View of a Common Marmoset (C

EVOLUTIONARY MORPHOLOGY OF THE MASTICATORY APPARATUS IN TREE GOUGING MARMOSETS

A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Amy Lovejoy Mork

August, 2012

Dissertation written by Amy Lovejoy Mork B.A., Pitzer College, 1997 M.A., Kent State University, 2002 Ph.D., Kent State University, 2012

Approved by

______, Chair Doctoral Dissertation Committee Christopher J. Vinyard

______, Members, Doctoral Dissertation Committee Anne M. Burrows

______, Walter E. Horton, Jr.

______, Richard S. Meindl

______, Steven C. Ward

Accepted by

______, Chair, School of Biomedical Sciences Robert V. Dorman

______, Dean, College of Arts and Sciences Raymond A. Craig

ii TABLE OF CONTENTS

LIST OF FIGURES ...... v

LIST OF TABLES ...... vii

ACKNOWLEDGEMENTS ...... viii

CHAPTERS Page

I INTRODUCTION ...... 1

Marmosets and Exudativory ...... 3 The Marmoset Masticatory Apparatus and Gouging at Wide Jaw Gapes .. 5 A Comparative Analysis of the Articular Cartilage in the Temporomandibular Joint of Gouging And Non-Gouging New World Monkeys ...... 8 Ontogenetic Allometry and Heterochrony of the Marmoset Masticatory Apparatus ...... 9 Comparative Analysis of Masticatory Morphology in Neonatal Common Marmosets (C. jacchus) and Cotton-Top Tamarins (S. oedipus) ...... 11

II A COMPARATIVE ANALYSIS OF THE ARTICULAR CARTILAGE IN THE TEMPOROMANDIBULAR JOINT OF GOUGING AND NON-GOUGING NEW WORLD MONKEYS ...... 15

Introduction ...... 15 Materials and Methods ...... 21 Results ...... 29 Discussion ...... 35

III ONTOGENTIC ALLOMETRY AND HETEROCHRONY OF CRANIAL FEATURES ASSOCIATED WITH WIDE JAW GAPES IN COMMON MARMOSETS (CALLITHRIX JACCHUS) ...... 49

Introduction ...... 49 Materials and Methods ...... 61 Results ...... 67 Discussion ...... 84

iii IV COMPARATIVE ANALYSIS OF MASTICATORY MORPHOLOGY IN NEONATAL COMMON MARMOSETS (C. JACCHUS) AND COTTON-TOP TAMARINS (S. OEDIPUS) ...... 103

Introduction ...... 103 Materials and Methods ...... 110 Results ...... 117 Discussion ...... 140 Conclusions ...... 146

V SUMMARY AND CONCLUSIONS ...... 148

Articular Cartilage of the Temporomandibular Joint ...... 148 Ontogenetic Allometry and Heterochrony ...... 151 Neonatal Morphological Patterns ...... 154 Conclusions and Future Directions ...... 155

REFERENCES ...... 160

iv LIST OF FIGURES

Figure Page

1.1 A marmoset gouging an Anacardium tree ...... 4

1.2 Marmosets use wide gapes when gouging ...... 6

1.3 Measurement of maximum jaw gape using gouging field data ...... 6

1.4 Lateral view of a common marmoset (C. jacchus) and saddleback tamarin (S. fuscicollis) jaw...... 7

2.1 A lateral-view schematic through the midline of a primate TMJ ...... 25

2.2 A lateral-view section through the midline of a Saguinus oedipus TMJ stained with 0.1% Thionin...... 27

2.3 Lateral-view histological slides of (a.) Callithrix jacchus, (b.) Cebuella pygmaea, (c.) Saguinus oedipus and (d.) Saimiri sciureus...... 30

2.4 Boxplots comparing the anteroposterior (AP) ratios for (a.) area, (b.) depth and (c.) percent metachromasia in the condyle and glenoid articular cartilage of gouging and non-gouging platyrrhines...... 34

3.1 Explanatory plots (a) allometry and (b) heterochrony...... 58

3.2 Measurements of gape-related features including (a) AP condyle length, (b) condyle height, (c) AP glenoid length and (d) AP mandible length...... 62

3.3 Measurements of load resistance-related features including (a) M1 depth, (b) M1 width and (c) symphysis length...... 64

3.4 Measurement of jaw length ...... 64

3.5 Allometric plots of ontogenetic series of marmosets and tamarins for cranial features associated with wide gape versus jaw length ...... 69

v LIST OF FIGURES (CONTINUED)

Figure Page

3.6 Allometric plots of ontogenetic series of marmosets and tamarins for cranial features associated with load resistance versus jaw length .... 72

3.7 Growth curves for gape-related features of ontogenetic series of marmosets and tamarins ...... 77

3.8 Growth curves for load resistance related features of ontogenetic series of marmosets and tamarins ...... 82

3.9 Box plots comparing shape differences of gape-related features around the time of weaning ...... 94

4.1 Box plots of absolute masseter and temporalis measurements ...... 118

4.2 Box plots of relative masseter and temporalis measurements and ratios ...... 123

4.3 Corporal and symphyseal cross-sections of the jaw ...... 129

4.4 Absolute measurements of mandibular corpus cross-sections ...... 130

4.5 Relative cross-sectional geometry of the mandibular corpus ...... 132

4.6 Example of in vitro simulation of wishboning stress/strain curve ...... 138

4.7 Box plots of in vitro simulation of wishboning data ...... 138

vi LIST OF TABLES

Table Page

2.1 Gouging and Non-Gouging Platyrrhine Sample...... 23

2.2 Condyle and Glenoid Cartilage Arc Lengths and Ratios of Arc Length to Jaw Length...... 31

2.3 Comparison of Area Measurements and Ratios ...... 33

2.4 Comparison of Depth Measurements and Ratios ...... 36

2.5 Comparison of Percentage Areas of Metachromasia Measurements and Ratios ...... 37

3.1 Allometry Results for Reduced Major Axis and Least Squares Comparisons for Slope Difference and Transposition ...... 68

3.2 Growth Curve Parameters for Gape-Related Features ...... 75

3.3 Growth Curve Parameters for Load Resistance Related Features ...... 81

3.4 Interspecific Shape Differences (Feature/Jaw Length) of Gape-Related Features Around the Time of Weaning ...... 93

4.1 Masseter and Temporalis Architectural and Fiber Length Variables ...... 120

4.2 Cross-Sectional Geometry of the Mandible ...... 125

4.3 Simulated Wishboning at the Mandibular Symphysis ...... 137

vii ACKNOWLEDGEMENTS

This dissertation would not have been possible without the support of many people. I would like to express my gratitude to my amazingly patient advisor, Dr. Chris Vinyard, who offered invaluable assistance, support, and guidance. I am also deeply grateful to the members of my dissertation committee, Drs. Walter Horton, Steve Ward, Anne Burrows and Richard Meindl, without whose knowledge and assistance this study would not have been successful. I am truly grateful to Joseph Bernard and Steve Ward, who made me laugh every day, and for sharing their immense knowledge of human anatomy and love of teaching. Special thanks to Tim Smith and Andrea Taylor for inviting me into their labs and sharing their knowledge and technical expertise.

I am truly indebted to my fellow graduate students Lisa Cooper, Meghan

Moran, Tobin Hieronymous, and Alison Doherty for all of their friendship, support, and assistance. I also want to thank Denise McBurney and Sharon Usip for their indispensible assistance in laboratory techniques.

Thank you also to Margaret Weakland, Debbie Heeter, Diane Kehner, and

Judy Wearden for the administrative support that made this dissertation so much easier to write. I greatly enjoyed and relied on my interactions with the library staff at NEOUCOM, including Denise Cardon, Laura Colwell, and Lisa Barker.

viii Thank you also to the amazing women of NEO Roller Derby, for welcoming me into the derby sisterhood and providing endless hours of fun and much needed stress release.

I am most indebted to my remarkable parents, Michael and Barbara, and to the wonderful people who agreed to marry them, Kristen and Hubert. Thanks for the love, the support, the long phone calls, and the cash. Thank you also to my amazing grandparents, Bonnie and Roland Boddy, who are the smartest people I have had the privilege to know. Thank you to Monika Flaschka for being my best friend for the past 15 years, even though I drive her insane, and for being the closest thing to a sister that I have ever had. Finally, thank you to my beautiful children, Connor and Owen, for supporting and understanding how important this endeavor was, for providing a break from the stress of school work, and for keeping me young and immature enough to enjoy our life.

CHAPTER I

INTRODUCTION

Diet and feeding behavior is one of the fundamental factors underlying variation among primates and have a pervasive influence on behavior, life history, morphology and evolution (Fleagle, 1999). There has been a long- standing interest in the relationships among diet, feeding behaviors and functional morphology of the masticatory apparatus in primates. Numerous studies have addressed variation in primate dental morphology to gain a functional understanding of how tooth shape relates to ingestion and the mechanical breakdown of foods in living primate species. These results are frequently extended to fossil primates to gain an understanding of diet and behaviors in these extinct species (e.g. Kay, 1975, 1978; Hershkovitz, 1977;

Szalay and Delson, 1979; Gingerich et al., 1982; Lucas, 1982, 2004; Oxnard,

1987; Plavcan, 1993; Kay and Williams, 1994; Ungar 1998; Jernvall and Jung,

2000; Teaford et al., 2000; McCollum and Sharpe, 2001; Swindler, 2002).

Analyses of in vivo jaw muscle recruitment patterns as well as investigations into jaw muscle architecture help to describe masticatory muscle recruitment and activity patterns and their relationship to loading patterns throughout the skull during feeding (e.g. Hylander, 1979a,b, 1984, 1985;

1 2

Hylander and Johnson, 1994,1997; Hylander et al., 1987, 1992, 1998, 2000,

2005; Ross and Hylander, 2000; Vinyard et al., 2005; Wall et al., 2006; Taylor and Vinyard; 2008; Eng et al., 2009; Taylor et al., 2009; Vinyard and Taylor,

2010). Comparative analyses of the associations between jaw form and diet have further expanded our understanding of the function and evolution of the primate masticatory apparatus relative to diet and feeding behaviors (e.g.

Bouvier, 1986; Cole, 1992; Deagling, 1992; Antón, 1996; Taylor, 2002, 2005,

2006a,b; Vinyard et al., 2003, 2009). Collectively, these research agendas have made the feeding apparatus one of the best studied regions in primates.

Primate diets are broadly categorized as frugivorous (i.e., fruit), folivorous (i.e., leaves) and/or insectivorous (i.e.,fauna) with associated craniodental and gastrointestinal features necessary for processing and digesting foods from each dietary category (Fleagle, 1999). An additional dietary class used by a number of primate species is exudativory (gummivory). Exudativory involves the ingestion of gums that are exuded by trees as a protective response to damage and is correlated with behavioral and morphological features of the diverse primate species that include gums in their dietary repertoire (Nash, 1986;

Nash and Burrows, 2010). Exudate feeding is widespread across primates, with nearly every major primate family having an exudate feeding representative

(Nash, 1986; Smith, 2010). Moreover, it has been hypothesized as a dietary staple for primitive primates (Martin, 1972).

Marmosets and Exudativory

Callitrichids are an ecologically diverse radiation of small-bodied primates

(100-750 grams) inhabiting Central and South America, and include three groups, Goeldi’s monkeys (Callimico), tamarins (Saguinus, Leontopithecus), and marmosets (Callithrix, Cebuella) (Fleagle, 1999; Rylands et al., 2009). Members of all three groups are adept at clinging to large vertical supports in order to access a high-energy diet of fruit, insects and tree exudates (Sussman and

Kinzey, 1984; Rosenberger, 1992; Garber et al., 1996; Ford and Davis, 2009).

Marmosets differ from other callitrichids in that they actively and habitually stimulate tree exudate flow by biting trees with their anterior teeth (Figure 1.1)

(Kinzey et al., 1975; Coimbra-Filho and Mittermeier, 1977). This type of biting behavior has been defined as gouging (Stevenson and Rylands, 1988). The mechanics of tree gouging involves anchoring the maxillary incisors into the tree and using the mandibular incisors to remove layers of bark from the tree’s surface, resulting in the flow of gum from the tree as a protective response

(Coimbra-Filho and Mittermeier, 1977). Marmosets return to feed on these exudates. Seasonally, exudates comprise a significant part of the marmoset diet suggesting the possibility of natural selective and/or functionally adaptive changes in masticatory apparatus form related to this feeding behavior (Nash,

1986; Nash and Burrows, 2010).

Tamarins share a similar diet with marmosets (e.g., fruits, insects, and exudates), but do not actively gouge trees (Fleagle, 1999). Garber (1992)

Figure 1.1: A marmoset gouging an Anacardium tree

suggests that of the extant callitrichids, Saguinus appears to be the most ecologically generalized member of the callitrichids in both behavior and anatomy and provides a useful model for reconstructing adaptations of early callitrichids.

For our purposes, the derived anatomy and behavior of tree gouging in marmosets can be examined relative to the closely related and more ecologically generalized tamarin with a limited influence of phylogeny. The overall dietary similarity including exudativory coupled with different ingestive behaviors, tree gouging versus non-gouging, provides a compelling natural experiment for assessing morphological consequences of this derived feeding specialization in marmosets.

The Marmoset Masticatory Apparatus and Gouging at Wide Jaw Gapes

It has been demonstrated in both the laboratory and the field that marmosets gouge using wide gapes, but not necessarily relatively large bite forces (Figures 1.2 & 1.3) (Vinyard et al., 2009). Marmoset skulls and jaw muscles exhibit musculoskeletal features that facilitate biting at wide jaw gapes during gouging compared to closely-related, non-gouging taxa (Figure 1.4)

(Vinyard et al., 2003, 2009; Taylor and Vinyard, 2004; Taylor et al., 2009). Skull features facilitating tree gouging at wide gapes include lower condylar heights relative to the tooth row and anterior-posterior elongated mandibular condyles

and glenoid (i.e., temporal) articular surfaces. Lower relative condylar heights have been posited to reduce the amount of masseter muscle stretch at wide

Figure 1.2: Marmosets use wide gapes when gouging

Figure 1.3: Measurement of maximum jaw gape using gouging field data.

Figure 1.4: Lateral view of a common marmoset (C. jacchus) and Saddle-back tamarin (S. fuscicollis) jaw.

gapes, thus potentially increasing maximum gapes (Herring and Herring, 1974 ) as well as maximum bite force at wide gapes (Eng et al., 2009). The anteroposterior (AP) elongation of the mandibular condyle and glenoid articular surfaces facilitates wide gapes by increasing the articular surface area available for condylar rotation and translation, respectively (Vinyard et al., 2003). Jaw- muscle features facilitating tree gouging at wide gapes include relatively longer jaw-muscle fiber lengths that allow increased whole-muscle stretch (Taylor and

Vinyard, 2004; Taylor et al., 2009).

A Comparative Analysis of the Articular Cartilage in the Temporomandibular Joint of Gouging and Non-Gouging New World Monkeys

Tree-gouging marmosets load their temporomandibular joints (TMJ) at wide gapes (Vinyard et al. 2003, 2009). When biting at these wide gapes, the mandibular condyle is likely rotated to contact the glenoid on the posterior condylar surface and translated onto the anterior extent of the glenoid. This wide-gaped position may create novel loading environments in the posterior portion of the condyle and anterior extent of the glenoid articular cartilage.

In Chapter 2, I use comparative histomorphometrics to determine whether loading of the TMJ at wide jaw gapes elicits modifications of articular cartilage depth, area or proteoglycan density in two habitual gouging species, common

(Callithrix jacchus) and pygmy marmosets (Cebuella pygmaea), compared with

non gouging cotton-top tamarins (Saguinus oedipus) and squirrel monkeys

(Saimiri sciureus).

I test the hypothesis that regions of the articular cartilage potentially subjected to increased loading (either percentage or magnitude) during gouging will exhibit relatively increased cartilage thickness and total area as well as relatively higher densities of proteoglycans in gouging species when compared to non gouging species. I hope to add to the understanding of the way in which the soft tissues of the temporomandibular joint adapt and respond to the differential loading associated with gouging behaviors at wide gapes in tree-gouging marmosets.

Ontogenetic Allometry and Heterochrony of the Marmoset Masticatory Apparatus

Ontogeny refers to the developmental history of an organism (Futuyma,

1998). Heterochrony is a change in the developmental timing of a character relative to its appearance or development in an ancestral form (De Beer, 1930;

Gould, 1977, 2000; Alberch et al., 1979; Alba, 2002; McNamara, 2002; Berge and Penin, 2004). Ontogenetic analysis of the functional morphology of primate crania provides a view of the developmental steps involved in achieving adult form. Heterochronic analysis considers shape and size as they relate to time and is key in determining whether shape changes observed throughout ontogeny are a product of changes in the timing or rate of developmental events (or some

combination of the two). Therefore, it is possible to further consider how findings from allometric comparisons may result from divergent patterns of growth.

In Chapter 3, I analyze the ontogenetic allometry and heterochrony of marmoset skulls to determine how the derived craniofacial morphology of adult marmosets arises. Using an ontogenetic approach to compare patterns of relative craniofacial growth among species, I hypothesize that cranial features associated with wide gapes will be present early in C. jacchus ontogeny as compared to non-gouging S. fuscicollis. Features hypothesized to be larger in marmosets are predicted to exhibit a higher slope value or be transposed above the same features in tamarins. The opposite is predicted for those features hypothesized to be smaller in marmosets than in tamarins. To establish whether gape-related traits are part of a pervasive change in craniofacial growth patterns in marmosets, I compared ontogenetic patterns of growth in features associated with load resistance between marmosets and tamarins

By considering heterochronic variation, I also examine variation in the timing of shape changes in craniofacial features during ontogeny in C. jacchus as compared to the closely-related S. fuscicollis. Because C. jacchus possess a specialized skull morphology to gouge trees at wide gapes, I hypothesize that cranial features associated with wide gapes that are larger in marmosets will be peramorphic displaying earlier onset in growth or a faster rate of growth in order to achieve functional competency for gouging at the time of weaning.

Alternatively, traits that are predicted to be smaller will be paedomorphic displaying delayed onset or a slower rate of growth. Cranial features associated with load resistance are also analyzed to determine whether marmosets show a pervasive pattern of growth in their skull form relative to tamarins. I hope to expand the understanding of the influence of allometry and heterochrony in the development of the cranial features associated with tree gouging at wide gapes in marmosets.

Comparative Analysis of Masticatory Morphology in Neonatal Common Marmosets (C. jacchus) and Cotton-top Tamarins (S. oedipus)

Marmoset jaw muscles appear to be designed to facilitate stretching and by extension wide jaw gapes as well as the ability to generate sufficient incisal bite forces at wide gapes (Taylor and Vinyard, 2004; Taylor et al., 2009; Eng et al., 2009). Because of an architectural tradeoff between fiber length and force production, adult marmoset masseter and temporalis muscles have relatively smaller physiologic cross-sectional areas (PSCA) and smaller proportions of tendon suggesting relatively reduced capacity for force production compared to cotton-top tamarins (Taylor and Vinyard, 2004, 2008; Taylor et al., 2009).

Previous analyses of the external metrics and cross-sectional geometry of the mandibular corpus and symphysis suggests that adult tree-gouging marmosets do not possess jaw morphologies that offer increased load resistance ability compared to non-gouging tamarins and squirrel monkeys (Vinyard et al., 2003;

Vinyard and Ryan, 2006; Hogg et al., 2011). In fact, these analyses suggests that marmosets possess a jaw shape that is less effective for resisting certain jaw loads relative to non-gouging platyrrhines (Vinyard and Ryan, 2006; Hogg et al.,

2011).

Chapter 4 addresses the question of whether morphological differences between adult marmosets and tamarins are present at birth. The essential logic is that if features are present at birth that confer functional advantages to marmosets (i.e., prior to any lifetime adaptation), then they are more likely to be evolutionary adaptations to tree gouging. Chapter 4 involves an analysis of the fiber architecture of the masseter muscle including physiological cross-sectional area, fiber orientation and fiber length, analysis of the cross-sectional morphology of the mandibular corpus, and an assessment of the strength of the mandibular symphysis between species during in vitro loading of neonatal marmoset and tamarin specimens. Based on adult comparisons, I predict that common marmoset neonates will have relatively longer masseter and temporalis muscle fibers and will have relatively smaller PCSAs as part of the tradeoff between fiber length and force production. Neonatal marmosets will exhibit no differences in cortical bone cross sectional areas or biomechanical shape ratios compared to the jaws of neonatal tamarins. Finally, common marmoset neonates will show no significant difference in symphyseal strength during in vitro loading compared to neonate cotton-top tamarins. I hope to add to the currently

limited understanding of how adult features of the masticatory apparatus that are associated with adult dietary demands are expressed in the masticatory apparatus of neonates, prior to the need for adult-like functional capabilities.

In the final chapter, I summarize the research findings and discuss major conclusions of the dissertation. In this dissertation, I use multiple methodological approaches to examine features of the bone, cartilage and muscles of the marmoset masticatory apparatus. I examine the effect of differential loading during gouging at wide gapes on the articular cartilage of the marmoset temporomandibular joint. I explore the ontogenetic and heterochronic patterns that underlie the development of the adult marmoset masticatory apparatus.

Finally, I analyze the fiber architecture of masticatory muscles, cross-sectional corpus geometry and in vitro loading at the mandibular symphysis in neonatal marmosets to determine if adult features are present at the time of birth.

The essential question at the core of each of these analyses is “what morphological features characterize the masticatory apparatus of tree-gouging marmosets and how do they arise during ontogeny?” Answering these questions involves furthering our understanding of the form-function relationships in the marmoset masticatory apparatus as well as the extent of functional integration of features that facilitate gouging at wide gapes. Moreover, these results should improve our understanding of what morphological features characterize exudate feeders. Improved functional knowledge of masticatory features associated with

exudativory aids in our understanding of the evolutionary significance of these features and expands our insight into the process of adaptive evolution of the skull in primates relative to dietary and behavioral specializations. Ultimately, this research seeks to add to our understanding of the function and evolution of the primate masticatory apparatus relative to diet and feeding behaviors.

CHAPTER II

A COMPARATIVE ANALYSIS OF THE ARTICULAR CARTILAGE IN THE TEMPOROMANDIBULAR JOINT OF GOUGING AND NON-GOUGING NEW WORLD MONKEYS

Introduction

Callitrichids are an ecologically diverse radiation of small-bodied primates

(100-750 grams) inhabiting Central and South America (Fleagle 1999).

Callitrichids include three groups, Goeldi’s monkeys (Callimico), tamarins

(Saguinus, Leontopithecus), and marmosets (Callithrix, Cebuella). Members of all three groups are adept at clinging to large vertical supports in order to access a high-energy diet of fruit, insects and tree exudates (Sussman and Kinzey,

1984; Rosenberger, 1992; Garber et al., 1996).

