log TIA − 0.075

Log Masseter PCSA = 0.897 × Log MIA − 0.027

Medial Pterygoid (Tupaiid)Regression Formula: Log MP PCSA

= 0.644 × Log MPIA − 0.161

Medial Pterygoid (Strepsirrhine)Regression Formula: Log MP PCSA

= 1.397 × Log MPIA − 0.219

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5.4.2 Plesiadapiform PCSA reconstruction

Table 5.6 reports the insertion areas by muscle group measured from the plesiadapiform specimens.

Using a common regression line containing strepsirrhine and tupaiid data, temporalis and masseter PCSA were estimated for 13 specimens of plesiadapiforms; medial pterygoid PCSA was estimated using either the strepsirrhine or the tupaiid regression line. The insertion area measurements from the fossils were input into the regression equation calculated from the corresponding regression line, which was solved for PCSA. Plesiadapiform PCSA data for each muscle group can also be found in Table 5.6.

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Table 5.6: Reconstructed plesidapiform muscle insertion areas (cm²) and PCSA (cm²) Mass. Temp. MP Specimen Taxon MIA TIA MPIA PCSA PCSA PCSA UM 86273 C. simpsoni 0.512 0.838 0.108 0.516 0.690 0.165 UM 41870 D. szalayi 0.149 0.319 0.012 0.170 0.235 0.040 USNM 425583 T. graybulliensis 0.121 0.296 0.015 0.141 0.216 0.046

AMNH 17390 Nav. kohlhassae 0.192 0.438 0.028 0.214 0.335 0.069 AMNH 35546/35547 Pa. depressidens 0.421 0.716 0.091 0.433 0.579 0.147 AMNH 35459/ 35453/ 89502 Pi. silberlingi 0.316 0.559 0.054 0.334 0.440 0.105 Chapter 5: PLESIADAPIFORM RECONSTRUCTION UM 87990 Ple. cookei 5.599 5.277 1.803 4.416 5.369 1.376

204 AMNH 17379 Nan. gidleyi 1.710 2.247 0.623 1.523 2.073 0.312 St 45822 Ple. tricuspidens 7.071 6.833 2.001 5.446 7.161 1.592

St 45825 Ple. tricuspidens 4.787 4.787 1.875 3.837 4.816 1.453 St 45580 Pla. antiquus 7.428 6.814 2.260 5.692 7.139 1.887 UALVP 46764 Pr. gaoi 1.346 1.849 0.474 1.228 1.628 0.213 Wa/351 S. crepaturae 0.553 0.915 0.120 0.553 0.762 0.176

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5.5 Discussion

5.5.1 PCSA

When comparing the reconstructed PCSA in plesiadapiforms to PCSA in the strepsirrhines and tupaiids, plesiadapiforms display patterns similar to strepsirrhines. This is unsurprising, however, as data from tupaiids and strepsirrhines were used to estimate the muscle dimensions in plesiadapiforms. The plesiadapiforms follow the pattern of the strepsirrhines as compared to the tupaiids.

This is likely due to the fact that more plesidapiforms overlap with extant strepsirrhine body size than tupaiid.

Like the strepsirrhines, plesiadapiforms display isometry when looking at each muscle group PCSA. Figure 5.12-Figure 5.14 illustrate PCSA regressed onto jaw length for each sample group, broken out by muscle group. Treeshrews are shown in blue, strepsirrhines in purple, and plesiadapiforms in black. The high level of positive allometry displayed by the treeshrews is probably due to the longer jaws of

(especially the smaller) treeshrews relative to strepsirrhine and plesiadapiform specimens of roughly the same overall body size.

Specimens and their assigned dietary categories can be found in Table 5.7. It should be noted that all dietary inferences are made using published data on teeth and any mismatch between dietary signals in the muscles and the teeth might be due, at least partly, to the teeth.

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Figure 5.12: Comparing masseter PCSA in tupaiids, strepsirrhines and plesiadapiforms

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Figure 5.13: Comparing temporalis PCSA in tupaiids, strepsirrhines andplesiadapiforms

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Figure 5.14: Comparing medial pterygoid PCSA in tupaiids, strepsirrhines and plesiadapiforms

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Table 5.7: Plesiadapiform jaw adductor PCSA percentages

Dietary Dietary Mass. Temp. MP Ratio Specimen Taxon Category Category PCSA% PCSA% PCSA% Mass:Temp Citations UM 86273 C. simpsoni Frugivore Biknevicius, 1986 37.62 50.37 12.01 0.75 Chester and UM 41870 D. szalayi Insectivore 38.24 52.77 8.99 0.72 Bloch, 2013 Insectivore- Chester and

USNM 425583 T. graybulliensis 34.97 53.59 11.44 0.65 frugivore Bloch, 2013 Kay and Cartmell, AMNH 17390 Nav. kohlhassae Insectivore 34.60 54.22 11.17 0.64 1977 Chapter 5: PLESIADAPIFORM RECONSTRUCTION AMNH 35546/ Unknown - Gingerich, 1974 Pa. depressidens 37.31 49.97 12.72 0.75 35547 omnivore? 209 AMNH 35459/ Szalay, 1968 Pi. silberlingi Frugivore 38.02 50.00 11.98 0.76

35453/ 89502 Omnivore- Boyer, 2010 UM 87990 Ple. cookei 39.57 48.10 12.33 0.82 folivore AMNH 17379 Nan. gidleyi Omnivore Gingerich, 1976 38.97 53.05 7.98 0.73 St 45822 Ple. tricuspidens Omnivore Boyer et al, 2010 38.35 50.44 11.21 0.76 St 45825 Ple. tricuspidens Omnivore Boyer et al., 2010 37.96 47.66 14.38 0.80 Folivore- Boyer et al., St 45580 Pla. antiquus 38.67 48.51 12.82 0.80 omnivore 2012a UALVP 46764 Pr. gaoi Omnivore Gingerich, 1976 40.02 53.05 6.94 0.75 Wa/351 S. crepaturae Frugivore Biknevicius, 1986 37.08 51.10 11.82 0.73

Chapter 5:PLESIADAPIFORM RECONSTRUCTION

Figure 5.15 shows a comparison between the percentage of total PCSA in each functional muscle group for tupaiids, strepsirrhines, and plesiadapiforms. Blue dots represent the average PCSA of the tupaiids, purple triangles represent average

PCSA in strepsirrhines, and black Xs represent average PCSA in plesiadapiforms as grouped by diet.

Figure 5.15: Comparing jaw adductor PCSA in tupaiids, strepsirrhines, and plesiadapiforms

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As in Chapter 4, PCSA for each taxonomic group is broken out by dietary group (e.g., omnivore, folivore, frugivore).

Supporting the prediction made earlier, the insectivorous plesiadapiforms favor the temporalis muscle more than any other group, as seen in Figure 5.17, having temporalis muscles that comprise more than half of their total PCSA (53.5%).

The ratio of their masseter muscle to temporalis is only 0.67, substantially lower than any other group, fossil or extant (closest is frugivorous-folivorous strepsirrhines at 0.73). This shows a greater reliance on the temporalis muscle.

Insectivorous plesiadapiforms do not group with any of the extant specimens when considering the percentage of PCSA across muscle groups. This is probably due to a diet based on a greater degree of insectivory than any of the living species practice today. This suggests that this group placed greater emphasis on their temporalis muscles more than any other group, fossil or extant. Based on these data, these insectivorous plesiadapiforms show a high degree of specialization towards insectivory not seen in living primates. However, this is only in comparison to living primates, as possessing a large temporalis muscle might also simply be the primitive condition for placental mammals, given that primitive placentals probably mainly ate insects (Sussman, 1991; Szalay, 1968a).

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Figure 5.16: Comparing masseter PCSA by dietary category

Dietary categoriy abbreviations -IR: Insectivore-Frugivore, RI: Frugivore-Insectivore, L: Folivore, RL: Frugivore-Folivore, R: Frugivore, GO: Gummivore-Omnivore, I: Insectivore, O: Omnivore

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Figure 5.17: Comparing temporalis PCSA by dietary category

Dietary categoriy abbreviations -IR: Insectivore-Frugivore, RI: Frugivore-Insectivore, L: Folivore, RL: Frugivore-Folivore, R: Frugivore, GO: Gummivore-Omnivore, I: Insectivore, O: Omnivore

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Figure 5.18: Comparing medial pterygoid PCSA by dietary category

Dietary categoriy abbreviations -IR: Insectivore-Frugivore, RI: Frugivore-Insectivore, L: Folivore, RL: Frugivore-Folivore, R: Frugivore, GO: Gummivore-Omnivore, I: Insectivore, O: Omnivore

Chapter 5:PLESIADAPIFORM RECONSTRUCTION

Supporting the hypothesis concerning greater PCSA in folivorous plesidiapiforms, those plesiadapiforms with specializations for folivory show the greatest reliance on the masseter muscle (39.1%) relative to all other plesiadapiforms (Figure 5.16). This difference is not very great though, as it is similar to the percentage reported for omnivorous plesiadapiforms (38.5%) as well as folivorous strepsirrhines (39.7%).

These folivorous plesiadapiforms also do not show the same degree of specialization towards folivory as the extant strepsirrhines, seen primarily in the difference in reliance on the medial pterygoid muscle (Figure 5.18). In folivorous strepsirrhines, the medial pterygoid comprises a stunning 21% of overall muscle

PCSA, compared to 12.6% in folivorous plesiadapiforms. While this 12.6% for the medial pterygoid is larger than in any of the other plesiadapiform dietary groups, supporting my hypothesis, it does not indicate the same degree of folivorous specialization as the extant strepsirrhines. Of course, the argument can also be made that they are approaching folivory differently than extant folivores. I find this unlikely as the specimens in this sample show similar adaptations for folivory as extant strepsirrhines: teeth with chearing crests and a large body size.

Additionally, while the frugivorous strepsirrhines exhibit a masseter:temporalis ratio greater than one, the folivorous plesiadapiforms exhibit a ratio of only 0.8, which is the same ratio seen in the folivorous-frugivorous strepsirrhines.

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Frugivorous plesiadapiforms exhibit similar percentages in their muscle

PCSA when compared to frugivorous strepsirrhines. A slightly higher percentage of temporalis muscle suggests that these plesiadapiforms either incorporated more insects into their diet or otherwise practiced feeding behaviors that include relatively more incisal biting. Omnivorous plesiadapiforms do not group with the omnivorous treeshrews, having a greater percentage of temporalis PCSA and a lower percentage of masseter PCSA. Instead, they group with the insectivorous and frugivorous strepsirrhines, meaning they might have included more fruit or insects into their omnivorous diet than do the treeshrews; by contrast, leaves are probably a greater proportion of the diet in tupaiids based on their greater masseter PCSA relative to strepsirrhines.

