Adaptation and Exaptation in the Evolution of the Upper Molar Talon in Microbats

Home , Exaptation, Leporinus, Mammal tooth, Megaderma

(Suborder Microchiroptera)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Sarah Colleen Gutzwiller

Graduate Program in Evolution, Ecology and Organismal Biology

The Ohio State University

2015

Dissertation Committee:

John P. Hunter, Advisor

Debra Guatelli-Steinberg

W. Scott McGraw

Ian M. Hamilton

Sarah Colleen Gutzwiller

2015

Abstract

The talon, the distolingual extension to the upper tribosphenic molar, has evolved convergently many times in therian mammals. In the form of the hypocone cusp, the talon has been identified as a key innovation allowing for the evolution of derived herbivory. However, the adaptive and exaptive significance of the talon prior to it being coopted in derived herbivores is not as well understood. This dissertation explores the potential function(s) of the talon, as it originated as a novel structure (small cingulum or cusp) and expanded into the diversity of shapes and sizes that we see today. This research uses extant microbats (suborder Microchiroptera) as a case study for therian talon evolution, due to the group’s diversity in molar shape and diet.

The first study examines the role of the talon in the balance of crushing and shearing performed by the tribosphenic molar during food breakdown. This project tests the hypothesis that the talon increases crushing function of the molar. Using 3- dimensional computer renderings attained from microcomputed tomography scans of the upper first molars of 26 microbat species, crushing function was estimated using Relief

Index. Relief Index quantifies the relationship between occlusal surface area and projected area, resulting in an estimate of the overall occlusal topography and crushing ability. Talons across all dietary (frugivore, insectivore, etc.) and hardness (hard or soft

ii food) specialists were found to increase the crushing function performed by the tribosphenic molar, supporting the hypothesis. The crushing function of the talon may provide a benefit to a frugivorous mammal that relies greatly on crushing for the breakdown of fruit pulp. However, the benefit of a crushing-dominated talon to other dietary groups is not yet fully understood.

The second study examines talon size evolution, primarily focusing on the role of the talon in mammalian body size evolution. This research tests the hypothesis that the talon functions to increase occlusal area in order to meet the metabolic demands of larger body size. Using a scaling approach that regressed projected and surface area datasets with body mass estimates attained from the literature, the results suggest that in many cases, talon size scales with body size. This relationship lends support to the hypothesis that increasing talon size helps to meet the occlusal requirements of a larger body size.

The effects of dietary group and body mass range are discussed.

The third study explores the role of the talon in the maintenance of tooth strength, testing the hypothesis that the talon functions in decreasing the likelihood of enamel cracking. Using a computer simulation technique called Finite Element Analysis, I measured the amplitude and distribution of maximum stress (an estimate of likelihood of tooth cracking) in theoretical and extant species models with a variety of talon morphologies. These analyses were performed for both hard food and soft food chewing simulations. Results suggest that the presence of the talon variably affects tooth strength, depending on talon size and simulation type. The talon provides the most benefit to tooth

iii strength during the chewing of soft food, supporting the hypothesis that the talon may function in the maintenance of tooth strength.

These studies suggest that both food breakdown and tooth strength functions are plausible for the talon, but depend on talon size, in addition to species’ dietary group and body mass. In the context of therian molar evolution, the convergent origins and elaborations of the talon likely represents a series of adaptations and exaptations reflecting changes in diet and body size.

Dedication

This dissertation is dedicated to my husband, Daniel Hill. Thank you for your never-ending support and love along the way.

Acknowledgments

I would like to thank my advisor, John Hunter, for his patience, support, and general good nature throughout my PhD. His guidance and insight helped me through many a “crisis”, to ultimately be able to finish this dissertation.

Thank you to my committee for always being happy to offer your time and feedback. Thank you to the Department of Evolution, Ecology, and Organismal Biology and to the College of Arts and Sciences. Thank you to my fellow graduate students for your intellectual support, not-so-intellectual good laughs, and general friendship. Thank you to David Gutzwiller for your advice and willingness to jump in and save the day.

Thank you to Zachary Weisberg for help with data collection.

Specimens were kindly provided by Angelika Nelson, curator of The Ohio State

University, Museum of Biological Diversity; Cody Thompson, collections manager of the

University of Michigan, Museum of Zoology; and Sue McLaren, collections manager of the Carnegie Museum of Natural History. Thank you to the staff of the Wright Center for

Biomedical Imaging at The Ohio State University, with special consideration to Michelle

Williams, who microCT scanned my specimens.

Vita

2006...... Walnut Hills High School

2010...... B.S. Biology, Ohio University

2010 to present ...... Graduate Teaching Associate, Department

of Evolution, Ecology, and Organismal

Biology, The Ohio State University

Publications

Gutzwiller, S., O’Connor, P., and Su, A. 2013. Postcranial pneumaticity and bone structure in two clades of neognath birds. The Anatomical Record 296:867–876.

Fields of Study

Major Field: Evolution, Ecology and Organismal Biology

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Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1. Introduction: The Upper Molar Talon ...... 1

Objectives ...... 6

Figures ...... 7

Literature Cited ...... 11

Chapter 2. Occlusal Relief and Function of the Upper Molar Talon ...... 14

Introduction ...... 14

Materials and Methods ...... 18

Results ...... 24

Discussion ...... 26 viii

Figures ...... 31

Tables ...... 35

Literature Cited ...... 38

Chapter 3. Upper Molar Talon Size Evolution ...... 42

Introduction ...... 42

Materials and Methods ...... 49

Results ...... 53

Discussion ...... 56

Figures ...... 62

Tables ...... 66

Literature Cited ...... 70

Chapter 4. Upper Molar Talon and Tooth Strength ...... 73

Introduction ...... 73

Materials and Methods ...... 79

Results ...... 85

Discussion ...... 88

Figures ...... 96

Tables ...... 99

Literature Cited ...... 101

Chapter 5. Conclusions: Adaptation and Exaptation ...... 104

Comprehensive Literature Cited ...... 109

Appendix A: Raw RFI Data ...... 117

Appendix B: Mean RFI and CV ...... 119

Appendix C: Faunivore Projected Area Scatterplot ...... 122

Appendix D: Frugivore Projected Area Scatterplot ...... 123

Appendix E: Faunivore Surface Area Scatterplot ...... 124

Appendix F: Frugivore Surface Area Scatterplot ...... 125

Appendix G: Faunivore Trigon Projected Area and Body Mass PIC ...... 126

Appendix H: Faunivore Talon Projected Area and Body Mass PIC ...... 127

Appendix I: Frugivore Trigon Projected Area and Body Mass PIC ...... 128

Appendix J: Frugivore Talon Projected Area and Body Mass PIC ...... 129

Appendix K: Faunivore Trigon and Talon Surface Area and Body Mass PIC ...... 130

Appendix L: Frugivore Trigon and Talon Surface Area and Body Mass PIC ...... 131

List of Tables

Table 1. Chapter 2 Species Information ...... 35

Table 2. RFI Descriptive Statistics ...... 36

Table 3. Dietary Comparison P-values ...... 37

Table 4. Chapter 3 Species Information ...... 66

Table 5. Phylogenetic Signal P-values...... 69

Table 6. Theoretical Model Max Stresses...... 99

Table 7. Extant Model Max Stresses ...... 100

Table 8. Raw RFI Data ...... 117

Table 9. Mean RFI and CV ...... 119

List of Figures

Figure 1. Tribosphenic Molar with a Talon ...... 7

Figure 2. Upper and Lower Tribosphenic Molar ...... 8

Figure 3. Character Map of Talon Shape ...... 9

Figure 4. Character Map of Talon Size ...... 10

Figure 5. Representative Molars with Talons ...... 31

Figure 6. Molar in Functional View ...... 32

Figure 7. Relief Index and Diet...... 33

Figure 8. Relief Index and Hardness...... 34

Figure 9. Molar Area Scaling...... 62

Figure 10. Projected Area Measurement ...... 63

Figure 11. Projected Area Scaling Coefficients ...... 64

Figure 12. Surface Area Scaling Coefficients...... 65

Figure 13. FEA Design ...... 96

Figure 14. Hard Food Contour Maps ...... 97

Figure 15. Soft Food Contour Maps ...... 98

Figure 16. Faunivore Projected Area Scatterplot ...... 122

Figure 17. Frugivore Projected Area Scatterplot ...... 123

xii

Figure 18. Faunivore Surface Area Scatterplot...... 124

Figure 19. Frugivore Surface Area Scatterplot ...... 125

Figure 20. Faunivore Trigon Projected Area and Body Mass PIC ...... 126

Figure 21. Faunivore Talon Projected Area and Body Mass PIC...... 127

Figure 22. Frugivore Trigon Projected Area and Body Mass PIC ...... 128

Figure 23. Frugivore Talon Projected Area and Body Mass PIC ...... 129

Figure 24. Faunivore Trigon and Talon Surface Area and Body Mass PIC ...... 130

Figure 25. Frugivore Trigon and Talon Surface Area and Body Mass PIC ...... 131

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Chapter 1. Introduction: The Upper Molar Talon

Adaptation (a trait brought about by natural selection that enhances fitness) and exaptation (a trait previously evolved, coopted for a new use) are concepts fundamental to the study of evolution (Gould and Vrba, 1982;Van Valen, 2009). While these processes can be difficult to directly observe, the study of functional morphology allows for a comparative framework in which adaptative and exaptive hypotheses can be tested.

This dissertation is an exploration of adaptation and exaptation in the succession of tooth forms in therian mammals (the living metatherians and eutherians, and their extinct relatives). Using extant microbats (suborder Microchiroptera) as a case study, this research investigates the adaptive and exaptive implications of the origin and elaboration of the talon, a distolingual addition to the upper tribosphenic molar complex (Fig. 1).

The talon has evolved more than 20 times independently in therian mammals

(Van Valen, 1994; Hunter and Jernvall, 1995; Jernvall, 1995; Butler, 2001). Small shelf- like talons, also called cingula or hypocone shelves, first appeared in the late Cretaceous

(Hunter and Jernvall, 1995). Since that time, the talon has expanded in some clades, taking on a variety of shapes and sizes, including large hypocone basins and cusps

(Hunter and Jernvall, 1995). Previous work has found that clades possessing talons tend to have higher species richness through time than clades without them (Hunter and

Jernvall, 1995). Additionally, fully-formed hypocone cusps have often been further

1 coopted into more specialized morphology (specifically, the addition of elongate ridges between the cusps), suggesting that the hypocone is a key innovation for derived herbivory (Hunter and Jernvall, 1995; Jernvall, 1995; Hunter, 1998; Crompton et al.,

2010). However, little is understood about this diverse region’s adaptive and exaptive significance prior to specialization in derived herbivores. How did the talon originate as a novel molar feature and subsequently elaborate into the diverse morphology seen in living therians? What function(s) does the talon perform within the therian molar?

In order to explore the evolution of the talon, we must first understand the shape and function of the ancestral condition. The primitive therian upper molar is a three- cusped (“tritubercular”) molar that performs a combination of shearing and crushing during food breakdown (“tribosphenic”; Simpson, 1936; Hillson, 1986). The trigon of the upper molar consists of three primary cusps (the paracone, metacone, and protocone), their associated crests, and a basin in the center (Fig. 2; Osborn, 1888; Bown and Kraus,

1979; Hillson, 1986; Butler, 1990; Butler, 2000). Each cusp has two shearing crests extending from it, totaling six shearing surfaces on the upper tribosphenic molar

(Crompton and Kielan-Jaworowska, 1978). In between the cusps on the upper molar lies the trigon basin that occludes with an extension of the lower molar (the talonid). The lower molar consists of the primitive trigonid and later-evolving talonid, each including three cusps and their shearing surfaces (Crompton and Kielan-Jaworowska, 1978). The paraconid, protoconid, and metaconid cusps lay on the trigonid. The entoconid, hypoconid, and hypoconulid cusps surround the talonid basin.