Marmosets differ from other callitrichids in that they actively stimulate tree exudate flow by biting trees with their anterior teeth (Kinzey et al., 1975;

Coimbra-Filho and Mittermeier, 1977). This type of biting behavior has been defined as gouging (Stevenson and Rylands, 1988). The mechanics of tree gouging involve anchoring the maxillary incisors into the tree and using the mandibular incisors to remove layers of bark from the tree’s surface, resulting in the flow of sap or gum from the tree as a protective response (Coimbra-Filho and

Mittermeier, 1977). Marmosets return to feed on these exudates. Seasonally,

exudates comprise a significant part of the marmoset diet suggesting the possibility of natural selective and/or functional adaptive changes in masticatory apparatus form related to this feeding behavior.

The Marmoset Masticatory Apparatus and Gouging at Wide Jaw Gapes

It has been demonstrated in both the laboratory and the field that marmosets gouge using wide gapes, but not necessarily relatively large bite forces (Vinyard et al., 2009). Marmoset skulls and jaw muscles exhibit musculoskeletal features that facilitate biting at wide jaw gapes during gouging compared to closely-related, non-gouging taxa (Vinyard et al., 2003, 2009; Taylor and Vinyard, 2004; Taylor et al., 2009). Skull features facilitating tree gouging at wide gapes include lower condylar heights relative to the tooth row and anterior- posterior elongated mandibular condyles and glenoid (i.e., temporal) articular surfaces. Lower relative condylar heights have been posited to reduce the amount of masseter muscle stretch at wide gapes, thus potentially increasing maximum gapes (Herring and Herring,1974 ) as well as maximum bite force at wide gapes (Eng et al., 2009). The anteroposterior (AP) elongation of the mandibular condyle and glenoid articular surfaces facilitate wide gape by increasing the articular surface area available for condylar rotation and translation, respectively (Vinyard et al., 2003). Jaw-muscle features facilitating tree gouging at wide gapes include relatively longer jaw-muscle fiber lengths that allow increased whole-muscle stretch (Taylor and Vinyard, 2004; Taylor et al.,

2009).

Form and Function of Temporomandibular Joint Articular Cartilage and Gouging

The observed differences in musculoskeletal morphology between tree- gouging and closely-related, non-gouging species provide expectations for subsequent functional comparisons of temporomandibular joint (TMJ) cartilage form. The goal of this study is to determine whether the TMJ articular cartilage of marmosets exhibits morphological differences, relative to non-gouging platyrrhines, that can be functionally linked to tree gouging. Habitual tree gouging at wide gapes likely involves novel articular surface contacts and hence different loading patterns in the TMJ relative to non-gouging primates. These novel patterns of load distribution throughout the TMJ may affect the structural composition of the articular cartilage layers that overlie the condylar and glenoid articular surfaces.

Articular cartilage is a specialized connective tissue with a large extracellular matrix component comprised of a dense collagen fiber network, high concentration of proteoglycans, as well as a smaller amount of other matrix proteins (Mow et al., 1992; Dijkgraaf et al., 1995; Benjamin and Ralphs, 2004).

The physical and mechanical properties of articular cartilage are dependent upon the integrity of the collagen network as well as the synthesis and retention of a high concentration of proteoglycans (Dijkgraaf et al., 1995; Silver, 2006; Kuroda et al., 2009; Singh and Detamore, 2009a). The articular cartilage of the TMJ provides both the condylar and the glenoid articular surfaces with joint mobility at

low frictional coefficients and the ability to withstand compressive forces (Mow et al., 1992; Knudson and Knudson, 2001; Kuroda et al., 2009). This capacity to resist compressive forces during routine loading is achieved by safely distributing loading stresses throughout the tissue without incurring permanent tissue damage (Freeman and Kempson, 1973). Physical properties of connective tissues depend on differing proportions of proteoglycans and structural proteins

(e.g. collagen) and how these constituent molecules are organized within the extracellular matrix (Kuettner and Kimura, 1985; Mow et al., 1992; Kuroda et al.,

2009; Singh and Detamore, 2009a). Whereas the tensile strength of articular cartilage is primarily ascribed to the collagen fiber network, the compressive stiffness of articular cartilage is often linked to the percentage of proteoglycans

(Kempson et al., 1970; Mizoguchi et al., 1996; Huang et al., 2001; Singh and

Detamore, 2009a).

Proteoglycans provides hydration and swelling pressure to the tissue to enable it to withstand compressive forces (Mow et al., 1992; Yanagishita, 1993;

Dijkgraaf et al., 1995; Knudson and Knudson, 2001; Silver, 2006). Proteoglycans are macromolecules composed of glycosylated core proteins and covalently attached highly anionic glycosaminoglycans. The highly negatively charged glycosaminoglycans attract and bind water molecules within the articular cartilage. The osmotic swelling pressure of the proteoglycan-laden cartilage enables the tissue to resist compressional forces in load bearing joints

(Hardingham and Bayliss, 1990; Mow et al., 1992).

Cartilage subjected to high (but not pathological) levels of loading can display increased proteoglycan content compared to cartilage exposed to low levels of loading (Slowman and Brandt, 1986; Kiviranta et al., 1987). Articular cartilage can respond to regular loading by increasing thickness at the site of loading (Kiviranta et al., 1987; 1988). Thickening of the articular cartilage has been interpreted as a localized tissue adaptation to loading (Tanaka et al., 2006;

Singh and Detamore, 2009b) and may result from stimulation of cartilage cells to increase the synthesis of extracellular matrix components (Mussa et al., 1999;

Singh and Detamore, 2009a). Furthermore, Singh and Detamore (2009b) report that increased cartilage thickness is correlated with increased stiffness in pig condylar cartilage. Proteoglycan distribution and compressive stiffness differ across regions of TMJ cartilage (Mizoguchi et al., 1996; Hu et al. 2001; Patel and

Mow, 2003; Tanaka et al., 2006; Singh and Detamore, 2009a,b). Collectively, these functional studies highlight the possibility of differential loading within the joint as well as tissue responses to loading involving increased cartilage thickness and proteoglycan content. As a caveat, however, it is important to recognize that articular cartilage in the TMJ can also degrade both in quantitative dimensions and proteoglycan content as loading persists and/or becomes pathological (Ravosa et al., 2007; Chen et al., 2008).

Hypothesis and Predictions

We use histomorphometrics to compare TMJ articular cartilage in tree- gouging common (Callithrix jacchus) and pygmy marmosets (Cebuella pygmaea) to non-gouging cotton-top tamarins (Saguinus oedipus) and squirrel monkeys

(Saimiri sciureus). Both common and pygmy marmosets habitually gouge trees to elicit exudate flow prior to consumption of the gums and saps (Nash 1986).

Like marmosets, cotton-top tamarins rely on fruit and insects for large portions of their diet, but only opportunistically feed on previously exuded gums and saps

(Nash, 1986; Fleagle, 1999). Squirrel monkeys are almost exclusively frugivorous and insectivorous, consuming little if any sap or gum except on an opportunistic basis (Sussman and Kinzey, 1984; Garber, 1980, 1992; Ferrari,

1993). Therefore, while marmosets and tamarins both consume tree exudates in their diets, methods of procurement differ in such a way to create a natural experiment for determining the influence of habitual tree-gouging on TMJ articular cartilage form by comparing marmosets to tamarins and squirrel monkeys.

We test the hypothesis that regions of the articular cartilage potentially subjected to increased loading (either percentage or magnitude) during gouging will exhibit relatively increased cartilage thickness and total area as well as relatively higher densities of proteoglycans in gouging species compared to non- gouging species. Tree-gouging marmosets load their temporomandibular joints at wide gapes (Vinyard et al., 2003, 2009). When biting at these wide gapes, the

mandibular condyle is rotated to contact the glenoid on the posterior condylar surface and translated onto the anterior extent of the glenoid. This wide-gaped position may create novel loading environments in the posterior portion of the condyle and anterior extent of the glenoid articular cartilage. Relatively thicker and/or proteoglycan rich articular cartilage in these regions of gouging species would suggest a relatively increased capacity to resist compressive loads. Based on these functional observations, we examine three predictions:

Prediction One: Gouging primates will exhibit relatively larger total articular cartilage area in the posterior condyle and anterior glenoid regions of the

TMJ compared to non-gouging tamarins and squirrel monkeys.

Prediction Two: Gouging primates will display relatively thicker cartilage in the posterior condyle and anterior glenoid portions of the TMJ compared to non-gouging taxa.

Prediction Three: Gouging primates will display relatively increased proteoglycan densities in the posterior condyle and anterior glenoid articular surfaces of the TMJ compared to non-gouging tamarins and squirrel monkeys.

Materials and Methods

Samples

We analyzed ten individuals from gouging species (five Callithrix jacchus, five Cebuella pygmaea), and eight individuals from non-gouging species (five

Saguinus oedipus, three Saimiri sciureus). A near equal ratio of males and

females was examined in each species. All specimens were captive bred adults with known ages ranging from 4 to 15 years (Table 2.1).

Histology

Temporomandibular joints were removed en bloc from formalin-fixed specimens and decalcified in 10% EDTA for an average of six weeks. Following decalcification, one TMJ from each individual was selected for paraffin embedding based on which joint demonstrated the most intact association of the joint capsule after being trimmed of excess tissue. We then processed and embedded TMJs in paraffin using standard histological techniques (Presnell et al., 1997).

Temporomandibular joints were serially sliced in a sagittal orientation into

10µm thick sections. Every other set of three sections was slide mounted. We stained TMJ sections with Thionin (0.1%) to observe basic cartilage morphology and to determine the relative distribution of proteoglycans in the articular cartilage (Bulstra et al., 1993). Thionin has been demonstrated to be an effective cationic dye for proteoglycan density analysis (Kiraly et al., 1996). We identified a representative section for analysis that was within five percent of the midline based on serial sectioning data and morphological details. We created a 10x montage image for this section for subsequent measurement using BioQuant

Osteo II version 8.00.20 software.

Table 2.1. Gouging and Non-Gouging Platyrrhine Sample

Species1 Sex2 Age at death (in years)

Callithrix jacchus M 12 Callithrix jacchus M 5 Callithrix jacchus F 11 Callithrix jacchus F 12 Callithrix jacchus F 10

Cebuella pygmaea M 4 Cebuella pygmaea F 4 Cebuella pygmaea F - Cebuella pygmaea M - Cebuella pygmaea F 4

Saguinus oedipus F 4 Saguinus oedipus M 15 Saguinus oedipus M > 14 years Saguinus oedipus F 14 Saguinus oedipus - -

Saimiri sciureus F >12 years Saimiri sciureus F - Saimiri sciureus M -

1 Gouging species include C. jacchus and C. pygmaea. Non-gougers include S. oedipus and Sa. sciureus. 2 M=male, F=female, “-“=unknown.

Histomorphometric measurements of temporomandibular joint cartilage. Measurements were performed on 10x digital montages of each histological section. We measured length, area and depth of the articular cartilage, as well as proteoglycan density, multiple times and reported the average for each. The anterior and posterior limits of the functional articular surface for both the condyle and glenoid were defined as the anterior and posterior insertion points of the temporomandibular joint disc, providing morphological boundaries for the measurements included in this analysis (Fig.

2.1a).

Cartilage arc lengths. We measured the arcs of the condylar and glenoid articular surfaces as the linear distance between anterior to posterior fibrous insertions of the disc (Fig. 2.1b). This arc was then used to equally divide the articular cartilage of the condyle and glenoid surfaces into anterior, middle, and posterior segments (Fig. 2.1a). We used these regional designations to divide the articular cartilage into functional zones for subsequent measurement and hypothesis testing.

Cartilage area. We measured condylar cartilage cross-sectional area in lateral view as the area bounded by the anterior and posterior disc insertions, the superior articular surface of the condylar cartilage, and the inferior depth of the articular cartilage

Figure 2.1: A lateral-view schematic through the midline of a primate TMJ depicting our (a.) designation of cartilage regions and (b.) metric dimensions. In (a.), the anterior and posterior boundaries for the cartilage articular surface are taken from the anterior and posterior attachment sites of the articular disc to the articular cartilage, respectively (white arrows). Based on these defined limits for the articular cartilage, we divided the condylar and glenoid cartilage into anterior, middle and posterior zones (black arrows). In (b.), the arc length of the cartilage is demonstrated for the glenoid (dotted white line) as the distance between anterior and posterior attachment sites. Schematic representations of cartilage area (hatched space) in the anterior condylar zone and cartilage depth throughout the posterior condylar zone (spaced white arrows) are also shown.

as distinguished from subchondral bone (Fig. 2.1b). The glenoid cartilage area was measured as the area bounded by the anterior and posterior disc insertions, the inferior articular surface of the articular cartilage, and the superior border that distinguishes articular cartilage from subchondral bone. We measured condylar and glenoid cartilage areas for the total cartilage area, as well as anterior, middle, and posterior zones. The above stated boundaries were traced with a mouse and the interior area was then calculated using the Bioquant program.

Cartilage depth. We measured cartilage depth for the condylar and glenoid articular surfaces as the distance from the articular surface of the cartilage to the margin of the transition from articular cartilage to subchondral bone (Fig. 2.1b). Depths were taken at 10µm intervals beginning at the anterior insertion of the disc. The mean of these values was used to estimate average depth for the total articular cartilage, as well as for each of the three designated zones.

Proteoglycan Density. Metachromasia, a chromatic shift of Thionin staining that occurs in the presence of proteoglycans (Bulstra et al., 1993; Kiraly et al., 1996), was evaluated following the establishment of a standardized specimen-specific threshold, using maximum staining intensity observed within the anterior zone of each specimen as a minimum criterion for presence of metachromasia (Fig. 2.2). The area of metachromatic proteoglycan staining was measured, based on the specimen specific threshold, for the total condylar

Figure 2.2: A lateral-view section through the midline of a Saguinus oedipus TMJ stained with 0.1% Thionin. The arrow points to a more darkly stained region in the condylar articular cartilage demonstrating metachromasia. (Magnification = 10x)

and glenoid articular cartilage, as well as within each of the divisional zones.

Proteoglycan staining density is reported as the percentage of the area of the cartilage that is metachromatic.

Analysis. We created ratios of anterior to posterior cartilage dimensions for each specimen and used these ratios in testing our hypothesis rather than absolute dimensions. By analyzing ratios created from the same individual, we controlled for variation in body size (which was largely unavailable for these specimens), age-related changes in joint morphology, tissue preservation and fixation, and histological staining. Our approach assumes that areas of the TMJ not routinely loaded during gouging at wide gapes (i.e., the anterior surface of the condyle and posterior surface of the glenoid) are relatively similar across gouging and non-gouging species.

Initially, we applied a one-way analysis of variance (ANOVA) to determine whether the two gouging species (C. jacchus and C. pygmaea) and the two non- gouging species (S. oedipus and Sa. sciureus) differed from each other in their anteroposterior (AP) ratios for different cartilage dimensions. In both cases, we found no significant differences within gouging or within non-gouging species.

Therefore, we grouped the two gouging species for comparison with the grouped non-gouging species for hypothesis testing. We used one-way ANOVA to determine whether anteroposterior (AP) ratios differed significantly between gougers versus non-gougers as predicted by our hypothesis. All statistical tests were performed in Systat (11.0).

Results

Histomorphology of the TMJ and Cartilage Arc Length

The four monkey species share a basic similarity in the histomorphology of their temporomandibular joints (TMJ) (Fig. 2.3). Like most other primates, the articular surface of the glenoid is markedly longer and flatter than the articular surface of the condyle in each species (Table 2.2). The articular disc maintains a narrow central region that fans out both anteriorly and posteriorly at the discal attachments. The lateral pterygoid muscle attaches to the anterior extent of this disc. In summary, we see little evidence that tree-gouging marmosets have undergone a major reorganization of the soft tissue structures in their TMJs.

A more in depth examination of the quantitative details of articular cartilage form begins to uncover subtle differences between gouging and non- gouging species. While the average arc length of the articular cartilage does not differ significantly among species for the condyle (P=0.89) or glenoid (P=0.24)

(Table 2.2), the differences in absolute size suggest that gougers often have relatively longer arc lengths. Despite their small size, the absolute length of the condylar articular surface is equally long in C. pygmaea and S. oedipus (Table

2.2). Thus, the ratio of condylar arc length to jaw length is much larger in C. pygmaea compared to the other species. Both gouging species maintain

Figure 2.3: Lateral-view histological slides of (a.) Callithrix jacchus, (b.) Cebuella pygmaea, (c.) Saguinus oedipus and (d.) Saimiri sciureus. (Note the tear in the posterior articular disc of this specimen). In each slide, anterior is to the left and superior is at the top. All slides were stained with 0.1% Thionin. (Magnification = 10x).

Table 2.2. Condyle and Glenoid Cartilage (a.) Arc Lengths and (b.) Ratios of Arc Length to Jaw Length for Gouging and Non-Gouging Species

a. Arc lengths

Arc Length Callithrix Cebuella Saguinus Saimiri ANOVA2 (mm) jacchus pygmaea oedipus sciureus Condylar Arc 1.86 (0.89)1 2.22 (0.40) 2.22 (0.97) 1.87 (1.22) P = 0.89 Glenoid Arc 4.23 (1.07) 3.04 (0.55) 4.14 (1.39) 4.41 (0.52) P = 0.24

b. Ratios of arc length to jaw length

Variable Callithrix Cebuella Saguinus Saimiri - (mm) jacchus pygmaea oedipus sciureus Jaw Length3 26.78 19.53 30.41 32.30 - Condyle Arc / 4 0.069 0.114 0.073 0.058 - Jaw Length Glenoid Arc / 0.158 0.156 0.136 0.137 - Jaw Length

1 Mean (S.D.) 2 P-values from one-way ANOVA comparing arc lengths among species. 3 Jaw length data taken from adult skeletons of C. jacchus (n=30), C. pygmaea (n=26), S. oedipus (n=22) and Sa. sciureus (n=19). 4 Arc length to jaw length ratios are based on species means as jaw length measurements were unavailable for the histological sample.

relatively longer glenoid articular surfaces compared to non-gouging species.

These relatively longer articular surfaces may facilitate increased range of motion at the TMJ related to generating wide jaw gapes during gouging.

Area. The anteroposterior (AP) ratio for condylar area shows no significant difference in articular cartilage area between gouging and non- gouging species (Table 2.3a; Fig. 2.4a). These results fail to support the predicted relative increase in articular cartilage area in the posterior condyle of tree gouging marmosets. Only C. jacchus among marmosets follows the predicted pattern in displaying the lowest anteroposterior (AP) condylar area ratio

(Fig. 2.4a).

Glenoid anteroposterior (AP) area ratios differ significantly between gouging and non-gouging species (ANOVA, P<0.0007) (Table 2.3b; Fig. 2.4a).

The significantly larger ratio in marmosets, indicating a relatively increased anterior cartilage area, supports the predicted increase in load resisting ability along the anterior surface of the glenoid linked to tree gouging at wide gapes.

Depth. Prediction Two states that gouging primates will display increased relative articular cartilage thickness in the posterior condyle and anterior glenoid, when compared to non-gouging primates. Ratios of anteroposterior (AP) articular cartilage depth for the condyle follow the pattern observed for anteroposterior area ratios and fail to demonstrate any statistical difference

Table 2.3. Comparison of (a.) Condyle and (b.) Glenoid Area Measurements and Ratios for Gouging and Non-Gouging Species a. Condyle areas Area Callithrix Cebuella Saguinus Saimiri Measurements ANOVA2 jacchus pygmaea oedipus sciureus (mm2) Anterior Area 0.059 (0.04)1 0.063 (0.02) 0.062 (0.04) 0.061 (0.05) - Posterior Area 0.103 (0.08) 0.068 (0.01) 0.059 (0.03) 0.070 (0.05) - Total Area 0.25 (0.20) 0.21 (0.03) 0.19 (0.10) 0.22 (0.20) - Anterior/Posterior 0.61 (0.28) 0.96 (0.37) 0.99 (0.23) 0.81 (0.12) P = 0.17 Ratio b. Glenoid areas Area Callithrix Cebuella Saguinus Saimiri Measurements ANOVA jacchus pygmaea oedipus sciureus (mm2) Anterior Area 0.158 (0.06) 0.132 (0.04) 0.056 (0.03) 0.230 (0.07) - Posterior Area 0.143 (0.03) 0.104 (0.04) 0.073 (0.04) 0.402 (0.23) - Total Area 0.45 (0.10) 0.34 (0.11) 0.18 (0.09) 0.94 (0.35) - P= Anterior/Posterior 1.08 (0.16) 1.31 (0.29) 0.79 (0.10) 0.67 (0.30) 0.0007 Ratio G>NG

1 Mean (S.D.) 2 P-values from one-way ANOVA comparing anteroposterior (AP) area ratios between gouging versus non-gouging species. Bold p-values are significant. “G”= gouging species. “NG” = non- gouging species.

Figure 2.4: Boxplots comparing the anteroposterior (AP) ratios for (a.) area, (b.) depth and (c.) percent metachromasia in the condyle and glenoid articular cartilage of gouging and non-gouging platyrrhines.

between gouging and non-gouging groups (Table 2.4b, Fig. 2.4b). Common marmosets exhibit posterior condylar depths that are nearly twice the anterior depth, while the remaining species maintain near equal ratios of cartilage depths between regions.

The glenoid anteroposterior (AP) depth ratios support the predicted pattern as the gouging species exhibit a significantly higher ratio compared to the non-gouging platyrrhines (Table 2.4b; Fig. 2.4b). Both relative depth and area of the glenoid articular cartilage would provide increased load resistance ability for loading along the anterior extent of the glenoid articular surface.

Proteoglycan density (metachromasia). Prediction Three states that gouging primates will display relatively greater proteoglycan density in the articular cartilage of the posterior condyle and the anterior glenoid. Neither the anteroposterior (AP) ratios for condyle or glenoid metachromasia exhibit significant differences between gouging and non-gouging species (Table 2.5, Fig.

2.4c). We see little evidence linking variation in proteoglycan density to novel loading patterns related to tree gouging.