Insectivorous plesiadapiforms do not group with any of the extant specimens when considering the percentage of PCSA across muscle groups, including the insectivorous strepsirrhines (here M. murinus). This is probably due to a diet based on a greater degree of insectivory than any of the living species in the strepsirrhine sample practice today.

5.5.2 Implications for Plesiadapiform Diet

I hypothesized that plesiadapiform PCSA would reflect the dietary inferences reached by previous authors based on teeth. The soft tissue evidence presented here supports prior broad dietary hypotheses made about plesiadapiforms while offering

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION further insights about dietary habits than available from dental evidence alone. In looking more closely at individual specimens, new insights can be made regarding specific plesiadapiform families.

5.5.2.1 Omnivory in Paromomyids

The paromomyid family is one of the largest and most diverse of the plesiadapiform groups, with dental evidence supporting a generalized omnivorous diet. However, it has been demonstrated by Lopez-Torres and Colleagues (2017) that while on the whole they demonstrate omnivorous tendencies, some species incorporate relatively more insects and fruit into their diet. Here, I assigned Pa. depressidens to the same dietary category they assigned I. fremontensis, omnivore, based on similar DNE values in the two species. I hypothesized that Pa. depressidens would display PCSA values similar to those of other omnivores (e.g., treeshrews, omnivorous strepsirrhines, and other omnivorous plesiadapiforms).

Reconstructing PCSA, however, revealed values very similar to the frugivorous plesiadapiforms (C. simpson, Pi. silberlingi, and S. crepaturae) and does not support my hypothesis of an omnivorous diet. In fact, Pa. depressidens displayed values very similar to those of Pi. silberlingi, considered to have been a specialized frugivore by some authors (Burger, 2013; Szalay, 1968b). Both C. simpson and S. crepaturae have temporalis PCSA values of 50%, while Pa. depressidens has a masseter PCSA value of 37% and Pi. silberlingi 38%. Also similar are their medial

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION pterygoid values, Pa. depressidens having a medial pterygoid value of 13% and Pi. silberlingi 12%.

Both Lopez-Torres (2017) and Bown and Rose (1976)have hypothesized a potentially close relationship between Pa. depressidens and the relatively frugivorous omnivores of the genus Ignacius. The results presented here, while not able to give weight to any phylogenetic interpretations, do at least argue for a shared frugivorous diet and broadly support the findings of Lopes-Torres et al.

(2017).

5.5.2.2 Frugivorous Convergence

Many authors have recognized the convergent evolution in plagiaulacoid dentition between saxonellids and carpolestids (e.g., Fox, 1991; Russell, 1964) and argued for a diet heavy in fruit (Biknevicius, 1986). I hypothesized that this convergence would be reflected in their reconstructed PCSA, where they would share a similar muscle percentage breakdown. This hypothesis was supported by the results, where S. crepaturae and C simpsoni share very similar PCSA percentages

(refer to Table 5.7). For specimens of a very similar size and dentition, this further suggests a diet comprising similar foods. Additionally, the pattern of muscle PCSA percentages is shared by Pa. depressidens and Pi. silberlingi, although of smaller body size, implying a static muscle PCSA ratio across frugivorous plesiadapiforms.

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5.5.2.3 Insectivorous Niche Partitioning

Concerning the niche overlap between the insectivorous Dryomomys and

Tinimomys, previous researchers (Chester and Beard, 2012) suggested they occupied the same broad dietary niche but displayed differences in their tooth and jaw morphology that suggest further niche partitioning: Dryomomys has larger distal premolars while Tinimomys has a reduced dental count and a deeper mandible inferior to the fourth premolar. Chester and Beard speculated that dietary or behavioral differences are responsible but do not hypothesize as to what these could be and leave further testing to future workers.

I hypothesized that reconstructing PCSA of their muscles of mastication would illustrate that Dryomomys would present greater masseter cross-sectional area and Tinimomys would present greater temporalis cross-sectional area. The results indicate that Dryomomys presents with a temporalis PCSA of 52.8% while

Tinimomys presents with 53.6%. While this indicates a very slight difference in reliance on the temporalis muscle in Tinimomys, the difference is negligable.

Furthermore, the masseter PCSA percentages also support my hypotheses, with

38.2% of jaw adductor PCSA allocated to the masseter in Dryomomys and 35% in

Tinimomys. While the results do support my hypotheses, data were collected for only one specimen each of Dryomomys and Tinimomys; differences were only quite small and so any conclusions drawn here should be read with caution.

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The medial pterygoid is larger in Tinomomys (11.4%; 9% in Dryomomys) which suggests that Tinimomys practiced greater insectivory, similar to the diet hypothesized for Navajovius. Dryomomys possesses an extremely long and thin angular process of the mandible for the insertion of the medial pterygoid and superficial masseter which implies a small area of insertion for these muscles. This, when combined with the PCSA percentages noted above, supports the inference that

Dryomomys did not rely as heavily on these muscles as compared to other plesiadapiforms.

Comparing D. szalayi to O. crassicaudatus, N coucang and P. potto, all of which practice gummivory, D. szalayi exhibits similar temporalis PCSA values to O. crassicaudatus and similar masseter PCSA to P. potto. D. szalayi may very well have practiced a diet similar to one of these extant strepsirrhines.

Alternatively, Dryomomys’ body size and skeletal adaptations for arboreality are similar to Ptilocercus lowii, the pen-tailed treeshrew, suggesting a functional similarity (Bloch et al., 2007) which may in turn suggest dietary similarities. P. lowii is the most primitive member of the Scandentia (Sargis, 2002a), and the sister to the remaining scandentian taxa. It is relatively smaller and more arboreal than tupaiid treeshrews and considered by many to represent a living analog for early euarchontans (Sargis, 2001a, 2002e; Silcox, 2014; Silcox et al., 2005). Due to its small size and nocturnal habits (Le Gros Clark, 1926), P. lowii has yielded little dietary information. A paucity of P. lowii specimens included in a field survey of

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION treeshrews conducted by Louise Emmons (2000) suggests only that the individuals encountered eat insects as readily as tupaiids do and eat fruit sporadically.

5.5.2.4 Plesiadapid Evolution

Reconstructed PCSA in plesiadapids only partially supported the hypotheses

I outlined above. I hypothesized that the most folivorous plesiadapid, Pla. antiquus - based on dental evidence - would present with the greatest masseter and medial pterygoid PCSA relative to the next folivorous plesiadapid, Ple. cookei, and the omnivorous plesiadapids, Ple. tricuspidens, Pr. gaoi, and Nan. gidleyi. However, the two most folivorous plesiadapids, Pla. antiquus and Ple. cookei, presented with the same masseter PCSA as the two omnivorous, basal plesiadapids, Nan. gidleyi and Pr. gaoi, at 39% and 40%, respectively. The two latter specimens also presented with the greatest temporalis PCSA (>50%). In comparing between the folivorous and omnivorous plesiadapids, the discrepancy in masseter PCSA is largely a product of the reduced medial pterygoid proportions in omnivorous specimens.

Looking further at the medial pterygoid, my hypothesis that the folivorous plesiadapids would have the greatest medial pterygoid PCSA was only partially supported. Ple. cookei did not have the greatest medial pterygoid PCSA (12.2%), but rather a specimen of the omnivorous Ple. tricuspidens (14.4%), followed by the folivorous Pla. antiquus (12.8%).

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Aside from the unusual results for the aforementioned Ple. tricuspidens, the

PCSA values reported here do support prior dietary hypotheses by Boyer and colleagues concerning plesiadapids (Boyer et al., 2012a, 2010). They concluded Pla. antiquus may be the descendent of Ple. cookei, with further dental specializations for folivory. The similarity in both medial pterygoid and masseter PCSA between these two indicate a similar diet. Optimistically, this may indicate that specializations for folivory begin in the dental region, followed by adaptations in the mandible.

Reservedly, this may indicate that these two individuals partook of the same diet, regardless of Pla. antiquus’ more specialized dental morphology.

Ple. tricuspidens presented with greater masseter PCSA than either of the two supposedly more folivorous plesiadapiforms (Pla. antiquus and Ple. cookei). This may be the result of a phenotypically plastic mandible and especially the muscles in response to folivory.

5.5.2.5 Caveats

One limitation placed on the results that can be drawn from this study concerns how PCSA is calculated and the impact this has on choosing extant analogs.

Increases to PCSA originate with either increasing muscle mass or the makeup of muscle fibers; however, the PCSA reported here does not indicate from which element the increase originates. Thus, while the PCSA for plesiadapiforms can be plotted alongside strepsirrhine and tupaiid PCSA for easy comparisons, the

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION underlying differences are not transparent. This discrepancy must be taken into account when deciding how and which extant analogs to use for reconstructing fossil PCSA. Since an extant regression line does not discern between muscle mass and muscle fiber number in estimating PCSA, this will have an impact on how refined any predictions on fossil PCSA can be. The functional significance of these differences will affect future reconstructions because these differences are likely due to different feeding regimes.

In considering sample size, a combined comparative sample of treeshrews and strepsirrhine primates was chosen to try to accommodate the large range of body sizes found in plesiadapiform primates. However, the smallest plesiadapiforms included in this study were smaller than the smallest treeshrew specimens included.

Unfortunately, even though one of the smallest treeshrews, T. minor, was included, the tupaiid comparative sample is not sufficient to make dietary claims about the smallest plesiadapiforms. I therefore suggest that the comparative sample be expanded to include the smallest treeshrews (Dendrogale murina and Ptilocercus lowii) as well as other specimens that fall within their size range, for example insectivorous, frugivorous, and omnivorous bat species (e.g., Freeman, 1979).

Although I included plesiadapiform specimens from several families in this study, I was nonetheless limited to only one specimen for each of five different families, followed by two specimens to represent one family, and five specimens to represent one family. This limitation was due to the constraints of the fossil record:

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION very nearly complete mandibular rami are required for this protocol, but are rare in the fossil record of plesiadapiforms. This creates a bias in the reporting of results because I present single data points, each representing a family, as equal to a mean score of five data points, representing only one family. Were one of the family- representative specimens to be an anomaly for its species, or its mandible poorly reconstructed, this would heavily influence the inferences I have made, thus misrepresenting the dietary aspects of an entire family. While the inferences I have made concerning the plesiadapid family are more balanced, having a greater number of specimens, this is still a low number. I think the best way forward for approaching these data is to ensure results are reported on an individual basis.