The study of wear facets on the fossil teeth of Cretaceous therians and chewing behaviors of living species has allowed for an understanding of the occlusal patterns of tribosphenic molars (Crompton and Hiiemae, 1969; Butler, 1972; Crompton and Kielan-

Jaworowska, 1978). During the Phase I power stroke, the lower molars move upwards, anteriorly, and medially towards the upper molars (Crompton and Hiiemae, 1969;

Crompton and Kielan-Jaworowska, 1978). The shearing crests of the trigon and talonid move past each other and cut the food (Crompton and Kielan-Jaworowska, 1978). A second wave of shearing occurs when the trigonid moves past the anterior crest of the paracone and into the space anterior to the upper molar. Movement halts at centric occlusion, when the protocone of the upper molar fits into the talonid basin of the lower, resulting in crushing of the food (Crompton and Hiiemae, 1969). Phase II occurs after centric occlusion, as the lower molar moves anteriorly, medially, and downwards, resulting in the grinding of the protocone against the talonid (Kay and Hiiemae, 1974).

Several authors have suggested that in certain species, including bats, food breakdown is minimal during Phase II grinding and instead the majority of food breakdown is due to the shearing and crushing performed in Phase I (Crompton and Hiiemae, 1969; Teaford and Walker, 1984; Hylander et al., 1987; Wall et al., 2006). The shearing function of the tribosphenic molar is useful for the breakdown of tough insect cuticle. Primitive tribosphenic therians are thought to have been insectivores, specialized for the chewing of soft-bodied insects, such as moths (Lucas, 2004). The crushing function provided by the protocone occluding in the talonid basin may have allowed for increased consumption of hard insects, such as beetles, as well (Lucas, 2004). Suborder Microchiroptera is a

3 representative focal group of therian talon evolution, because the primitive condition for microbats is insectivory with a tribosphenic molar shape and microbats have since diversified into a variety of dietary specializations and tooth morphologies (Gunnell and

Simmons, 2005).

Order Chiroptera originated around 65 million years ago (Rose, 2006; Baker et al., 2012). There are now over one thousand living species of bats in eighteen families

(Gunnell and Simmons, 2005). Traditionally, two chiropteran suborders are recognized.

Suborder Microchiroptera (microbats) includes seventeen families and suborder

Megachiroptera (megabats) consists of one family (Pteropodidae). Microbats and megabats diverged relatively early in the evolution of bats (probably in the Eocene), evolving dietary specializations independently (Phillips, 2000; Rose, 2006; Giannini et al., 2012).

The diversity in microbat diet, body size, and molar shape allows for a comparative study to explore the factors influencing talon origination and elaboration within therian mammals. Thirteen of the seventeen microbat families are strict insectivores, suggesting that strict insectivory is the primitive diet for microbats (Van

Cakenberghe et al., 2002; Baker et al., 2012). Species within Megadermatidae,

Nycteridae, Noctilionidae, and Vespertilionidae evolved derived carnivory. One family,

Phyllostomidae, experienced vast dietary diversification and now includes insectivore, omnivore, nectivore, carnivore, sangivore, and frugivore specialists.

Microbats are generally small-bodied, especially when compared to some megachiropteran species. However, body mass varies within living microchiropterans,

4 ranging from 2 g (Craseonycteris thonglongyai) to 169 g (Vampyrum spectrum; Giannini et al., 2012). Early Eocene microbats were small to mid-ranged when compared to living microbats, with estimates varying from 7 g (Palaeochiropteryx tupaiodon) to 90 g

(Hassainycteris magna). Giannini et al. (2012) estimated that the primitive microbat ancestor weighed between 10 and 14 g at the time of diversification from megabats. A unidirectional trend in change to body size, however, is not observed in microchiropterans, and changes in body size instead reflect clade specific behavioral and ecological factors (aerial foraging, prey size, etc.).

Due to the poor fossil record for bats, determining when the first talon evolved in bats is difficult. However, by the early Eocene several North American and European bats possessed a hypocone shelf (Hunter and Jernvall, 1995). Extant microbats primarily retain the tribosphenic shape, but many species (especially within the phyllostomids) now exhibit a wide range of molar shapes. These changes in shape not only include the addition of talons of various shapes and sizes, but also changes in the shape of the trigon for specialization in derived diets. Figures 3 and 4 are character maps showing the wide diversity of talon shapes and sizes across the family level phylogeny of microbats

(created from a personal survey of over 200 species, using the Jones et al., 2002 tree).

These maps show that despite the majority of microbat families being strict insectivores, talon diversification has occurred across the microbat phylogenetic tree. The diversity of talons observed in microbats begs the question: What role is the talon performing in microbats? How might the addition of this diverse array of talons affect the functional balance of the molar complex within microbats?

Objectives

This dissertation aims to explore three possible functions of the talon in microbats. Two studies focus on the role of the talon in food breakdown. The first study

(Ch. 2) examines what talon shape can reveal about its effect on the crushing and shearing functional balance of the tribosphenic molar. The second study (Ch. 3) explores talon size evolution, specifically focusing on the talon’s potential role in maintenance of occlusal function at larger body sizes. Finally, the third study examines an alternative talon function, unrelated to food breakdown. This study (Ch. 4) uses theoretical modeling techniques to examine the talon’s role in the maintenance of tooth strength in the face of the high impact of chewing. The results of this research help to characterize the function of the talon in microbats and, in doing so, better understand the adaptive and exaptive significance of the talon, as it originated as a novel structure and subsequently expanded into a diversity of shapes during therian upper molar evolution.

Figures

Figure 1. Tribosphenic Molar with a Talon A representative tribosphenic molar with a talon (Rhinolophus ferrum-equinum, UMMZ 88609) in functional view. Abbreviations: (An) anterior, (Bu) buccal, (ec) ectoloph, (Li) lingual, (me) metacone, (pa) paracone, (Po) posterior, (popc) postprotocrista, (pr) protocone, (prpc) preprotocrista, (ta) talon, (tr) trigon, (trb) trigon basin, (UMMZ) University of Michigan Museum of Zoology. Scale = 1mm.

Figure 2. Upper and Lower Tribosphenic Molar The upper and lower tribosphenic molar in functional view. Abbreviations: (An) anterior, (Bu) buccal, (End), entoconid, (Hyd) hypoconid, (Hyld) hypoconulid, (Li) lingual, (Me) metacone, (Med) metaconid, (Pa) paracone, (Pad) paraconid, (Po) posterior, (Pr) protocone, (Prd) protoconid, (TaB) talon basin, (TrB) trigon basin.

Figure 3. Character Map of Talon Shape Character map of microbat talon shape, using the Jones et al. (2002) phylogenetic tree.

Figure 4. Character Map of Talon Size Character map of microbat talon size, using the Jones et al. (2002) phylogenetic tree.

Literature Cited

Baker RJ, Bininda-Emonds ORP, Mantilla-Meluk H, Porter CA, Van Den Bussche RA. 2012. Molecular time scale of diversification of feeding strategy and morphology in New World Leaf-Nosed bats (Phyllostomidae): A phylogenetic perspective. In: G.F. Gunnel and N.B. Simmons, editors. Evolutionary history of bats: fossils, molecules, and morphology. Cambridge: Cambridge University Press. p385-409.

Bown TM, Kraus MJ. 1979. Origin of the tribosphenic molar and Metatherian and Eutherian dental formulae. In: J.A. Lillegraven, A. Keilan-Jaworowska, and W.A. Clemens, editors. Mesozoic mammals: the first two-thirds of mammalian history. Berkeley: University of California Press. p172-181.

Butler PM. 1972. Some functional aspects of molar evolution. Evol 26:474-483.

Butler PM. 1990. Early trends in the evolution of tribosphenic molars. Biol Rev 65:529- 552.

Butler PM. 2000. The evolution of tooth shape and tooth function in primates. In: M.F. Teaford, M.M. Smith, and M.W.J. Ferguson, editors. Development, function, and evolution of teeth. Cambridge: Cambridge University Press. p201-211.

Butler PM. 2001. Evolutionary transformations in the mammalian dentition. Zoosyst and Evol 77:167-174.

Crompton AW, Hiiemae K. 1969. Functional occlusion in tribosphenic molars. Nature 222:678-679.

Crompton AW, Kielan-Jaworowska Z. 1978. Molar structure and occlusion in Cretaceous Therian mammals. In: B.M. Butler and K.A. Joysey, editors. Development, function, and evolution of teeth. New York: Academic Press Inc. p249-287.

Crompton AW, Owerkowics T, Skinner J. 2010. Masticatory motor pattern in the koala (Phascolarctos cinereus): a comparison of jaw movements in marsupial and placental herbivores. J Exper Zool 313:1-15.

Giannini NP, Gunnel GF, Habersetzer J, Simmons NB. 2012. Early evolution of body size in bats. In: G.F. Gunnel and N.B. Simmons, editors. Evolutionary history of bats: fossils, molecules, and morphology. Cambridge: Cambridge University Press. p353- 384.

Gould SJ, Vrba ES. 1982. Exaptation – a missing term in the science of form. Paleobiol 8:4-15.

Gunnel GF, Simmons NB. 2005. Fossil evidence and the origin of bats. J Mammal Evol 12:209-246.

Hillson S. 1986. Teeth. Cambridge: Cambridge University Press.

Hunter JP. 1998. Key innovations and the ecology of macroevolution. Trends Ecol Evol 13:31-36.

Hunter JP, Jernvall J. 1995. The hypocone as a key innovation in mammalian evolution. Proc Natl Acad Sci 92:10718-10722.

Hylander WL, Crompton AW, Johnson KR. 1987. Loading patterns and jaw movements during mastication in Macaca fascicularis: a bone-strain, electromyographic, and cineradiographic analysis. Amer J Phys Anthro 72:287-312.

Jernvall J. 1995. Mammalian molar cusp patterns: developmental mechanisms of diversity. Acta Zoologica Fennica 198:1-61.

Jones KE, Purvis A, MacLarnon A, Bininda-Emonds ORP, Simmons NB. 2002. A phylogenetic supertree of the bats (Mammalia: Chiroptera). Biol Rev 77:223-259.

Kay RF, Hiiemae KM. 1974. Jaw movement and tooth use in recent and fossil primates. Amer J Phys Anthro 40:227-256.

Lucas PW. 2004. Dental function morphology: how teeth work. Cambridge: Cambridge University Press.

Osborn HF. 1888. The evolution of the mammalian molar to and from the tritubercular type. Amer Naturalist 22:1067-1079.

Phillips CJ. 2000. A theoretical consideration of dental morphology, ontogeny, and evolution in bats. In: R. Adams and S. Pedersen, editors. Ontogeny, functional ecology, and evolution of bats. Cambridge: Cambridge University Press. p247-274.

Rose KD. 2006. The beginning of the age of mammals. Baltimore: The Johns Hopkins University Press.

Simpson GG. 1936. Studies of the earliest mammalian dentitions. Dental Cosmos 78:791- 800.

Teaford MF, Walker A. 1984. Molar microwear and diet in the genus Cebus. Amer J Phys Anthro 66:363–370.

Van Cakenberghe V, Herrel A, Aguirre LF. 2002. Evolutionary relationships between cranial shape and diet in bats (Mammalia: Chiroptera). In: P. Aerts, K. A. D’Août, A.

Herrel, and R. Van Damme, editors. Topics in functional and ecological vertebrate morphology. Shaker Publishing. p205-236.

Van Valen LM. 1994. Serial homology: the crests and cusps of mammalian teeth. Acta Palaeontologica Polica 38:145-158.

Van Valen LM. 2009. How ubiquitous is adaptation? A critique of the epiphenomenist program. Biology and Philosophy 24:267-280.

Wall CE, Vinyard CJ, Johnson KR, Williams SH, Hylander WL. 2006. Phase II jaw movements and masseter muscle activity during chewing in Papio anubis. Amer J Phys Anthro 129:215-224.