Discussion

Tree gouging marmosets load the temporomandibular joints (TMJ) at wide jaw gapes when biting trees to remove bark layers and provoke exudate flow

(Vinyard et al., 2009). Because opening the jaw involves condylar rotation and

Table 2.4. Comparison of (a.) Condyle and (b.) Glenoid Depth Measurements and Ratios for Gouging and Non-Gouging Species

a. Condyle depths Depth Callithrix Cebuella Saguinus Saimiri Measurements ANOVA2 jacchus pygmaea oedipus sciureus (mm) Anterior Depth 0.089 (0.05)1 0.100 (0.03) 0.088 (0.02) 0.101 (0.01) - Posterior Depth 0.169 (0.11) 0.101 (0.03) 0.088 (0.01) 0.099 (0.02) - Total Depth 0.131 (0.08) 0.103 (0.02) 0.090 (0.01) 0.113 (0.03) - Anterior/Posterior 0.60 (0.27) 1.04 (0.38) 0.99 (0.13) 1.04 (0.19) P = 0.20 Ratio

b. Glenoid depths

Depth Measurements Callithrix Cebuella Saguinus Saimiri ANOVA (mm) jacchus pygmaea oedipus sciureus Anterior Depth 0.137 (0.05) 0.143 (0.02) 0.073 (0.03) 0.153 (0.05) - Posterior Depth 0.129 (0.05) 0.127 (0.03) 0.081 (0.04) 0.279 (0.16) - Total Average Depth 0.130 (0.05) 0.131 (0.02) 0.074 (0.03) 0.209 (0.08) - Anterior/Posterior P = 0.046 1.05 (0.11) 1.19 (0.43) 0.94 (0.14) 0.67 (0.32) Ratio G>NG

1 Mean (S.D.) 2 P-values from one-way ANOVA comparing anteroposterior (AP) depth ratios between gouging versus non-gouging species. Bold P-values are significant. “G”= gouging species. “NG” = non- gouging species.

Table 2.5. Comparison of (a.) condyle and (b.) glenoid percentage areas of metachromasia measurements and ratios for gouging and non-gouging species

a. Condyle metachromasia

Percentage Callithrix Cebuella Saguinus Saimiri ANOVA2 Metachromasia (%) jacchus pygmaea oedipus sciureus Anterior 1 0.46 (0.18) 0.43 (0.10) 0.34 (0.06) 0.41 (0.04) - Metachromasia Posterior 0.27 (0.15) 0.30 (0.11) 0.22 (0.08) 0.14 (0.05) - Metachromasia Total 0.38 (0.16) 0.41 (0.06) 0.32 (0.09) 0.23 (0.05) - Metachromasia Anterior/Posterior 1.92 (0.65) 1.63 (0.91) 1.82 (0.91) 3.17 (0.87) P = 0.22 Ratio

b. Glenoid metachromasia

Percentage Callithrix Cebuella Saguinus Saimiri ANOVA Metachromasia (%) jacchus pygmaea oedipus sciureus Anterior 0.09 (0.07) 0.29 (0.10) 0.22 (0.16) 0.19 (0.14) - Metachromasia Posterior 0.34 (0.23) 0.46 (0.19) 0.37 (0.22) 0.20 (0.09) - Metachromasia Total 0.24 (0.21) 0.40 (0.09) 0.33 (0.17) 0.22 (0.12) - Metachromasia Anterior/Posterior 0.27 (0.04) 0.86 (0.78) 0.60 (0.31) 0.91 (0.51) P = 0.53 Ratio

1 Mean (S.D.) 2 P-values from one-way ANOVA comparing anteroposterior (AP) % metachromasia ratios between gouging versus non-gouging species.

anterior translation on the glenoid, gouging at wide gapes likely results in a relatively unusual loading pattern in the TMJ where the posterior surface of the condyle and the anterior surface of the glenoid experience an increased frequency of loading compared to non-gouging primates. Based on these functional and behavioral scenarios, we predicted that the articular cartilage in the posterior condyle and anterior glenoid would be relatively larger, thicker and show a higher percentage of proteoglycans to provide increased load resistance abilities in gouging marmosets.

Our comparative analysis of articular cartilage morphology in gouging and non-gouging platyrrhines yielded mixed results. Marmosets demonstrated relatively larger and thicker articular cartilage in the anterior glenoid compared to non-gougers. The posterior condylar articular cartilage did not differ consistently between gouging and non-gouging species. The relative density of proteoglycans did not differ in either the condyle or glenoid cartilages.

As typical of many comparative studies, it is difficult to provide a single straightforward interpretation of this mixed pattern of results. In part, we lack sufficient comparative data from other species to assess whether these findings are characteristic of primates exhibiting similar variation in feeding behaviors.

Additionally, experimental studies have yielded a range of different, and sometimes conflicting, results when addressing how loading impacts TMJ articular cartilage form (e.g. Bouvier and Hylander, 1982; Ravosa et al., 2007).

We consider both of these issues in assessing how tree gouging impacts TMJ

articular cartilage. In the end, we acknowledge that the strength of our results will be documenting the interspecific variation in articular cartilage form and highlighting areas for future work studying the evolutionary morphology of TMJ articular cartilage in primates.

The Evolutionary Morphology of TMJ Articular Cartilage in Primates

In vivo studies demonstrate that the TMJ is a load-bearing joint with the largest loads often occurring during incision (Hylander, 1979a; Hylander and

Bays, 1979; Boyd et al., 1990). Numerous comparative analyses of TMJ skeletons across primates have correlated dietary and behavioral variation with the relative area, width and length of the condyle as well as anteroposterior (AP) glenoid length (Hylander, 1979b; Smith et al., 1983; Bouvier, 1986; Wall, 1999;

Williams et al., 2002; Vinyard et al., 2003; Taylor, 2005). As a relevant example, marmoset skulls possess relatively elongated condyles and glenoids compared to tamarins (Vinyard et al., 2003). There have been far fewer studies that consider morphological variation in TMJ articular cartilage of primates.

A significant percentage of the studies examining TMJ articular cartilage across primates assess a single species, such as macaques (Tong and

Tideman, 2001), baboons (Milam et al., 1991), or marmosets (Wilson and

Gardner, 1982; Robinson and Poswillo, 1994), often considering their potential as clinical models for studying human TMJs. There have been relatively few studies of articular cartilage growth with available studies documenting embryonic and/or postnatal ontogeny in macaques (Kanouse et al., 1969;

Carlson et al., 1978; Hinton and Carlson, 1983; Luder and Schroeder, 1990,

1992) and marmosets (Wilson and Gardner, 1982; Robinson and Poswillo,

1994). Burrows and Smith (2007) provide the only other interspecific analysis of articular cartilage in primates. They compare articular cartilage in Otolemur crassicaudatus and O. garnetti and generate hypotheses relating variation between species to differences in diet and joint loading. Quantitative data on articular cartilage morphology only exists for macaques (Carlson et al., 1978;

Bouvier and Hylander, 1982; Hinton and Carlson, 1983).

Given the paucity of descriptive studies of TMJ articular cartilage, we have limited opportunities to compare results from these four platyrrhines to other primates. The major qualitative differences observed between galago species by

Burrows and Smith (2007) involved relative cartilage thickness and cellularity between midline and lateral regions. We focused on variation along an anteroposterior (AP) axis making it difficult to compare results between these two studies.

We can compare quantitative measures of articular depth at homologous sites in these four platyrrhines to similar data collected on rhesus macaques

(Macaca mulatta) (Carlson et al., 1978; Hinton and Carlson, 1983). Adult rhesus macaques exhibit much larger condylar cartilage depths at anterior (0.26 mm) and posterior (0.25 mm) sites along the midline (Carlson et al., 1978; see also

Bouvier and Hylander, 1982) compared to the four platyrrhines (Table 2.4a).

This is a reasonable outcome given the larger size of macaques. With the

exception of the low anteroposterior (AP) condylar depth ratio in C. jacchus

(0.60), macaques maintain a similar anteroposterior ratio (1.05) to the remaining platyrrhines (Table 2.4a). When scaled relative to jaw length (80 mm for adult rhesus macaques; n=19), the relative anteroposterior depth in macaques

(0.0031) is similar to S. oedipus (0.0029) and Sa. sciureus (0.0031), but noticeably smaller than both C. jacchus (0.0063) and C. pygmaea (0.0052).

These results suggest a relative similarity among macaques, tamarins and squirrel monkeys and point to a potential increase in load resistance ability relative to an external load arm during incisal biting (i.e., jaw length) in the two marmosets.

The glenoid of rhesus macaques is absolutely thin anteriorly (0.077 mm) and intermediate posteriorly (0.184 mm) compared to the four platyrrhines (Table

1.4b). This translates into a much lower anteroposterior glenoid depth ratio

(0.42) in M. mulatta compared to New World monkeys (Table 2.4b). When scaled to jaw length, the relative anterior depth of macaques (0.00096) is markedly smaller than S. oedipus (0.0024), Sa. sciureus (0.0047), C. jacchus

(0.0051) and C. pygmaea (0.0073). Interestingly, marmosets exhibit the largest anterior depths relative to jaw length supporting the hypothesis tests comparing anteroposterior depth ratios. In sum, we see preliminary evidence suggesting a size-related decrease in relative articular depth across anthropoids coupled with a relative increase in load resistance ability in marmosets compared to other monkeys.

Despite these interesting preliminary comparisons, we still know very little about the evolutionary morphology of TMJ articular cartilage in primates. In fact, most of what we know comes from non-histological studies. In vivo experiments demonstrate that articular cartilage bears loads and comparative morphometrics of the underlying TMJ skeleton suggest that morphological variation relates to diet and function. Given that it has been at least 40 years since the earliest descriptions of primate articular cartilage (Kanouse et al., 1969) and this study nearly doubles the interspecific primate sample, future progress in understanding the evolutionary morphology of primate TMJ articular cartilage hinges on gathering additional data across primates.

Unraveling the Complexity of Articular Cartilage Function

The mosaic nature of our hypothesis test results lends itself to multiple interpretations. We can reasonably argue that the increased relative thickness and size of the anterior glenoid in tree gouging marmosets provides increased load-resisting ability compared to non-gouging platyrrhines. Although we cannot definitively identify where the glenoid is loaded during gouging at wide gapes, these results support the hypothesis that relative increases in anterior cartilage size and thickness are responses to repetitive gouging at wide gapes.

Relative to our hypothesis, we can interpret this similarity to indicate that

condylar articular cartilage is not loaded as we hypothesize, that the cartilage is overbuilt relative to the increased loading frequency experienced during gouging, and/or that the magnitude of loading in the posterior condyle during gouging is not sufficient to elicit a physiological or evolutionary response in these tissues.

Deciding among these various interpretations relies on incorporating our results with existing comparative and experimental evidence describing articular cartilage form and function. We have already noted the lack of comparative data from primates. Experimental studies on articular cartilage form and function also leave a number of questions that need to be addressed to further our understanding of articular cartilage function. In large part, these questions relate to identifying where loads are in the joint, quantifying their magnitude and frequency as well as how they impact cellular responses, and determining how cellular responses change with age.

There is no doubt the articular cartilage in the primate TMJ is load bearing

(Hylander, 1979a; Hylander and Bays, 1979; Boyd et al., 1990). It is, however, unclear as to the magnitude, location and distribution of these loads during various behaviors. Early strain gauge studies suggested reaction forces are unevenly distributed throughout the TMJ during chewing (Hylander and Bays,

1979). More recent work has used a combination of in vivo data and modeling to approximate stress concentrations within the joint and document how these stresses shift locations during chewing (Gallo et al., 2000; Gallo, 2005).

Furthermore, there is some relationship between condyle morphology and both

stress location and distribution (Colombo et al., 2008). These data, however, are only available for humans. It should be possible to conduct similar studies in non-human primates attempting a relatively straightforward kinematic behavior such as tree gouging. Evidence for an anteroposterior (AP) migration of stresses along the articular cartilage with changing jaw gape and a characterization of mediolateral (ML) location of these stresses would greatly benefit our ability to identify appropriate sites in the joint for histomorphological comparison.

A number of studies attempt to manipulate the loading environment in the joint, primarily through altering dietary properties, to study how cartilage responds to these changes (Bouvier and Hylander, 1982; Bouvier, 1987;

Sasaguri et al., 1998; Pirttiniemi et al., 1996; Huang et al., 2002; Ravosa et al.,

2007; Chen et al., 2008). Not unexpectedly, the results from these studies can vary markedly. While it is certainly possible that the articular cartilage may display multiple responses to alterations in loading, there are several potential influencing factors that are often unaccounted for in these studies. Foremost among these limitations is the failure to determine where peak loading is occurring. Without knowing where maximum stresses are, given that they can change with shifts in dietary properties, then it is nearly impossible to determine functionally homologous regions of cartilage for comparison (as noted here). We also know little about the thresholds for loading magnitudes and frequency that elicit physiological responses from chondrocytes (Ravosa et al., 2007). What is

clear is that articular cartilage responses can change over time and the extent of loading.

Finally, changes in articular cartilage throughout ontogeny also influence the ability of articular tissue to resist and respond to loading stresses. In macaques, the articular and growth (i.e., chondroblastic) layers of the condylar and glenoid cartilages change in absolute and relative thickness during ontogeny

(Carlson et al.,1978; Hinton and Carlson, 1983). Metric changes are mirrored by age-related changes in collagens, proteoglycans and chondrocytes (Luder and

Schroeder, 1990, 1992; Haskin et al., 1995; Klinge, 2001). These age-related changes impact how the zones of articular cartilage respond to loading stresses

(Hinton and McNamara, 1984).

Future studies, likely on model rodent species, are needed that identify how and where loads are changing with experimental manipulation followed by examining the time course of cartilage response throughout ontogeny.

Comparative studies can use these results as a baseline for interpreting morphological and histological variation in articular cartilage across primate

TMJs.

Limitations and Future Directions

It was impossible to control several factors known to influence articular cartilage morphology in this comparative study of primate cadavers. Thus, we suffer from several limitations typical of comparative work on non-laboratory animal models. Sample sizes were regrettably small for each species limiting our

statistical power. All of the animals used in this analysis were captive bred. We do not know how frequently these marmosets gouged in captivity (if at all).

Similarly, we had no control over diet and related physiological responses in the

TMJ to masticatory loading. Given the small sample sizes, we included a range of ages, despite known trends for age-related decreases in cartilage thickness, proteoglycans and cellularity (Haskin et al., 1995). We found no association between specimen age and our metric dimensions suggesting that age was not a major covariate in our analysis.

We identify several future analyses comparing articular cartilage in gouging and non-gouging primates that would build on the results presented here. First, it would be beneficial to explore articular cartilage regions outside of the midline. While we predicted major differences along an anteroposterior (AP) axis, we do not know where peak loads are located and may find different patterns at medial or lateral locations in the joint. Because metachromatic staining is an indirect measure of proteoglycan density, we need to employ immunohistochemical approaches to determine if the patterns observed here persist with a more direct assessment. We also would extend these immunohistochemical approaches to examine collagen content across the different zones of articular cartilage. Finally, we can improve our age control in comparing articular cartilages across species. Here we are aided by the natural history of callitrichids in that these species frequently triplet in captivity with one individual often stillborn. Examination of these perinatal tissues would allow us to

control loading and behavior at this specific age to provide insights into largely genetic differences established early in development between gouging and non- gouging species during callitrichid evolution.

Conclusions

Experimental in vivo evidence, behavioral field data and mechanical properties estimates from gouging trees collectively suggest that tree gouging in marmosets involves significant mandibular excursion but not necessarily relative large bite forces (Vinyard et al., 2009). Both mandible and jaw-muscle morphology in marmosets facilitate jaw opening compared to non-gouging tamarins (Vinyard et al., 2003; Taylor and Vinyard, 2004; Taylor et al., 2009).

Alternatively, mandible and jaw-muscle morphologies do not facilitate load resistance or force production compared to tamarins. Based on these comparative morphological patterns, we have concluded that the marmoset masticatory apparatus evolved to increase jaw opening linked to gouging performance (Vinyard et al., 2009).

While this overarching conclusion may accurately represent much of the masticatory apparatus, it is rare that an entire complex system is adapted to a single mechanical function. More reasonably, the morphology of the marmoset masticatory apparatus represents compromises among multiple demands placed on it during various feeding behaviors. Our results suggest that the articular cartilage of the anterior glenoid represents one of these compromise morphologies by providing increased load resistance abilities linked to novel

loading regimes in TMJ during gouging. The derived, wedge-like morphology of the anterior dentition of marmosets (Rosenberger, 1978) linked to improved cutting performance provides a second example of a morphological change unrelated to jaw excursion. Collectively, these morphologies highlight the complex functional challenges that tree gouging poses to marmosets and their masticatory apparatus.

CHAPTER III

ONTOGENTIC ALLOMETRY AND HETEROCHRONY OF CRANIAL FEATURES ASSOCIATED WITH WIDE JAW GAPES IN COMMON MARMOSETS (CALLITHRIX JACCHUS).

Introduction

Functional Morphology of Primate Cranial Form

There has long been a significant interest in the relationships among diet, feeding behaviors and functional morphology of the masticatory apparatus in primates. Several approaches, including experimental research (e.g. bone strain, kinematics and electromyography) and comparative morphological analysis have been utilized to identify and explain these associations.

Comparative morphological analysis of cranial form is one commonly used approach for the study of morphological adaptations to feeding behaviors.

Comparative analyses include both interspecific and intraspecific adult and ontogenetic assessments of form hypothesized to be functionally linked to specific feeding behaviors. Numerous interspecific and intraspecific examinations of primate functional morphology have been undertaken to relate cranial form to specific feeding behaviors and diets. Hypotheses concerning the relationship between primate mandibular form and function are often explored

using a combination of both experimental and comparative analyses (e.g.

Hylander, 1979, 1984; Ravosa, 1990, 1999; Daegling, 2003).

Comparative functional morphology of marmoset skulls has helped to identify morphological features related to the unusual tree gouging behavior practiced by marmosets. Marmosets are unique among callitrichids, in that they actively gouge trees with their anterior teeth to stimulate exudate flow (e.g.,

Kinzey et al., 1975; Coimbra-Filho and Mittermeier, 1977). Previous interspecific morphological analyses of adult samples have determined that common marmosets have a jaw form (Vinyard et al., 2003) and jaw-muscle architecture

(Taylor & Vinyard, 2004; Eng et al., 2009; Taylor et al., 2009) that facilitate gouging at large jaw gapes. It is not known, however, how features that facilitate gouging develop during marmoset growth. Anatomical comparisons between closely-related species that utilize either entirely different food sources or the same resources but in different ways are valuable for exploring questions of functional adaptation of the masticatory apparatus, separate from the influence of phylogeny (Fleagle, 1979; Anapol and Lee, 1994). Comparisons of the ontogeny of morphological features are also useful for understanding the developmental patterns that result in anatomical differences observed between adult specimens of different species. The aim of this analysis is to determine how the derived craniofacial morphology of adult marmosets arises throughout ontogeny.

Therefore, allometric and heterochronic analyses of callitrichid growth patterns

are considered here to determine size-related shape changes and the timing of development of cranial features associated with wide gape.

Why Study Ontogeny?

Ontogeny refers to the developmental history of an organism (Futuyma,

1998). Cock (1966) viewed this developmental history as a continuum of shapes as he argued that ignoring ontogeny in the analysis of shape was to view only a cross section of the development of the organism. Shea (1985) argues that ontogenetic allometry provides an approach to the problems of size adjustment and provides a method for reading the actions of natural selection by comparing the growth allometry patterns of closely-related species. Ontogenetic analysis of the functional morphology of primate cranial form provides a view of the developmental steps involved in achieving adult form. Not surprisingly, shapes observed during growth may not be readily anticipated from adult form.

Weaning is a time of potentially high selection pressure requiring functional competency for independent acquisition of food (e.g. Janson & van

Schaik, 1993; Kennedy, 2005). Therefore in primates, as in all mammals, the morphology of the masticatory apparatus must be functionally competent to ensure continued survival of the individual, as juvenile primates often ingest food similar to adults (Watts, 1985). Mammalian studies of the transition from suckling to independent food acquisition suggest that the majority of neural, behavioral and anatomic changes necessary for independent food acquisition and mastication achieve adult levels prior to weaning, yet the functional abilities

of weanlings lack adult-like proficiency (Herring, 1985; Westneat and Hall, 1992;

Huang et. al., 1994; Fragaszy and Adams-Curtis, 1997).

Independent food acquisition by juvenile marmosets requires a masticatory apparatus that is functionally capable of accessing resources such as exudates. The juvenile period is commonly considered to be the time between age at weaning and age at first reproduction (Janson & van Schaik,

1993). The juvenile period in marmosets begins at approximately four months of age with the end of weaning and continues to approximately one and a half years of age when marmosets achieve adult size and begin reproducing (Stevenson and Rylands, 1988; Yamamoto, 1993; Kendal, Coe and Laland, 2005; Marroig

2007). Therefore, ontogenetic changes in marmoset cranial morphology associated with tree gouging are expected to be early in ontogeny, and achieve adult size prior to the end of the juvenile stage. Ontogenetic analysis of tree- gouging marmosets can provide information about growth of the masticatory apparatus and the subsequent implications for gouging. Two common approaches utilized in the analysis of morphological changes that occur throughout ontogeny are allometry and heterochrony.

Allometry

Allometry is “the study of size and its consequences” (Gould, 1966:587).

In his definition of allometry Gould includes both allometry and isometry, as a more general description of scaling studies. Isometry is an indication of maintenance of shape with size increase (McKinney and McNamara, 1991).

Jungers et al. (1995), however, define allometry as scaling that is non-isometric indicating a size-correlated change in shape. McKinney and McNamara (1991) define allometry as the observed change in shape with changing size.

Deviations from the allometric trend line indicate a change in shape that is independent of change in size. Ultimately, shape changes that are independent of change in size may be an indication of adaptive processes (Huxley, 1932;

Gould, 1975; Cheverud, 1982).

The primary mathematical equation for the evaluation of the relationship between shape and size is the power function y=axb (Figure 3.1a) wherein y is a variable whose increase is considered relative to another variable x which commonly represents a measure of body size (Gould, 1966). The linear bivariate plot of this relationship utilizes the logarithmic transformation log y=log a + b log x. The slope of the plot, or ratio of the specific growth rates of y and x is represented by b, and a represents the value of y when x is equal to one.

Therefore, a value for b that is greater than one (when x and y are similar in dimensional rank) indicates a positive allometric relationship and differential increase of y relative to x. Alternately, if b is less than one, a negative allometric relationship is indicated with a decrease in the ratio of y relative to x accompanying an increase in x. Finally, two variables share an isometric relationship with a value of b that is equal to one, when measurements are at the same scale, indicating that the ratio of y to x maintains geometric similarity with size increase.

Three distinct types of allometry analysis are 1) static, 2) evolutionary and

3) ontogenetic allometry. Static or intraspecific adult allometry examines patterns of variation among individuals of the same growth stage within a population (Cock, 1966; Gould, 1966; Klingenberg and Zimmermann, 1992;

Klingenberg, 1998). A static allometric approach enables examination of individual variability while narrowing the influence of ontogenetic or evolutionary factors (Klingenberg, 1998). Evolutionary allometry utilizes a static allometric approach, involving a single growth stage, to determine the extent of character variation among individuals from several evolutionary lineages or species that share a common ancestor (Klingenberg and Zimmermann, 1992). This evolutionary approach attempts to discern phylogenetic transformations through the exploration of changes in size and/or shape that may have resulted from the actions of natural selection.