To increase the sample size, I propose an approach that will allow future researchers to include more data on plesiadapiform masseter and medial pterygoid muscles. Of the plesiadapiform specimens surveyed for this study, there were many that included a large portion of the ramus of the jaw, but lacked the coronoid process and frequently the anterior portion of the jaw; these specimens usually included the molar teeth. By using the length of the second lower molar as a scaling variable (Boyer, 2008), this would allow mandibles to be used that were not complete anteriorly but still preserved a portion of the back of the mandible. While a missing coronoid process would not increase data on the temporalis muscle, it would allow further research into the masseter and medial pterygoid in

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION plesiadapiforms, potentially for use in studying load transfer between balancing and non-balancing sides of the mandible, and stress and strain.

5.6 Conclusions

Before tupaiid muscles of mastication could be used to estimate dietary parameters in fossil specimens, it was first necessary to ascertain that muscle parameters can be faithfully reconstructed from linear mandibular measurements.

Therefore, osteological correlations between skeletal and soft tissue measurements were tested on an extant sample of treeshrew specimens before applying the same correlations to fossil specimens.

Results of regressing insertion area and calculated physiological cross- sectional area onto jaw length for each muscle group studied (temporalis, masseter, medial pterygoid) returned statistically significant p-values, showing a correlation between these variables and “body size”. Regression analyses of PCSA on muscle insertion sites also returned significant p-values for each group (p-values < 0.01).

From these positive correlations, predictive equations for estimating plesiadapiform

PCSA were generated: temporalis and masseter equations were created from data on tupaiids and strepsirrhines plotted together, while separate medial pterygoid equations were created from the tupaiid and strepsirrhine data.

Hypotheses regarding general trends in PCSA were tested as well as specific hypotheses regarding plesiadapiform dietary groups and current hypotheses. As

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION predicted, insectivorous plesiadapiforms exhibit greater PCSA in their temporalis muscle group and folivorous plesiadapiforms exhibit greater masseter and medial pterygoid PCSA.

The results presented here also allow me to weigh in on plesiadapiform dietary inferences based on dental evidence. Here, I found support from temporalis proportions for the hypothesis that T. graybulliensis and D. szalayi exhibit niche partitioning. Furthermore, I found support paralleling dental similarities between paromomyids Pa. depressidens and Ignacius species suggesting that Pa. depressidens likely incorporated more fruit into its diet, similar to the frugivorous Ignacius.

Similar levels of frugivory were also inferred in C. simpsoni and S. crepaturae, hypothesized to have convergently adapted to frugivory through specialized plagiulacoid dentition. Increasing levels of folivory is also supported in plesiadapid plesiadapiforms, paralleling previous hypotheses based on dental evidence.

Future work investigating plesiadapiform diet should aim to combine multiple methods and theoretical approaches. As shown here, inferences based primarily on dental evidence do not tell the whole story. It would be best to combine dental methods, like second molar relief index, pioneered by Boyer (2008), with geometric morphometric analyses of plesiadapiform mandibles to assess muscle insertion areas. Ideally, further work would look to reconstruct plesiadapiform jaw adductors themselves, similar to work by Slade (2018), who reconstructed chewing musculature in Smilodectes gracilis based on strepsirrhine data. This would allow a

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Chapter 5:PLESIADAPIFORM RECONSTRUCTION model to be built to test functional hypotheses for studying both extant and fossil species dietary behaviors, including assigning major dietary categories, refining paleobiological niche partitioning, and estimating bite force.

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Chapter 6: Bite Force

Chapter 6: Biomechanically modelling the plesiadapiform

mandible and estimating bite force

6.1 Introduction

Many researchers have approached the primate masticatory apparatus from a functional point of view and sought to explain form and function as it relates to evolution. (e.g., Beecher, 1977, 1979; Bouvier, 1986; Daegling, 1992, 2001;

Hylander, 1979; Hylander et al., 1987, 1998, 2000; Kay, 1975; McNamara Jr, 1974;

M. J. Ravosa, 1996; M. J. Ravosa and Hylander, 1994; Ross and Hylander, 2000;

Taylor, 2002, 2006; Vinyard and Ravosa, 1998; Wall, 1999). Further studies have tried to elucidate the role of the masticatory apparatus as it relates to primate taxonomy (e.g., Daegling, 2002; Daegling and McGraw, 2001; Smith, 1983; Wood and

Aiello, 1998).

A comprehensive survey of these studies was addressed by Ross and colleagues (2012), where they concluded that mandibular morphological variation was only indirectly related to dietary categorization. They argued that the strength of any correlation between diet and mandibular morphology differs depending on the scope of a study and the taxonomic sample of focus. However, they, along with many other researchers (e.g., Daegling, 2002; Hylander, 1977), have established clear links between mandibular morphology and the stress and strain imparted on

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Chapter 6: Bite Force the mandible. They argue that instead of interpreting diet directly from the shape and size of a mandible, one needs to interpret it indirectly, through examining the loading regime of the masticatory apparatus.

The muscles of mastication exert stress on the mandible during the act of chewing and these forces have clear consequences on the plastic nature of the mandible. When low strain is placed on the mandible, this reduces masticatory forces and leads to less bone apposition in the corpus and decreased growth of the condyle (Daegling, 1992). Research on mice and rats clearly illustrates the effects of a soft food diet on the (Bouvier and Hylander, 1984; Kiliaridis et al., 1999; Kono et al., 2017). Much research has also been conducted on bats concerning their dietary diversity and the morphology of their mandible and skull with similar results. For example, work performed by Santana has investigated the interplay between PCSA and diet (Santana, 2018; Santana et al., 2010), dietary hardness and skull form

(Santana and Dumont, 2009; Santana et al., 2011, 2012) and the role biomechanical models play in estimating bite force (Davis et al., 2012; Santana, 2016).

Given the clear relationships between diet and mandibular morphology in primates (Bouvier, 1986; Daegling, 1992; Ravosa, 1991, 1996, 2000; Taylor, 2002;

Taylor and Vinyard, 2013; Vinyard et al., 2003; Williams et al., 2002, 2005), one way to study the strain placed on the mandible is via bite force, which has been shown to correlate with jaw length in primates. For example, in looking at platyrrhines, Taylor and colleagues (2015) found a negatively allometric correlation between bite force

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Chapter 6: Bite Force and jaw length. This is in contrast to the isometry found by Perry (2008) in strepsirrhine primates, and the positively allometric correlation seen in bats

(Aguirre et al., 2002).

Most research conducted on estimates of bite force have been narrow in focus, largely due to the invasive nature of in vivo measurements. For example,

Vinyard and colleagues (2009)measured bite force in the marmoset Callithrix jacchus to investigate exudate feeding, Chazeau and colleagues studied the mouse lemur Microcebus murinus (2013), Ledogar and colleagues (2018) the pitheciine monkeys, and Perry the strepsirrhine primates (Perry, 2008; Perry et al., 2011a).

Here, estimates of bite force will be calculated for plesiadapiform as well as strepsirrhine and tupaiid specimens, using a Class III lever as a biomechanical model. This is the most common way to model the chewing system in mammals

(Greaves, 1978; Ravosa, 1990). In this system, the force and load are both located on the same side of the fulcrum. Additionally, the force is located between the load and fulcrum. By modelling the strepsirrhine and tupaiid specimens in this way, I can estimate bite force at three chosen points along the occlusal plane (refer to the

Methods section 6.3.6).

Following predictions made by Spencer (1998)and upheld by Perry (2008), I hypothesize that for all specimens measured, bite force will be relatively greater the farther distal along the tooth row it is measured.

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Chapter 6: Bite Force

Secondly, I hypothesize that bite force will correspond to body size (here jaw length) with isometry as found by Perry (2008) in strepsirrhine primates. This is in opposition to the negative allometry predicted by Lucas (2004) in his fracture scaling hypothesis whereby larger pieces of food, eaten by larger individuals, requires less force to break down than does smaller food. Pursuant to Lucas’ model is my third hypothesis, that all folivorous specimens, extant and fossil, as the largest specimens included in this study, will exhibit the greatest bite force and insectivores the least, as the smallest specimens.

Fourth, for their size, I hypothesize that frugivorous specimens, fossil and extant, will exhibit less bite force compared to other specimens. This is due to the smaller degree of toughness associated with fruit and therefore presumably less strain placed on the mandible during processing (Anapol and Lee, 1994; Ravosa,

1996; Taylor, 2006).

Fifth, regarding specific plesiadapiforms, my predictions on bite force will follow those from Chapter 5. I predict that inferred-frugivorous Carpolestes simpsoni and Saxonella crepaturae will exhibit similar bite force to each other due to their plagiaulacoid dentition, similar inferred diets, and similar size (Biknevicius, 1986). I predict similar results for the picrodontid, Pi. silberlingi, having a characteristically expanded m1 hypothesized as a platform for shearing fruit (Burger, 2013). As part of this prediction, I hypothesize that they will exhibit greater bite force at the mesial premolar than other specimens of a similar size.

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Chapter 6: Bite Force

Finally, reconstructing PCSA for the sympatric Dryomomys szalayi and

Tinimomys graybulliensis, showed that T. graybulliensis has slightly greater temporalis PCSA than does D. szalayi, presumably due to a greater degree of insectivory (Chester and Bloch, 2013). Therefore, I hypothesize that bite force measurements will follow suit, where T. graybulliensis will present with greater anterior bite force due to a more insectivorous diet than D. szalayi.

6.2 Materials

Specimens included in this study are the same specimens used in previous chapters. Refer to Chapter 4, Table 4.1 for a complete list of specimens.

6.3 Methods

Calculating bite force requires measuring and calculating the components to construct a Class III lever system:

1. PCSA for each muscle group

2. Orientation (angle) of each muscle group

3. Jaw adductor resultant vector

4. Moment arms of the muscles (lever arms)

5. Magnitude of the moment arm of the jaw adductor resultant vector

6. Moment arm of bite force (load arm) for each point along the tooth

row.

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6.3.1. PCSA

Regarding the first requirement, PCSA for tupaiid and strepsirrhine specimens can be found in Chapter 4, Table 4.4, whereas PCSA for plesiadapiforms will use the estimated PCSA as found in Chapter 5, Table 5.6.