Chapter 2. Occlusal Relief and Function of the Upper Molar Talon

*in press in the Journal of Morphology

Introduction

Comparative morphology provides a framework for identifying evolutionary adaptations, traits built by natural selection (see Van Valen, 2009). Here, I provide a case study of adaptation in the evolution of molar tooth forms in therian mammals (the living metatherians and eutherians, and their extinct relatives). The traditional views of mammalian molar evolution highlight transitions in molar shape and function that ultimately resulted in key evolutionarily changes (clade diversification, expansion into new niches, etc.; Butler, 1972; Hunter and Jernvall, 1995). One such transition is the expansion of the talon, a distolingual extension to the tribosphenic upper molar. The best known and studied example of talon expansion is the convergent evolution of the hypocone (Hunter and Jernvall, 1995; Sanchez-Villagra and Kay, 1996). But expansion of the talon into a well-developed main cusp is only one of several evolutionary paths realized among therian mammals. This study examines the effect of the expansion of the talon on the function of the tribosphenic upper molar, in order to explore its adaptive significance in therian mammals.

The tribosphenic molar is thought to have evolved twice, with origins in both the australosphenidans (extant monotremes and their extinct relatives) on the southern continents in the late Jurassic and in the therians on the northern continents in the early

Cretaceous (Luo et al., 2001; see Davis, 2011 for an alternative view). The three-cusped upper molar is defined by its signature W-shaped ectoloph and when brought together with the lower molar performs a dual-function in food breakdown (Fig. 2; Simpson,

1936). The protocone cusp of the upper molar and the talonid basin of the lower molar come together to perform a crushing function (apply a compressive stress to food), whereas the ectoloph and the crests running from the protocone (pre- and postprotocrista) mark sites of shearing (applying a shear stress to the food) against the lower corresponding features (Butler, 1972, 1990; Sibbing, 1991; Spears and Crompton, 1996).

It should be noted that this dual-function seen in the tribosphenic molar is analogous to molars of more primitive lineages. Shuotheriids possessed molars that were remarkably tribosphenic-like, but with the lower “pseudotalonid” forming on the mesial surface instead of the distal one (Luo et al. 2007; Davis, 2011). Docodont molars possessed a large upper lingual cusp that occluded with a lower basin, resulting in a similar dual- function as the tribosphenic molar (Patterson, 1956; Keilan-Jaworowska et al., 2004;

Davis, 2011).

The tribosphenic molar is retained in many extant therians and, in others, constitutes the baseline to which a variety of modifications, such as additions to the distolingual region, have occurred. Postcingulum, lingual cingulum, hypocone basin, hypocone wing, hypocone, metaconule, and talon are all terms that have been used to

15 describe various features in this quadrant of the upper molar (Hunter and Jernvall, 1995;

Jernvall, 1995; Freeman, 1998; Czaplewski et al., 2008). In order to examine the vast diversity of features in this region, the present study uses the regional term “talon” to describe any extension posterior and lingual to the postprotocrista (see Fig. 1, as in

Osborn, 1907 and Czaplewski et al., 2008). Under this definition, the talon may include all varieties of features in this region, from a small cingulum to a large cusp. The use of the term talon in this manner facilitates the use of the corresponding term “trigon”, describing the portion of the molar containing the three primitive tribosphenic cusps (Fig.

1). Being able to separate each molar into the trigon and talon regions allows for an explicit examination of the functional evolution of the distolingual region with respect to the rest of the molar.

Beginning small and cingulum-like or as a displaced metaconule, there was very little in the way of expansion of the talon among therians in the late Cretaceous (Butler,

1972; Hunter and Jernvall, 1995; Jernvall, 1995). A diversity of opinions has been offered regarding the function of the early stages of talon expression. For example,

Crompton and Keilan-Jaworowska (1978) suggested that the small postcingulum in

Kennalestes shears against the protoconid of the lower molar, whereas the larger postcingulum of Gypsonictops crushes against the paraconid of the lower molar. Kay and

Hiiemae (1974) proposed that the small hypocone cusp of Palenochtha functions as part of a “compression cylinder” complex to maximize crushing performed by the protocone and hypoconid. In some clades during the Paleogene, the talon expanded to completely fill in the distolingual space as a large cusp (e.g. primates) or shelf (e.g. shrews and some

16 bats; Butler, 1972; Hunter and Jernvall, 1995). With this expansion, it has been hypothesized that the presence of the talon squares off the molar and increases the occlusal surface area dedicated to crushing (Butler, 1972; Hunter and Jernvall, 1995;

Jernvall, 1995).

A transition in molar function with the expansion of the talon may have been influenced by changes in ecological factors, especially diet. A growing wealth of research has uncovered clear links between dental shape, dental function, and dietary factors, including diet type (frugivore, insectivore, etc.) and diet physical properties (hardness, toughness, etc.). For example, frugivore molars tend to have blunt occlusal surfaces (low curvature, low relief, and an overall flat topography) and perform a crushing-dominated function, optimized for compressing as many of the fluid-filled cells of fruit pulp as possible with each chew (Freeman, 1988; Lucas, 2004; Boyer, 2008; Bunn and Ungar,

2009; Boyer et al., 2010; Bunn et al., 2011). In contrast, insectivore molars possess shearing crests with high relief capable of cutting insect cuticle for proper digestion by gut enzymes (Strait, 1997; Lucas, 2004; Boyer, 2008; Hogue and ZiaShakeri, 2010; Bunn et al., 2011). Additionally, research has found that species specializing in hard foods tend to have molars of low curvature and relief, where load concentration on the food is located at the cusp tips without compromising tooth strength (Lucas, 2004; Winchester et al., 2014). With this understanding, it seems most likely that if the talon expanded to increase the crushing function of the tribosphenic upper molar in the Paleogene, as hypothesized for a fully cuspal hypocone, its bulbous shape would also be associated with a frugivorous and/or hard-food specialist diet.

The present research uses suborder Microchiroptera, an extant therian clade that illustrates diversity in molar morphology and ecological traits, as a case study for talon evolution in therian mammals. This research aims to test two hypotheses related to talon evolution: (1) the talon increases crushing function of the tribosphenic molar and (2) a crushing talon is associated with a frugivorous and/or hard food diet. Both hypotheses will be addressed by measuring molar Relief Index. Relief Index, a measure of the degree of relief in the crown surface, has been shown to correlate with tooth crushing and shearing function and varies according to dietary factors (Ungar and Williamson, 2000;

Boyer, 2008; Bunn and Ungar, 2009; Boyer et al., 2010; Bunn et al., 2011). By measuring molar Relief Index, this research will (1) confirm the relationship between whole molar relief, function, and diet for microbats, as seen in other mammals, (2) quantify the relief and function of the two components of the upper molar, the trigon and talon, and explore how each component individually relates to diet, and (3) examine the direct impact of possessing a talon by exploring how the presence of a talon of any given shape influences the crushing or shearing functional balance of the entire molar within each species. Ultimately, the main goal of this research is to illuminate the adaptive implications of the evolution of the talon as a component of the tribosphenic upper molar complex, both within the microbats and among the therian mammals.

Materials and Methods

Twenty-six species across seven families were selected for the presence of a talon and in order to capture phylogenetic and dietary diversity within microbats. The diet of

18 each species was categorized according to Van Cakenberghe et al. (2002) and citations therein, unless otherwise cited (see Table 1 for species information). The five dietary groups are carnivore, insectivore, omnivore, nectivore, and frugivore. The frugivores and insectivores were also categorized according to hardness: hard diet and soft diet

(animaldiversity.org; Van Cakenberghe et al., 2002).

Micro-computed Tomography Scanning and 3D Rendering

One to three specimens of each species were borrowed from The Ohio State

University, Museum of Biological Diversity (OSUM) and the University of Michigan,

Museum of Zoology (UMMZ; Table 1). After preliminary results showed minimal intraspecific variation in molar shape, the sampling strategy focused on maximizing the number of species included in the study in order to examine as many microchiropteran molar shapes as possible (see intraspecific variation in Table 9 in Appendix B). Each specimen was selected based on minimal molar wear. An impression of the upper dental arcade of each specimen was made using President Plus, Regular Body Impression

Material (Coltène/Whaledent Inc.). A subsequent cast of the upper left first molar (M1) was created using Four to One, Super Hard Epoxy Resin, Hardener, and Dye (TAP

Plastics Inc.). Each M1 cast was scanned using a micro-computed tomography scanner

(microCT; Inveon, Siemens) at the Wright Center of Innovation in Biomedical Imaging

(The Ohio State University). Scans were performed at 100 kV voltage, 200 mA current, and an effective pixel size of 19.4. The resulting digital volume was imported into Amira

5.4.5 visualization software (Visualization Sciences Group) for three-dimensional rendering.

For each M1, a surface rendering was created in Amira using the Isosurface function. Each rendering was surface-smoothed with 100 iterations following Boyer

(2008) in order to smooth out small artifacts of the scanning process and attain a more accurate model. Each model was then imported into MeshLab 1.3.3

(meshlab.sourseforge.net) for removal of any additional artifacts and for cropping of the surface. In order to minimize the impact of non-occluding parts of the crown on relief measurements, models were cropped to only include surfaces of the upper molar that come into close contact with the lower molar. This process ultimately resulted in the removal of the buccal edge of the molar (which is most likely involved in preventing food slipping away from the occlusal surface rather than actual food breakdown; see

Clemens, 1979) and any under-hanging portions of the cusps (Fig. 5). For each model, two portions of the M1, the trigon and the talon, were separated in order to examine the contribution of each individually to molar function.

In order to gauge the food breakdown function of the talon and trigon, each molar was assessed based on its relative contribution to shearing and crushing. Many metrics have been developed that relate tooth shape to function, including shearing quotient, crest sharpness, and cusp sharpness (Strait, 1993a,b; Ungar and Kay, 1995; Evans, 2005).

However, to some degree these metrics rely upon identification of homologous crests and cusps, which can be difficult to accomplish when species vary greatly in tooth shape.

Instead, the present study used Relief Index (RFI) as an estimate of the shearing and crushing potential of the surface, because this method does not rely upon the definitive location of cusps, crests, and basins, which is greatly useful when examining such a

20 variable feature as the talon (Ungar and Williamson, 2000; Boyer, 2008; Bunn and

Ungar, 2009; Boyer et al., 2010; Bunn et al., 2011). RFI has been shown to be one of the most reliable techniques for differentiating tooth shapes by diet, can be used to examine possible diets of extinct mammals, and correlates with other morphological metrics, such as Dirichlet Normal Surface Energy (DNE; Bunn et al., 2011). Relief Index results in a range of values representing a crushing-dominated function to a shearing-dominated function. A lower RFI means that there is a lesser degree of relief in the crown (i.e. the occlusal surfaces tend to be more perpendicular to the occlusal vector), resulting in a higher crushing function performed. A higher RFI represents a greater degree of relief in the crown (i.e. the occlusal surfaces tend to be more parallel to the occlusal vector), resulting in a higher shearing function performed. Following Boyer (2008), RFI was calculated as:

RFI = ln[√Surface Area / √Projected Area]

For each RFI measurement, surface area was defined as the sum of the areas of all tetrahedral faces of the model surface and was measured for the trigon and talon of each

M1 using Amira. Projected area was defined as the two dimensional area of the model in functional view (with the tooth surface perpendicular to the occlusal vector during Phase

I of the chewing cycle; Fig. 6b). In order to attain a projected area consistent with functional view, attricial wear facets of a representative specimen of each species were examined under a Hirox digital microscope (hirox-usa.com) and functional view was defined as the position in which vertical Phase I shear facets are out of view (as in Fig.