Ontogenetic or growth allometry examines the variation of features, within species, among multiple individuals recorded at different growth stages (cross- sectional data) or of individuals recorded throughout their growth trajectories

(longitudinal data) (Cock, 1966; Klingenberg and Zimmermann, 1992;

Klingenberg, 1998). Cross-sectional data provide a species average of ontogenetic allometry, whereas longitudinal data reveal individual variability of the ontogenetic trajectory (Klingenberg, 1998).

The aim of this analysis is to determine how the derived craniofacial morphology of adult marmosets arises throughout ontogeny. Ontogenetic

allometry enables an examination of morphological changes in marmoset cranial shape over a developmental growth trajectory to determine if changes observed in adult shape comparisons between gouging marmosets and non-gouging tamarins are present throughout their ontogeny. As modifications of ontogenetic trajectories are often correlated with shifts in phylogeny, the outcome of selection can often be seen via the interspecific comparison of growth allometries of closely-related species (Shea, 1985). Therefore, comparison of the growth allometries of tree-gouging marmosets and non-gouging tamarins can provide information concerning the functional development of the masticatory apparatus relative to gouging.

Ontogenetic Allometry of Primate Cranial Form as Related to Diet

Interspecific analyses of ontogenetic allometry have been utilized in numerous studies of primate cranial form to test predictions or develop hypotheses concerning the relationship between cranial form and diet.

Deviations from ontogenetic scaling of masticatory apparatus features among or between species are often interpreted to reflect differences in morphology linked to diet (Leigh, 2006). These types of comparisons have been used to link differences in masticatory apparatus morphology to hard-object feeding (Cole,

1992; Masterson, 1997; Leigh, 2006), folivory (Ravosa, 1998, 2007; Ravosa and

Ross, 1994; Ravosa and Daniel, 2010), variation in levels of folivory and frugivory (Taylor, 2002, 2005) and insectivory (Shea, 1992). Absent from all of

these analyses, however, is a method for determining the underlying variations of timing or rate of growth that lead to the observed ontogenetic trajectories.

Limitations of Ontogenetic Allometry

Ontogenetic allometry analysis fails to provide a means for identifying the specific underlying alterations in growth trajectories that lead to the changes in shape that are often observed in comparisons of adult morphologies. Shifts in rate and timing of growth can be extrapolated from comparisons of ontogenetic allometry, but are unverified in the absence of individual age data. Ultimately, allometry considers size and shape only and addresses time indirectly as it pertains to the assumption that older individuals will be larger than younger individuals (Klingenberg, 1998). The direct analysis of growth related to age, timing and rate requires the use of heterochrony techniques.

Heterochrony

Heterochrony is a change in the developmental timing of a character relative to its appearance or development in the ancestral form (De Beer, 1930;

Gould, 1977, 2000; Alberch et al., 1979; Alba, 2002; McNamara, 2002; Berge and Penin, 2004). Shea (1983) identifies heterochrony as shifts in either the rate or timing of developmental patterns in a descendent that result in evolutionary modifications. Therefore, with the explicit inclusion of the additional dimension of time, heterochrony permits the comparison of growth parameters such as onset, offset and rate of growth. Therefore, using heterochrony analysis, it is possible

to further investigate how results garnered via allometric comparisons may result from divergent patterns of growth.

The primary designations of heterochronic transformations are paedomorphosis and peramorphosis (Figure 3.1b). Paedomorphosis refers to a descendent whose later age stages retain characters of an earlier stage in the ancestral form. Paedomorphic characters may result from developmental changes in timing such as late onset (postdisplacement) or early offset

(progenesis) of growth, or from a decrease in the rate (neoteny) of growth.

Conversely, peramorphosis refers to a descendent character that goes beyond the stage observed in the ancestral condition. Peramorphic characters may result from developmental shifts in timing such as early onset (predisplacement) or late offset (hypermorphosis) of growth, or from an increase in the rate

(acceleration) of growth.

Heterochrony Analysis of Functional Craniofacial Form in Primates as Related to Diet

Fewer analyses of the heterochrony of primate functional craniofacial form exist, when compared to the number of studies utilizing ontogenetic allometry.

This relative lack is due to the fact that heterochrony analyses require accurate age data which are not usually available for wild or museum populations. Only a limited number of heterochronic studies of primate craniofacial morphology have been conducted with functional implications linked to diet.

Figure 3.1: Explanatory plots (a) allometry and (b) heterochrony.

Both interspecific and intraspecific analyses of primate craniofacial form have been pursued in an attempt to reveal the role of developmental timing as it impacts evolutionary change. These types of comparisons have been used to link differences in the rate or timing of growth of features of the masticatory apparatus to hard-object feeding (Cole, 1992), folivory (Ravosa, 1992; Ravosa and Ross, 1994; Ravosa et al., 1995), as well as an experimental analysis of the influence on growth rate and timing in a high versus low protein diet (Rozzi et al.,

2005).

Hypotheses and Predictions

Marmosets are unique among callitrichids, in that they actively gouge trees with their anterior teeth to stimulate exudate flow (e.g., Kinzey et al., 1975;

Coimbra-Filho and Mittermeier, 1977). Previous morphological analyses of skull form and jaw-muscle architecture have demonstrated features that facilitate gouging at wide gapes in adult common marmosets. It is not known, however, how features that facilitate gouging develop during marmoset ontogeny.

Allometric and heterochronic analyses of callitrichid growth patterns are used to determine shape changes and timing of the development of cranial features associated with wide gape.

Using an ontogenetic approach to compare patterns of relative craniofacial growth among species, we hypothesize that cranial features associated with wide gapes will be present early in ontogeny, as compared to non-gouging S. fuscicollis; given that juvenile and sub-adult marmosets gouge trees (Soini, 1988;

Stevenson and Rylands, 1988). Features hypothesized to be larger in marmosets are predicted to exhibit a higher slope value or be transposed above the same features in tamarins. The opposite is predicted for those features hypothesized to be smaller in marmosets than in tamarins. To establish whether gape-related traits are part of a pervasive change in craniofacial growth patterns in marmosets, we compared ontogenetic patterns of growth in features associated with load resistance between marmosets and tamarins. No predicted change is given in those features associated with load resistance as gouging does not appear to involve relatively large bite forces (Vinyard et al., 2009).

Utilizing heterochrony analysis, we also examine variation in the timing of shape changes in craniofacial features during ontogeny in C. jacchus as compared to the closely-related S. fuscicollis. Because C. jacchus possess a specialized skull morphology to gouge trees at wide gapes, we hypothesize that cranial features associated with wide gapes that are larger in marmosets will be peramorphic and display earlier onset in growth or a faster rate of growth in order to achieve functional competency for gouging at the time of weaning. Similarly, the traits that are predicted to be smaller will be paedomorphic and display delayed onset or a slower rate of growth. Cranial features associated with load resistance are also analyzed to determine whether marmosets show a pervasive pattern of growth in their skull form relative to tamarins.

Material and Methods

Samples

This study was conducted using cross-sectional data from an ontogenetic skull series of captive common marmosets (Callithrix jacchus) and saddle- backed tamarins (Saguinus fuscicollis). The skulls were from individuals originally housed at Oak Ridge Associated Universities’ Colony. Specimen ages were taken from collection records and nearly evenly distributed from neonates through adults for C. jacchus (~320g) (N=129) and S. fuscicollis (~350g) (N=135)

(Fleagle, 1999). Specimens chosen for analysis displayed no observable pathologies. Condyle height measurement was performed on a truncated portion of the overall sample (C. jacchus N=68, S. fuscicollis N=54).

Cranial Measurements

The measurements used for both the analysis of allometry and heterochrony include four cranial dimensions associated with wide gape (Vinyard et al., 2003) and three cranial dimensions related to load resistance (Hylander,

1988). Cranial dimensions were measured to the nearest 0.01 mm using digital calipers (Fowler UltraCal III). All measurements, excluding condyle height, were taken on dry skulls. Condyle height was measured from lateral-view digital images of dry skulls with SigmaScan Pro 5 software.

The four cranial dimensions associated with wide gapes are condyle length, condyle height above the alveolar plane, glenoid length and anterioposterior (AP) mandible length (Figures 3.2a-d). Condyle length is a

b a

c d

Figure 3.2: Measurements of gape-related features including (a) AP condyle length, (b) condyle height, (c) AP glenoid length and (d) AP mandible length.

measurement of the AP length of the condyle articular surface. Condyle height measures the vertical and perpendicular distance between a line drawn along the alveolar surface of the mandible and extending to the superior articular surface of the mandibular condyle. Glenoid length describes the maximum AP length of the glenoid fossa from the base of the postglenoid process to the anterior limit of the articular surface. AP mandible length is a measurement of the distance from infradentale to the posterior margin of the ramus, parallel to the occlusal plane

(Vinyard, 1999; Vinyard et al., 2003).

The three load-resistance related features recorded for this study include

M1 depth, M1 width and symphysis length (Figures 3.3 a-c). M1 depth is a measure of the superioinferior depth of the mandibular corpus at M1. M1 width describes the mediolateral measurement of the mandibular corpus at M1.

Symphysis length is the distance between the incisor alveolus and the inferior limit of the symphysis (Hylander, 1985; Vinyard et al., 2003).

Jaw length is used in this analysis as a mechanically relevant size estimate for both jaw gape, as a component of linear gape (Smith, 1984), and skull loading, as an estimate of load arm for incisive biting (Hylander, 1988)

(Figure 3.4). This combined functional relevance makes jaw length an appropriate scaling measurement for all aspects of this analysis. Jaw length is defined as the distance between infradentale and the posterior extent of the condyle (Vinyard, 1999; Vinyard et al., 2003).

Figure 3.3: Measurements of load resistance-related features including (a) M1 depth, (b) M1 width and (c) symphysis length.

Figure 3.4: Measurement of jaw length.

Allometry and Heterochrony Data Collection

We use cross-sectional data to model an entire growth curve with measurements of different individuals of varying ages. The assumption of cross- sectional data is that the individuals in the sample are following the same basic growth trajectory and therefore sample estimates represent the average of the population (Kaufmann, 1981).

Allometry Data Analysis

We calculated reduced major axis (RMA) and least squares (LS) regressions for each cranial feature within species and then compared growth trajectories between species. Significant differences between slopes and/or transpositions were calculated for RMA results with (S)MATER, Version 1.0 software (Falster et al., 2003). Least squares regression results were compared using ANCOVA. Results for both RMA and LS regressions are reported for this analysis. However, RMA regression is likely the more appropriate analysis for this data as the possibility of measurement error is present in both the x and y variables (Smith, 2009). Therefore, precedence is given to results of the RMA analyses throughout the text. As stated previously, cranial dimensions were regressed upon jaw length as a mechanically relevant size estimate for both gape and skull loading measurements.

Heterochrony Data Analysis

The Gompertz curve was used to model growth in this analysis of craniofacial dimensions as mammalian growth is non-linear (Kaufmann, 1981;

German et al., 1994). Linear models are not suited to mammalian growth as they require a constant rate of growth and do not reach an asymptote or the point at which the absolute value of growth is maximized. The Gompertz curve is sigmoidal in shape and is used to mathematically model a time series where growth is slowest at the beginning and the end of the series. This model can be used to calculate changes in growth rate over time, as well as initial and final size of each cranial feature. The growth of each cranial feature was characterized using a sigmoidal Gompertz equation (Reichling and German, 2000):

-be-kt Y=Ae (1) where Y is the craniofacial measurement, t represents age at death (in days), k reflects the rate of exponential growth decay, b represents the initial growth trajectory impacting the delay in the start of the spurt of growth and the time when the curve reaches its asymptote. The variable A is the final size achieved for the feature in the growth trajectory. We also calculated w, the initial size of Y at t = 0 (i.e., birth), and I, the initial growth rate at t = 0 based on the following equations (Reichling and German, 2000):

-b w=Ae (2) I=bk (3)

WALD confidence intervals were calculated for each growth parameter (A, K, W,

I) using SYSTAT 11. Significance between species in each growth parameter is

determined with calculated WALD confidence intervals for each growth parameter and assigning significance for those intervals that do not overlap

(Moscarella et al., 2001).

Results

Allometry Gape-Related Results

Two gape-related features, condyle length and AP mandible length, reveal primarily a negatively allometric relationship relative to jaw length in marmosets and tamarins. Glenoid length, however, did demonstrate a positive allometric association in C. jacchus and a nearly isometric scaling pattern in S. fuscicollis.

Both LS and RMA regression of gape-related features indicate that marmosets possess a significantly higher slope or are significantly transposed above tamarins. The only exception is the regression for condyle height which shows

S. fuscicollis to be significantly transposed above C. jacchus.

Regression analysis of AP condyle lengths in both C. jacchus (RMA slope

= 0.85, LS slope = 0.59) and S. fuscicollis (RMA slope = 0.80, LS slope = 0.29) indicate negative allometry relative to jaw length (Table 3.1a, Figure 3.5a).

According to RMA regressions, C. jacchus is significantly transposed (p<0.001) above S. fuscicollis for this dimension. The LS regression indicates that the slope for C. jacchus is significantly higher than S. fuscicollis (ANCOVA, p<0.005).

Ultimately, the AP condyle lengths of C. jacchus appear to be relatively larger for a given jaw length throughout ontogeny when compared to S. fuscicollis.

Table 3.1: Allometry results for Reduced Major Axis and Least Squares Comparisons for Slope Difference and Transposition of (a) Gape-Related Features and (b) Load Resistance Related Features of both Marmosets and Tamarins. a ANCOVA ANCOVA RMA Test Least Test for Test for Reduced Major for Common RMA Test for Squares Common Transpositio Axis Slope Slope Tranposition Slope Slope n Condyle Length 0.85 (0.76- 0.59 (0.49- C.jacchus 0.95)* p<0.454 p<0.000 0.68)* p<0.000 n/a 0.80 (0.70- C. jacchus 0.29 (0.18- S. fuscicollis 0.91) above 0.39) Condyle Height 3.68 (3.33- 3.36 (2.99- C.jacchus 4.08) p<0.578 p<0.002 3.74) p<0.182 p<0.003 3.51 (3.01- S. fuscicollis 2.93 (2.40- S. fuscicollis S. fuscicollis 4.09) above 3.47) above Glenoid Length 1.16 (1.07- 0.96 (0.87- C.jacchus 1.26) p<0.005 n/a 1.06) p<0.031 n/a 0.99 (0.91- 0.82 (0.74- S. fuscicollis 1.07) 0.90) AP Mandible Length 0.95 (0.94- 0.95 (0.93- C.jacchus 0.97) p<0.890 p<0.000 0.96) p<0.531 p<0.000 0.95 (0.93- C. jacchus 0.94 (0.92- C. jacchus S. fuscicollis 0.97) above 0.96) above * 95% confidence intervals b ANCOVA Reduced RMA Test Least Test for ANCOVA Test Major Axis for Common RMA Test for Squares Common for Slope Slope Tranposition Slope Slope Transposition M1 Depth 1.10 (1.06- 1.06 (1.02- C.jacchus 1.14)* p<0.286 p<0.000 1.11)* p<0.016 n/a 1.06 (1.01- S. fuscicollis 0.98 (0.92- S. fuscicollis 1.12) above 1.03) M1 Width 0.63 (0.55- 0.30 (0.22- C.jacchus 0.71) p<0.045 n/a 0.38) p<0.100 p<0.000 0.75 (0.66- 0.40 (0.31- S. fuscicollis S. fuscicollis 0.84) 0.49) above Symphysis Length 1.12 (1.08- 1.08 (1.03- C.jacchus 1.17) p<0.001 n/a 1.12) p<0.021 n/a 1.30 (1.23- 1.18 (1.10- S. fuscicollis 1.38) 1.26) * 95% confidence intervals

a c

b d

Figure 3.5: Allometric plots of ontogenetic series of marmosets and tamarins for cranial features associated with wide gape versus jaw length including (a) AP condyle length, (b) condyle height, (c) AP glenoid length and, (d) AP mandible length.

Regression analysis of condyle height above the tooth row in both C. jacchus (RMA slope = 3.68, LS slope = 3.36) and S. fuscicollis (RMA slope =

3.51, LS slope = 2.93) indicate positive allometry relative to jaw length (Table

3.1a, Figure 3.5b). The C. jacchus growth trajectory is transposed below that of

S. fuscicollis, according to comparisons of RMA regressions (p<0.002). Slope transposition of C. jacchus below that of S. fuscicollis is also indicated in the

ANCOVA (p<0.003). C. jacchus maintains a relatively lower condyle height above the tooth row during ontogeny compared to S. fuscicollis.

Regression slopes for AP glenoid lengths differ in their allometric relationships according to the method of calculating the slope (Table 3.1a, Figure

3.5c). LS regression for C. jacchus (LS slope= 0.96) is slightly negatively allometric, but confidence intervals also point to the possibility of an isometric, or slightly positively allometric, relationship between AP glenoid length and jaw length. RMA regression of AP glenoid length for C. jacchus (RMA slope = 1.16), however, indicates positive allometry. S. fuscicollis displays equally variable results with LS regression (LS slope = 0.82) indicating a negatively allometric relationship, and RMA regression (RMA slope = 0.99) indicating isometry with confidence intervals that include positive and negative allometry as well.

Comparison of RMA regressions demonstrates that the slope for C. jacchus is significantly higher than S. fuscicollis (p<0.006) for relative AP glenoid length.

Similarly, LS ANCOVA indicates that the slope for C. jacchus is significantly transposed above that for S. fuscicollis (ANCOVA, p<0.001). Overall, C. jacchus

AP glenoid length appears to increase more rapidly for a given jaw length throughout ontogeny, when compared to S. fuscicollis.

Regression slopes for AP mandible lengths of C. jacchus (RMA slope =

0.95, LS slope= 0.95) and S. fuscicollis (RMA slope = 0.95, LS slope = 0.94) both exhibit negative allometry relative to jaw length (Table 3.1a, Figure 3.5d). RMA regression comparison indicates that the C. jacchus growth trajectory is transposed above that of S. fuscicollis (p<0.001). The LS ANCOVA presented the same transposition relationship between the two species (p<0.001). AP mandible length in C. jacchus is relatively elongated throughout ontogeny when compared to S. fuscicollis.

Allometry of Load Resistance Related Features

Allometric analysis of features associated with load resistance demonstrates multiple allometric relationships for the regression of these features on jaw length for both marmosets and tamarins. Both RMA and LS regression indicate that marmosets possess a significantly lower slope for load resistance related features or are significantly transposed below the tamarin slope.

Regression analysis of M1 depth in both C. jacchus (RMA slope = 1.10, LS slope = 1.06) and S. fuscicollis (RMA slope = 1.06, LS slope = 0.98) indicate a scaling pattern of slight positive allometry, while still including isometry in some instances (Table 3.1b, Figure 3.6a). According to RMA comparisons, C. jacchus is significantly transposed below S. fuscicollis (p<0.001). LS ANCOVA indicates

a b

Figure 3.6: Allometric plots of ontogenetic series of marmosets and tamarins for cranial features associated with load resistance versus jaw length including (a) M1 depth, (b) M1 width and (c) symphysis length.

that the slope for C. jacchus is significantly lower than S. fuscicollis (p<0.016). In comparison to C. jacchus, saddle-back tamarins maintain a relatively deeper corpus throughout ontogeny.

Regression analysis of M1 width in both C. jacchus (RMA slope = 0.63, LS slope = 0.30) and S. fuscicollis (RMA slope = 0.75, LS slope = 0.40) indicate negative allometry of corpus width relative to jaw length (Table 3.1b, Figure

3.6b). The C. jacchus slope is significantly lower than that of S. fuscicollis according to RMA regression comparisons (p<0.045). However, the LS

ANCOVA indicates a slope transposition of C. jacchus below that of S. fuscicollis

(p<0.001). C. jacchus maintains a relatively narrower corpus width during ontogeny as compared to S. fuscicollis.

Regression slopes for symphysis length of C. jacchus (LS slope= 1.08,

RMA slope = 1.12) and S. fuscicollis (LS slope = 1.18, RMA slope = 1.30) both indicate positive allometry relative to jaw length (Table 3.1b, Figure 3.6c). RMA regression comparisons establish a significantly higher slope for S. fuscicollis symphysis length compared to C. jacchus (p<0.001). ANCOVA also indicates a comparable significant difference between slopes for C. jacchus and S. fuscicollis

(ANCOVA, p<0.021). Overall, relative symphysis length in C. jacchus does not increase as rapidly throughout ontogeny as observed for S. fuscicollis.

Heterochrony of Gape-related Features

Results for the analysis of heterochrony indicate a significant increase in the asymptote or final size (A) for C. jacchus in three of the four gape-related

features, as compared to S. fuscicollis. Specifically, a significant increase in asymptote for C. jacchus was observed for AP condyle length, AP glenoid length and AP mandible length. The calculated asymptote for condyle height, however, was larger in S. fuscicollis than in C. jacchus. This significant increase in final size for three of the four gape-related features lends support to the hypothesis of peramorphosis for these relatively larger cranial features in C. jacchus as compared to S. fuscicollis. Condyle height, however, appears to be paedomorphic for C. jacchus relative to S. fuscicollis.

Adult AP condyle length in C. jacchus is significantly larger (A) compared to S. fuscicollis (Table 3.2a, Figure 3.7a). Initial growth rate (I) is increased to an extent that approaches significance in C. jacchus and the initial growth trajectory

(b) is significantly lower for C. jacchus indicating that marmosets begin their growth spurt and reach the asymptote earlier than observed in tamarins. Values for both initial size (w) and the rate of growth decay (k) are elevated in S. fuscicollis as compared to C. jacchus. These results indicate that marmosets accelerate early growth in order to achieve their elongated glenoid.