6.3.2. Calculating Muscle Group Orientation

Regarding the second requirement, muscle orientation is calculated from dissected tupaiid specimens as outlined below and taken from the literature for strepsirrhines (Perry, 2008, Table 6.1) . In keeping with the method of PCSA reconstruction, those plesiadapiforms falling within the size range of the strepsirrhine primates (all plesiadapid plesiadapiforms; jaw length > 3.448cm) will have their angle of orientation for the medial pterygoid estimated from the strepsirrhine average, and those falling within the size range of the tupaiid specimens (all non-plesiadapid plesidadpiforms; jaw length < 2.297cm) will have their medial pterygoid angle values estimated from the tupaiid average.

Calculating orientation of muscle groups must be done against a standard so as to compare all specimens equally. A functionally important reference plane is the occlusal plane; however, reliable determination of an occlusal plane can be problematic because the tooth row curves in many specimens. Therefore, a more precise reference plane was chosen: the Frankfurt Horizontal (FH), here defined for

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Chapter 6: Bite Force use in lateral photographs as a plane through the inferior border of the orbit and the upper margin of the external auditory meatus.

The first step towards this calculation is done using anterior and posterior limits of the attachment sites for each muscle group in the extant samples.

Dissection photographs were oriented to align with the FH and the insertion areas for each muscle group identified. Next, the anterior landmarks are identified on the cranium and mandible, after which a line is drawn between them; the same is done with the posterior landmarks (Figure 6.1). The angles of these lines will be averaged together, giving a direction of pull to each muscle group. To create a vector for each muscle group, this angle of orientation must be combined with a force, here the PCSA of the same muscle group.

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Figure 6.1: Calculating the orientation of the temporalis muscle group

Arrows delineate the anterior and posterior bounds of the temporalis muscle used to measure the orientation of the temporalis muscle. It will be used to calculate the jaw adductor resultant

6.3.3. Calculating the Jaw Adductor Resultant

Once the angle of orientation is calculated for each muscle group, the overall pull of the different muscle groups (jaw adductor resultant) must be determined.

This is done by summing together the vectors of all three muscle groups. The vector of this common force is calculated by breaking down the orientations of each muscle group into both a vertical and horizontal component with respect to the FH.

Therefore, for each muscle group, the following equations were calculated:

푉푒푟푡푖푐푎푙 푣푒푐푡표푟 (푌) = PCSAsin θ

퐻표푟푖푧표푛푡푎푙 푣푒푐푡표푟 (푋) = PCSAcos θ

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With these orientations broken down into their component parts, the vectors of each direction can be summed together. I therefore add together the sum of the vertical components and horizontal components to create a jaw adductor resultant

(JAR), the sum of the vectors for all adductors. This is calculated as:

퐽푎푤 퐴푑푑푢푐푡표푟 푅푒푠푢푙푡푎푛푡 (퐽퐴푅)

= √(TempY + MassY + MPY)2 + (TempX + MassX + MPX)²

The orientation of the jaw adductor resultant can be calculated as:

퐽퐴푅° = 푡푎푛−1 Y/X

The JAR is positioned on the mandible such that its tail is at the centroid location of the common jaw adductor muscle insertion area, an area covering most of the ramus of the mandible. This point is therefore the focus of the pull of these adductors on the mandible. (Figure 6.2).

6.3.4. Measuring the Lever Arm

Regarding the fourth requirement, the lever arm is the shortest

(perpendicular) distance between the fulcrum and the jaw adductor resultant. It is measured from the condyle at a right angle to the vector of the jaw adductor resultant (refer to Figure 6.2).

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Figure 6.2: Lever and load arm measurements and placing the jaw adductor resultant

A represents the Jaw Adductor Resultant. B is the lever arm, measured as the perpendicular distance from the fulcrum to the JAR. I represents the first bite point (at the mesial premolar), 2 the second bite point (at m1), and 3 the third bite point (at m3).

6.3.5. Measuring the magnitude of the moment arm of the jaw adductor resultant

The magnitude of the jaw adductor resultant is directly proportional to its perpendicular distance from the fulcrum (condyle) and is the product of the force

(JAR) and moment arm (lever arm).

퐽푎푤 푎푑푑푢푐푡표푟 푚푢푠푐푙푒 푚표푚푒푛푡 =

(magnitude of JAR) x (constant value of muscle force per unit cross −

sectional area) x (lever arm)

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Where the magnitude of the JAR is taken from the PCSA, the constant is given as 3kg/cm2 (Close, 1972) and the lever arm is measured as the perpendicular distance from the center of the mandibular condyle to the jaw adductor resultant.

6.3.6. Calculating the moment arm of bite force

The moment arm for bite force (load arm) is the shortest distance from the fulcrum (condyle) to the bite point. Three load arms will be measured for this study

– the mesial edge of the first premolar, the protoconid of the first molar, and the distal-most aspect of the third molar (refer to Figure 6.2). In instances where the tooth is missing or broken, the closest approximation was used. All measurements were calculated using Fiji software (Schindelin et al., 2012). Calculating these moment arms is necessary for the estimation of bite force.

6.3.7. Calculating Bite Force

Bite Force is calculated as:

퐵푖푡푒 푓표푟푐푒 = Jaw adductor muscle moment/Load arm

RMA regressions will be used to assess the correlations between bite force and jaw length among different groups of specimens at the three bite points along the tooth row.

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6.4 Results

Tables 6.1, 6.2, and 6.3 list the measurements and calculations necessary to estimate bite force in an extant sample of treeshrews, strepsirrhine primates, and a selection of plesiadapiforms, respectively. Tables 6.4, 6.5, and 6.6 list the estimated bite force for the same individuals at three chosen points along the tooth row

(mesial premolar, first molar, and distal molar).

The greatest absolute bite force, extant or fossil, belongs to the strepsirrhine

Propithecus diadema, the diademed sifaka, having a bite force of 301.07N at its distal molar. The greatest bite force found in the fossil sample is the third molar bite force of a specimen of Ple. tricuspidens at the third molar. The weakest absolute bite force belongs to the plesiadapiform D. szalayi, at 2.16N, followed closely by T. graybulliensis (2.49N) at their mesial premolar. The smallest extant bite force belongs to T. minor, having a bite force of 5.78N at the mesial premolar.

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Table 6.1 Measurements and calculations for estimating bite force in tupaiids

Jaw Length Lever Arm Muscle Moment Specimen Taxon JARa (cm²) JAR Angle (°) (cm) (cm) (kg·cm)

JHUTS2 R T. belangeri 3.48 1.71 86 0.44 2.26 734BEF2 R T. belangeri 3.32 1.65 93 0.39 1.93 734BABA L T. belangeri 3.12 1.56 90 0.43 2.00 Chapter 6: BITE FORCE USNM 497016 T. glis 3.49 1.72 84 0.61 3.14

240 YBL 9011 T. minor 2.44 1.22 93 0.31 1.14

USNM 292532 T. montana 3.31 1.65 97 0.39 1.92 USNM 296647 T. montana 3.31 1.64 95 0.39 1.92 USNM 111783 T. nicobarica 3.81 1.84 94 0.48 2.65 USNM 546344 T. tana 4.31 2.01 94 0.52 3.13 USNM 580476 T. tana 4.23 1.98 98 0.57 3.38

aJaw Adductor Resultant

Table 6.2: Measurements and calculations for estimating bite force in strepsirrhines

Jaw Length JAR Lever Muscle Moment Specimen Taxon JAR (cm²) (cm) Angle (°) Arm (cm) (kg·cm) AMNH 170501 A. lanager 3.45 9.42 77 0.32 9.10 DUPC 5800m E. collaris 6.45 14.61 86 0.43 18.90 DUPC 6251m E. coronatus 5.86 18.24 93 0.43 23.50 DUPC 6394f E. macaca flavifrons 5.27 13.87 99 0.30 12.60 DUPC 6468f E. mongoz 5.95 13.34 90 0.38 15.40

DUPC 6631m E. rubriventer 6.05 18.00 89 0.39 21.10 DUPC 2041m G. moholi 2.38 3.52 83 0.19 2.00 DUPC 1322f/ DUPC 1353f H. griseus 4.60 12.77 87 0.40 15.50 Chapter 6: BITE FORCE BAA C2f L. catta 5.94 12.93 92 0.46 18.00 AMNH 170790 L. leucopus 3.34 6.53 85 0.33 6.40

241 DUPC 889f/ DUPC 874f M. murinus 2.60 1.83 93 0.16 0.90 DUPC 363f M. coquereli 3.12 3.51 90 0.20 2.10 BAA C6m N. coucang 3.84 12.03 99 0.31 11.10 DUPC 1931f N. pygmaeus 3.19 7.17 92 0.27 5.90 DUPC 1731m O. crassicaudatus 5.29 35.90 90 0.32 35.00 AMNH 200640f P. potto 4.32 8.11 88 0.31 7.60 DUPC 6560m/ DUPC 6110f P. coquereli 5.63 28.87 88 0.49 42.50 DUPC 6563m P. diadema 6.39 56.61 86 0.36 61.60 DUPC 6197m P. tattersalli 6.34 44.92 85 0.45 60.40 AMNH 201395/ DUPC 6769m V. rubra 7.48 15.79 95 0.57 26.90

Table 6.3: Measurements and calculations for estimating bite force in plesiadapiforms

Jaw Length JAR JAR Lever Arm Muscle Moment Specimen Taxon (cm) (cm²) Angle (°) (cm) (kg·cm) UM 86273 C. simpsoni 2.22 1.01 96 0.38 1.15 UM 41870 D. szalayi 1.49 0.33 99 0.29 0.28

USNM 425583 T. graybulliensis 1.38 0.30 100 0.31 0.28 AMNH 17390 Nav. kohlhassae 1.57 0.46 100 0.31 0.42 AMNH 35546/35547 Pa. depressidens 2.12 0.85 96 0.44 1.13 AMNH 35459/ 35453/ 89502 Pi. silberlingi 1.75 0.65 96 0.33 0.64 Chapter 6: BITE FORCE UM 87990 Ple. cookei 6.87 8.08 93 1.44 34.90 242 AMNH 17379 Nan. gidleyi 4.15 2.83 98 0.90 7.64

St 45822 Ple. tricuspidens 7.79 10.27 95 1.97 60.70 St 45825 Ple. tricuspidens 7.03 7.32 92 1.71 37.55 St 45580 Pla. antiquus 9.52 10.65 93 2.12 67.74 UALVP 46764 Pr. gaoi 3.45 2.22 98 0.76 5.07 Wa/351 S. crepaturae 2.30 1.10 97 0.63 2.07