6a; Butler, 1972; Wood and Clemens, 2001; Polly, 2005; Rook et al., 2010). Given that

21 there is likely little, if any, lingual phase (Phase II) during the chewing cycle in bats

(Crompton and Hiiemae, 1969), examining the models in functional view adequately characterizes the functions involved in food breakdown by tribosphenic molars. In

Amira, each rendering was manually positioned in order to match proper functional view and a 2D jpeg “snapshot” of each model was exported into ImageJ (imagej.nih.gov/ij/) for the projected area measurement. For each individual, RFI was calculated for the trigon alone, the talon alone, and the whole tooth (trigon and talon together).

Analyses

Shearing and crushing function

In order to examine the degree of intraspecific variability in molar shape of those species with more than one representative individual (see Appendix B), the

Coefficient of Variation (CV) of the RFI of the talon, trigon, and whole tooth was calculated in Excel 2010 (office.microsoft.com). Species mean values of the RFI of the trigon, talon, and whole tooth were calculated in order to account for any intraspecific variability in molar shape. Mean RFI of the whole tooth, trigon, and talon was also calculated for the entire dataset (no dietary groupings) and for each dietary and hardness group. In order to examine how the function of the whole tooth, and of the trigon and talon individually, vary according to dietary factors, differences in RFI among dietary and hardness groups were analyzed using pairwise phylogenetic ANOVAs in the Geiger package (version 1.99-3.1) in R (version 2.15.2; cran.r-project.org) with the Jones et al.

(2002) chiropteran phylogenetic tree. During this analysis, significance is assessed with a randomization test in which new sets of dependent variables are simulated on the

22 phylogenetic tree under a Brownian-motion model. This process allowed us to account for phylogenetic relationships that may influence molar shape (Garland, 1993; Harmon,

2008).

It should be noted that a higher alpha level for the phylogenetic ANOVA was needed to make dietary distinctions in RFI, reflecting the way that diets have evolved in microbats. While there is great dietary diversity represented within Microchiroptera, frugivores are present in only one family, Phyllostomidae (Nowak, 1994; Van

Cakenberghe et al., 2002). This phylogenetic grouping of frugivores suggests that frugivory (and potentially any dental morphology associated with it) arose only once in basal phyllostomids (Phillips, 2000; Dumont et al., 2012). Thus, the correlation between overall molar shape and preferred diets (particularly frugivory and nectivory) hasn’t arisen independently in microbats. As a result, discerning patterns in the evolution of molar shape and diet is difficult in microbats, as a lack of phylogenetic diversity in diet reduces statistical power. Given the reduced statistical power for this microbat dataset, an alpha level of 0.10 was used in all phylogenetic ANOVAs.

Effect of the talon on molar function

While RFI estimates shearing and crushing function according to molar shape, it cannot account for the relative size of the trigon and talon, which determines the extent to which both components contribute to overall molar function. Thus, to examine if the presence of a talon of any given shape significantly affects overall molar function,

RFI was compared within each species for models with the talon present (the whole tooth) and models with the talon removed (the trigon alone), using a paired samples t-test

23 in SPSS. This analysis was conducted for the entire dataset (no dietary groupings), and for each dietary and hardness group. A significant difference between whole tooth and trigon RFI suggests that, when present, the talon significantly changes the shearing/crushing function of the tooth.

Results

Raw specimen RFI data is presented in Appendix A. Intraspecific variability in

RFI for the trigon, talon, and whole tooth is generally low, with CV ranging from ~0% in

Asellia tridens to 26% in Macrotus waterhousii (Appendix B). For the entire dataset (no dietary groupings), species mean RFI of the whole tooth ranges from 0.41 to 0.74 with a mean of 0.56 (Table 2). RFI of the trigon alone ranges from 0.44 to 0.76, with a mean of

0.62. RFI of the talon alone ranges from 0.17 to 0.63, with a mean of 0.32.

Shearing and Crushing Function

Diet

See Table 2 for mean RFI of the whole tooth, trigon, and talon for all dietary and hardness groups. RFI of the whole tooth is significantly lower in frugivores than in insectivores (p=0.059) and omnivores (p=0.078; Fig. 7; Table 3). RFI of the trigon is significantly lower in frugivores than in carnivores (p=0.059) and insectivores

(p=0.02) and significantly lower in nectivores than in carnivores (p=0.098) and insectivores (p=0.02). There is no significant difference in the RFI of the talon across dietary groups (Fig. 7; p=0.489). It should be noted, however, that all phylogenetic

ANOVA p-values among diet groups are nonsignificant if post-hoc p-value adjustments are used (see discussion).

Food Hardness

The RFI of the whole tooth and RFI of the trigon do not significantly differ between hard food eaters and soft food eaters (Fig. 8; whole tooth p≈1; trigon p=0.921). However, RFI of the talon is higher in hard food eaters than soft food eaters

(p=0.098).

Effect of the Talon on Whole Tooth Function

For each species, whole tooth RFI is significantly lower than trigon RFI, suggesting that the presence of the talon significantly lowers RFI of the molar (by an average of 0.05 across species, p<0.001). This result also generally holds true when species are categorized by diet. With the presence of the talon, RFI is lowered to the greatest degree in carnivores (mean reduction in RFI of 0.10, p=0.04) and insectivores

(mean reduction in RFI of 0.06, p=0.001). RFI is lowered to a lesser extent in frugivores

(mean reduction of 0.03, p<0.001) and nectivores (mean reduction of 0.01, p=0.04). RFI is not significantly affected by talon presence in omnivores (p=0.213). Both hard and soft food eaters also exhibit a significant reduction in RFI with the presence of the talon (hard food eater RFI mean reduction of 0.04, p<0.001; soft food eater RFI mean reduction of

0.06, p=0.008).

Discussion

Whole Tooth Function and Diet

The patterns in microchiropteran molar shape observed in this study are consistent with the results of previous work on other mammal groups, including tree shrews, flying lemurs, prosimian primates, Old World monkeys, and New World monkeys (Boyer,

2008; Bunn and Ungar, 2009; Bunn et al., 2011; Winchester et al., 2014). Relief Index is sufficient to distinguish dietary groups, with the most notable trend being that frugivores consistently exhibit lower molar relief than some other dietary groups (including insectivores and omnivores in this study and insectivores, omnivores, and folivores in other studies). Indeed the mean whole tooth RFI of the frugivorous microbats in this study (0.46) is very similar to the mean RFI (0.411) for frugivorous prosimians observed in Winchester et al. (2014). This suggests that frugivorous molars are more crushing- dominated in function, whereas the molars of other dietary groups, such as insectivores and omnivores, rely more upon shearing. Unlike previous work, the present study also examined carnivores and nectivores. Results suggest that carnivores have high molar relief quite similar to insectivores and omnivores, consistent with carnivores relying on shearing for the breakdown of tough vertebrate tissue (Freeman, 1984, 1998).

Conversely, nectivores represent an intermediate RFI for the whole molar among the dietary groups. Overall molar reduction with the evolution of long protruding tongues and a decreased reliance on mastication may account for the intermediate molar relief, not dominated by a crushing or shearing function, observed in nectivores (Freeman,

1988; Dumont, 1997). Additionally, the supplementation of insects in the nectivorous diet

26 may also account for intermediate molar relief (Nowak, 1994). No differences in whole tooth RFI were observed for hard and soft food eaters, suggesting that food hardness does not significantly influence molar relief for these microbats.

Trigon Function and Diet

Whereas clear dietary distinctions can be observed when examining the molar as a whole, only the trigons exhibited this dietary partitioning when these microbat molars were separated into trigons and talons. Indeed the relief of the trigon alone was able to distinguish dietary groups better than the whole molar, additionally differentiating nectivores from carnivores and insectivores and frugivores from carnivores. This suggests that the shape of the trigon has been evolutionarily influenced by the functional demands of different diets and likely plays a greater role than the talon in the shearing and crushing functional balance of the molar. Again, no differences in trigon relief between hard food and soft food eaters were observed, suggesting that food hardness does not greatly influence trigon relief.

Talon Function and Diet

Unlike the trigon, variation in talon shape did not relate to dietary category in this study. Instead, the consistent low relief of the talon suggests that it generally performs a crushing function, even across different dietary groups. In fact, in all dietary groups other than omnivores, when the talon is manually removed from the model, molar relief increases, resulting in a reduction of crushing function. This effect suggests that when the talon is present, the combination of its shape and relative size significantly contributes to the crushing function of the entire molar, consistent with my hypothesis. Whereas, talons

27 were generally found to exhibit low relief and a crushing function, there was a slight, but statistically significant, difference in talon relief between hard food eaters and soft food eaters. Contrary to the hypothesis that hard food eaters would exhibit lower talon relief, hard food eaters actually had higher talon relief than soft food eaters. However, given that there is no difference in Whole Tooth RFI between the hardness groups, this slight difference in talon relief between hardness groups likely doesn’t result in a meaningful change in the function of the entire tribosphenic complex. Thus, it seems that in this study any evolutionary benefit of a crushing talon relates primarily to dietary category instead of dietary hardness.

The evolutionary benefit of a crushing-dominated talon to a frugivorous bat is easy to envision. If the addition of the talon increases the surface area of the molar, as one might expect, it would mean increased occlusal surface dedicated to crushing. Even if the molar was somehow remodeled, so that the addition of the talon did not add surface area to the molar, crushing function would still increase to a small extent due to the low relief of the talon relative to the rest of the molar. This increase in crushing function would allow for increased chewing efficiency, as more fruit could be crushed in each chew.

However, the evolutionary benefit of a crushing talon to a carnivorous or insectivorous bat is less obvious. Indeed, the presence of a talon has an even greater effect on carnivore and insectivore molar function than the other diets. This apparent paradox makes sense, however, considering the high relief of the trigons in these carnivores (mean = 0.68) and insectivores (mean = 0.69). For example, adding a talon

28 with a crushing function to a shearing-dominated trigon of a carnivore will result in a greater change in overall molar function than adding the same talon to a crushing- dominated trigon of a frugivore. Since carnivores and insectivores primarily depend on the shearing action of their molars for food breakdown, it seems unlikely that the crushing function of a talon is of strong evolutionary benefit. Instead, the talon more likely represents a tradeoff for insectivores and carnivores, potentially adding a benefit to the molar unrelated to food breakdown efficiency, but correlated with a crushing shape.

These alternative benefits of the talon were not examined in the present study, but may include increased retention of food near the occlusal surface or increased molar bulk to promote tooth strength.

Phylogenetic Context and Post-hoc Corrections

As stated in the Methods section, the single origin of frugivory in the microchiropteran phylogeny has likely resulted in a strong phylogenetic correlation between molar shape and diet and, thus, reduced statistical power. However, even with reduced statistical power, frugivores and nectivores have markedly lower molar relief than the other groups. It is only after post-hoc p-value adjustments to the phylogenetic

ANOVA (including Holm-Bonferroni, Hochberg, or Hommel corrections) are used on this dataset that already has reduced statistical power that these patterns are obscured.

While it is true that p-value adjustments are needed to reduce the likelihood of a false positive, it seems more likely that in this case it has actually resulted in a false negative, due to the similar trends in molar relief and diet observed in other mammal groups

(Boyer, 2008; Bunn and Ungar, 2009; Bunn et al., 2011; Winchester et al. 2014).

Conclusions

The present research used suborder Microchiroptera as a case study to examine the functional factors influencing talon evolution in therian mammals. The results lend support to my first hypothesis that the talon is an adaptation to increase crushing function of the tribosphenic molar, due to its shape and its relative effect on whole tooth function.

However, my second hypothesis, that a crushing talon is associated with a frugivorous and/or hard food diet, is only partially supported, given that talons across all diet types perform crushing. As such, the evolutionary benefit of a crushing talon is feasible for some groups, such as frugivores, and, for other groups such as insectivores, is still unknown.

Figures

Figure 5. Representative Molars with Talons Representative molars of a variety of dietary and hardness groups, cropped for the RFI measurement. (A) Artibeus lituratus, UMMZ 126732, (B) Nyctinomops laticaudatus, UMMZ 166622, (C) Noctilio leporinus, UMMZ 124383 (D) Vampyrodes caraccioli, UMMZ 112022, (E) Megaderma spasma, UMMZ 160295, (F) Phyllostomus hastatus, UMMZ 105769, (G) Lonchophylla robusta, UMMZ 112037. Abbreviations: (An) anterior, (Bu) buccal, (Li) lingual, (Po) posterior, (UMMZ) University of Michigan Museum of Zoology. Scale = 1mm.