The asymptote for condyle height is larger for S. fuscicollis than for C. jacchus, but not significantly (Table 3.2b, Figure 3.7b). Initial size (w) and the rate of growth decay (k) are elevated in S. fuscicollis while the initial rate of growth (I) observed for C. jacchus condyle height is increased as compared to S. fuscicollis. Initial growth trajectory (b) is elevated in S. fuscicollis indicating a longer period of time before the growth spurt is initiated and before final size in

Table 3.2: Growth Curve Parameters for Gape-Related Features including (a) AP Condyle Length, (b) Condyle Height, (c) AP Glenoid Length, and (d) AP Mandible Length a Parameter AP Condyle Length Species Value Lower CI Upper CI Final Size (A) C. jacchus 2.02310 1.97583 2.07051 S. fuscicollis 1.70900 1.65106 1.76696 Rate of Growth Decay (k) C. jacchus -0.00260 -0.00379 -0.00137 S. fuscicollis -0.00120 -0.00254 -0.00016 Initial Size at t=0 (w) C. jacchus 1.46494 1.34237 1.58751 S. fuscicollis 1.48588 1.40675 1.56500 Initial Growth Rate at t=0 (I) C. jacchus 0.00080 0.00030 0.00137 S. fuscicollis 0.00020 -0.00004 0.00037 Initial growth trajectory (b) C. jacchus -0.32284 -0.40561 -0.24007 S. fuscicollis -0.13991 -0.19509 -0.08473 c

Parameter Condyle Height Species Value Lower CI Upper CI Final Size (A) C. jacchus 5.25190 4.77391 5.72998 S. fuscicollis 5.77763 5.36451 6.19076 Rate of Growth Decay (k) C. jacchus -0.00490 -0.00707 -0.00291 S. fuscicollis -0.00503 -0.00769 -0.00239 Initial Size at t=0 (w) C. jacchus 1.32390 0.72930 1.91856 S. fuscicollis 1.65971 1.08929 2.23015 Initial Growth Rate at t=0 (I) C. jacchus 0.00688 0.00241 0.01135 S. fuscicollis 0.00628 0.00165 0.01092 Initial growth trajectory (b) C. jacchus -1.37799 -1.82039 -0.93560 S. fuscicollis -1.24735 -1.57845 -0.91625

Table 3.2 (Continued) c

Parameter AP Glenoid Length Species Value Lower CI Upper CI Final Size (A) C. jacchus 5.73920 5.64667 5.83174 S. fuscicollis 4.95986 4.89140 5.02834 Rate of Growth Decay (k) C. jacchus -0.00375 -0.00503 -0.00249 S. fuscicollis -0.00549 -0.00806 -0.00293 Initial Size at t=0 (w) C. jacchus 3.66976 3.21249 4.12704 S. fuscicollis 2.95403 2.55709 3.35099 Initial Growth Rate at t=0 (I) C. jacchus 0.00167 0.00071 0.00265 S. fuscicollis 0.00284 0.00085 0.00484 Initial growth trajectory (b) C. jacchus -0.44719 -0.57004 -0.32435 S. fuscicollis -0.51821 -0.65135 -0.38506 d

Parameter AP Mandible Length Species Value Lower CI Upper CI Final Size (A) C. jacchus 28.23699 27.91201 28.56198 S. fuscicollis 27.21166 26.99975 27.42359 Rate of Growth Decay (k) C. jacchus -0.00520 -0.00592 -0.00448 S. fuscicollis -0.00448 -0.00529 -0.00367 Initial Size at t=0 (w) C. jacchus 14.24076 13.43860 15.04294 S. fuscicollis 15.32504 14.63169 16.01841 Initial Growth Rate at t=0 (I) C. jacchus 0.00356 0.00288 0.00424 S. fuscicollis 0.00257 0.00195 0.00320 Initial growth trajectory (b) C. jacchus -0.68452 -0.74108 -0.62797 S. fuscicollis -0.57416 -0.61910 -0.52921

(Figure Continues)

Figure 3.7: Growth curves for gape-related features of ontogenetic series of marmosets and tamarins including (a) AP condyle length, (b) condyle height, (c) AP glenoid length and (d) AP mandible length.

achieved. Despite accelerated initial growth in marmosets as observed for AP condyle length a smaller final condyle height is achieved when compared to tamarins.

Adult AP glenoid length in C. jacchus is significantly larger (A) compared to S. fuscicollis (Table 3.2c, Figure 3.7c). Initial size (w) is very nearly significantly larger in C. jacchus, but values for both initial growth rate (I) and the rate of growth decay (k) are elevated in S. fuscicollis. Initial growth trajectory (b) is higher in marmosets indicating a delayed initiation of the growth spurt and an extension of the time period prior to reaching the asymptote. These results indicate that marmosets extend their growth period in order to achieve their elongated glenoid.

The AP mandible lengths of C. jacchus reach a significantly larger final size (A) compared to S. fuscicollis (Table 3.2d, Figure 3.7d). Initial AP mandible length (w) for S. fuscicollis is larger, but the rates for both initial growth (I) and growth decay (k) are elevated in C. jacchus. Initial growth trajectory (b) is significantly larger in S. fuscicollis indicating that C. jacchus is initiating an earlier growth spurt, as well as reaching final size sooner. These results point toward marmosets accelerating initial growth rate and trajectory within a truncated growth period to achieve their AP elongated mandibles.

Heterochrony of Load-Resistance Related Features

Growth patterns for load-resistance features diverge from the largely peramorphic pattern observed in growth patterns for gape-related features.

Overall, the cranial features associated with load resistance in C. jacchus appear paedomorphic relative to S. fuscicollis. These results suggest that there is not a uniform underlying growth pattern that explains the species differences in these features.

The asymptote (A) for M1 depth is significantly smaller for C. jacchus as compared to S. fuscicollis (Table 3.3a, Figure 3.8a). C. jacchus M1 depth is somewhat reduced in initial (w) size as well as slower in the initial rate of growth

(I), relative to S. fuscicollis. The rate of growth decay (k) and the initial growth trajectory (b) for C. jacchus, however, are slightly faster than for S. fuscicollis.

These results indicate that marmoset corpus depth is not growing as early, quickly or as long as in tamarins.

The asymptote (A) for the width of the corpus at M1 in C. jacchus is slightly larger than in S. fuscicollis (Table 3.3b, Figure 3.8b). The initial growth trajectory

(b) is elevated in marmosets indicating tamarins begin growth and reach adult size later than marmosets. Initial growth rate (I) and the rate of growth decay (k) are, however, significantly larger in S. fuscicollis with the addition of a larger initial size (w) that approaches significance. These results suggest that corpus width in marmosets grows more slowly and continues later than in tamarins.

Adult symphysis length (A) in C. jacchus is significantly shorter than in S. fuscicollis (Table 3.3c, Figure 3.8c). Although C. jacchus displays a more rapid initial growth rate (I), the rate of growth decay (k), initial growth trajectory (b) and initial length (w) are decreased in C. jacchus as compared to S. fuscicollis.

Table 3.3: Growth Curve Parameters for Load Resistance Related Features including (a) M1 Depth, (b) M1 Width, and (c) Symphysis Length a Parameter M1 Depth Species Value Lower CI Upper CI Final Size (A) C. jacchus 5.62851 5.53848 5.71856 S. fuscicollis 5.81697 5.73260 5.90136 Rate of Growth Decay (k) C. jacchus -0.00620 -0.00734 -0.00507 S. fuscicollis -0.00736 -0.00933 -0.00539 Initial Size at t=0 (w) C. jacchus 2.51901 2.29781 2.74022 S. fuscicollis 2.80272 2.49165 3.11380 Initial Growth Rate at t=0 (I) C. jacchus 0.00498 0.00373 0.00625 S. fuscicollis 0.00537 0.00326 0.00749 Initial growth trajectory (b) C. jacchus -0.80397 -0.89192 -0.71603 S. fuscicollis -0.73019 -0.84126 -0.61912 b Parameter M1 Width Species Value Lower CI Upper CI Final Size (A) C. jacchus 2.61427 2.17402 3.05454 S. fuscicollis 2.43934 2.39714 2.48155 Rate of Growth Decay (k) C. jacchus -0.00042 -0.00080 -0.00005 S. fuscicollis -0.01802 -0.02545 -0.01060 Initial Size at t=0 (w) C. jacchus 1.81829 1.74058 1.89602 S. fuscicollis 1.59398 1.40073 1.78724 C. jacchus Initial Growth Rate at t=0 0.00015 0.00007 0.00024 (I) S. fuscicollis 0.00766 0.00293 0.01240 Initial growth trajectory (b) C. jacchus -0.36309 -0.51762 -0.20855 S. fuscicollis -0.42549 -0.54718 -0.30380 c Parameter Symphysis Length Species Value Lower CI Upper CI Final Size (A) C. jacchus 8.35183 8.19869 8.50498 S. fuscicollis 9.01589 8.87012 9.16167 Rate of Growth Decay (k) C. jacchus -0.00640 -0.00778 -0.00504 S. fuscicollis -0.00586 -0.00764 -0.00410 Initial Size at t=0 (w) C. jacchus 3.74003 3.36047 4.11961 S. fuscicollis 4.13921 3.64586 4.63256 C. jacchus Initial Growth Rate at t=0 0.00514 0.00364 0.00666 (I) S. fuscicollis 0.00456 0.00262 0.00652 C. jacchus Initial growth trajectory -0.80339 -0.90499 -0.70178 (b) S. fuscicollis -0.77848 -0.89737 -0.65960

(Figure Continues)

Figure 3.8: Growth curves for load resistance related features of ontogenetic series of marmosets and tamarins including (a) M1 depth, (b) M1 width and, (c) symphysis length.

These results suggest that a shorter adult symphysis length in marmosets is due primarily to a shortened period of growth.

Discussion

Allometry and Heterochrony of Gape-Related Features in Marmosets

Allometric and heterochronic analyses of callitrichid growth patterns indicate that cranial features associated with wide gapes exhibit changes in size- related shape and developmental timing consistent with increased jaw opening ability throughout postnatal ontogeny compared to non-gouging tamarins.

Previous analyses have demonstrated that the adult jaw form (Vinyard et al.,

2003) and jaw-muscle architecture of common marmosets (Taylor and Vinyard,

2004; Taylor et al., 2009) facilitate the production of relatively large jaw gapes.

The results of this current analysis are consistent with the hypothesis that marmosets exhibit morphological adaptations for tree gouging at large jaw gapes early in ontogeny. In addition, allometric and heterochronic analysis of features associated with gape-related features also demonstrates that marmosets and tamarins exhibit mosaic patterns of growth throughout their skulls during ontogeny.

Marmoset anteroposterior (AP) condyle length exhibits a negatively allometric relationship relative to jaw length. More significantly, allometric analysis indicates that the marmoset growth trajectory is significantly transposed above the tamarin trajectory for this feature. This transposition indicates that for a given jaw length, AP condyle length is longer in marmosets compared to

tamarins throughout ontogeny. Heterochronic analysis suggests that the increased relative length of this feature results from an earlier onset of growth and more rapid initial rate of growth in combination with a later offset of growth relative to S. fuscicollis. The higher initial growth rate likely reflects the continuation of an accelerated embryonic growth period. This more rapid embryonic growth rate is manifest as a predisplacement in the heterochronic assessment of postnatal ontogeny. The early onset and late offset of growth suggest that time hypermorphosis may also play a role in the relatively increased adult length of the marmoset condyle. The observed allometric pattern for AP condyle length in marmosets is likely related to a peramorphic pattern of growth in marmosets. This pattern follows the prediction that marmosets will achieve a relatively elongated condyle earlier in ontogeny to promote functional competency for gouging at wide gapes around the time of weaning.

Condyle height above the tooth row likely exhibits a negatively allometric relationship for both C. jacchus and S. fuscicollis throughout ontogeny. C. jacchus condyle height, however, is significant in its transposition below that of S. fuscicollis condyle height during growth. Heterochrony analysis suggests a slightly accelerated initial rate of growth for marmosets, however, the increased rate of growth decay observed for C. jacchus denotes that the rate of growth for the remainder of ontogeny will decrease more rapidly leading to the attainment of adult size occurring earlier in C. jacchus than observed in S. fuscicollis.

Therefore, condyle height in S. fuscicollis achieves a larger final adult size likely

due to larger initial condyle height in combination with a later offset of growth.

This observed downward transposition of C. jacchus below S. fuscicollis, for condyle height above to tooth row, likely results from a paedomorphic pattern of growth for this feature in marmosets due to postdisplacement.

Allometric analysis of AP glenoid length indicates a significant slope difference between marmosets and tamarins for this wide gape-related feature.

AP glenoid length is positively allometric in C. jacchus and approximately isometric in S. fuscicollis relative to jaw length. Heterochronic analysis suggests that C. jacchus achieves its significantly larger adult size primarily as a result of an increased initial size likely due to an accelerated embryonic rate of growth when compared to S. fuscicollis. The increase in final adult size for AP glenoid length in marmosets as compared to tamarins is achieved despite an increased rate of growth decay and decreased initial rate of growth observed for C. jacchus.

The significantly higher slope observed for marmosets combined with the achievement of a significantly larger final adult size suggests a peramorphic pattern of growth by acceleration likely due to the extension of a relatively higher embryonic growth trajectory.

AP mandible length exhibits a negatively allometric relationship relative to jaw length in both marmosets and tamarins. However, comparison of allometric patterns indicates that the C. jacchus growth trajectory is significantly transposed above S. fuscicollis for this feature. Heterochronic analysis suggests that the relative increase in this gape-related feature results from a more rapid initial

growth rate as well as a later offset of growth relative to S. fuscicollis. The higher initial growth rate likely represents the final phase of an accelerated embryonic growth period. This more rapid embryonic growth rate is manifest as a predisplacement in the heterochronic assessment of postnatal ontogeny. The late offset of growth observed for C. jacchus suggest that time hypermorphosis may also play a role in the relatively larger adult shapes observed for this feature.

The observed allometric pattern for AP mandible length in marmosets is likely related to a peramorphic pattern of growth in marmosets. This pattern follows the prediction that marmosets will achieve relatively elongated mandibles earlier in ontogeny to promote functional competency for gouging at wide gapes around the time of weaning.

Cole (1992) has suggested that when comparing allometric patterns of growth a difference in slope implies a postnatal change in growth trajectory whereas a transposition suggests the continuation of a prenatally established trajectory of growth. Gape-related AP condyle length and AP mandible length both demonstrate transposition of marmosets over tamarins suggesting an extension of an accelerated prenatal growth trajectory. Marmoset AP glenoid length, however, suggests a postnatal difference in growth trajectory with a higher slope observed in marmosets compared to tamarins. Condyle height in marmosets is transposed below tamarins suggesting extension of a relatively decreased prenatal growth trajectory.

Allometry and Heterochrony of Load Resistance-Related Features in Marmosets

Allometric analysis of features associated with load resistance demonstrates that marmosets and tamarins do not exhibit a distinct, pervasive pattern of cranial growth allometry as compared to tamarins. The width of the corpus at M1 demonstrates negative allometry for both species; however, the slope for C. jacchus is shown to be significantly lower than the slope for S. fuscicollis. Heterochrony analysis suggests that the smaller final adult size for M1 corpus width in C. jacchus is paedomorphic by way of neoteny as compared to corpus width in S. fuscicollis. The smaller adult dimensions observed for corpus width in C. jacchus likely reflect a more shallow growth curve due to a significantly higher rate of growth decay combined with a significantly smaller initial size. S. fuscicollis likely achieves an increased final adult size as a result of both a larger initial size and relatively increased initial growth rate.

The depth of the corpus at M1 exhibits a positively allometric relationship relative to jaw length in marmosets. More significantly, the comparative allometric analysis indicates that the marmoset growth trajectory is significantly transposed below the observed tamarin trajectory. This transposition indicates that for a given jaw length, the marmoset corpus is more shallow than in tamarins. Heterochrony analysis suggests this feature is paedomorphic by way of postdisplacement in C. jacchus. The shallower adult corpus depth observed for C. jacchus likely results from a smaller initial size for this feature combined with a decreased initial growth rate. A somewhat higher rate of growth decay

also contributes to a decrease in marmoset adult corpus depth as the observed rate of growth in C. jacchus appears to decrease more rapidly than for S. fuscicollis.

Marmoset symphysis length likely exhibits a positively allometric relationship relative to jaw length. More notably, comparison of allometric patterns indicates that the slope for the marmoset growth trajectory is significantly lower than the tamarin trajectory. This slope difference indicates that for a given jaw length, symphysis length is shorter in marmosets compared to tamarins. Heterochronic analysis suggests that the relative decrease in this load resistance feature results from a paedomorphic pattern of growth via neoteny relative to S. fuscicollis. C. jacchus achieves a significantly smaller final adult symphysis length despite a higher initial rate of growth and a slower rate of growth decrease throughout ontogeny. The smaller initial symphysis length in marmosets is likely the result of a relative decrease in the rate of embryonic growth that may have established the postnatal slope differences for this feature.

The early offset of growth observed for C. jacchus suggest that time hypomorphosis may also play a role in the relatively smaller adult marmoset shape for this feature.

M1 width and symphysis length both display a lower allometric slope during growth for marmosets compared to tamarins. These observations suggest a potential shift in the postnatal timing of the growth trajectory between the two species. Conversely, M1 depth allometry indicates extension of a

prenatal growth difference in the transposition of the marmoset allometric growth pattern below that observed for tamarins. Therefore, comparison of allometric patterns of growth between marmosets and tamarins do not exhibit any single, consistent pattern between either prenatal or postnatal growth patterns that would differentiate gape-related and load resistance-related features for these two species. These findings suggest a potential for mosaic patterns of growth within the callitrichid skull that permit the achievement of shape changes for features that facilitate tree-gouging at wide gapes separate from the patterns of growth observed for features associated with load resistance.

The Ontogeny of C. jacchus Jaw Morphology and Implications for Weaning

Weaning is a demanding period involving the transition from suckling to independent food acquisition and occurs at a time when masticatory functional abilities may still lack maturity (Tanner et al., 2010). In discussing the transition from suckling to independent feeding Herring (1985:342) states: “In general, anatomy precedes function”, suggesting that although initial incision and mastication are largely functionally inefficient the morphology to achieve functional competency is in place prior to full weaning. Numerous investigations of masticatory abilities in mammals at the time of weaning support the suggestion that the functional abilities of weanlings are still immature during this transitional period (Herring, 1985; Wainwright and Reilly, 1994; Monteiro et al., 1999; Herrel and Gibb, 2006; Tanner et al., 2010). Therefore, it has been theorized that

selection would favor those individuals that are able to shorten the duration of this transitional stage by achieving adult-like masticatory abilities at younger ages

(Tanner et al., 2010).

Reports of age at weaning are likely to be quite variable as investigators define weaning differently (Harvey and Clutton-Brock, 1985). Yamamoto (1993) reports that weaning in C. jacchus occurs between nine and thirteen weeks (63-

91 days) followed by a period in which the majority of ingested food is obtained through sharing or stealing. Garber and Leigh (1997) report weaning at three months of age (90 days) for both C. jacchus and S. fuscicollis. A post-weaning period from four to eight months (120-240 days) has been reported for tamarins and marmosets in which juveniles are foraging but continue to be provisioned by adults of the group (Ferrari, 1992; Goldizen, 1987; Snowdon and Soini, 1988). It has been previously demonstrated that a significant association exists between higher body weight at 120 days of age and survivorship at six months in captive born marmosets (Jaquish et al., 1997). This link to survivorship may be significant as 120 days may coincide with the typical end of the weaning and the beginning the independent feeding period in C. jacchus (de Castro Leão et al.

2009).

To evaluate cranial features around this weaning period comparisons were made of the gape-related cranial feature shape values for marmosets versus tamarins at ages coinciding with active weaning(30-120 days), immediately subsequent to weaning (121-240 days) and young

adulthood/adulthood (241-2161 days) (Table 3.4, Figures 3.9a-d). Observed differences where C. jacchus exhibits more adult-like shapes for gape-related morphology (i.e. functionally improved for wide gapes) than S. fuscicollis at the time of weaning suggest a key shift in form related to gaining functional competency around the start of independent feeding.

Comparisons of marmoset and tamarin shapes for gape-related features demonstrate a trend for shape changes in the directions predicted for improved gape-related functional abilities in marmosets (Table 3.4, Figures 3.9a-d).

Although generally not significant, the para-weaning group of individuals (31 to

120 days) suggest a greater deviation toward adult-like shape values for marmosets relative to tamarins than found for the post-weaning groups. These result in lower p-values, but ones only approaching significance in the predicted direction (Mann-Whitney U-test, p<0.05).

This pattern of early development of adult-like gape-related shapes in the para-weaning marmosets suggests an early acquisition of shape changes necessary to facilitate wide jaw opening ability. The subsequent decrease in significant shape difference between species for the post-weaning period indicates a period where C. jacchus and S. fuscicollis shapes more closely resemble each other (i.e. a catch-up phase in tamarins). By the time animals reach young adulthood and into later adult stages, however, marmosets and tamarins once again significantly diverge in the direction of improved facilitation of gape-related functional abilities for C. jacchus. The presence of adult-like

Table 3.4: Interspecific Shape Differences (Feature/Jaw Length) Of Gape- Related Features around the Time of Weaning

Para- Post- Young (241-2161 Weaning (31-120 Days) Weaning (121-240 Days) Adult/Adult Days) Mann-Whitney Mann-Whitney Mean Mann-Whitney Mean U Mean U (s.d.) U (Interspecific) (s.d.) (Interspecific) (s.d.) (Interspecific) AP Condyle Length 0.087 0.080 0.070 (0.017), (0.013), (0.006), C. jacchus N=4 0.223 N=6 0.591 N=72 0.001 0.080 0.074 0.060 (0.008), (0.006), (0.007), S. fuscicollis N=19 N=4 N=48 Condyle Height 0.104 0.173 0.175 (0.029), (0.030), (0.031), C. jacchus N=6 0.008 N=5 0.462 N=44 0.001 0.150 0.149 0.203 (0.037), (0.053), (0.028), S. fuscicollis N=18 N=4 N=27 AP Glenoid Length 0.197 0.203 0.203 (0.015), (0.021), (0.012), C. jacchus N=5 0.226 N=6 0.748 N=72 0.001 0.185 0.193 0.178 (0.019), (0.032), (0.012), S. fuscicollis N=19 N=4 N=47 AP Mandible Length 1.001 0.995 0.996 (0.036), (0.022), (0.019), C. jacchus N=5 0.095 N=6 0.134 N=72 0.001 0.976 0.959 0.969 (0.026), (0.027), (0.017), S. fuscicollis N=19 N=4 N=48

Figure 3.9: Box plots comparing shape differences of gape-related features around the time of weaning including (a) AP condyle length, (b) condyle height, (c) AP glenoid length, and (d) AP mandible length.

shapes for gape-related cranial features at the shift to independent feeding would suggest that the observed shape changes in marmoset cranial features are the result of functional adaptations to gouging at wide gape rather than the result of functional plasticity.

Functional Implications for C. jacchus Jaw Morphology throughout Ontogeny

Ontogenetic results lend support to previous analyses of adult data

(Vinyard et al., 2003) demonstrating allometric and heterochronic differences during growth that support functional hypotheses for wide gape-related features in C. jacchus. The ontogenetic data lends further support to the assertion that these features are morphological adaptations to tree gouging at wide gapes, rather than arising as the result of functional plasticity in response to this behavior.

Comparisons of shape changes throughout ontogeny and into adulthood suggest that many of the relative shape differences in jaw morphology observed between marmosets and tamarins around the time of weaning are maintained throughout ontogeny and into adulthood. AP glenoid length and AP mandible length both demonstrate maintenance of shape values that remain consistent across age groupings. This consistency of shape values from the early stages of weaning into adulthood suggest that adult-like functional competency is attained early in ontogeny for these gape-related features.