Table 6.4: Load arm measurements and estimations of bite force in tupaiids

Load Arm 1 Load Arm 2 Load Arm 3 Specimen Taxon BF (N) BF (N) BF (N) (cm) (cm) (cm)

JHUTS2 R T. belangeri 2.57 8.64 1.96 11.31 1.29 17.20 734BEF2 R T. belangeri 2.76 6.85 2.04 9.28 1.18 16.02

734BABA L T. belangeri 2.57 7.65 1.87 10.50 1.01 19.49 USNM 497016 T. glis 2.80 11.00 2.06 14.93 1.20 25.66 YBL 9011 T. minor 1.93 5.78 1.46 7.64 0.83 13.44 Chapter 6: BITE FORCE USNM 292532 T. montana 2.57 7.34 1.86 10.15 1.10 17.16 USNM 296647 T. montana 2.67 7.07 1.97 9.56 1.21 15.57

243 USNM 111783 T. nicobarica 3.17 8.19 2.34 11.09 1.41 18.40 USNM 546344 T. tana 3.09 9.93 2.28 13.45 1.46 21.01 USNM 580476 T. tana 3.20 10.37 2.33 14.25 1.51 21.98

Table 6.5: Load arm measurements and estimations of bite force in strepsirrhines

Load Arm 1 Load Arm 2 Load Arm 3 Specimen Taxon BF (N) BF (N) BF (N) (cm) (cm) (cm) AMNH 170501 A. lanager 3.64 24.52 2.79 31.97 1.58 56.39 DUPC 5800m E. collaris 5.61 33.05 4.01 46.19 2.70 68.55 DUPC 6251m E. coronatus 5.65 40.80 4.12 55.99 2.78 82.87 DUPC 6394f E. macaca flavifrons 5.65 21.87 3.87 31.97 2.41 51.29 DUPC 6468f E. mongoz 5.33 28.34 3.69 40.89 2.41 62.76 DUPC 6631m E. rubriventer 5.34 38.74 3.64 56.78 2.19 94.34

DUPC 2041m G. moholi 2.35 8.34 1.75 11.18 1.18 16.57 DUPC 1322f/ DUPC 1353f H. griseus 5.10 29.81 4.09 37.16 2.37 64.24 BAA C2f L. catta 6.29 28.05 4.63 38.15 3.02 58.55 Chapter 6: BITE FORCE AMNH 170790 L. leucopus 4.00 15.69 3.06 20.49 1.93 32.56 DUPC 889f/ DUPC 874f M. murinus 2.05 4.32 1.53 5.79 0.91 9.71

244 DUPC 363f M. coquereli 3.00 6.86 2.26 9.12 1.37 15.00 BAA C6m N. coucang 3.40 31.97 2.58 42.26 1.65 65.80 DUPC 1931f N. pygmaeus 3.16 18.34 2.49 23.24 1.67 34.72 DUPC 1731m O. crassicaudatus 3.59 95.72 3.05 112.67 1.91 179.27 AMNH 200640f P. potto 3.64 20.50 2.84 26.28 2.00 37.27 DUPC 6560m/ DUPC 6110f P. coquereli 6.78 61.49 5.38 77.47 3.04 137.20 DUPC 6563m P. diadema 4.89 123.57 3.91 154.35 2.01 301.07 DUPC 6197m P. tattersalli 5.48 108.07 4.31 137.48 2.74 216.15 AMNH 201395/ DUPC 6769m V. rubra 7.89 33.44 5.66 46.58 3.41 77.48

Table 6.6: Load arm measurements and estimations of bite force in plesiadapiforms Load Arm 1 Load Arm 2 Load Arm 3 Specimen Taxon BF (N) BF (N) BF (N) (cm) (cm) (cm) UM 86273 C. simpsoni 1.67 6.75 1.46 7.72 0.96 11.74

UM 41870 D. szalayi 1.29 2.16 0.91 3.06 0.63 4.42 USNM 425583 T. graybulliensis 1.09 2.49 0.93 2.92 0.63 4.31 AMNH 17390 Nav. kohlhassae 1.26 3.30 1.14 3.65 0.74 5.62 AMNH 35546/35547 Pa. depressidens 1.81 6.11 1.53 7.22 1.00 11.05 Chapter 6: BITE FORCE AMNH 35459/ 35453/ 89502 Pi. silberlingi 1.45 4.33 1.41 4.45 0.71 8.85 245 UM 87990 Ple. cookei 5.01 68.32 4.28 79.97 2.68 127.72

AMNH 17379 Nan. gidleyi 3.23 23.20 2.80 26.76 1.90 39.44 St 45822 Ple. tricuspidens 5.49 108.43 4.38 135.90 3.08 193.26 St 45825 Ple. tricuspidens 5.09 72.35 4.45 82.76 2.99 123.17 St 45580 Pla. antiquus 6.51 102.05 5.57 119.28 3.79 175.29 UALVP 46764 Pr. gaoi 2.69 18.47 2.49 19.96 1.65 30.11 Wa/351 S. crepaturae 1.94 10.48 1.66 12.25 1.12 18.15

Chapter 6: Bite Force

Figures 6.3, 6.4, and 6.5 illustrate bite force as regressed onto jaw length for each sample, treeshrews (Figure 6.4), strepsirrhines (Figure 6.5), and plesiadapiforms (Figure 6.6). Circles represent bite force at Load 1 (mesial premolar), triangles represent bite force at Load 2 (first molar), and squares represent bite force at Load 3 (distal molar).

Table 6.7 lists the regression statistics associated with Figures 6.3, 6.4, and

6.5. All specimens return statistically significant p-values (p<0.05) indicating bite force is correlated with jaw length. Treeshrews demonstrate negative allometry at each bite point (b=0.63, 0.64, and 0.58, respectively, moving distally along the tooth row). Strepsirrhines exhibit positive allometry (b=1.27, 1.25, 1.28) while plesiadapiforms exhibit isometry (b=1.03, 1.03, 1.02). However, a non-significant difference from the line of isometry as well as a confidence interval that includes the slope of isometry (1), indicates that isometry may also be a possible interpretation for strepsirrhines, as found by Perry (2008), ans plesiadapiforms. Additionally, an analysis of covariance between the three bite force regressions (ANCOVA) returns non-significant p-values (p = 0.47, 0.44, 0.32), also indicating that these slopes are not statistically significantly different from each other. Correlations are moderate for treeshrews (R²=0.65, 0.67, 0.47) and strepsirrhines (R²=0.61, 0.66, 0.64) but high for plesiadapiforms (R²=0.98, 0.99, 0.99).

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Figure 6.3: RMA regression of tupaiid bite force at three points along the tooth row

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Figure 6.4: RMA regression of strepsirrhine bite force at three points along the tooth

row

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Figure 6.5: RMA regression of plesiadapiform bite force at three points along the tooth row

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Table 6.7: Regression statistics for bite force estimations

y- Allometric p- H0 Taxon Y Variable Slope LCL UCL RSE(%) R² intercept Trend valuea p-value Mesial Incisor 0.63 0.121 0.79 -0.755 6.45 Negative 0.65 0.005 0.050 Tupaiid First Molar 0.64 0.122 0.81 -0.640 6.58 Negative 0.67 0.004 0.055 Chapter 6: BITE FORCE Distal Molar 0.58 -0.109 0.77 -0.351 6.07 Negative 0.47 0.029 0.057 Mesial Incisor 1.27 0.931 1.66 -1.242 19.25 Positive 0.61 <0.001 0.113

250 Strepsirrhine First Molar 1.25 0.906 1.59 -1.088 18.16 Positive 0.66 <0.001 0.120 Distal Molar 1.28 0.937 1.65 -0.928 19.78 Positive 0.64 <0.001 0.089 Mesial Incisor 1.03 0.948 1.11 -0.880 33.54 Isometric 0.99 <0.001 0.383 Plesiadapiform First Molar 1.03 0.958 1.10 -0.813 34.23 Isometric 0.99 <0.001 0.352 Distal Molar 1.02 0.946 1.09 -0.613 33.60 Isometric 0.99 <0.001 0.622

aSignificant values in bold

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6.5 Discussion

6.5.1 Bite Force

Supporting the first prediction made earlier, bite force increases as the bite point moves distally along the toothrow in each sample of specimens studied. While the allometry remains stable between jaw length and bite force (~0.62 for treeshrews, ~1.27 for strepsirrhines, and ~1.03 for plesiadapiforms), bite force will be relatively stronger in the more distal of any two bite points in the same sample group (refer to Figures 6.3, 6.4, and 6.5).

This pattern has to do with the leverage obtained at each bite point. The further distal the bite point moves along the tooth row, the shorter the load arm becomes relative to the lever arm (which remains stable around the fulcrum). The decrease in distance between the load and fulcrum brings the load closer to the action of the jaw adductor resultant, reduces the amount of work needed to move the load, and reduces the joint reaction force produced by the temporomandibular joint (Terhune, 2011). This pattern has been observed by several authors investigating primate bite patterns (Curtis et al., 2010; Koolstra et al., 1988).

Because plesiadapiforms have an extremely high correlation between bite force and jaw length (R2>0.98 for each bite point), this may suggest that their bite force is more directly a function of jaw length than in either strepsirrhines or treeshrews. However this is much more likely a reflection of the fact that muscle

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Chapter 6: Bite Force insertion area, which is highly correlated to jaw length, was used to estimate their

PCSA.

Figures 6.6, 6.7, and 6.8 illustrate bite force for each bite point against jaw length, grouped by taxon. The treeshrews have the greatest degree of negative allometry between groups, while the plesiadapiforms exhibit a positive allometric trend and plesiadapiforms isometry. This means that treeshrews will generate less force than either strepsirrhines or plesiadapiforms as the length of the jaw increases. This is likely related to the different ways treeshrews achieve leverage as discussed in Chapter 4, and probably due to having longer jaws relative to their body than the other taxa. Therefore, my second hypothesis, where I predicted that bite force would grow weaker with larger individuals, only holds true for treeshrews.