Figure 6. Molar in Functional View (A) An example of a cropped M1 (Rhinolophus ferrum-equinum, UMMZ 88609) in functional view used for the measurement of (B) the two-dimensional projected area. Abbreviations: (An) anterior, (Bu) buccal, (Li) lingual, (Po) posterior, (UMMZ) University of Michigan Museum of Zoology. Scale = 1mm.

Figure 7. Relief Index and Diet Relief Index of the whole tooth, trigon, and talon for carnivores, insectivores, omnivores, nectivores, and frugivores.

Figure 8. Relief Index and Hardness Relief Index of the whole tooth, trigon, and talon for hard food eaters and soft food eaters.

Tables

Table 1. Chapter 2 Species Information Dietary categories: (F) frugivore, (C) carnivore, (N) nectivore, (I) insectivore, (O) omnivore. Food Hardness: (H) hard, (S) soft. All diets were assigned according to Van Cakenburghe (2002), excluding those assigned according to 1Nowak (1994) and 2Animal Diversity Web (animaldiversity.ummz.umich.edu). Family Species Diet Hardness n Phyllostomidae Artibeus lituratus F H 3 Phyllostomidae Platyrrhinus helleri F H 3 Phyllostomidae Uroderma bilobatum F H 3 Phyllostomidae Platyrrhinus brachycephalus F S 3 Phyllostomidae Artibeus cinereus F S 1 Phyllostomidae Artibeus obscurus F S 3 Phyllostomidae Vampyrodes caraccioli F S 2 Noctilionidae Noctilio leporinus C - 2 Phyllostomidae Chrotopterus auritus C - 1 Phyllostomidae Trachops cirrhosus C - 2 Vespertilionidae Myotis vivesi 1 C - 2 Phyllostomidae Anoura geoffroyi N - 3 Phyllostomidae Lonchophylla robusta2 N - 2 Noctilionidae Noctilio albiventris I H 2 Vespertilionidae Pipistrellus tenuis I H 3 Vespertilionidae Miniopterus schreibersii I H 2 Molossidae Molossops temminkii2 I H 2 Molossidae Nyctinomops laticaudatus2 I H 2 Molossidae Tadarida brasiliensis2 I H 3 Vespertilionidae Nyctalus noctula I S 2 Megadermatidae Megaderma spasma I S 2 Rhinolophidae Rhinolophus ferrumequinum I S 1 Hipposideridae Asellia tridens I S 2 Phyllostomidae Macrotus waterhousii2 I S 2 Phyllostomidae Phyllostomus hastatus O - 2 Vespertilionidae Antrozous pallidus1 O - 2

Table 2. RFI Descriptive Statistics Descriptive statistics of the RFI of the whole tooth, trigon, and talon of the entire dataset, and of select dietary and hardness groups. RFI of… Min Max Mean SD n Whole 0.41 0.74 0.56 0.09 Tooth Entire Dataset Trigon 0.44 0.76 0.62 0.10 26 Talon 0.17 0.63 0.33 0.11 Whole 0.55 0.64 0.58 0.04 Tooth Carnivores Trigon 0.61 0.72 0.68 0.05 4 Talon 0.18 0.43 0.28 0.11 Whole 0.54 0.74 0.62 0.06 Tooth Insectivores Trigon 0.60 0.76 0.69 0.06 11 Talon 0.17 0.63 0.36 0.15 Whole 0.41 0.53 0.46 0.04 Tooth Frugivores Trigon 0.44 0.57 0.49 0.04 7 Talon 0.24 0.35 0.29 0.04 Whole 0.54 0.71 0.62 0.12 Tooth Omnivores Trigon 0.61 0.75 0.68 0.10 2 Talon 0.24 0.33 0.28 0.07 Whole 0.48 0.53 0.50 0.03 Tooth Nectivores Trigon 0.49 0.54 0.52 0.03 2 Talon 0.38 0.44 0.41 0.04 Whole 0.44 0.67 0.56 0.08 Tooth Hard Diet Trigon 0.49 0.71 0.60 0.08 9 Talon 0.30 0.49 0.38 0.08 Whole 0.41 0.74 0.56 0.12 Tooth Soft Diet Trigon 0.44 0.76 0.62 0.14 9 Talon 0.17 0.63 0.29 0.14

Table 3. Dietary Comparison P-values P-values from multiple comparison phylogenetic ANOVAs of dietary comparisons of whole tooth and trigon RFI. Asterisks denotes diet groups that are statistically distinct at the 0.10 alpha level (*) and at the 0.05 alpha level (**). RFI of… Frugivore Carnivore Insectivore Nectivore Whole 0.216 Carnivore Trigon 0.059* Whole 0.059* 0.235 Insectivore Trigon 0.020** 0.902 Whole 0.451 0.216 0.196 Nectivore Trigon 0.706 0.098* 0.020** Whole 0.078* 0.490 1.000 0.373 Omnivore Trigon 0.137 1.000 0.902 0.196

Literature Cited

Boyer DM. 2008. Relief index of second mandibular molars is a correlate of diet among Prosimian primates and other Euarchontan mammals. J Hum Evol 55:1118-1137.

Boyer DM, Evans AR, Jernvall J. 2010. Evidence of dietary differentiation among Late Paleocene-Early Eocene Plesiadapids (Mammalia, Primates). Am J Phys Anthropol 142:194-210.

Bunn JM, Ungar PS. 2009. Dental topography and diets of four Old World monkey species. Am J Primatol 71:466-477.

Bunn JM, Boyer DM, Lipman Y, St. Clair EM, Jernvall J, Daubechies I. 2011. Comparing dirichlet normal surface energy of tooth crowns, a new technique of molar shape quantification for dietary inference, with previous methods in isolation and in combination. Am J Phys Anthropol 145:247-261.

Butler PM. 1972. Some functional aspects of molar evolution. Evolution 26:474-483.

Butler PM. 1990. Early trends in the evolution of tribosphenic molars. Biol Rev 65:529- 552.

Clemens WA, Lillegraven JA, Lindsay EH, and Simpson GG. 1979. Where, when, and what-a survey of known Mesozoic mammal distribution. In: Lillegraven JA, Kielan- Jaworowska Z, and Clemens WA, editors. Mesozoic mammals- the first two-thirds of mammalian history. Berkley: University of California Press. p 7-58.

Crompton AW, Hiiemae K. 1969. Functional occlusion in tribosphenic molars. Nature 222:678-679.

Crompton AW, Kielan-Jaworowska Z. 1978. Molar structure and occlusion in Cretaceous therian mammals. In: P. M. Butler and K. A. Joysey, editors. Studies in the development, function and evolution of teeth. London and New York: Academic Press. p249-287.

Czaplewski NJ, Morgan GS, McLeod SA. 2008. Chiroptera. In: C. M. Janis, G. F. Gunnell, and M. D. Uhen, editors. Evolution of tertiary mammals of North America, vol. 2. Cambridge: Cambridge University Press. p174-197.

Davis BM. 2011. Evolution of the tribosphenic molar pattern in early mammals, with comments on the “dual-origin” hypothesis. J Mam Evol 18:227-244.

Dumont ER. 1997. Cranial shape in fruit, nectar, and exudate feeders: implications for interpreting the fossil record. Am J Phys Anthropol 102:187–202.

Dumont ER, Dávalos LM, Goldberg A, Santana SE, Rex K, Voigt CC. 2012. Morphological innovation, diversification, and invasion of a new adaptive zone. Proc R Soc Lond B doi:10.1098/rspb.2011.2005.

Evans AR. 2005. Connecting morphology, function, and tooth wear in microchiropterans. Biol J Lin Soc 85:81-96.

Freeman PW. 1984. Functional cranial analysis of large animalivorous bats (Microchiroptera). Mammalogy Papers: University of Nebraska State Museum 7:387- 408.

Freeman PW. 1988. Frugivorous and animalivorous bats (Microchiroptera): dental and cranial adaptations. Biol J Lin Soc 33:249-272.

Freeman PW. 1998. Form, function, and evolution in skulls and teeth of bats. Papers in Natural Resources 9:140-156.

Garland T, Dickerman AW, Janis CM, Jones JA. 1993. Phylogenetic analysis of covariance by computer simulation. Syst Biol 42(3): 265-292.

Harmon LJ, Weir JT, Brock CD, Glor RE, Challenger W. 2008. GEIGER: investigating evolutionary radiations. Bioinformatics 24(1):129-131.

Hogue AS, ZiaShakeri S. 2010. Molar crests and body mass as dietary indicators in marsupials. Austr J of Zool 58:56-68.

Hunter JP, Jernvall J. 1995. The hypocone as a key innovation in mammalian evolution. Proc Natl Acad Sci 92:10718-10722.

Jernvall J. 1995. Mammalian molar cusp patterns: developmental mechanisms of diversity. Acta Zool Fen 198:1-61.

Jones KE, Purvis A, MacLarnon A, Bininda-Emonds ORP, Simmons NB. 2002. A phylogenetic supertree of the bats (Mammalia: Chiroptera). Biol Rev 77:223-259.

Kay RF, Hiiemae KM. 1974. Jaw movement and tooth use in recent and fossil primates. Am J Phys Anthrop 40:227-256.

Kielan-Jaworowska Z, Cifelli RL, Lou Z-X. 2004. Mammals from the age of dinosaurs: structure, relationships, and paleobiology. New York: Columbia University Press. 633 p.

Lucas PW. 2004. Dental function morphology: how teeth work. Cambridge UK: Cambridge University Press. 372 p.

Luo ZX, Cifelli RL, Kielan-Jaworowska Z. 2001. Dual origin of tribosphenic mammals. Nature 409:53-57.

Luo ZX, Ji Q, Yuan CX. 2007. Convergent dental adaptations in pseudo-tribosphenic and tribosphenic mammals. Nature 450: 93–97.

M’Kirera F, Ungar PS. 2003. Occlusal relief changes with molar wear in Pan troglodytes troglodytes and Gorilla gorilla gorilla. Am J Primatol 60:31–41.

Nowak RM. 1994. Walker's bats of the world. Baltimore: The John Hopkins University Press. 288 p.

Osborn HF. 1907. Evolution of mammalian molar teeth. New York: Macmillan. 250 p.

Patterson B. 1956. Early cretaceous mammals and the evolution of mammalian molar teeth. Fieldiana: Geology 13(1). Chicago: Chicago Natural History Museum. 105 p.

Phillips CJ. 2000. A theoretical consideration of dental morphology, ontogeny, and evolution in bats. In: Adams R and Pedersen S, editors. Ontogeny, functional ecology, and evolution of bats. Cambridge: Cambridge University Press. p 247-274.

Polly PD, Le Comber SC, Burland TM. 2005. On the occlusal fit of tribosphenic molars: are we underestimating species diversity in the Mesozoic? Journal of Mammalian Evolution 12:283-299.

Rook DL, Hunter JP, Pearson DA, Bercovici A. 2010. Lower jaw of the Early Paleocene mammal Alveugena and its interpretation as a transitional fossil. J Paleontol 84: 1217-1225.

Sanchez-Villagra MR, Kay RF. 1996. Do phalangeriforms (Marsupialia: Diprotodontia) have a "hypocone"? Austr J Zool 44:461-467.

Sibbing FA. 1991. Mastication in cyprinid fish in feeding and the texture of food. Vincent JF and Lillford PJ, editors. New York: Cambridge Press. p 63.

Simpson GG. 1936. Studies of the earliest mammalian dentitions. Dental Cosmos 78:791- 800.

Spears IR, Crompton RH. 1996. The mechanical significance of the occlusal geometry of Great Ape molars in food breakdown. J Hum Evol 31:517-535.