Marmoset AP condyle length and condyle height both demonstrate maintenance of shape values, or isometry, beyond the age of 121 days.

Inclusion of shape values prior to the age of 121 days, however, suggest a negatively allometric trend of growth for AP condyle length and a positively allometric trend of growth for condyle height. These deviations from isometric shape values across age groups suggest that adult-like functional competency of the mandibular condyle in marmosets is achieved later in ontogeny than is found for the other gape-related features under analysis. It is of interest that these opposing trends in shape both involve growth aspects of the mandibular condyle and may suggest independent growth trends within this component of the jaw.

This somewhat later attainment of adult shape, however, still occurs within the late stages of weaning and the shift to independent feeding.

Modularity of the Masticatory Apparatus

The results of this study suggest that patterns of development for gape- related features in marmosets differ from those related to load resistance. These different patterns of development do not support the existence of a single pervasive pattern of development in the marmoset skull. The means for understanding the observed differences in development of masticatory apparatus features throughout ontogeny may be found in the concept of modularity.

Morphological modules are internally integrated elements that are hypothesized to develop primarily independently from other modules (Klingenberg, 2008).

The mandible is composed of two primary functional units; the ascending ramus and the corpus. Rodent studies have suggested that the ascending ramus and the corpus develop as separate modules, though not entirely independent of each other (Klingenberg et al., 2003). In turn, these regions are each composed of multiple anatomical units arising from distinct cell populations that differentiate at varying times (Atchley and Hall, 1991; Atchley, 1993; Miyake et al., 1997; Klingenberg et al., 2003, 2004). Mandibular modularity based on morphogenetic components of distinct cell condensations may point to the existence of as many as six modules within the mammalian mandible (Duarte et al. 2000; Hall, 2003). Ultimately, the modularity within the mandible may be a matter of degree rather than stark divisions (Klingenberg et al., 2003, 2004;

Monteiro et al., 2005).

One simplified assumption of modularity is that there is a universal model of growth within each module. It is possible, however, that heterochronic shifts in genes within modules may result in differing rates of development affecting the growth in one feature that therefore results in a seemingly independent shift in the morphology of another feature within that module (e.g., as the mandible elongates it becomes more shallow). It is of interest in the current analysis to note that both gape-related and load resistance features can be located within the same or can develop across multiple developmental modules of the masticatory apparatus and yet have been demonstrated to exhibit divergent growth patterns. These findings suggest the numerous developmental shifts

have occurred both within and across developmental modules of the marmoset masticatory apparatus throughout the evolution of this species. We hypothesize that many of the evolutionary changes support the functional integration of masticatory apparatus and its function in the dietary specialization of tree gouging to elicit exudate flow.

Modularity and Development of Features that Facilitate Gouging

The gape-related features of AP condyle length and AP glenoid length both exhibit peramorphosis via acceleration. This shared pattern of development is functionally consistent as the rotation and translation of the articular surface of the mandibular condyle occurs in conjunction with the articular surface of the glenoid (Hylander, 2006). Acting in concert as the functional bony components of the temporomandibular joint, the relative elongation of both of these features via an increase in rate of growth may enable increased rotation and translation of the mandible to facilitate gouging at wide gapes. Similar growth patterns suggest that despite the strong likelihood that the mandibular condyle and the glenoid fossa are elements of different developmental modules their development is integrated. The shared growth trajectory supports a hypothesis of developmental interaction in order to facilitate functional integration.

AP mandible length is a linear measurement that includes components of both the ramus and alveolar modules. Similar to both AP condyle and AP glenoid lengths, marmoset AP mandible length also appears to be peramorphic compared to tamarins. However, in contrast to the process of acceleration

observed for both AP condyle and glenoid lengths the elongation of the marmoset mandible appears to be achieved via predisplacement. The suggested earlier onset of growth facilitates gouging at wide gapes by providing a longer mandible for a given degree of mandibular rotation at the temporomandibular joint. This ontogenetic variation of growth pattern for AP mandible length relative to other gape-related features suggests that developmental interactions between the ascending ramus and alveolar modules drive the AP elongation of the mandible.

Condyle height develops within the module of the ascending ramus, but exhibits a relatively divergent pattern of development throughout ontogeny compared to tamarins. Whereas AP condyle length is peramorphic via acceleration indicating an increase in the rate of growth, condyle height is paedomorphic via postdisplacement indicating a delay in the timing of the onset of growth. The divergent patterns of development occur despite the inclusion of both condyle height and AP condyle length within the same functional and development module of the ascending ramus. These deviating growth patterns observed within the same developmental module for C. jacchus enable the relative AP elongation of the condyle to occur simultaneously with a relative decrease in the height of the condyle. Ultimately, the resulting increased rotation and translation of the marmoset mandible that occurs in conjunction with a relative decrease in condyle height may facilitate a reduction in masseter and

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medial pterygoid stretch during the wide jaw opening that accompanies tree gouging at wide gapes.

Modularity and Development of Load Resistance-Related Features

The load resistance features included for comparison in this study all develop within the mandibular corpus module. In contrast to the peramorphic pattern observed for the AP length of the mandible, load resistance features in marmosets appear to be paedomorphic compared to tamarins. The width of the marmoset corpus at M1 achieves paedomorphosis via neoteny indicating a decrease in the rate of growth compared to tamarins. This relative narrowing of the marmoset corpus suggests a decreased ability to resist twisting along the long axis of the mandibular corpus (Hylander, 1979, 1988).

The relative depth of the marmoset corpus at M1 also appears paedomorphic compared to tamarins. Despite inclusion in the alveolar module and close association to the dimension of M1 width, marmoset M1 depth achieves its relatively more shallow shape via postdisplacement. This delay in the onset of growth relative to tamarins results in a shallower mandibular corpus that is less able to resist parasagittal bending (Hylander, 1979, 1988).

Length of the mandibular symphysis at the anterior border of the alveolar module also appears to be paedomorphic in marmosets compared to tamarins.

Similar to M1 depth, this pattern seems to be achieved via postdisplacement through a relative delay in the onset of postnatal growth. The relatively shorter

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marmoset mandibular symphysis suggests a decreased ability for resisting vertical bending stress at the symphysis (Hylander, 1984, 1985, 1988).

Mosaic Development of the Marmoset Masticatory Apparatus

Mosaic evolution describes diverse rates of evolutionary change in different structures or functions of a species and provides an explanation for the evolution of a complex of characters (Raff, 1996; Wagner, 1996; Wagner and

Altenberg, 1996; Klingenberg et al., 2003; Rosas and Bastir, 2004; Bastir and

Rosas, 2009). The masticatory apparatus is a complex morphological structure that likely requires integration as a whole in order to function (Zelditch et al.,

2009). The absence of a singular pattern of developmental timing both among and within gape-related and load-resistance features despite inclusion within interdependent modules of the masticatory apparatus suggests a mosaic pattern of development for the marmoset masticatory apparatus. This mosaic pattern does not support an interpretation that features related to wide gapes in marmosets appear as a result of global changes to the masticatory apparatus.

The results of this study demonstrate differential patterns of development both within the masticatory apparatus of tree gouging marmosets and when compared to the masticatory apparatus of a closely-related non-gouging tamarin species. These results suggest a series of individual developmental changes to the marmoset masticatory apparatus that lead to functional integration that facilitates gouging at wide gapes.

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Essentially, the likely pattern of mosaic development implies that the observed suite of marmoset masticatory apparatus traits did not result from a single pleiotropic shift globally affecting the features of the marmoset masticatory apparatus, but rather suggests a series of distinct developmental shifts that facilitated morphological adaptation to tree gouging at wide gapes.

The mosaic nature of masticatory shape changes observed for marmosets additionally supports the potential for developmental interactions both within as well as between individual growth modules of the skull resulting in the integration of differential patterns of growth. This observed pattern of differential growth trajectories within both the ascending ramus and corpus modules of the mandible points toward the potential for individual features within a module to develop distinctively in facilitating the adaptation for gouging trees at wide gapes.

This array of divergent growth patterns for features of the masticatory apparatus suggest the existence of an overriding selective pressure in the direction of functional competence for the dietary requirements of gouging trees at wide gapes in order to elicit exudates flow. Essentially, selection appears to have acted upon multiple morphologies resulting in several, potentially separable adaptive developmental shifts in those features related to opening the jaw widely.

CHAPTER IV

COMPARATIVE ANALYSIS OF MASTICATORY MORPHOLOGY IN NEONATAL COMMON MARMOSETS (C. JACCHUS) AND COTTON-TOP TAMARINS (S. OEDIPUS)

Introduction

Tree gouging distinguishes marmosets from other platyrrhines in that they actively gouge trees with their anterior teeth to stimulate exudate flow. This derived tree gouging behavior presents functional morphologists with a compelling natural experiment for studying the morphological consequences of this feeding behavior for the masticatory apparatus (e.g., Kinzey et al., 1975;

Coimbra-Filho and Mittermeier, 1977). Tree gouging in marmosets has been demonstrated to involve relatively large jaw gapes that approach maximum jaw- opening ability for the individual, but this behavior does not appear to require relatively large bite forces (Vinyard et. al. 2003, 2009).

The mechanics of tree gouging involves anchoring the maxillary incisors into the tree substrate and using the mandibular incisors to remove layers of bark from the tree’s surface resulting in the flow of gum from the tree as its protective response (Coimbra-Filho and Mittermeier, 1977). Not surprisingly, the anterior dentition of tree gouging marmosets exhibits modifications that facilitate gouging including reduced lower canine height (bringing them in line with the lower incisors), thin enamel on the lingual surface of the incisors, a stronger enamel

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decussation pattern and “wedge-shaped” incisors that are both sharper and more robust. These modifications provide a stronger tooth with improved ability to remove tree substrate during gouging (Kinzey et al., 1975; Hershkovitz, 1977;

Eaglen, 1984; Nogami and Natori, 1986; Rosenberger, 1978, 1992; Propst, 1995;

Maas and Dumont, 1999; Hogg et al., 2011).

Comparative analyses of adults demonstrate that marmosets possess features of the masticatory apparatus that facilitate tree gouging at wide jaw gapes compared to closely-related, non-gouging callitrichids (Vinyard et al.,

2003). For the bony masticatory apparatus, these features include relatively lower condylar heights, anteroposteriorly (AP) elongated mandibles as well as

AP elongated temporal articular surfaces and mandibular condyles. Lower relative condylar heights are thought to reduce masseter muscle stretch during jaw opening which may facilitate larger maximum gapes (Herring and Herring,

1974 ) as well as bite force at wide gapes (Eng et al., 2009). Relative AP elongation of the marmoset mandible enables increased maximum gape and facilitates tree gouging (Vinyard et al., 2003). The AP elongation of the mandibular condyle and temporal articular surfaces assists wide gapes by increasing the articular surface area available for condylar rotation and translation, respectively (Vinyard et al., 2003).

Analysis of jaw-muscle architecture in adult marmosets has shown that they possess relatively longer jaw-muscle fibers that facilitate greater whole- muscle stretch compared to non-gouging tamarins. Seemingly, the increased

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stretching ability improves masticatory muscle performance during tree gouging at wide gapes. As an architectural tradeoff of increased fiber length, marmoset jaw muscles have relatively smaller physiological cross sectional areas (PCSA) suggesting decreased maximum force production abilities compared to non- gouging tamarins (Taylor and Vinyard, 2004; Taylor et al., 2009; Eng et al.,

2009). Eng et al. (2009), however, have demonstrated that marmoset masticatory muscle architecture appears to enable the production of relatively larger muscle forces at wide gapes compared to tamarins at similar gapes, even though overall maximum jaw-muscle force for marmosets is relatively lower than in tamarins.

The articular cartilage of the temporomandibular joint (TMJ) of adult marmosets is relatively larger and deeper in the anterior region of the glenoid.

Interestingly, these changes have occurred where the mandibular condyle is likely contacting the glenoid during gouging at large gapes. The derived features of the glenoid articular cartilage may demonstrate increased soft tissue load resistance ability at wide gape in marmosets compared to non-gouging tamarins

(Mork et al., 2010; see Chapter 2).

Previous comparative analysis of the ontogenetic allometry and heterochrony of the bony masticatory apparatus suggests that features associated with tree gouging at wide gapes are present early in ontogeny in marmosets compared to tamarins (Chapter 3). We hypothesize that neonatal common marmosets (C. jacchus) will display similar morphological patterns in

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the masticatory apparatus when compared to neonatal tamarins as previously demonstrated in adult marmosets compared to non-gouging species. We will examine this hypothesis by comparing the 1) fiber architecture of the masseter and temporalis muscles, 2) cross-sectional geometry of the mandibular corpus and 3) mandibular symphyseal strength between neonatal marmosets and tamarins. If this hypothesis is supported, it would suggest that derived features of the masticatory apparatus in marmosets that are present at birth are more likely the product of evolutionary adaptation rather than resulting from functional plasticity.

Muscles of Mastication

The jaw muscles play a critical role in generating jaw movements and bite forces during feeding behaviors. The fiber architecture of a muscle is a fundamental component of its ability to generate both movements and forces

(Gans, 1982). Two crucial elements that determine muscle function are the lengths of individual muscle fibers (Lf) and the muscle’s physiological cross sectional area (PCSA) (Lieber, 2002). Fiber length is theoretically proportional to a muscle’s maximum excursion and velocity of contraction as longer muscle fibers contain an increased number of sarcomeres in series. Sarcomeres are comprised of overlapping actin and myosin filaments and act as the fundamental unit of muscle contraction (Gans, 1982). Thus, the maximum excursion, or distance a muscle can move, as well as the velocity of contraction, or the distance per unit time that a fiber can shorten, are a function of the number of

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sarcomeres in series and by extension fiber length (Gans, 1982; Lieber, 2002;

Lieber and Ward, 2011). Physiological cross sectional area (PCSA) represents the sum of the cross-sectional areas of all muscle fibers within a muscle and is proportional to its maximum force generating capacity (Gans and Bock, 1965;

Powell et al., 1984; Lieber and Ward, 2011).

Adult marmosets have absolutely and relatively longer masseter fibers

(relative to an incisal biting load arm) than cotton-top tamarins (Taylor and

Vinyard, 2004; Taylor et al., 2009; Eng et al., 2009). Marmoset jaw muscles appear to be designed to facilitate stretching and by extension wide jaw gapes as well as the ability to generate sufficient incisal bite forces at wide gapes (Taylor and Vinyard, 2004; Taylor et al., 2009; Eng et al., 2009). Because of the architectural tradeoff between fiber length and force production, marmoset masseter and temporalis muscles have relatively smaller physiologic cross- sectional areas (PSCA), lower angles of pinnation and smaller proportions of tendon suggesting relatively reduced capacity for force production compared to cotton-top tamarins (Taylor and Vinyard, 2004; Taylor et al., 2009). We predict that common marmoset neonates will have relatively longer masseter and temporalis muscle fibers and will have relatively smaller PCSAs as part of the tradeoff between fiber length and force production.

Internal Bone Architecture of the Masticatory Apparatus

Numerous studies have used external measurements of skulls to investigate the functional morphology of the masticatory apparatus in tree-

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gouging primates (Burrows and Smith, 2005; Dumont, 1997; Viguier, 2004;

Vinyard et al., 2001, 2003; Williams et al., 2002). External measurements of jaw morphology, however, may fail to reveal the internal variation in cross-sectional morphology of the mandible that could have functional consequence among species (Daegling 1993, 2002; Vinyard and Ryan, 2006). Analysis of the cross- sectional geometry of the mandible may provide a more reliable estimate of the jaw’s ability to withstand the variety of stresses associated with feeding behaviors

(Daegling 1993, 2002; Vinyard and Ryan, 2006). Previous analyses, however, have demonstrated similar interpretations of jaw form when comparing both internal and external measurements of marmoset jaws (Vinyard and Ryan,

2006).

In vivo and field data suggest that adult marmosets do not generate relatively large bite forces during gouging (Vinyard et al., 2001, 2003, 2009).

Examination of the external metrics and cross-sectional geometry of the mandibular corpus and symphysis suggests that tree-gouging marmosets do not possess jaw morphologies that offer increased load resistance ability compared to non-gouging tamarins and squirrel monkeys (Vinyard et al., 2003; Vinyard and

Ryan, 2006; Hogg et al., 2011). In fact, these analyses suggests that marmosets possess a jaw shape that is less effective for resisting certain jaw loads relative to non-gouging platyrrhines (Vinyard and Ryan, 2006; Hogg et al., 2011). Based on adult comparisons, we predict that neonatal marmosets will exhibit no

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differences in cortical bone cross sectional areas or biomechanical shape ratios compared to the jaws of neonatal tamarins (Vinyard and Ryan, 2006).

Symphysis Strength

If marmosets generate relatively large bite forces to gouge trees, then the mandibular symphysis would likely need to be relatively robust compared to non-gouging callitrichids to withstand the increased force production and avoid structural failure. If marmosets are not recruiting relatively large bite forces during gouging, then there may not be any need for increased load resisting ability in the mandibular symphysis or the rest of the corpus. As argued above, previous morphological comparisons offer no evidence that marmosets have improved load resistance abilities in their jaws compared to tamarins (Vinyard et al., 2003;

Vinyard and Ryan, 2006). These morphological comparisons were extended by in vitro loading tests of the mandibular symphysis. These tests quantify symphyseal performance by recording the forces required to generate structural failure in the symphysis. These estimates of symphyseal performance suggest that adult marmosets exhibit relatively reduced symphyseal strength compared to tamarins and squirrel monkeys (Hogg et al., 2011). Based on these findings in adults, we predict that common marmoset neonates will show no significant difference in symphyseal strength during in vitro loading compared to neonate cotton-top tamarins.

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Materials and Methods

Samples

Samples used in these comparative analyses of the neonate masticatory apparatus include six common marmosets (Callithrix jacchus) from the Wisconsin

National Primate Research Center (WNPRC) and six cotton-top tamarins

(Saguinus oedipus) from the New England Primate Research Center (NEPRC).

Specimens were stillborn or died shortly after birth and range in age from 0 to 5 days. Intact specimens were preserved in 10% formalin prior to study.

Masseter and Temporalis Muscle Fiber Architecture Data Collection

The skin and overlying fascia were removed from each specimen to reveal the masseter and temporalis muscles. Lateral-view photographs of both the right and left sides of the specimens were taken to create a record of in situ morphology. Intact muscles and cranial landmarks were measured using calipers (0.1 mm). The muscle belly length of the superficial masseter was determined as the distance from the anterior attachment at the zygomatic arch to the distal attachment at the angle of the mandible. Temporalis muscle belly length was measured from the most superior attachment on the skull to the most anterior attachment on the coronoid process and anterior margin of the ramus.

Muscles were subsequently dissected from their bony attachments bilaterally, trimmed of excess fascia, blotted dry and weighed to the nearest 0.0001 g

(Denver Instruments A-200DS Digital Analytical Balance). Jaw length was

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measured as the distance from the posterior border of the mandibular condyle to infradentale.

Masseter and temporalis muscles were chemically digested in 30% HNO3 to facilitate the removal of individual fiber bundles. Digestion time was approximately 15 hours with the digestion status of the muscles assessed every

15 minutes for the first 9 hours and then every hour. The longer than expected time necessary for muscle digestion relative to the small size of the muscles is likely due to a higher concentration of connective tissue per muscle fiber content in these neonatal specimens. Following digestion, the muscles were placed in

1X PBS and small fiber bundles dissected under a stereomicroscope (10-20X magnification). From each muscle, we isolated and measured 10 fiber bundles from anterior, middle and posterior regions of the muscle to the nearest 0.1mm, respectively.

Variables

We computed an average fiber length for the anterior, middle and posterior regions of the muscle as well as an average fiber length for the muscle incorporating all fiber lengths. Physiological cross-sectional area (PCSA) was calculated for both the masseter and temporalis using the equation:

muscle mass (gm) * cos θ

average fiber length (cm) * 1.0564 (gm/cm3)

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where θ is the pinnation angle and 1.0564 represents the specific density of muscle (Mendez and Keys, 1960; Murphy and Beardsley, 1974).

Muscle mass/predicted effective maximal tension (M/Po) estimates how much of a muscle’s mass is due to longer fibers, and hence dedicated to excursion, versus fibers in parallel that benefit force production (Sacks and Roy,

1982; Anapol and Barry, 1996):

-2 2 M/Po = muscle mass (g) / 22.5 N cm * PCSA (cm )

To calculate M/Po, the specific tension of muscle is estimated as ~22.5 N

-2 cm (Powell et al., 1984). A higher M/Po ratio suggests that a muscle is facilitating excursion/contraction velocity relative to its mass over force production.

The priority index for force (I) was computed as PCSA/V2/3 (where V= wet muscle weight). The priority index examines a muscle’s force production ability relative to muscle volume. For two muscles of equal volume, the muscle with shorter more pinnate fibers will enhance force at the expense of excursion whereas a muscle with longer, parallel fibers will increase excursion over force

(Woittiez et al., 1986; Weijs et al., 1987; Van Eijden et al., 1997). Therefore, a higher ratio of I is indicative of a muscle facilitating force production at the expense of excursion.

Statistical Analyses

Analysis of variance (ANOVA) (α=0.05) was used to test whether common marmoset neonates differ from saddleback tamarin neonates for masseter and

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temporalis muscle architecture variables. One-tailed tests were used following the prediction of higher values for variables related to muscle excursion (e.g., fiber length) for marmosets compared to tamarins and lower values for variables associated with force production (e.g., PCSA).

Cross-Sectional Geometry of the Mandibular Corpus and Symphysis Data Collection

All specimens were scanned in the Scanco vivaCT 75 micro-computed tomography scanner at the Department of Anatomy and Neurobiology,

Northeastern Ohio Medical University (NEOMED). Each skull was scanned while secured with gauze with the occlusal surface of the mandible perpendicular to the scan plane and slices recorded perpendicular to the occlusal plane. Serial cross-sectional scans began at the anterior tip of the incisors and included the entire skull. Scans were collected with source energy settings of 70 kV and 114 mA. Scans had a pixel size of 20.5 µm and slice thickness was 20.5 µm resulting in a 20.5 µm voxel resolution. Scans were collected at a standard density threshold of -209.233 mg/cm3. Raw scan image data were converted to

2,048 x 2,048, 8-bit TIFFs using the Scanco software. TIFF images were cropped in Adobe Photoshop to isolate the mandible and stacked for three- dimensional reconstruction and analysis in Avizo 6.3 software.