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Figure 6.6: Allometry of bite force estimations at the mesial premolar

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Figure 6.7: Allometry of bite force estimations at the first molar

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Figure 6.8: Allometry of bite force estimations at the third molar

Strepsirrhines have greater bite forces compared to plesiadapiforms with similar jaw lengths at larger body sizes. Due to the way the PCSA values were estimated from extant specimens for plesiadapiforms, tupaiid values should not be pulling the plesiadapiform bite force regression line down to underestimate bite force in larger specimens. Instead, this should be a true reflection of further folivorous adaptations in extant strepsirrhines compared to plesiadapiforms of the

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Chapter 6: Bite Force same size, supporting my third hypothesis. I argue the same pattern for plesiadapiforms of small body size, which show smaller bite forces than what might be extrapolated for strepsirrhines.

6.5.2 Implications for plesiadapiform diet

The results of this study support my third hypothesis, that folivorous specimens would demonstrate greater bite force than non- folivorous species (for dietary categories, refer to Tables 4.6, 4.7, and Table 5.7). As seen in Figures 6.9,

6.10, and 6.11, the specialized folivorous strepsirrhines (P. diadema and P. tattersalli) produce the greatest bite forces at each point along the tooth row. While several plesiadapid plesiadapiforms (Ple. tricuspidens; Pla. antiquus), are of a similar size to these specimens, they do not produce as great a bite force; these plesiadapiforms are not as specialized for folivory as the strepsirrhine species (See

Chapter 5, section 5.5.2.4) but rather are omnivores with specializations for folivory.

It is possible that were these plesiadapiforms as specialized for folivory as the smaller forms are for insectivory, the plesiadapiform bite force results would more closely match the strepsirrhine results.

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Figure 6.9: Bite force estimations at the mesial premolar

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Figure 6.10: Bite force estimations at the first molar

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Figure 6.11: Bite force estimations at the third molar

6.5.2.1 Frugivory in the fossil record

The results found here also support my fourth prediction that more frugivorous primates would display a weaker bite force for their size, but only the extant specimens follow this pattern. Here, the frugivorous-folivorous strepsirrhines Varecia rubra, Eulemur macaco flavifrons, and E. mongoz, underperform in terms of force production compared to other similarly sized

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Chapter 6: Bite Force strepsirrhines (refer to Figures 6.9, 6.10, and 6.11). The plesiadapiforms labelled as frugivorous, C. simpsoni, S. crepaturae, Pi. silberlingi, and Pa. depressidens (now considered frugivorous, refer to Chapter 5, 5.5.2.1) did not show a lower than expected bite force. The most probable explanation for this disparity is that the extant forms are eating fleshy fruits with low toughness, whereas the fossil forms relied more heavily on nuts and seeds.

These results also lead me to reject my fifth hypothesis, predicting that C. simpsoni and S. crepaturae would exhibit similar bite forces and, along with Pi. silberlingi, exhibit relatively greater bite force at the mesial premolar. Instead, S. crepaturae presents with great bite force for its size (10.48 N) while the similarly sized C. simpsoni and Pa. depressidens exhibit bite forces in line with a correlation to jaw length (6.75 N and 6.11 N), as does the smaller Pi. silberlingi (4.33 N). Therefore, although C. simpsoni and S. crepaturae present with convergent evolution in the development of a large plagiaulacoid premolar, the two species retain differences in their dietary adaptions from one another.

S. crepaturae is the only plesiadapiform that consistently demonstrates relatively higher bite forces at each bite point along the tooth row. It may be achieving these forces through a shorter jaw in relation to the other specimens of a similar size, thus increasing its mechanical advantage. Pi. silberlingi also exhibits relatively higher bite forces at the third bite point, m3 (8.85 N), perhaps also the result of a shorter jaw. The weaker bite forces reported for the first and third bite

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Chapter 6: Bite Force points may be a consequence of the expanded m1: its relatively greater length causing the bite point measurements to be shifted distally down the tooth row, thereby increasing the load arm and decreasing bite force. This suggests Pi. silberlingi may have been adapted to processing tough foods proximally along the tooth row to increase leverage.

6.5.2.2 Exudate and nectar feeding in the fossil record

Interestingly, the plesiadapiform D. szalayi presented with a pattern similar to the frugivorous strepsirrhines, having an estimated bite force at the first bite point lower than otherwise expected for a plesiadapiform of its size. While this supports my fifth prediction that T. graybulliensis would display greater anterior bite force (2.49 N) than D. szalayi (2.16 N), this was based on an assumed diet of insectivory for both T. graybulliensis and D. szalayi.

However, insectivory may not be the true diet of D. szalayi. D. szalayi has a longer mandible by over 1mm (~7.5% greater) and greater temporalis PCSA (~8% larger) compared to T. graybulliensis. T. graybulliensis also has an estimated bite force at the mesial premolar approximately 13% higher than D. szalayi (refer to

Figure 6.6). Given the earlier conclusion that bite force is heavily correlated with jaw length in plesidapiforms (R2>0.99), this result for D. szalayi is unusual.

One explanation for this result is the possibility that D. szalayi was ingesting a greater amount of fruit than previously thought and was somewhat specialized as

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Chapter 6: Bite Force an exudate or nectar feeder. D. szalayi displays some of the same specializations for exudate feeding as Otolemur crassicaudatus in particular. As found by Smith and

Burrows (2005) in their comparison between O. crassicaudatus and the frugivorous

O. garnetti and by Williams et al. (2002) in their study of exudate feeding galagids, O. crassicaudatus displays different adaptations for exudate feeding adaptations when compared to other galagids. These adaptations are specifically for resisting higher masticatory loads and include a high condyle and shorter tooth row relative to jaw length. While other studies (Eng et al., 2009; Ravosa, 1990; Vinyard et al., 2003;

Williams et al., 2002) reveal that lowering the mandibular condyle and generating maximum jaw gapes are the most advantageous adaptations to facilitate exudate feeding, O. crassicaudatus is an outlier in this regard. Thereofore, if D. szalayi was an exudate feeder, it may have practiced feeding behaviors more similar to O. crassicaudatus than to other exudate feeders.

Additionally, D. szalayi and O. crassicaudatus both exhibit the same percentage of temporalis muscle (~52%, refer to Tables 5.7 and 4.7), while O. crassicaudatus has a greater masseter percentage (+4%). D. szalayi, however has a reduced bite force while the exudate feeders in the sample, including O. crassicaudatus and Nycticebus coucang exhibit greater bite forces, perhaps from a reduced mandible length in relation to their size.

Furthermore, the shallow and narrow mandible seen in D. szalayi may also point to a diet higher in fruit or nectar rather than exudates due to the mandibular

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Chapter 6: Bite Force morphological changes imparted by low masticatory stresses (Muchlinski and

Perry, 2011). Anapol and lee (1994) and Daegling (1992) illustrate that mandibular corpus depth is functionally linked to the strain imparted on the mandible, with primates that eat tougher foods exhibiting deeper mandibles. D. szalayi presents with a long and narrow mandible that is not very deep.

To aid in interpreting diet in the fossil record, Dumont (1997) created a discriminant function analysis to compare extant fruit, exudate, and nectar feeders.

In comparing D. szalayi to this selection of taxa, D. szalayi presents with features found in both nectar (long narrow dentary) and exudate (high coronoid process, high condyle height) feeders. I therefore argue that D. szalayi is a facultative exudate and nectar feeder, supplementing a diet of insects and fruit, further supporting the argument that Ptilocercus lowii, known to feed on nectar (Wiens et al., 2008) would make a suitable functional analog, as has already been suggested based on postcrania (Bloch et al., 2007).

6.5.3 Fallback foods and the importance of food mechanical properties

The interpretations presented here are only as sound as the information available to analyze. For example, there is the argument against my hypothesis that

D. szalayi is specialized not for exudate or nectar feeding as its primary diet, but rather as an adaptation for accessing fallback foods (Dumont, 1997). These are not foods eaten routinely by a species, perhaps varying by season or availability

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(Marshall and Wrangham, 2007). They are not the preferred food and are often relied upon during seasons when their food of choice is unavailable. Sometimes these fallback foods are very similar to the species’ usual diet (i.e., leaves but from a different plant species) but often they are completely different (i.e., gums or leaves instead of fruit) and often more difficult to process (Kinzey, 1974; Lambert et al.,

2004; Wright et al., 2009). This poses the problem whereby it is unknown for fossil forms whether the mandible is optimized for a usual diet, or to enable them to partake in fallback foods, or some combination of the two (Marshall et al., 2009;

Ravosa et al., 2015).

Work by Norconk and colleagues (2009) on platyrrhine primates demonstrates that in several species, specializations may be present for accessing fallback foods and not necessarily for the regular diet. For example, Cebus apella partakes mainly of food of low toughness, but has a fallback food (palm fruit) of great toughness. Yet C. apella exhibits large molar occlusal area that Anapol and Lee

(1994)associated with a greater ability to masticate relatively tougher foods.

This is concerning for some plesiadapiforms, such as the carpolestids and saxonellids, because their plagiulacoid dentition is frequently considered an adaptation for breaking tough foods, such a fruit rind, seed, or nut (Biknevicius,

1986)It is assumed the members of these families routinely ate these tough foods as a regular part of their diet, and that this selected for specialized dental adaptations.

However, these specializations may have instead been an adaptation to access

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Chapter 6: Bite Force fallback foods, driving their evolution by increasing access to foods unavailable to other species. The same argument can be made concerning Pi. silberlingi, with its expanded lower first molar.

6.5.4 Limitations of the model and future work

There are a number of factors inherent in the design of this study that influenced the results. For example, the mesial premolar bite point was not homologous between species, being the p4 premolar in C. simpsoni but p3 in S. crepaturae. Although these teeth likely functioned similarly, this cannot be assumed.

Furthermore, the results modelled only one side of the mandible, and did not take into account the effects of balancing side muscles. The beam model presented here is a simplified version of a complicated system with many variables acting upon it.

Arguably it is easier to compare the kind of data presented here due to the simplicity of the design.

I discussed the outcome of a static system with limited inputs, yet in reality there would have been a number of external factors influencing the plesiadapiform masticatory apparatus. In order to fully understand the plesiadapiform chewing system, more research into these variables is needed. By collecting data on both treeshrew and strepsirrhine variables including gape angle, muscle orientations, joint constraints, and material properties of both the foods and mandibular bone, just to name a few, the accuracy of such a model would increase.

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These data then need to be combined with behavioral data, such as fallback food usage in tupaiids and strepsirrhines. This begins with more research into food mechanical properties. While some research has been conducted on food mechanical properties in platyrrhines (Norconk et al., 2009) and select few other primates (Lambert et al., 2004; Yamashita, 2003; Yamashita et al., 2009), future work needs to focus on collecting data for food items processed by tupaiids and strepsirrhines (Hartstone-Rose et al., 2015; Perry and Hartstone-Rose, 2010).