Strait SG. 1993a. Molar morphology and food texture among small-bodied insectivorous mammals. J Mam 72:391-402.

Strait SG. 1993b. Differences in occlusal morphology and molar size in frugivores and faunivores. J Hum Evol 25:471-484. 40

Strait SG. 1997. Tooth use and the physical properties of food. Evol Anthropol 5:199- 211.

Ungar PS, Kay RF. 1995. The dietary adaptations of European Miocene Catarrhines. Proc Natl Acad Sci 92:5479-5481.

Ungar PS, Williamson M. 2000. Exploring the effects of toothwear on functional morphology: a preliminary study using dental topographic analysis. Palaeontol Electron 3(1):1-18.

Van Cakenberghe V, Herrel A, Aguirre LF. 2002. Evolutionary relationships between cranial shape and diet in bats (Mammalia: Chiroptera). In: Aerts P, D’Août K, Herrel A, Van Damme R, editors. Topics in functional and ecological vertebrate morphology. Maastricht: Shaker Publishing. p 205-236.

Van Valen LM. 2009. How ubiquitous is adaptation? A critique of the epiphenomenist program. Biol Philosophy 24:267-280.

Winchester JM, Boyer DM, St. Clair EM, Gosselin-Ildari AD, SB Cooke, JA Ledogar. 2014. Dental topography of platyrrhines and prosimians: convergence and contrasts. Am J Phys Anthropol 153:29–44.

Wood CB, Clemens WA. 2001. A new specimen and a functional reassociation of the molar dentition of Batodon tenuis (Placentalia, Incertae sedis), latest Cretaceous (Lancian), North America. Bull Mus Comp Biol 156:99-118.

Chapter 3. Upper Molar Talon Size Evolution

Introduction

When exploring the origin and elaboration of novel anatomy, such as the upper molar talon in therian mammals, researchers must consider size evolution. The size of a feature can lend clues to several important evolutionary factors, including its effect on the functional complex it participates in and what evolutionary constraints are acting upon it.

Examining changes in size across evolutionary time and across closely related species can tell us a great deal about the factors influencing anatomical evolution.

The therian upper molar talon is a prime example of a feature that has varied greatly in size throughout evolutionary time and across living mammals. The talon is defined as the derived distolingual region of an upper tribosphenic molar, often flanked anteriorly by the postprotocrista (Osborn, 1907; Czaplewski et al., 2008; see Fig. 1).

When the talon is defined regionally (as opposed to the typical morphological terms of cingulum, cusp, etc.), the remaining primitive portion of the tooth is called the trigon.

Separating the molar into these two regions, trigon and talon, has allowed for an examination of talon function with respect to the rest of the molar (Gutzwiller and

Hunter, in press; Ch. 2). The talon first originated as a small postcingulum or metaconule in therian mammals of the late Cretaceous, constituting a small component of the tribosphenic complex (Butler, 1972; Hunter and Jernvall, 1995; Jernvall, 1995). Many

42 living species retain the primitive trigon, consisting of a shearing-dominated trigon that is specialized for a faunivorous diet (Strait, 1997: Lucas, 2004; Boyer, 2008; Hogue and

ZiaShakeri, 2010). These teeth may lack a talon or have talons of varying sizes. Since the

Cretaceous, some talons have expanded into large features that constitute a significant component of the tribosphenic complex, such as a fully-formed hypocone cusp (Butler,

1972; Hunter and Jernvall, 1995; Jernvall, 1995). These derived molars can exhibit large talons and similarly derived trigons, both of which may have modifications reflecting dietary specializations (e.g., hard food or fruit; Lucas, 2004; Boyer, 2008; Bunn et al.,

2011; Winchester et al., 2014).

Previous work has examined what the shape of the talon can tell us about its function within the upper molar complex (Gutzwiller and Hunter, in press). Most

Microchiropteran (microbat) talons are low in occlusal relief (generally flat occlusal surface) when compared to the trigon, and this low relief significantly increases the crushing function performed by the tooth (Gutzwiller and Hunter, in press). The low relief of the talon supports the long-proposed hypothesis that the talon squares off the tribosphenic molar and represents an adaptation for increased crushing (Butler, 1972;

Hunter and Jernvall, 1995; Jernvall, 1995; Gutzwiller and Hunter, in press). This increase in crushing function may provide the benefit of increased chewing efficiency to a frugivorous bat, which relies greatly on crushing for food breakdown (Freeman, 1998;

Lucas, 2004; Boyer, 2008; Bunn and Ungar, 2009; Boyer et al., 2010; Bunn et al., 2011;

Gutzwiller and Hunter, in press). However, the benefit of a crushing talon to a faunivorous (insectivorous or carnivorous) bat, which relies primarily on shearing

43 function for food breakdown, is not fully understood. Differences in talon size may help to explain the role of the talon in faunivores.

Thus, a thorough examination of talon size, in addition to its shape, may help to further illuminate the functional role that it plays in faunivorous and frugivorous mammals. The present research examines two facets of talon size evolution. The first explores how talon size is related to its food breakdown function within the tooth. Are differences in talon crushing or shearing function across species attributable to talon size?

Examining if relief varies according to talon size will illuminate how potential selective pressures could change according to talon size. Understanding the relationship between talon size and its crushing or shearing function across living therians may reveal how its role in the tooth has changed as the talon became larger, any potential constraints acting on its evolution, and how talon specialization has occurred in different dietary groups.

In addition to exploring the relationship between talon size and function, it is also important to examine if other factors, unrelated to crushing and shearing, have affected talon size evolution. A primary alternative factor that may explain much of the variability in talon size observed in mammals is body size scaling. In the study of scaling, a wealth of research has explored the relationship between anatomical and physiological features, such as mammalian postcanine tooth size and body size. These scaling studies have allowed us to understand trends in the way organisms have changed over evolutionary time, while also providing a variety of explanatory tools, such as estimating the body size of fossil mammals given only a single tooth (e.g., Gingerich et al., 1982; for discussion, see Fortelius, 1990a).

It has long been understood that mammalian postcanine tooth size scales with body size, in order to maintain food intake to match the increasing metabolic demand of a larger body size (Pilbeam and Gould, 1974). The majority of previous research on mammal tooth size scaling has focused on exploring the precise nature of the relationship between tooth size and body size, using slope estimates from regression analyses (i.e. scaling coefficients). However, the scaling of tooth size with body size continues to provide debate, because a diversity of work has found scaling relationships varying from positive allometry to isometry to negative allometry (Gould, 1975; Corruccini and

Henderson, 1978; Gingerich and Smith, 1984; see Ungar, 2014 for a review of dental allometry). Isometry reflects a one-to-one relationship between tooth size and body size.

However, when comparing a two-dimensional measurement (such as tooth area) to a three-dimensional measurement (such as body mass), an isometric relationship would

rds 2/3 scale to the 2/3 power (Mb ; or a scaling coefficient of 0.67). Positive allometry reflects any scaling relationship greater than isometry (>0.67) and negative allometry reflects any scaling relationship less than isometry (<0.67). Researchers have attempted to reconcile these varied results in light of a simple premise: the scaling of metabolism with body size (Pilbeam and Gould, 1974; Fortelius, 1985; 1990b). As mammals increase

th 3/4 in size, metabolic rate increases to the 3/4 power (Mb ; negative allometry), resulting in larger mammals having an absolutely higher, but relatively lower, metabolic rate than smaller mammals (Kleiber, 1947). Because tooth morphology is so closely linked with food intake (and thus metabolism), we would expect tooth size scaling to reflect the negative allometric scaling of metabolism. Some simple solutions have been posed (for

45 example, accounting for the scaling of chewing rate; see Fortelius, 1985; 1990b), but it seems that the precise nature of mammalian postcanine scaling remains a bit of an enigma (Ungar, 2014).

Other studies have approached scaling with a different goal in mind: understanding the scaling of tooth shape in order to address functional questions. For example, Popowics and Fortelius (1997) and Evans et al. (2005) examined the scaling of tooth sharpness in herbivorous and faunivorous mammals. Kay (1975) and Ungar and

Kay (1995) explored the scaling of molar adaptations for shearing, crushing, and grinding in extant and extinct primates. These studies reveal how scaling relationships could reflect functional adaptations and constraints in tooth shape across a variety of molar and body sizes. Additionally they show how scaling relationships could vary depending on dietary groups and the range in body size that is examined. Although the present study does attempt to characterize the scaling of talon size with the traditional scaling coefficient approach, my research is more similar to these functional studies, because I am ultimately concerned with the functional and evolutionary implications of talon size.

Not often explored in scaling studies, however, is the functional significance of the scaling of individual components of a tooth in order to understand how body size scaling might influence the evolution of a new structure. Exploring the scaling of individual components can allow researchers to delve into the factors affecting the origin of a novel anatomical feature within a tooth and the functional implications of its expansion in size. Does the talon scale with body size in the same manner as the rest of the tooth? See Figure 9 for two possible answers to this question. A tooth can increase in

46 size either by increasing the size of its existing morphology (9a) or by adding new morphology, such as the talon (9b). It is possible that a novel feature may evolve as an adaptation to increase occlusal area, in order to meet the metabolic demand of a larger body size. In this case, the talon may not be specialized for a particular diet (a result observed in Ch. 2), but instead be a more generalized structure. Ultimately, examining the scaling of talon size with body size will help us to further understand its role within the molar functional complex. Additionally, we can explore if this role changes depending on the size of the mammal and if any evolutionary constraints affect talon size scaling.

Goals

The goal of the present study is to address these two facets of talon size evolution, in order to explore the functional significance of the origin and expansion of the upper molar talon in therian mammals. This research uses the morphologically and ecologically diverse suborder Microchiroptera (microbats) as a case study for therian mammals.

Hypotheses and Predictions

The first hypothesis (H1) pertains to the relationship between talon size and its crushing or shearing function (here represented by relief; see Chapter 2). There are two alternatives:

H1a: There is no relationship between talon relief and talon size. This result would suggest that crushing and shearing function is size-independent and could occur at any talon size. Given the overall trend for talons to be low in relief (thus more specialized for crushing) in previous work, this hypothesis is expected to be supported. This result

47 would suggest that a factor other than crushing and shearing functional specialization is the driving force in talon size evolution. Alternatively,

H1b: There is a relationship between talon relief and talon size. This result would suggest that crushing or shearing functional specialization is dependent on talon size.

Exploring this relationship may reveal potential evolutionary constraints on talon function (e.g. perhaps functional specializations occur only at larger sizes when the talon may be more likely to participate in food breakdown). Although talons generally are low in relief and this hypothesis is not expected to be supported, any relationship between talon size and relief could explain the remaining variability that does exist in talon relief.

These results may help illuminate the functional role or evolutionary constrainst of the talon in faunivorous microbats, where a crushing function of the talon may not provide a benefit to food breakdown.

The second hypothesis (H2) pertains to the scaling of talon size with body size.

Again, there are two alternative hypotheses:

H2a: Talon size does not scale with body size. This result would suggest that the talon is unrelated to maintaining occlusal function with the increasing metabolic demand of large body sizes. In this case, we would instead expect that trigon size would scale with body size (as seen in 9a), allowing for occlusal function to match metabolic need of larger body sizes. We would conclude that the talon performs a role in the tooth that is unrelated to body size, not specifically tested here (such as specialization for a particular diet or maintenance of tooth strength).

H2b: Talon size does scale with body size (with a isometric, positive allometric, or negative allometric relationship). This scaling would suggest that the talon is a vital component to food breakdown and performs a role of maintaining overall tooth function as metabolic demands increase with body mass (as in Fig. 9b). In this case, the trigon may also scale with body mass to some extent. Comparing the scaling coefficients of the trigon versus the talon in relation to an isometric scaling expectation could help to inform the degree to which either component performs this role. Again, these results may help illuminate the functional role or evolutionary constraints of the talon in faunivorous microbats.