To quantify cross-sectional geometric properties of mandibular cortical bone, slices were obtained perpendicular to the occlusal plane (Daegling, 1993) at the midline of the mandibular symphysis as well as the mesiodistal midpoint of

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di1, di2, dc, dp1, dp2, dp3 and M1 in the corpus (Figure 4.3). We oriented the slice through each tooth to obtain a minimum cross-section for analysis (Daegling,

1992, 1993). We did not include tooth crowns in the cross-sectional analysis as our goal is to investigate cortical bone distribution in the mandibular corpus and symphysis. Cortical bone measurements for the mandibular symphysis and corpus were recorded using the freeware ImageJ 1.44p (National Institutes of

Health; http://imagej.nih.gov/ij) and MacroMoment software.

Variables

Several cross-sectional measures of cortical bone were taken for the midline symphysis and midline of each tooth (Daegling, 1993, 2002). We calculated cortical cross-sectional area, area moments of inertia at the transverse

(Ixx) and superior inferior axes (Iyy), and both the maximum (Imax) and minimum

(Imin) area moment of inertia at each location. Two standard biomechanical indices, Ixx/Iyy and Imax/Imin, were calculated for each section to describe the relative ability of a cross-section to resist specific bending regimes.

Several shape variables describing a cross-section’s ability to resist a specific loading regime were also calculated. To create a dimensionless shape variable, the nth root of the geometric variable was divided by jaw length

(Daegling, 1992, 1993, 2002). Jaw length was measured as the linear distance between infradentale and the midpoint of a line between the posterior borders of the two mandibular condyles. Cross-sectional area0.5 was divided by jaw length

(CA0.5 /JawL) for all sections, as were the area moments of inertia at the

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.25 .25 transverse (Ixx /JawL) and superior inferior axes (Iyy /JawL) (Daegling, 1992,

2002). An additional relative shape variable was calculated by dividing

.25 Ixx by bicondylar breadth (Ixx /BiconBr), measured as the coronal plane distance between the two condyles (Daegling, 1992, 1993, 2002).

Statistical Analyses

Analysis of variance (ANOVA) (α=0.05) was used to test whether common marmoset neonates differ from saddleback tamarin neonates for each cross- sectional shape. Two-tailed tests were used following the prediction of no significant difference between species. In addition, the Bonferroni correction for multiple comparisons along tooth positions (n=8) was utilized to guard against inappropriate rejection of the null hypothesis yielding a corrected α=0.00625 (α/n,

0.05/8=0.00625) (Sokal and Rohlf, 1995).

Strength of the Mandibular Symphysis During In Vitro Loading

The mandibular symphyses of neonatal specimens were loaded to structural failure in an in vitro wishboning loading regime to assess variation in symphyseal joint strength. The mandibular corpus was removed by carefully isolating it at the ramus and stripping off the attached soft tissue. Care was taken to avoid stressing the mandibular symphysis during isolation. To simulate wishboning a loop of braided fishing line (Spiderwire® Stealth Braid™) was secured around each side of the corpus with a Uni-knot. The knot was positioned at the posterior sympheseal border to minimize the bending moment

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arm. Loops were secured with super glue to prevent the line from sliding along the corpus during loading. Care was taken to avoid glue leaking onto the symphyseal joint. The free ends of the lines were attached to the stationary grip of a Universal Testing Machine (Electropuls E3000 Instron, Norwood, MA). The symphysis was loaded to structural failure in wishboning at a constant rate of

4mm/minute with a load cell size of 250 Newtons. We recorded force at failure

(N) and displacement (mm) at 10 Hz or 100 Hz from initial loading until structural failure. We also verified for each specimen that failure occurred at the symphyseal midline. Data was discarded for one specimen (S. oedipus, #169) where structural failure occurred along the corpus rather than at the mandibular symphysis.

Variables

Variables associated with strength of the mandibular symphysis include load at structural failure (N) and the elastic modulus (E) (i.e., stiffness) which describes the linear portion of the stress/strain curve prior to the accumulation of permanent deformation. To calculate stress at failure (N/mm4) during wishboning, force at failure (N) was divided by the area moment of inertia for resisting wishboning (Iyy) calculated from the CT scan of the midline symphysis

(Hylander, 1985; Vinyard and Ravosa, 1998; Daegling and McGraw, 2009; Hogg et al., 2011).

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Statistical Analyses

Analysis of variance (ANOVA) (α=0.05) was used to test whether common marmoset neonates differ from saddleback tamarin neonates for load at failure

(N), elastic modulus (stiffness) and stress at failure (N/mm4). Two-tailed tests were used following the prediction of no significant difference between species.

The Bonferroni correction for multiple comparisons (n=3) was utilized to guard against inappropriate rejection of the null hypothesis yielding a corrected

α=0.0167 (α/n, 0.05/3=0.0167) (Sokal and Rohlf, 1995).

Results

Masseter and Temporalis Muscle Fiber Architecture

Average values for weight and length of both the masseter and temporalis muscles are smaller for neonatal marmosets compared to tamarins, and significantly smaller for masseter mass and temporalis length (Table 4.1a,

Figure 4.1b). In addition, jaw length is significantly shorter in neonatal marmosets relative to tamarins (Figure 4.1a). Masseter and temporalis absolute physiological cross sectional areas (PCSA) are smaller in marmosets relative to tamarins and significantly so for the masseter (Figure 4.1c). There is no significant difference, however, in average muscle fiber length in either the masseter or temporalis between C. jacchus and S. oedipus (Table 4.1a, Figure

4.1d). In addition, regional comparisons of average anterior, middle and

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(Figure Continues)

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Figures 4.1: Box plots of absolute masseter and temporalis measurements

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Table 4.1 Masseter and Temporalis Architectural and Fiber Length Variables

a Callithrix Masseter muscle jacchus Saguinus oedipus ANOVA1 Cj vs. So2

Muscle length (mm) 7.100 ± 0.5663 8.233 ± 1.147 0.055 Cj < So Mass (g) 0.051 ± 0.007 0.081 ± 0.021 0.007 Cj < So

Fiber length (Lf) (mm) 2.067 ± 0.154 2.484 ± 0.499 0.079 Cj < So Anterior fiber length 1.977 ± 0.151 2.351 ± 0.470 0.092 Cj < So Middle fiber length 2.085 ± 0.448 2.613 ± 0.557 0.100 Cj < So Posterior fiber length 2.131 ± 0.092 2.510 ± 0.563 0.134 Cj < So PCSA (cm2) 0.230 ± 0.036 0.287 ± 0.048 0.043 Cj < So

Callithrix Temporalis muscle jacchus Saguinus oedipus ANOVA Cj vs. So Muscle length (mm) 9.517 ± 0.445 10.950 ± 0.997 0.009 Cj < So Mass (g) 0.057 ± 0.009 0.074 ± 0.023 0.132 Cj < So

Fiber length (Lf) (mm) 2.808 ± 0.310 2.826 ± 0.452 0.939 Cj < So Anterior fiber length 2.745 ± 0.236 2.681 ± 0.352 0.717 Cj > So Middle fiber length 2.730 ± 0.417 2.849 ± 0.541 0.680 Cj < So Posterior fiber length 2.963 ± 0.389 2.944 ± 0.534 0.945 Cj > So

PCSA (cm2) 0.207 ± 0.034 0.261 ± 0.068 0.112 Cj < So Jaw length (mm) 14.933 ± 0.470 16.867 ± 0.939 0.001 Cj < So

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Table 4.1 (Continued)

b Callithrix Masseter muscle jacchus Saguinus oedipus ANOVA1 Cj vs. So2 Masseter Length/JawL 0.476 ± 0.0443 0.487 ± 0.052 0.694 Cj < So 4 Lf/JawL 0.139 ± 0.012 0.146 ± 0.023 0.475 Cj < So 1/3 Lf/MW 5.609 ± 0.491 5.701 ± 0.943 0.836 Cj < So

M/Po 0.010 ± 0.001 0.013 ± 0.003 0.029 Cj < So PCSA0.5/JawL 0.032 ± 0.003 0.032 ± 0.004 0.892 Cj < So I 1.673 ± 0.147 1.554 ± 0.254 0.345 Cj > So

Temporalis Callithrix muscle jacchus Saguinus oedipus ANOVA Cj vs. So Temporalis Length/JawL 0.637 ± 0.028 0.648 ± 0.031 0.536 Cj < So

Lf/JawL 0.188 ± 0.021 0.167 ± 0.021 0.109 Cj > So 1/3 Lf/MW 7.303 ± 0.782 6.465 ± 0.806 0.098 Cj > So

M/Po 0.012 ± 0.001 0.013 ± 0.002 0.870 Cj < So PCSA0.5/JawL 0.030 ± 0.002 0.030 ± 0.003 0.855 Cj < So I 1.391 ± 0.147 1.495 ± 0.212 0.348 Cj < So

1 Column reports p-values of the ANOVA for that variable between the two species. The p-values in bold are significantly less than α = 0.05. 2 Callithrix jacchus vs. Saguinus oedipus with > or < to indicate directionality of results. Directionality of a significant difference is indicated in bold. 3 Mean ± S.D. 4 1/3 Lf = fiber length; JawL = jaw length; MW = cube root of muscle weight; M/P0 = muscle mass/predicted effective maximal tetanic tension; PCSA = physiologic cross-sectional area; I = priority index of force

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posterior muscle fiber lengths do not demonstrate any significant differences between the two species.

No significant differences were found for relative fiber length comparisons between neonatal marmosets and tamarins for the masseter or temporalis muscles. Masseter fiber lengths relative to jaw length (Lf/JawL) are smaller for marmoset neonates compared to tamarins (Table 4.1b, Figure 4.2b).

Alternatively, relative fiber lengths of temporalis appear larger on average in marmosets. Relative physiological cross sectional areas (PCSA0.5/JawL) are similar for both masseter and temporalis between species (Table 4.1b, Figure

4.2b). These findings for the masseter muscle fiber lengths and PCSAs run counter to the prediction that marmosets would possess relatively longer masseter fiber lengths and relatively smaller PCSAs for both the temporalis and the masseter.

The ratio of excursion/contraction to force production (M/Po) is significantly smaller for the marmoset masseter compared to the tamarin masseter suggesting that relative to mass the tamarin masseter is facilitating excursion/contraction compared to marmosets (Table 4.1b, Figure 4.2c). The same pattern is found for the temporalis muscle, but the two species do not differ significantly. The priority index of force (I) is larger for the marmoset masseter compared to tamarins, but the opposite pattern is observed between species for the temporalis (Table 4.2b, Figure 4.2d). The smaller ratio of excursion/

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(Figure Continues)

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Figure 4.2: Box plots of relative masseter and temporalis measurements and ratios

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Table 4.2: Cross-Sectional Geometry of the Mandible

Univariate Statistics

Callithrix jacchus

.5 .25 .25 CA /Ja I /Bic .25 I /Ja CA I I I /I I /I xx I /JawL yy xx yy xx yy max min wL onBr xx wL Midline 6.143 3.287 4.679 0.716 1.927 0.203 0.100 0.110 0.109 sym- (0.275)2 (0.482) (0.778) (0.135) (0.182) (0.004) (0.007) (0.005) (0.011) physis 2.856 1.895 1.471 1.291 1.361 0.138 0.087 0.096 0.090 i 1 (0.153) (0.213) (0.137) (0.123) (0.152) (0.004) (0.006) (0.003) (0.003)

2.175 1.110 2.910 1.083 1.469 0.120 0.083 i 2 (0.391) (0.294) (1.305) (0.311) (0.260) (0.011) (0.005)

1.471 0.556(0. 0.690 0.988 1.720 0.098 0.070 c (0.472) 205) (0.498) (0.385) (0.374) (0.016) (0.007)

1.425 0.550 0.502 1.138 1.478 0.097 0.070 dp 1 (0.163) (0.081) (0.127) (0.217) (0.264) (0.005) (0.003)

1.110 0.332 0.336 1.023 1.385 0.086 0.062 dp 2 (0.123) (0.068) (0.103) (0.189) (0.284) (0.005) (0.004)

1.095 0.326 0.327 1.040 1.265 0.085 0.061 dp 3 (0.216) (0.110) (0.135) (0.282) (0.243) (0.009) (0.006)

0.965 0.251 0.168 1.775 2.463 0.080 0.057 M 1 (0.179) (0.106) (0.130) (0.586) (0.671) (0.008) (0.007)

1 4 2 Ixx and CA are in mm and mm , respectively 2 Mean (S.D.)

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Table 4.2 (Continued)

Univariate Statistics1

Saguinus oedipus

CA.5/Ja I .25/Bic I .25/Ja I .25/Ja CA I I I /I I /I xx xx yy xx yy xx yy max min wL onBr wL wL

Midline 6.467 6.036 5.590 1.046 4.332 0.174 0.098 0.106 0.097 sym- (1.578)2 (3.027) (1.991) (0.248) (1.575) (0.021) (0.010) (0.013) (0.006) physis

4.386 3.357 3.006 1.140 1.231 0.143 0.085 0.092 0.090 i 1 (0.016) (0.999) (1.063) (0.166) (0.135) (0.016) (0.004) (0.006) (0.008)

4.240 2.985 1.107 1.104 1.436 0.141 0.090 i 2 (1.019) (0.997) (0.397) (0.427) (0.275) (0.019) (0.007)

2.129 1.315 1.352 0.986 1.971 0.099 0.072 c (0.630) (0.588) (0.629) (0.200) (0.655) (0.013) (0.008)

1.463 0.562 0.684 0.811 2.213 0.082 0.058 dp 1 (0.452) (0.291) (0.357) (0.206) (0.621) (0.012) (0.008)

0.843 0.164 0.214 0.699 3.456 0.062 0.042 dp 2 (0.293) (0.108) (0.114) (0.239) (1.335) (0.010) (0.008)

0.816 0.135 0.191 0.690 2.165 0.061 0.041 dp 3 (0.258) (0.070) (0.084) (0.129) (0.671) (0.009) (0.005)

1.156 0.447 0.218 2.032 2.344 0.073 0.054 M 1 (0.333) (0.283) (0.054) (1.033) (0.811) (0.009) (0.008)

1 4 2 Ixx and CA are in mm and mm , respectively 2 Mean (S.D.)

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Table 4.2 (Continued)

Species statistical comparisons

CA.5/ I .25/Bic I .25/Ja I .25/Ja CA I I I /I I /I xx xx yy xx yy xx yy max min JawL onBr wL wL Midline sym- 0.6021 0.036 0.286 0.011 0.0022 0.006 0.696 0.492 0.002 physis Cj < So2 Cj < So Cj < So Cj < So Cj < So Cj > So Cj > So Cj > So Cj > So

i1 0.003 0.003 0.003 0.086 0.132 0.437 0.612 0.224 0.950

Cj < So Cj < So Cj < So Cj > So Cj > So Cj < So Cj > So Cj > So Cj < So

i2 0.001 0.001 0.005 0.921 0.831 0.029 0.085 0.254

Cj < So Cj < So Cj < So Cj < So Cj > So Cj < So Cj < So Cj < So

c 0.054 0.008 0.058 0.992 0.404 0.917 0.625 0.924

Cj < So Cj < So Cj < So Cj > So Cj < So Cj < So Cj < So Cj < So

dp1 0.838 0.921 0.231 0.018 0.015 0.011 0.002 0.130

Cj < So Cj < So Cj < So Cj > So Cj < So Cj > So Cj > So Cj > So

dp2 0.049 0.006 0.067 0.020 0.002 0.001 0.001 0.001

Cj > So Cj > So Cj > So Cj > So Cj < So Cj > So Cj > So Cj > So

dp3 0.057 0.004 0.056 0.018 0.007 0.001 0.001 0.001

Cj > So Cj > So Cj > So Cj > So Cj < So Cj > So Cj > So Cj > So

M1 0.213 0.116 0.395 0.584 0.778 0.192 0.56 0.388

Cj < So Cj < So Cj < So Cj < So Cj < So Cj > So Cj > So Cj > So

1 The first row of each cell reports the p-value of the ANOVA for that variable and section. The p-values in bold are significantly less than α = 0.05. The p-values in bold italics are significantly less than α= 0.00625, using the Bonferroni correction for significance.

2 < or > indicates directionality of the means between species. Bold indicates a significant directional difference at p< 0.05 > Bonferroni correction, with bold italics indicating significance at α = 0.00625 (p< 0.00625) based on the Bonferroni correction for significance.

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contraction to force production (M/Po) in marmosets does not support the prediction that the masseter and temporalis in marmosets is designed for excursion/contraction rather than force production. The results for the priority index of force (I) support the prediction of decreased force production potential for the marmoset temporalis compared to tamarins, but fail to support the same prediction for the masseter. As both of these variables are contingent upon muscle fiber length for their calculation, it is not unexpected that the results for the comparison of the marmoset and tamarin masseter muscles would fail to support the original prediction as the masseter muscles fiber lengths are shorter in marmosets.

Cross-Sectional Geometry of the Mandibular Corpus and Symphysis

Absolute cortical area (CA) for both C. jacchus and S. oedipus neonates is largest at the midline of the mandibular symphysis (MS) and gradually decreases moving posteriorly along the corpus (Figure 4.4a). The absolute CA of the first

(di1) and second (di2) incisors is significantly smaller in C. jacchus as compared to S. oedipus, however, the relative CA (CA.5/JawL) of the mandibular symphysis is significantly larger in C. jacchus relative to S. oedipus (Table 4.2a-c; Figures

4.4a & 4.5a).

The variable Ixx describes resistance to coronal bending in the anterior corpus (MS, di1, di2) and resistance to parasagittal bending in the posterior corpus (dc-M1) (Figure 4.4b). The ability to resist coronal bending in the anterior corpus appears to be stronger in both species compared to the ability to resist

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Figure 4.3: Corporal and symphyseal cross-sections of the jaw, slice orientation for cross-sections at each tooth location examined.

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(Figure Continues) c

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Figure 4.4: Absolute measurements of mandibular corpus cross-sections.

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(Figure Continues)

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(Figure Continues)

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Figure 4.5: Relative cross-sectional geometry of the mandibular corpus.

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parasagittal bending in the posterior corpus. Resistance to coronal bending (Ixx) at the incisors and canine appears to be significantly smaller for marmosets as compared to tamarins. The opposite pattern for Ixx is observed posterior to the canine, as the ability to resist parasagittal bending is significantly larger for C. jacchus at the second and third premolars relative to S. oedipus. Values for resistance to lateral transverse bending (Iyy) are significantly lower for neonatal marmosets as compared to tamarin neonates at the mandibular symphysis and significantly so at the first and second deciduous incisors (Tables 4.2a-c, Figure

4.4c). Posterior to the incisors, values for Iyy are smaller for marmosets, but not significantly compared to tamarins for all remaining corpus measurements except for the second and third premolar.

.25 Relative ability to resist parasagittal bending (Ixx /JawL) in the posterior corpus (dp1-dp3) is significantly larger in neonatal marmosets compared to tamarins (Tables 4.2a-c, Figure 4.5b). Alternatively, relative resistance to coronal

.25 bending (Ixx /JawL) in the anterior region of the corpus demonstrates no significant differences between the two species. Relative resistance to

.25 transverse bending (Iyy /JawL) at the mandibular symphysis and at the second and third premolars is significantly higher for C. jacchus neonates compared to S. oedipus neonates (Tables 4.2a-c, Figure 4.5c). Despite a lack of significance, anterior to the first premolar the relative resistance to lateral transverse bending appears to be lower for marmosets, whereas the regions of the corpus at the first

.25 premolar and posterior demonstrate higher values for Iyy /JawL in marmosets.

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.25 Relative to bicondylar breadth, resistance to coronal bending (Ixx /BiconBrd) shows no significant difference between marmosets and tamarins in the anterior corpus (Tables 4.2a-c, Figure 4.5d).

The biomechanical ratio of vertical (coronal or parasagittal) to transverse bending resistance ability (Ixx/Iyy) for neonatal marmoset mandibles is variable and demonstrates no clear pattern (Tables 4.2a-c, Figure 4.5e). Tamarin neonates exhibit a gradual decrease in this ratio moving from anterior to posterior in the corpus. Posterior to the symphysis, C. jacchus tends to have a consistently increased ratio of vertical to transverse bending resistance ability compared to S. oedipus. The Imax/Imin ratio is variable for both species and fails to demonstrate a discernible trend. C. jacchus neonates, however, have a significantly smaller Imax/Imin ratio relative to S. oedipus in the regions of the mandibular symphysis and second premolar (Tables 4.2a-c, Figure 4.5f).

Collectively, these results run counter to the prediction that neonatal marmosets, compared to tamarins, display no differences in load resistance ability in the mandibular corpus. The lack of consistent differences in absolute dimensions coupled with the significantly longer jaw in tamarin neonates may contribute to the often relatively improved load resistances in C. jacchus neonates compared to S. oedipus neonates.

Strength of the Mandibular Symphysis During In Vitro Loading

C. jacchus and S. oedipus neonates did not differ significantly in load at failure (N) or stress at failure (N/mm4) during lateral transverse bending (i.e.

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simulated “wishboning”) (Table 4.3, Figures 4.7a & b). Alternatively, marmosets demonstrated a significantly higher modulus of elasticity (E) while undergoing lateral transverse bending (Table 4.3, Figure 4.7c). This suggests that the mandibular symphysis of neonatal marmosets is significantly stiffer than neonatal tamarins. These results are mixed in that they support the prediction that neonatal marmosets possess no significant differences in the ability to resist lateral transverse forces (wishboning) at the mandibular symphysis compared to tamarins. The significantly higher modulus of elasticity found for neonatal marmosets compared to tamarins, however, does not support the prediction of no difference in symphyseal mechanical properties.

Table 4.3: Simulated wishboning at the mandibular symphysis

Load at Stress Species n failure (N)2 (N/mm4)3 Modulus

Callithrix jacchus 6 4.78 (1.02)4 1.09 (0.29) 0.05 (0.02)

Saguinus oedipus 51 4.35 (1.26) 0.76 (0.21) 0.02 (0.01)

ANOVA 0.553 0.065 0.0045 Cj > So Cj > So Cj > So

1 One S. oedipus excluded from data as failure occurred on corpus and not at midline symphysis. 2 N = Newtons 3 Stress at failure in wishboning is estimated by dividing load (N) by and estimate of the second moment area for resisting wishboning (mm4 ) 4 Mean followed by standard deviation (in parentheses) 5 Italics indicate significance at α=0.0167 using Bonferroni adjustment

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Figure 4.6: Example of in vitro simulation of wishboning stress/strain curve

(Figure Continues)

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Figure 4.7: Box plots of in vitro simulation of wishboning data

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Discussion

Muscle Fiber Architecture, Jaw Gapes and Bite Force Production

I predicted that common marmoset neonates would possess relatively longer masseter and temporalis muscle fibers and would have relatively smaller

PCSAs as a tradeoff between fiber length and force production. This prediction was based on previous adult comparisons demonstrating that longer muscle fibers may facilitate increased ability for whole muscle stretch at wide gapes and therefore enable wider jaw gapes during tree gouging (Eng et al., 2009; Taylor et al., 2009). I found no significant difference for either absolute or relative masseter or temporalis muscle fiber lengths. In fact, neonatal marmosets tend to possess relatively shorter muscle fiber lengths (Lf/JawL) for the masseter muscle.