More information is needed concerning food processing as well, including building on the work performed by Emmons (1991). In her field study of tupaiids, she found that when eating fruit they reject the rind and other fibrous material in favor of swallowing the juice and pulp. Further analyses of jaw muscle electromyography during mastication, like that performed by Vinyard et al. (2005), as well as details about where along the tooth row different species place different foods during biting (Yamashita, 2003) is also necessary. Gathering this information may further influence future choices of extant analogs.

It would also be interesting to examine the role teeth play in the production of bite force. For example, Demes and Creel (1988) found a positive correlation between the molar occlusal area in fossil hominins and bite force. Perhaps this is a pattern that holds for non-human primates as well. It would be especially interesting to see how the expanded molars of picrodontid plesiadapiforms influence force production.

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Ideally, a truly holistic approach could be taken to modelling the crania of plesiadapiforms, where a 3D representation is reconstructed with muscles of mastication modelled on muscle firing patterns that in turn act on the mandible to create mandibular movement. Stress and strain could then be captured with variable inputs such as gape angle, food toughness and texture, and food placement in the mouth.

6.6 Conclusions

Dental evidence predominates plesiadapiform fossil remains, making dietary data from non-dental sources, such as mandibles of particular importance for making dietary inferences. As an integration of mandibular morphology and muscle parameters, bite force as a metric allows researchers to look more closely at dietary distinctions in primates beyond dental evidence alone. Here, bite force in a sample of extant tupaiids and strepsirrhines and fossil plesiadapiforms was estimated following a simple lever III beam model.

As predicted, bite force is relatively greater the farther distal along the tooth row it is measured in all specimens studied due to the increase in leverage caused by a shorter load arm. Bite force was also supported to have a statistically significant negatively allometric correlation with jaw length. Additional hypotheses regarding dietary groupings were also supported. I hypothesized that bite force would be greatest in folivorous strepsirrhines, and it was found to be greatest in the

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Chapter 6: Bite Force folivorous strepsirrhine species P. diadema, followed by P. tattersalli. I also predicted that insectivorous species would exhibit the least bite forces and frugivorous species lower bite force than expected for their size. These hypotheses were also supported by the insectivorous plesiadapiforms and frugivorous strepsirrhines.

Regarding specific plesiadapiforms, my predictions on bite force followed those outlined in Chapter 5 investigating muscle physiological cross-sectional area. I hypothesized that C. simpsoni and S. crepaturae, displaying convergent plagiaulacoid dentition in the mesial premolars, would exhibit similar, and high, bite forces at that point along the tooth row. However, while S. crepaturae demonstrated this pattern, possibly due to a relatively shorter mandible, C. simpsoni demonstrated bite forces more in line with the other frugivorous plesiadapiforms Pa. depressidens and Pi. silberlingi. The latter also displayed higher bite forces, although at the third bite point, which may also indicate adaptations for hard-object food processing.

Reconstructed PCSA for the sympatric D. szalayi and T. graybulliensis, showed that T. graybulliensis may have practiced insectivory to a greater degree than D. szalayi and therefore should have presented with greater anterior bite force for a more insectivorous diet. This hypothesis was supported, and the data present D. szalayi as having a weaker bite force than T. graybulliensis at each bite point studied.

In fact, bite force measurements combined with mandibular characteristics, such as a shallow narrow mandible and tall coronoid process, point to D. szalayi as

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Chapter 6: Bite Force potentially having a greater proportion of fruit, exudates, and nectar in its diet than previously inferred.

Furthermore, the dietary interpretations of D. szalayi, C. simpsoni, S. crepaturae, and Pi. silberlingi highlight the importance of food mechanical properties when investigating diet. In the future, more work needs to be conducted on variables associated with the masticatory system. These include aspects of the lever system itself, such as bone material properties and gape angle, food material properties, such as food toughness, and behavioral variables, including food processing and food availability.

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Chapter 7: Conclusions

7.1 Summary

As possible stem primates, plesiadapiforms have been intensively studied for the insights they might present on our own evolutionary and phylogenetic history.

Due to dental remains comprising a large part of plesidapiform fossil assemblages, diet has been a common topic of study. It is also a potential indicator of other life traits, such as locomotion. For example, frugivorous plesiadapiforms are more likely to have had adaptations for terminal branch feeding because fruits are often found in terminal branches. Dental remains of plesiadapiforms suggest that many of the smaller species had an omnivorous diet consisting largely of insects and fruit, similar to the diets of extant treeshrews. Larger individuals were hypothesized to rely more heavily on fruit and leaves.

7.1.2 Tupaiid musculature

Investigating muscles of mastication is one method by which to further interpret diet for plesiadapiforms beyond what is known from teeth. Muscles of mastication generate forces that mold the mandible to withstand the stresses imposed by food. Each muscle group within the jaw adductors acts on the mandible in a different way to optimize mandibular movement and forces. In primates, these patterns of movement and force are associated with different dietary categories,

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themselves based on nutritional needs and body size. By studying the relative sizes and strength of these muscles, inferences can be made about dietary behaviors of fossil primates as well.

Jaw adductors dissected from the treeshrews in this study show uniformity amongst specimens and differ in only minor aspects. For the most part, muscles conform to dissections made by previous researchers with the exception that the masseter muscle here was found to be composed ofthree layers in every case. The zygomatic temporalis proved to be the most contentious muscle, arguably with a function intermediate between that of the masseter and temporalis groups. More work needs to be performed on this muscle. Overall, the tupaiid jaw adductors are very similar to those of colugos and strepsirrhines, with major differences attributed to size differences between taxa. The greatest difference was found in the zygomatic temporalis, with relative uniformityin the superficial temporalis, deep temporalis, and medial pterygoid.

7.1.3 DiceCT

One method for investigating chewing muscle anatomy involves binding iodine to the muscles in order to visualize them in CT scans. DiceCT, as this method is known, is an emerging technique in the fields of evolutionary and comparative biology that should increase sample sizes and ease of access to specimens.

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DiceCT as a method to digitally isolate the muscles of mastication has the potential to provide more accurate measures of muscle tissue volume as compared to traditional dissection. Muscle fascicles were not measurable but may become so if more time is taken to properly reslice the image data. However, DiceCT offers greater detail and permits reversible manipulation of muscles, whereas dissection may, at least for the time being, offer more accurate muscle fiber measurements.

This method shows great promise for increasing accessibility to specimens and increasing the incorporation of muscle data into dietary reconstructions.

7.1.4 Osteological proxy correlations

PCSA is often used as a metric for the force output of a muscle, and is reliant on measuring muscle mass and the length of individual muscle fascicles. The traditional way to calculate PCSA in the muscles of mastication is through dissection. In fossil species, for which PCSA cannot be directly measured, it must be estimated based on living taxa that are closely related and that exhibit the same basic morphology.

Counter to my predictions, tupaiids show positive allometry in muscle mass when regressed onto jaw length, in opposition to strepsirrhine primates which display isometry. Fiber length measurements followed the same pattern as jaw adductor muscle mass when regressed against jaw length, displaying positive allometry, counter to my predictions that they would follow strepsirrhines and

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present with isometry. Therefore, for a given treeshrew, larger specimens will have relatively greater muscle mass and longer muscle fibers relative to their body size than smaller treeshrews.

Scaling calculations for muscle PCSA reveal that the temporalis muscle is emphasized in insectivorous and frugivorous strepsirrhines instead of insectivorous strepsirrhines and treeshrews as predicted. The folivorous strepsirrhines exhibited the greates masseter and medial pterygoid muscle PCSA. Overall, treeshrews group with omnivorous and insectivorous strepsirrhine primates. Future research should try to incorporate more individuals, especially larger individuals, to investigate further the phenomenon of relatively greater muscle mass.

7.1.5 Reconstruction of plesiadapiform PCSA

Once muscles of mastication have been studied and PCSA calculated for close living relatives of plesiadapiforms, including strepsirrhine primates and treeshrews, these same parameters can be reconstructed for plesiadapiforms. Results indicate correlations between insertion area and PCSA independent of body size in tupaiids and strepsirrhines, from which regression calculations were constructed. Using dental-dietary inferences as a basis for establishing plesiadapiform dietary categories, as predicted, insectivorous plesiadapiforms exhibit greater PCSA in their temporalis muscle group and folivorous plesiadapiforms exhibit greater masseter and medial pterygoid PCSA.

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Like the strepsirrhines, plesiadapiforms display isometry in PCSA compared to jaw length, whereas treeshrews exhibit positive allometry. The insectivorous plesiadapiforms favor the temporalis muscle more than any other group, supporting predictions, but do not group with any extant species including insectivorous strepsirrhines. Omnivorous plesiadapiforms and those with specializations for folivory show the greatest reliance on the masseter muscle relative to all other plesiadapiforms, also supporting predictions. These species also present with a smaller medial pterygoid muscle. Frugivorous plesiadapiforms exhibit similar muscle PCSA percentages to frugivorous strepsirrhines whereas omnivorous plesiadapiforms group with the insectivorous and frugivorous strepsirrhines.

Dietary inferences made about specific plesiadapiforms cover all dietary categories and support hypothesis. There is evidence of niche partitioning between the insectivorous T. graybulliensis and D. szalayi as well as support for frugivory in

Pa. depressidens. Furthermore there is evidence for similar levels of frugivory in the similarly specialized C. simpsoni and S. crepaturae. Increasing levels of folivory were also supported in plesiadapid plesiadapiforms, paralleling previous hypotheses based on dental evidence.

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7.1.6 Bite force estimations

From studying the forces and dimensions of the reconstructed plesiadapiform muscles, dietary inferences can be refined beyond what is known from teeth. Here, forces as imparted by the muscles of mastication were modeled as a simple lever III beam model in a sample of extant tupaiids, extant strepsirrhines, and fossil plesiadapiforms. From this model, bite force could be estimated and compared. In all specimens, bite force increased the farther distal along the tooth row it was estimated. It correlated with negative allometry to jaw length in treeshrews, unlike predictions, but with isometry in strepsirrhines and plesiadapiforms, as predicted. Bite force was greatest in the folivorous strepsirrhines and least in the insectivorous plesiadapiforms.