With this framework in mind, the present study quantified the relationship between talon crushing and shearing function (estimated by relief index, RFI; see Chapter

2) and talon size, in addition to the relationship between trigon and talon size with body size for faunivorous and frugivorous microbat species.

Materials and Methods

The body size scaling relationships of trigon and talon size were explored using two datasets: projected area (2-dimensional area in occlusal view) from dental photos and surface area (the sum of the areas of all tetrahedral faces) from 3-dimensional computer molar models. The relationship between talon size and function (RFI) was estimated using the surface area dataset only, due to relief being a 3-dimensional measurement.

Specimen Selection

Focal species were selected according to diet (see Table 4 for species information). The projected area dataset consists of 96 species, categorized as faunivorous (including insectivorous and carnivorous; n = 62) or frugivorous (n = 34; animaldiversity.org; Nowak, 1994). An occlusal-view photo of the upper dentition of a representative individual of each species was attained either through the Animal

Diversity Web database from the University of Michigan (animaldiversity.org; n = 81) or through personal photos taken at the Carnegie Museum of Natural History (CMNH; n =

15). Personal photos were taken at a similar resolution and field-of-view as those in the

Animal Diversity Web database, so subsequent measurements were comparable. The surface area dataset consists of 26 species, also categorized as faunivorous (n = 18) or frugivorous (n = 8; animaldiversity.org; Nowak, 1994; Van Cakenberghe et al., 2002).

For this dataset (as described in Chapter 2), one to three specimens for each species were collected from The Ohio State University, Museum of Biological Diversity (OSUM) and the University of Michigan, Museum of Zoology (UMMZ). Body size for both datasets was represented by species mean body mass estimates, attained from Animal Diversity

Web, Nowak (1994), and Genoways et al. (2007).

Area and RFI Measurements

For each occlusal view photo of the projected area dataset, the outline of the trigon and talon (if present) of the upper first molar was traced using a series of markers.

The areas outlined for the trigon and the talon were measured using ImageJ

(www.imagej.nih.gov/ij/; see Fig. 10).

For the surface area dataset, 3D computer models, and all subsequent surface area and relief index (RFI) measurements, were attained following the methods described in

Chapter 2.

Statistical Analyses

All projected and surface area measurements were increased by one millimeter, in order to include any talons with an area of zero in natural log calculations. Since I am ultimately concerned with the slope of the relationship between area and body mass, this process does not change the resulting scaling coefficients. All area measurements and body mass estimates were natural logged in order to increase normality and to be able to fit linear regression lines to the data. All variables (including area measurements, RFI, and body mass) were then tested for the presence of a phylogenetic signal using a pruned version of the Jones et al. (2002) microchiropteran phylogenetic tree in the Picante package of R, version 2.15.2. Since a phylogenetic signal was detected in many cases

(see Results), Felsenstein’s Phylogenetic Independent Contrasts (PIC) were calculated for each variable using the Ape package in R and were used in the following correlation and regression analyses. In order to examine the relationship of talon size with relief and trigon/talon size with body mass, Pearson’s Product Moment Correlation and Major Axis

Model II Regression through the origin were calculated using the Lmodel2 package in R for the following comparisons.

To address hypotheses H1a and H1b:

- talon RFI v. ln(talon surface area)

To address hypotheses H2a and H2b:

- ln(trigon projected area) v. ln(body mass)

- ln(talon projected area) v. ln(body mass)

- ln(trigon surface area) v. ln(body mass)

- ln(talon surface area) v. ln(body mass)

These analyses were computed for faunivores and frugivores separately, in order to examine the effect of diet on talon scaling.

Post-hoc Statistical Additions

After a preliminary examination of the projected area dataset, a potential body mass threshold was detected for the talon of faunivores, at ln(body mass) = 3 (or about 20 grams; see Appendix C, Fig. 16b). Thus, in addition to performing the correlations and regressions for the entire body mass range (as described above), the same analyses were repeated for subsets of the data consisting of those species with ln(body mass) less than 3 (n = 45 for faunivores and n = 23 for frugivores) and ln(body mass) greater than 3 (n = 17 for faunivores and n =11 for frugivores). This allowed for me to examine if the scaling relationships are different for species with small body sizes or large body sizes. These additional analyses were performed for the projected area dataset only, since the sample size allowed it.

Results

Talon Size and RFI

Talon RFI does not correlate with talon surface area for faunivores (p=0.75) or frugivores (p=0.60), suggesting there is no relationship between talon crushing or shearing function and talon size.

Body Size Scaling of Trigon and Talon Size

See Appendix C through F for scatterplots of area and body mass prior to phylogenetic correction using Phylogenetically Independent Contrasts.

Projected Area Dataset

Significant phylogenetic signals were detected for trigon projected area, talon projected area, and body mass for both faunivores and frugivores (see Table 5). See

Appendix G through J for scatterplots of phylogenetically independent contrasts and

Figure 11 for a comparison of the following regression slopes for the projected area dataset.

Faunivores

For the faunivores, trigon area correlates very highly significantly with body mass (p<0.001; r=0.93), with a negative allometric regression slope of 0.54

(slope 95% CI: 0.48-0.60; r2=0.86; Appendix G). Talon area also correlates very highly significantly with body mass (p<0.001; r=0.79), with a negative allometric regression slope of 0.40 (slope 95% CI: 0.32-0.48; r2=0.63; Appendix H). For species with a ln(body mass) less than 3, trigon area correlates very highly significantly with body mass

(p<0.001; r=0.77), with a negative allometric regression slope of 0.51 (slope 95% CI:

0.39-0.65; r2=0.60; Appendix G). At these small body masses, talon area also correlates very highly significantly with body mass (p<0.001; r=0.54). However, the regression reveals a negative allometric, but minimal, slope (0.19; slope 95% CI: 0.10-0.28) and that little of the variability in talon area is explained by body mass (r2=0.29; Appendix H). In contrast, for species with a ln(body mass) greater than 3, both trigon and talon area correlate very highly significantly with body mass (trigon p<0.001; trigon r=0.94; talon p<0.001; talon r=0.94), with much higher regression slopes that are approximately isometric for the trigon (slope = 0.70, CI: 0.56-0.86; Appendix G) and positive allometric for the talon (slope = 0.96, CI: 0.76-1.20; Appendix H). For species with a ln(body mass) greater than 3, body mass also explains much more of the variability in area (trigon r2 =

0.88; talon r2 = 0.87).

Frugivores

Across the entire body mass range of frugivores, trigon area correlates very highly significantly with body mass (p<0.001; r=0.76), with a regression slope of 0.69, or near isometry (95% CI: 0.49-0.93; r2=0.58; Appendix I). Talon area also correlates very highly significantly with body mass (p<0.001; r=0.63), with a negative allometric regression slope of 0.40 (95% CI: 0.23-0.59; r2=0.40; Appendix J). For species with a ln(body mass) less than 3, trigon area correlates highly significantly with body mass (p=0.01; r=0.54), with a positive allometric regression slope of 0.78 (95% CI: 0.32-

1.68; Appendix I). However, at this body mass range, body mass explains little of the variability in trigon area (r2=0.29). For species with a ln(body mass) less than 3, talon area also correlates significantly with body mass (p=0.03; r=0.46), with a negative

54 allometric regression slope of 0.26 (95% CI: 0.03-0.52; Appendix J), but, again, with little of the variability in talon area explained by body mass (r2=0.21). For species with a ln(body mass) greater than 3, neither trigon or talon area correlates with body mass

(trigon p=0.19; talon p=0.67).

Surface Area Dataset

For the surface area dataset, one of the variables (frugivore talon area) has a significant phylogenetic signal (see Table 5). See Appendix K and L for scatterplots of phylogenetically independent contrasts and Figure 12 for a comparison of the following regression slopes for the surface area dataset.

Faunivores

For the faunivore surface area dataset, trigon area correlates very highly significantly with body mass (p<0.001; r=0.76), with a negative allometric regression slope of 0.39 (95% CI: 0.22-0.59; r2=0.58; Appendix K). Talon area also correlates highly significantly with body mass (p<0.01; r=0.62), with a negative allometric regression slope of 0.29 (95% CI: 0.10-0.51; r2=0.39; Appendix K).

Frugivores

For frugivores, the surface area of the trigon correlates highly significantly with body mass (p<0.01; r=0.94), with a positive allometric regression slope of 0.78 (95% CI: 0.49-1.20; r2=0.88; Appendix L). The surface area of the talon also correlates highly significantly with body mass (p<0.01; r=0.92), with a positive allometric regression slope of 0.75. (95% CI: 0.43-1.23; r2=0.84; Appendix L).

Discussion

The presence of a phylogenetic signal in many parameters suggests that differences in tooth shape, tooth size, and body size are consistent with expectations according to phylogenetic relatedness (i.e. closely related species have similar tooth morphology and body size due to shared ancestry). For this reason, phylogenetic relatedness needed to be accounted for (by use of Phylogenetically Independent

Contrasts) when examining the scaling of talon size and relief.

Talon RFI and Size

Talon crushing and shearing function, estimated by occlusal relief, does not correlate with talon size, supporting hypothesis H1a. This result suggests that the food breakdown function of the talon is size-independent and can occur at any given size of the talon. Since most talons have low occlusal relief, and thus an overall crushing function, the lack of relationship between talon relief and size is not surprising. However, this does suggest that larger talons are not more likely to be specialized for crushing or shearing, as one might expect. Additionally, talon size cannot account for the remaining variability (although small) in talon shape. This variability may be explained by further examining the differences in talon relief for dietary groups not previously explored

(including those based on physical properties such as toughness) or exploring alternative functions of the talon that may correlate with talon relief (see Chapter 4).

Body Size Scaling of Trigon and Talon Area

Overall Trends

Across the projected area and surface area datasets, both trigon and talon size scale with body size to some extent, suggesting that both tooth components play a role in increasing occlusal area to meet the metabolic demand of a larger body size and supporting hypothesis H2b. The scaling coefficients are often negatively allometric, but some also represent isometry and positive allometry. However, there are some cases (see further discussion below) in which trigon or talon size does not scale with body size (e.g., trigons and talons in large frugivores) or where little of the variability in trigon or talon size is explained by body size (e.g., low r2 values in talons in small faunivores). This result lends some support for hypothesis H2a and suggests that in these cases factors other than body size have played a large role in the size evolution of the trigon or talon.

Body Mass Range

The greatest effect of body mass range is evident in faunivores. For faunivores in the small body mass range (ln of body mass less than 3; less than around 20 g), both trigon and talon size scale with negative allometry with body size, but little of the variability in talon size (only 29%) is actually explained by body size. This suggests that at these small body masses, both the trigon and talon do contribute to the scaling of tooth size to some extent. However, there is a good deal of variability of talon size that is not related to body size and more likely relates to another function of the talon (such as tooth strength for instance, see Chapter 4). For larger body sizes (greater than 20 g), much of the variability in talon size (87%) is explained by body size and the talon exhibits a much

57 higher regression slope (positively allometric). This suggests that for larger microbats, the talon significantly contributes to the increased occlusal area needed to match metabolic need for large body size. Thus, in faunivores, small talons may not be specialized for food breakdown, whereas large talons may be specialized for food breakdown. This threshold observed at a body mass of about 20 grams suggests that the primary factors contributing to talon functional evolution are different depending on the size range examined.

However, this difference in talon function across the body mass range examined here is not observed for the frugivore projected area dataset. Across the entire projected area dataset, frugivore talon size already exhibits a large spread from the regression line.

So, breaking up the dataset into small and large body mass ranges results in lower r2 values for low body masses and nonsignificance for high body masses. This ultimately reduces our ability to see any overall trend. Instead, at smaller body mass, factors other than body size that contribute to variability in trigon and talon size are highlighted. This difference in scaling trends observed in faunivorous versus frugivorous bats suggests that the function of the talon is under differing selection pressures according to diet.