Relative temporalis fiber length (Lf/JawL) is longer in neonatal marmosets, but not significantly longer than tamarins as predicted. These findings suggest that marmosets are not born with jaw-muscle fiber architectures designed to facilitate the wide gapes observed in adult marmosets.

Bite force related variables recorded for the temporalis muscle demonstrated no significant differences between neonatal marmosets and tamarins. The priority index of force (I) calculations for both the masseter and temporalis demonstrated no significant difference between marmosets and tamarins. The results do suggest, however, that the neonatal marmoset masseter is architecturally facilitating force production at the expense of excursion, while the opposite trade-off appears to be reflected in the architecture

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of the marmoset temporalis muscle. This discrepancy is likely a reflection of differing growth trajectories between the marmoset masseter and temporalis.

Whereas the neonatal marmoset temporalis is beginning to reflect the elongated condition observed for adult marmosets, with a significantly longer muscle (P<

0.009) compared to tamarins, the neonatal masseter is both absolutely and relatively shorter in contrast to its adult state.

Ultimately, the fiber architecture of the neonatal masseter and temporalis muscles of marmosets compared to tamarins do not display the predicted trade off in favor of muscle excursion over force production that is observed for adult comparative analyses. It is possible that birth may not be the best time for functional comparisons of masticatory muscles as adult feeding behaviors are not occurring at this time. Marmosets and tamarins are weaned and begin engaging in adult-like feeding behaviors at three months (Garber and Leigh,

1997; de Castro Leão et al., 2009). Prior to weaning, the functional requirements of the masseter and temporalis muscles differ from the requirements associated with the ingestion of solid food and the functional requirements associated with tree gouging. Therefore, the initiation of weaning and gouging behavior may be a more appropriate stage for evaluating differences in muscle fiber architecture that occur earlier in development. It is possible that deviations from the adult marmoset morphological pattern of the masticatory apparatus at birth may not have a major impact if the adult condition is achieved at the time of weaning when gouging behaviors become more critical. My results do show, however,

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that major changes in architecture are not establish during embryological development as these would have been apparent at birth. If there are heritable differences in marmoset muscle architecture related to tree gouging, then they are more likely to appear during postnatal growth based on this analysis. Future work should attempt to determine if these changes are apparent by weaning.

Cross-Sectional Geometry of the Mandibular Corpus and Symphysis

Based on adult comparisons, we predicted that neonatal marmoset jaws would exhibit no differences in relative cortical bone cross sectional areas or biomechanical shape ratios compared to the jaws of neonatal tamarins (Vinyard and Ryan, 2006). Contrary to this prediction, we found that many of the relative biomechanical shapes for the jaws of neonatal marmosets suggest increased robusticity and resistance ability compared to tamarins. These results imply that, at least in the neonatal marmoset, the ability to resist loading of the mandibular symphysis and premolar region during incisive biting and wishboning is increased compared to neonatal tamarins.

Differences in cortical area between neonatal marmosets and tamarins may reflect differences in the timing of dental development between the two species. Dental eruption patterns suggest that marmosets are comparatively delayed in the development and timing of the eruption of the permanent dentition compared to tamarins (Smith et al., 1994). For example, the eruption of marmoset permanent mandibular incisors has been shown to be delayed by approximately two months compared to tamarins (S. fuscicollis) (Smith et al.,

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1994). Comparison of the CT section for the first incisor in both species demonstrates a qualitative difference between marmosets and tamarins in the size of the tooth crypt and the density of compact bone. Marmosets possess a seemingly larger tooth crypt area, but display a visually more dense bone surrounding the crypts. The absolute CA of the anterior corpus at the first and second incisors is significantly smaller in C. jacchus compared to S. oedipus.

The distribution of cortical bone, however, is more densely concentrated to the inferior, labial and lingual borders of the corpus in the incisor region.

Alternatively, cortical bone in tamarins is visibly less dense and more uniformly distributed throughout the cross-section of the anterior corpus. This increase in marginal bone density of the corpus in the region of the mandibular symphysis and anterior corpus may contribute to the observation of increased robusticity and load resistance ability observed for marmosets in this cross sectional analysis. Moreover, it may also relate to the increased stiffness observed for the marmoset mandibular symphysis in the in vitro simulation of wishboning.

The premolar region of the neonatal marmoset corpus presents significantly increased values for multiple load resistance variables as well as significantly larger relative cortical areas when compared to tamarins (Table 4.2).

Comparative observations of the CT slices in the premolar/posterior corpus regions of marmosets and tamarins reveal a more uniform distribution of cortical bone in marmosets along the labial, inferior and lingual borders of the corpus.

The cortical bone of tamarins in the premolar region is almost entirely

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concentrated to the inferior aspect of the corpus with only thin layers present at the labial and lingual borders. On the whole, the neonatal marmoset mandible appears to be significantly more robust and increasingly more resistant to loading in the more posterior region of the mandibular corpus where parasagittal and forces are concentrated during mastication (Hylander, 1987). However, at the neonatal stage, it is unclear what functional consequences these morphological implications of increased load resistance would confer during the pre-weaning period.

The prediction that there would be no significant difference between neonatal marmosets and tamarins in the cross sectional geometry of the mandibular corpus is not supported by the results of this analysis. The functional loading requirements of the neonatal marmoset masticatory apparatus are not known, but it can be presumed that mandibular loading requirements involved in suckling differ from the functional demands involved in the ingestion of solid foods and tree gouging. It is possible, however, that the differences observed are not grounded in functional performance, but are rather due to divergence in growth trajectories of the mandible as well as variation in the timing of dental development between the two species. The lack of correspondence between these results and analyses of adult marmosets and tamarins suggest a possible role for plasticity in the development of the marmoset mandible during the weaning transition to adult dietary behaviors. In particular, tamarin jaws may

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experience a plasticity related increase in robusticity based on potential differences in diet between these two groups.

Strength of the Mandibular Symphysis During In Vitro Loading

I predicted that common marmoset neonates would show no significant difference in symphyseal strength during in vitro loading compared to neonate cotton-top tamarins. Lateral transverse bending (i.e., wishboning) has been shown to generate significant stress in the macaque mandibular symphysis during mastication (Hylander, 1984, 1985; Hylander et al. 1998, 2000, 2005).

Previous analyses suggest that the cross-sectional cortical bone distribution of the mandibular symphysis of tree-gouging marmosets does not provide increased load resistance ability as demonstrated by in vitro wishboning tests

(Vinyard et al., 2003; Vinyard and Ryan, 2006; Hogg et al., 2011). The only significant difference between neonatal marmosets and tamarins during in vitro loading at the mandibular symphysis is the significantly greater stiffness, or higher elastic modulus (E), of the marmoset mandibular symphysis compared to tamarins.

Differences in dental development or the degree of symphyseal fusion at birth may have played a role in the observation of significantly increased stiffness in the neonatal marmoset. Marmosets possess a seemingly larger tooth crypt area, but display a visually more dense bone surrounding the crypts.

Comparisons of the CT cross-sections at the first incisor (di1) demonstrate a differential distribution of bone between the neonatal marmosets and tamarins.

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Neonatal marmosets appear to possess dense, compact bone at the labial, lingual and inferior corpus whereas the neonatal tamarins show a more widespread distribution of cancellous bone throughout the cross section of the first incisor. Observations of the CT scans of the neonate mandibular symphyses clearly demonstrate varying degrees of mandibular fusion for both marmosets and tamarins with no clear qualitative differences between the two species.

The significantly increased stiffness of the marmoset mandibular symphysis may also reflect an accelerated pattern of mineralization linked to an earlier initial growth spurt in the mandible (see Chapter 3 for growth results).

Increased mineralization need only occur over a part of the symphysis to result in the observed differences in stiffness. Additional study of the CT images documenting regional variation in density could test this hypothesis.

Conclusions

Weaning marks the shift to independent feeding behaviors as well as a shift in the functional requirements of the infant masticatory apparatus. This event is likely the most appropriate functional stage for addressing morphological differences that arise throughout the early stages of ontogeny for these two species, especially in relation to features of the marmoset masticatory apparatus that are associated with gouging at wide gapes. We do not know how closely pre-weaning behaviors resemble adult feeding behaviors. There has been debate about whether adult–like masticatory function is acquired at weaning, but all recognized that a major shift in feeding behavior does occur with independent

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feeding (Herring, 1985; Ravosa and Vinyard, 2002). One of most relevant future steps is adding comparative functional analyses of muscle architecture, in vitro load resistance and cross-sectional studies using young, recently weaned animals.

It was hypothesized that the masticatory apparatus of neonatal common marmosets would display morphological patterns similar to those observed for adult comparisons of muscle fiber architecture, cross-sectional geometry and resistance to wishboning when compared to neonatal tamarins. The relatively few significant differences for comparisons of muscle fiber architecture and in vitro wishboning, suggest that functional plasticity during growth contributes to the morphological patterns found in adults. The mixed results of the cross- sectional geometry of the mandibular corpus combined with previous data of ontogenetic allometry and heterochrony (Chapter 3) suggests that a contribution from early embryological patterning cannot be discounted. Overall, these analyses underscore the mosaic nature of development of the adult marmoset masticatory apparatus combining features that likely developed early during embryonic patterning as well as those that arose due to functional plasticity associated with the shift to independent feeding behaviors.

CHAPTER V

SUMMARY AND CONCLUSIONS

The essential questions at the core of each of the preceding analyses are

“what specialized morphological features characterize the masticatory apparatus of tree-gouging marmosets and how do they evolve?” Chapter 2 addresses how the soft-tissues of the temporomandibular joint of marmosets differ from other non-gouging callitrichids, while Chapters 3 and 4 ask how musculoskeletal features arise during ontogeny? Answering these questions broadens our understanding of the form-function relationships in the marmoset masticatory apparatus, the evolutionary history of callitrichids as well as the extent of functional integration of features that facilitate gouging at wide gapes.

Articular Cartilage of the Temporomandibular Joint

Tree gouging marmosets load the temporomandibular joints (TMJ) at wide jaw gapes when biting trees to remove bark layers and provoke exudate flow

(Vinyard et al., 2009). Because opening the jaw involves condylar rotation and anterior translation on the glenoid, gouging at wide gapes likely results in a relatively unusual loading pattern in the TMJ where the posterior surface of the condyle and the anterior surface of the glenoid experience an increased frequency of loading compared to non-gouging callitrichids. Based on these

148 149

functional and behavioral scenarios, we predicted that the articular cartilage in the posterior condyle and anterior glenoid would be relatively larger, thicker and show a higher percentage of proteoglycans to provide increased load resistance abilities in gouging marmosets.

Comparative analysis of articular cartilage morphology in gouging and non-gouging platyrrhines yielded mixed results. Marmosets demonstrated relatively larger and thicker articular cartilage in the anterior glenoid compared to non-gougers. The posterior condylar articular cartilage, however, did not differ consistently between gouging and non-gouging species. The relative density of proteoglycans did not differ in either the condyle or glenoid cartilages.

The interpretation of our results comparing condylar articular cartilages is less clear. The lack of statistically significant differences suggests a similarity in relative load resisting capacity in the posterior condyle across gouging and non- gouging species. Relative to our hypothesis, the relative similarity between gouging and non-gouging species could indicate that condylar articular cartilage is not loaded as we hypothesize, that the cartilage is overbuilt relative to the increased loading frequency experienced during gouging, and/or that the magnitude of loading in the posterior condyle during gouging is not sufficient to elicit a physiological or evolutionary response in these tissues. Future additional analyses of the composition of the articular cartilage in gouging primates as well as further in vivo data concerning the nature of loading at the temporomandibular

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joint when biting at wide gapes will likely assist in clarifying articular cartilage responses to loading at wide gapes.

It is rare that an entire complex system such as the masticatory apparatus is adapted to a single mechanical function (e.g. Olson and Miller, 1958;

Cheverud 1996; Monteiro et al., 2005; Zelditch et al., 2008; Klingenberg, 2008,

2009). More reasonably, the morphology of the masticatory apparatus represents a derived suite of morphologies having different functional consequences that represent a series of compromises among the multiple functional demands placed on it during various feeding behaviors. My findings from the comparative analysis of TMJ articular morphology provide additional evidence that the marmoset masticatory apparatus is a complex structure reflecting multiple functional roles. Whereas numerous features of the marmoset masticatory apparatus have been shown to facilitate increased capacity for gape, other features have been demonstrated to provide increased load resistance abilitiy. Our results suggest that the articular cartilage of the anterior glenoid represents one of these derived morphologies providing increased load resistance abilities linked to novel loading regimes in TMJ during gouging.

Similarly, the derived wedge-like morphology of the anterior dentition of marmosets provides improved cutting performance (Vinyard et al., 2012) and increased enamel decussation provides increased load resistance abilities in the teeth (Rosenberger, 1978; Nogami and Natori, 1986; Hogg et al., 2011).

Collectively, these morphologies highlight the complex functional challenges that

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tree gouging poses to marmosets and their masticatory apparatus as well as the complex nature of potential morphological adaptations in the primate head.

Ontogenetic Allometry and Heterochrony

Using an ontogenetic approach to compare patterns of relative craniofacial growth among species, we hypothesize that cranial features associated with wide gapes will be present early in ontogeny as compared to non-gouging S. fuscicollis. This hypothesis follows reasonably from the observation that juvenile and sub-adult marmosets gouge trees (Soini, 1988; Stevenson and Rylands,

1988). Utilizing heterochrony analysis, we also examine variation in the timing of shape changes in craniofacial features during ontogeny in C. jacchus as compared to the closely-related S. fuscicollis. Because C. jacchus possess a specialized skull morphology to gouge trees at wide gapes, we hypothesize that cranial features associated with wide gapes that are larger in marmosets will be peramorphic and display earlier onset in growth or a faster rate of growth in order to achieve functional competency for gouging at the time of weaning. Similarly, the traits that are predicted to be smaller will be paedomorphic and display delayed onset or a slower rate of growth.

The results of this study demonstrate differential patterns of ontogenetic growth both within the masticatory apparatus of tree gouging marmosets and when compared to the masticatory apparatus of a closely-related non-gouging tamarin species. These results suggest a series of individual ontogenetic

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changes to the marmoset masticatory apparatus that facilitates gouging at wide gapes.

The absence of a singular, overarching change to the pattern of masticatory ontogeny both among and within gape-related and load-resistance features suggests a mosaic pattern of ontogenetic changes in the marmoset masticatory apparatus related to tree gouging. This hypothesis is further supported by the fact that several of these features with different ontogenetic alternations are included in the same functional and morphological modules of the masticatory apparatus. This mosaic pattern does not support an interpretation that features related to wide gapes in marmosets appear as a result of a single (or small number) of global change(s) to the masticatory apparatus.

Given these different changes in masticatory ontogeny, the likely pattern of mosaic development implies that the observed suite of marmoset masticatory apparatus traits did not result from a single pleiotropic shift globally affecting the features of the marmoset masticatory apparatus. Alternatively, a series of distinct developmental shifts that facilitated morphological adaptation to tree gouging at wide gapes appears more likely. It is likely, based on evidence of differential growth trajectories of the various features that facilitate gouging at wide gape, that these developmental shifts occurred over several events. All marmoset species examined to date appear to use wide gapes to habitually gouge trees. The extent of this behavior likely varies relative to overall dietary

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intake of exudates and morphological specialization to this derived feeding behavior. It is unknown, however, if tree-gouging is a hallmark adaptation for marmosets or the manner in which these morphological and behavior changes took place along the marmoset phylogeny. Mapping the morphological changes of the masticatory apparatus associated with gouging at wide gapes onto the marmoset phylogeny may help us to better understand the evolution of this feeding behavior and its impact of masticatory form. Additional data is needed to determine if the developmental patterns observed for marmosets are similarly expressed in other habitual tree gouging species of marmosets.

In addition, the mosaic nature of masticatory shape changes observed for marmosets also supports the potential for developmental interactions both within and between seemingly independent growth modules of the skull resulting in the eventual integration of differential patterns of growth. For example, the observed pattern of differential growth trajectories within both the ascending ramus and corpus modules of the mandible points toward the potential for individual features within a module to develop distinctively in facilitating the adaptation for gouging trees at wide gapes, despite inclusion in seemingly developmentally distinct modules. These results extend previous studies of integration in the mandible focused on spatial definitions of functional modules to include functional relationships from morphologies located at different regions in the mandible (e.g.,

Klingenberg et al 2004; Young and Badyaev, 2006).

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The array of divergent growth patterns observed throughout marmoset ontogeny for gape related features of the masticatory apparatus suggest the existence of an overriding selective pressure in the direction of functional competence for the dietary requirements of gouging trees at wide gapes to elicit exudates flow. Essentially, selection appears to have acted upon multiple morphologies resulting in several, potentially separable adaptive developmental shifts in those features related to opening the jaw widely. However, data concerning neonatal morphological patterns suggest that we cannot completely discount the contribution of physiological effects related to repetitive gouging.

Neonatal Morphological Patterns

It was hypothesized that the masticatory apparatus of neonatal common marmosets would display similar morphological patterns when compared to neonatal tamarins to those observed in comparisons of adults for muscle fiber architecture, cross-sectional geometry of the mandible and resistance to wishboning. The relatively few significant differences for comparisons of muscle fiber architecture and in vitro wishboning between neonatal marmosets and tamarins suggest that functional plasticity during growth contributes to the morphological patterns found in adults. Relative to previous data concerning the ontogenetic allometry and heterochrony of marmoset masticatory apparatus

(Chapter 3), however, the degree to which functional plasticity plays a role is not known. It is possible that the slice of time defined as birth occurs prior to the accelerations in the growth trajectories that result in adult morphological patterns

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of the masticatory apparatus related to facilitating tree gouging at wide gapes.

These results may also reflect differences in the growth trajectories of the different tissues (i.e., bone vs. muscle) that contribute to the adult masticatory apparatus as a whole. Thus, it is reasonable to speculate that the adult marmoset masticatory apparatus includes contributions from both embryological patterning and functional plasticity that vary across the different components of the system. Overall, these analyses underscore the mosaic nature of development of the adult marmoset masticatory apparatus combining features that likely developed early during embryonic patterning as well as those that arose due to functional plasticity associated with the shift to independent feeding behaviors.

Conclusions and Future Directions

The overarching conclusion of the preceding analyses is that the evolution of the masticatory apparatus to exudate feeding in marmosets is a mosaic process likely involving multiple adaptations. This mosaic process is evidenced in terms of the anatomical location, types and timing of changes in marmosets compared to tamarins. Ontogenetic and heterochronic analyses appear to discount the notion of a global, pervasive change to the marmoset masticatory apparatus in adapting to tree gouging at wide gapes. It appears likely that the marmoset masticatory apparatus experiences different functional demands at different stages throughout ontogeny as demonstrated by the structural dissimilarity of features of the neonatal marmoset relative to adult features. In

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addition, it is likely that different functional demands and responses exist throughout the marmoset masticatory apparatus. Overall, the tree gouging of marmosets is largely comprised of morphological changes related to improving gapes rather than increasing force production or resistance to loads. We cannot, however, assume an overall design of the marmoset masticatory apparatus for one functional outcome. Therefore, marmosets provide as an example suggesting that cranial adaptations in primates are the result of a complex interplay of functional demands related to diet and played out over a long evolutionary history.

Additional comparative analysis of the soft tissue of the temporomandibular joint of exudate feeders is needed to further distinguish the effects of differential loading of the joint as it relates to this derived feeding behavior. First, an ontogenetic analysis of the articular cartilage of the TMJ would provide an improved understanding of the roles of potential evolutionary adaptation versus functional plasticity underlying the observed morphologies in adult marmoset soft tissue of the TMJ. Second, understanding the effect of loading at wide gape on the articular cartilage of the TMJ would benefit from more detailed analysis of the cartilage components. To augment the existing findings, the collection of additional data such as collagen type (Collagen Type I vs. Collagen Type II) and fiber orientation, detailed immunohistochemical identification of specific glycosaminoglycan types associated with resistance to compression (i.e., chondroitin sulfate and aggregan) and their distribution as well

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as markers of over-use and degradation (i.e., MMPs, Aggrecanases, VEG-F) can further clarify the reaction of marmoset cartilage to differential loading at wide gapes (Kempson et al., 1970; Kuettner and Kimura, 1985; Mow et al., 1992;

Mizoguchi et al., 1996; Huang et al., 2001; Kuroda et al., 2009; Singh and

Detamore, 2009)..

A major change in feeding behavior and functional demands of the masticatory apparatus occurs at the shift from suckling to independent feeding.

One future step is adding comparative functional analyses of muscle architecture, in vitro load resistance and cross-sectional studies using young, recently weaned animals to bridge the available data for both neonatal and adult specimens. Future analysis of the masticatory muscles, soft tissue of the TMJ and anterior dentition of marmosets focusing on the features of the masticatory apparatus at the time of weaning will further clarify the development of masticatory features associated with gouging at wide gapes. These analyses can address the levels of functional competency for the transition to tree gouging at wide gapes at weaning. Identifying morphological patterns of features associated with gouging at wide gape will further aid in the distinction between the roles of adaptation and functional plasticity in the development of the adult marmoset masticatory apparatus. By focusing on a period when functional competency for gouging at wide gape may be crucial, but has not yet been frequently utilized, we can help clarify the contributions of early developmental

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and plasticity processes underlying the growth of those features associated with gouging at wide gapes.

Adult marmosets have been shown to possess numerous dental adaptations to tree gouging with their anterior teeth. In addition to wedge shaped incisors and incisiform canines, marmosets have been shown to possess an increased degree of dental enamel decussation compared to tamarins

(Rosenberger, 1978; Nogami and Natori, 1986; Hogg et al., 2011). This increased enamel complexity is believed to protect enamel from fracture and is likely a dental adaptation to increased loading of the anterior teeth during tree gouging at wide gapes. The shift to self-feeding in marmosets entails the achievement of functional competency for gouging with the anterior teeth at wide gapes. Weaning often represents a period of nutritional and physiological stress for primate infants. Analysis of dental markers of stress, such as enamel hypoplasia and localized hypoplasia of primary canines in a comparative examination of both tree-gouging marmosets and non gouging tamarins around the time of weaning will provide clues as to the level of competency in each species to adequately access their divergent diets (Lukacs, 2009).

Improved functional knowledge of masticatory features associated with exudativory aids to our understanding of the evolutionary significance of these features and expands our insight into the process of adaptive evolution of primate heads relative to dietary and behavioral specializations. Ultimately, this

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research seeks to add to our understanding of the function and evolution of the primate masticatory apparatus relative to diet and feeding behaviors.

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