Bite force was weaker than expected for frugivores based on allometry including the frugivorous plesiadapiforms Pa. depressidens and Pi. silberlingi. The possibly hard fruit-eating plesiadapiform S. crepaturae demonstrated greater bite forces than other frugivores, as predicted, but C. simpsoni did not. Further evidence suggests that T. graybulliensis was more insectivorous than the sympatric D. szalayi but also that the previously considered insectivorous D. szalayi may have, in fact, incorporated a greater degree of fruit, exudates, and nectar in its diet than previously inferred.

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7.2. Future research directions

While this study has introduced new data on tupaiid jaw adductor musculature and dietary inferences in plesiadapiforms, there is still a great more deal to do to integrate this information. Ideally, future work would also incorporate dietary, muscular, and osteological data of the primitive treeshrew Ptilocercus lowii.

Further work should try to increase the treeshrew sample size and include data from more tupaiids, including those from genera not sampled here. That way, aspects of the masticatory apparatus and further dietary behavior in tupaiids can be studied and understood before being applied to plesiadapiform primates. Future work would include studying mandibular stress and strain patterns, food material properties, muscle activity patterns, and food processing behaviors.

To approach this integration between extant and fossils forms, a 3-

Dimensionsal model of the tupaid masticatory apparatus could be constructed that can be modified to fit plesiadapiform parameters. DiceCT would allow digital representations of muscles of mastication to be incorporated as well as measurements of muscle fiber and muscle volume for calculating PCSA. Strain in the masticatory apparatus could be estimated through Finite Element Analysis, informing patterns of strain throughout the skull. This would then allow experiments to be performed, including estimating bite force more realistically than what was performed here, measuring differences in strain during cycles of chewing and how gape, bite point, and chewing frequency affect strain. 276

The next step would be to use this model to investigate the relationship between dietary toughness and the mandible. Most studies investigating food material properties are small in scale and focus on only a few species’ preferences.

Because the exact foods plesiadapiforms ate are unknown, the foods that tupaiids and strepsirrhines eat are of particular interest. Therefore, more work needs to be done to measure food material properties of a range of foods that could then be tested on a 3D model with plesiadapiform parameters.

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H L KRISTJANSON

CURRICULUM VITAE

EDUCATION 2019 Ph.D. Anatomy, Center for Functional Anatomy and Evolution Johns Hopkins University School of Medicine, Baltimore, MD, USA Dissertation Title: Tupaiid masticatory anatomy and the application of extant analogs to reconstructing plesiadapiform jaw adductors Dissertation Advisor: Dr. Jonathan MG Perry

2012 Hons. B.Sc. Biological Anthropology, University of Toronto, Toronto, ON, Canada

TEACHING EXPERIENCE Johns Hopkins University, Baltimore, MD Laboratory Instructor 2014 Scientific Foundations of Medicine: Human Anatomy – medical Taught whole body dissection-based lab sections (>100 hours); executed and marked practical examinations; contributed exam questions; tutored

2013, 2014 Anatomy for Undergraduates - Upper level undergraduate Taught prosection-based cadaver lab sections; prosected gross anatomical structures; presented bovine eye dissections; supervised review sessions; graded exams; tutored

Teaching Assistant 2015, 2016 Scientific Foundations of Medicine: Human Anatomy – medical Substitute laboratory instructor; graded exams

2016 Primate Adaptation and Evolution - Upper level undergraduate Lectured; contributed exam questions; marked exams and labs; held review sessions; proctored

2015 Mammalian Evolution - Upper level undergraduate Lectured; marked exams and quizes; held review sessions; proctored

2014 Human Gross Anatomy - Upper level undergraduate Created online lab modules

2013 Scientific Foundations of Medicine: Human Anatomy – medical Prosected gross anatomical structures 327

H L KRISTJANSON

MANUSCRIPTS IN PREPARATION Kristjanson, HL and JMG Perry. Jaw adductor muscle fiber architecture and estimated bite force in treeshrews (Mammalia: Scandentia).

Perry, JMG and HL Kristjanson. A new specimen of Smilodectes gracilis (Primates: Notharctidae) from the Bridger Formation, WY.

CONFERENCE PARTICIPATION 2017 Kristjanson, HL and JMG Perry Contrast Enhanced Imaging as an Alternative to Dissection in Treeshrew (Tupaiidae: Scandentia) Chewing Musculature. American Association of Anatomists.

2016 Kristjanson, HL and JMG Perry. Jaw adductor muscle fiber architecture and estimated bite force in Treeshrews (Mammalia: Scandentia). International Congress of Vertebrate Morphology, Washington, DC. Invited symposium speaker.

Kristjanson, HL and JMG Perry. Reconstructing jaw adductors in plesiadapid plesiadapiforms from Berru, France (Thanetian, ELMA). American Association of Anatomists.

Kristjanson, HL, Silcox, MT. and JMG Perry. 2016. A new partial cranium of Plesiadapis tricuspidens and insights into plesiadapiform cranial anatomy. (Contender for the Edwin H. and Margaret M. Colbert Prize for best student poster).

2015 Kristjanson, HL and JMG Perry. Estimating bite force in new plesiadapid material from Berru, France (Thanetian, ELMA) and considerations for reconstructing plesiadapiform jaw adductors from extant analogs. Journal of Vertebrate Paleontology, Program and Abstracts: 32.

Kristjanson, HL, Perry, JMG and EM St Clair. Occlusal Patterns as Evidence of Functional Properties in Molars of Afradapis longicristatus (Primates, Adapidae). American Journal of Physical Anthropology 156 (S60): 195. (Won the CG Turner/ Cambridge University Press poster prize for best student poster).

2014 Kristjanson, HL, Deleon, VB. and JMG Perry. A three-dimensional morphometric analysis of the basicranium in Parapithecus grangeri (Anthropoidea, Parapithecidae) and implications for anthropoid origins. Journal of Vertebrate Paleontology, Program and Abstracts: 60.

328

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Kristjanson, HL and JMG Perry. Jaw adductor muscles in treeshrews: Implications for Plesiadapiforms and primate origins. American Journal of Physical Anthropology 153(S58): 161.

2013 Kristjanson, HL and Deleon, VB. Retrodeformation of an Oligocene (33mya) skull of Parapithecus grangeri (Primates: Parapithecidae) using CT imaging. Hopkins Imaging Initiative.

2012 Kristjanson, HL, Prufrock, KA and MT Silcox. Body mass and shearing quotients of Microsyopidae (Mammallia, Primates) from the early Eocene, Bighorn Basin, WY. (Wasatchian, NALMA): paleoecological implications for diet. Journal of Vertebrate Paleontology, Program and Abstracts: 123A.

INVITED TALKS 2016 “Jaw adductor muscles in treeshrews: Implications for Plesiadapiforms and primate origins”. Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, Tulsa, OK.

AWARDS AND HONORS 2018 “Hopkins Hero” – one of 125 employees recognized for personifying Johns Hopkins Medicine’s core values in honor of the university’s 125th anniversary HTTPS://WWW.HOPKINSMEDICINE.ORG/SCHOOL-OF-MEDICINE-125- ANNIVERSARY/INDEX.HTML 2016, 2017 American Association of Anatomists Student Travel Award 2016 Society of Vertebrate Paleontology Student Travel Grant 2016 American Museum of Natural History Theodore Roosevelt Memorial Fund 2016 American Association of Anatomists Short-term Visiting Scholarship 2015 Dental Anthropology Association Cambridge University Press poster prize

MEDIA HTTPS://RESEARCHBLOG.DUKE.EDU/2018/02/02/RESEARCHERS-GET-SUPERMANS-X-RAY- VISION/

SOCIETY MEMBERSHIPS 2013 American Association of Physical Anthropologists 2012 American Association of Anatomists 2011 Society of Vertebrate Paleontology

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UNIVERSITY SERVICE Johns Hopkins University School of Medicine, Baltimore, MD 2016-2017 Member - Committee on the Biomedical Scientific Workforce Task force recommending policy initiatives to ease funding pressures and other difficulties facing early career investigators, and to facilitate the career development of graduate student and postdoctoral trainees HTTPS://PROVOST.JHU.EDU/ABOUT/BIOMEDICALWORKFORCE/REPORT/

2015-2016 Member - Mountcastle Auditorium Art Panel Committee

2014-2016 Member - School of Medicine Ma/PhD Committee Participated in overseeing graduate education policies and initiatives. Accomplishments include establishing the need for increased faculty mentorship training and co-leading the initiative for a graduate student leave policy

2015 Member - Professional Development Office Search Committee 2015 Member - Office of Assessment and Evaluation Search Committee

STUDENT GOVERNMENT Johns Hopkins University School of Medicine, Baltimore, MD 2015-2016 President - Graduate Student Association Chief executive officer; student liaison with School of Medicine deans, registrar and medical students. Accomplishments include initiating fundraising efforts; increasing funds for student travel; leading sustainability efforts

2014-2016 Executive Member - White Coat Ceremony Co-organizer of an awards ceremony honoring graduate students in the Schools of Medicine and Public Health advancing to candidacy 2014-2016 Executive Member - Interschool Committee Co-organizer of social events between professional and graduate students to foster interdisciplinary research.

2014-2015 President- Elect - Graduate Student Association

2013-2014 Vice President of Events - Graduate Student Association

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H L KRISTJANSON

PROFESSIONAL DEVELOPMENT 2015-2016 Leadership Development Series Received personalized training in negotiation, conflict management, effective communication, and management of leadership styles from university Deans.

IN THE SCIENCE COMMUNITY 2016 - pres. Compelling Science Fiction Science advisor and copyeditor for the hard science fiction magazine Compelling Science Fiction.

2016 Research Remix Participant in a unique collaboration between Johns Hopkins scientists and local Baltimore art students to reinterpret research projects as art for public education.

FIELDWORK 2014, 2015 Mammals of SE Saskatchewan: Cyprus Hills Formation, Saskatchewan, Canada 2012-2014 Mammals of Wyoming: Willwood Formation, Bighorn Basin, Wyoming, USA

SKILLS AND QUALIFICATIONS Software: CT imaging segmentation (Avizo/Amira), databases and queries (Access, Excel), data visualization (ImageJ), graphic design and illustration (Adobe Creative Suite), MS Office suite, photogrammetry (AgriScan), geometric morphometrics (MorphoJ), statistical (R, JMP, SPSS)

Laboratory: MicroCT scanning and image reconstruction, human and animal cadaver dissection, iodine staining as a contrast agent, landmarking, light microscopy

Administrative: Educational (Blackboard), survey development and distribution (Qualtrics)

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