Faunivores vs. Frugivores

Interestingly, the differences between faunivores and frugivores depend on the dataset examined. For the projected area dataset, faunivores generally have higher r2 values (varying from 0.29 to 0.88) than frugivores (varying from non-significance to

0.58), suggesting that body size explains a greater amount of the variability in trigon and talon size for faunivores than for frugivores. This observation may be explained by

58 considering talon relief. In frugivores, a large crushing talon would be beneficial not only for maintaining occlusal area with increasing body size, but also increasing the crushing function of the tooth to maximize the efficiency of fruit breakdown. In faunivores, however, a large crushing talon is not expected to provide the same food breakdown benefit (since faunivores instead generally rely on shearing of their molars for insect/tissue breakdown). In the case of faunivores, it makes sense that more of the variability in the size of the talon can be explained by body size per se, suggesting that the metabolic need of a large body size plays a greater role in the generalized increased occlusal area provided by large talons of faunivores.

However, in the surface area dataset, the opposite result is found. A greater degree of the variability in the surface area of the trigon and talon is explained by body size in frugivores (trigon r2 = 0.88; talon r2 = 0.84) than in faunivores (trigon r2 = 0.58; talon r2 =

0.39). It is possible that the relationship of trigon and talon size with body size may simply be better explained using projected area for faunivores and surface area for frugivores. Frugivores may rely to a greater degree on surface area for the entrapment of food for compression, whereas faunivores do not. Alternatively, the lower r2 values in talon surface area versus projected area in faunivores may reflect the greater variability in talon RFI in faunivores (especially, insectivores) than frugivores. Note this higher variability in Figure 7 (Chapter 2). Greater variability in talon relief in faunivores may ultimately represent variability in talon height. Thus, while talon projected area may scale with body size, variability in talon height may actually be explained by an alternative

59 function for the talon in faunivores, such as maintenance of tooth strength. This idea will be further explored in Chapter 4.

Conclusions – Talon Functional Evolution

When examining the evolution and subsequent expansion of novel anatomy, such as the talon, it is important to explore evolution of size. Talon size is less related to its ideal crushing or shearing function and more related to body size scaling. My results support the hypothesis that the talon does indeed scale with body size, suggesting a function in increasing occlusal area to match the chewing efficiency needed for higher metabolic rates associated with the evolution of larger body size. This functional role of the talon is most prominent in the projected area of faunivores, especially for those species with large body sizes (greater than about 20 g). In these large faunivores, the talon is low in relief, but may instead act as a generalized area of increased occlusal function, perhaps allowing the trigon to continue to specialize in shearing required for a faunivorous diet. Small-bodied faunivores, on the other hand, may not be able to use their talons as effectively for increased occlusal area. This may suggest an evolutionary constraint in which faunivore talons can only perform this scaling function once they reach adequate size to participate in food breakdown.

Overall, less of the variability in talon projected area is explained by body size in frugivores, suggesting that factors other than body size, not tested here, have more greatly influenced the evolution of talon projected area in this group. Instead, surface area is more closely tied with talon size in frugivores, perhaps suggesting that increased surface

60 occlusal area is necessary to allow for proper compression of food to match the metabolic need with increasing body size in this group.

These results lend support for the hypothesis that talon size can function to increase occlusal area. However, this function of the talon is dependent on the size range and diet examined. Faunivores likely represent the best example. With the origin of a small cingulum or cusp-shaped talon in the late Cretaceous faunivorous therians, the talon may have represented an adaptation completely unrelated to body size. However, subsequent body size evolution may have resulted in the expansion of larger talons and the exaptation of the talon functioning as a more vital component of the tribosphenic chewing complex. In the end, size evolution of the talon is likely a complicated issue, influenced by many factors. The functional role of the talon in body size evolution is not mutually exclusive with other functional hypotheses, including functional specialization for crushing (i.e., in frugivores; as explored in Chapter 2) or providing a benefit for increasing tooth strength (to be discussed in Chapter 4).

Figures

Figure 9. Molar Area Scaling Theoretical proportional changes occurring with the increase in molar size in response to body size. (A) Talon does not evolve. Trigon size increases with body size. (B) Talon evolves. Trigon size remains the same and talon size increases with body size. Abbreviations: ta, talon; tr, trigon.

Figure 10. Projected Area Measurement Example projected area measurement of the upper molar trigon (Tr) and talon (Ta) of Lonchorhina aurita, UMMZ 122294. Photo taken from the Animal Diversity Web database (contributor: Phil Myers, Museum of Zoology, University of Michigan-Ann Arbor; license: http://creativecommons.org/licenses/by-nc-sa/3.0/) and edited to reflect area of trigon and talon. Area measurement taken in ImageJ.

Figure 11. Projected Area Scaling Coefficients … for (A) faunivores and (B) frugivores according to tooth component. Abbreviations: (lnBM<3), ln(Body Mass) less than 3; (lnBM>3), ln(Body Mass) greater than 3; Ta, talon; Tr, trigon.

Figure 12. Surface Area Scaling Coefficients …for (A) faunivores and (B) frugivores according to tooth component. 65

Tables

Table 4. Chapter 3 Species Information Taxon names assigned according to Koopman (1993). Dietary categories assigned according to animaldiversity.org and Nowak (1994). Abbreviations: (P), projected area dataset; (S), surface area dataset. Family Species Diet Dataset Emballonuridae Peropteryx kappleri Faunivore P Emballonuridae Rhynchonycteris naso Faunivore P Emballonuridae Saccopteryx bilineata Faunivore P Emballonuridae Taphozous melanopogon Faunivore P Hipposideridae Asellia tridens Faunivore S Hipposideridae Hipposideros diadema Faunivore P Hipposideridae Hipposideros fulvus Faunivore P Megadermatidae Lavia frons Faunivore P Megadermatidae Megaderma spasma Faunivore S Molossidae Eumops dabbenei Faunivore P Molossidae Eumops perotis Faunivore P Molossidae Molossops temminckii Faunivore P,S Molossidae Nyctinomops femorosaccus Faunivore P Molossidae Nyctinomops laticaudatus Faunivore S Molossidae Nyctinomops macrotis Faunivore P Molossidae Tadarida aegyptiaca Faunivore P Molossidae Tadarida brasiliensis Faunivore P,S Mormoopidae Mormoops megalophylla Faunivore P Mormoopidae Pteronotus davyi Faunivore P Mormoopidae Pteronotus parnellii Faunivore P Noctilionidae Noctilio albiventris Faunivore P,S Noctilionidae Noctilio leporinus Faunivore P,S Phyllostomidae Ametrida centurio Frugivore P Phyllostomidae Anoura geoffroyi Faunivore P Phyllostomidae Ardops nichollsi Frugivore P Phyllostomidae Artibeus cinereus Frugivore S Phyllostomidae Ariteus flavescens Frugivore P Phyllostomidae Artibeus jamaicensis Frugivore P Phyllostomidae Artibeus lituratus Frugivore P,S Continued 66

Table 4 Continued Phyllostomidae Artibeus obscurus Frugivore S Phyllostomidae Artibeus phaeotis Frugivore P Phyllostomidae Brachyphylla cavernarum Frugivore P Phyllostomidae Carollia perspicillata Frugivore P,S Phyllostomidae Centurio senex Frugivore P Phyllostomidae Chiroderma villosum Frugivore P Phyllostomidae Choeronycteris mexicana Frugivore P Phyllostomidae Chrotopterus auritus Faunivore P,S Phyllostomidae Ectophylla alba Frugivore P Phyllostomidae Erophylla sezekorni Frugivore P Phyllostomidae Glossophaga commissarisi Frugivore P Phyllostomidae Glossophaga soricina Frugivore P Phyllostomidae Hylonycteris underwoodi Frugivore P Phyllostomidae Lonchophylla thomasi Frugivore P Phyllostomidae Lonchorhina aurita Faunivore P Phyllostomidae Macrotus waterhousii Faunivore S Phyllostomidae Mesophylla macconnelli Frugivore P Phyllostomidae Monophyllus plethodon Frugivore P Phyllostomidae Monophyllus redmani Frugivore P Phyllostomidae Phylloderma stenops Frugivore P Phyllostomidae Phyllonycteris aphylla Frugivore P Phyllostomidae Phyllostomus discolor Frugivore P Phyllostomidae Phyllostomus hastatus Faunivore P Phyllostomidae Platyrrhinus brachycephalus Frugivore S Phyllostomidae Platyrrhinus helleri Frugivore P,S Phyllostomidae Platyrrhinus lineatus Frugivore P Phyllostomidae Platyrrhinus vittatus Frugivore P Phyllostomidae Pygoderma bilabiatum Frugivore P Phyllostomidae Sturnira lilium Frugivore P Phyllostomidae Sturnira magna Frugivore P Phyllostomidae Trachops cirrhosus Faunivore S Phyllostomidae Uroderma bilobatum Frugivore P,S Phyllostomidae Uroderma magnirostrum Frugivore P Phyllostomidae Vampyressa pusilla Frugivore P Phyllostomidae Vampyrodes caraccioli Frugivore P,S Phyllostomidae Vampyrum spectrum Faunivore P Rhinolophidae Rhinolophus ferrumequinum Faunivore P,S Rhinolophidae Rhinolophus hipposideros Faunivore P Continued

Table 4 Continued Rhinolophidae Rhinolophus inops Faunivore P Rhinolophidae Rhinophylla pumilio Frugivore P Thyropteridae Thyroptera tricolor Faunivore P Vespertilionidae Antrozous pallidus Faunivore P,S Vespertilionidae Eptesicus fuscus Faunivore P Vespertilionidae Idionycteris phyllotis Faunivore P Vespertilionidae Kerivoula lanosa Faunivore P Vespertilionidae Lasionycteris noctivagans Faunivore P Vespertilionidae Lasiurus borealis Faunivore P Vespertilionidae Lasiurus cinereus Faunivore P Vespertilionidae Lasiurus ega Faunivore P Vespertilionidae Lasiurus intermedius Faunivore P Vespertilionidae Lasiurus seminolus Faunivore P Vespertilionidae Miniopterus australis Faunivore P Vespertilionidae Miniopterus schreibersi Faunivore S Vespertilionidae Murina cyclotis Faunivore P Vespertilionidae Myotis austroriparius Faunivore P Vespertilionidae Myotis californicus Faunivore P Vespertilionidae Myotis evotis Faunivore P Vespertilionidae Myotis grisescens Faunivore P Vespertilionidae Myotis keenii Faunivore P Vespertilionidae Myotis leibii Faunivore P Vespertilionidae Myotis myotis Faunivore P Vespertilionidae Myotis mystacinus Faunivore P Vespertilionidae Myotis sodalis Faunivore P Vespertilionidae Myotis thysanodes Faunivore P Vespertilionidae Myotis vivesi Faunivore S Vespertilionidae Myotis velifer Faunivore P Vespertilionidae Myotis volans Faunivore P Vespertilionidae Myotis yumanensis Faunivore P Vespertilionidae Nyctalus leisleri Faunivore P Vespertilionidae Nyctalus noctula Faunivore P,S Vespertilionidae Nycticeius humeralis Faunivore P,S Vespertilionidae Pipistrellus hesperus Faunivore P Vespertilionidae Pipistrellus pipistrellus Faunivore P Vespertilionidae Pipistrellus subflavus Faunivore P,S Vespertilionidae Pipistrellus tenuis Faunivore P,S Vespertilionidae Plecotus auritus Faunivore P Vespertilionidae Tylonycteris pachypus Faunivore P

Table 5. Phylogenetic Signal P-values

Trigon Talon Body Mass Faunivores 0.001 0.001 0.001 Projected Area Dataset Frugivores 0.002 0.002 0.010 Faunivores 0.293 0.077 0.059 Surface Area Dataset Frugivores 0.103 0.036 0.107

Literature Cited

Boyer DM. 2008. Relief index of second mandibular molars is a correlate of diet among Prosimian primates and other Euarchontan mammals. J Hum Evol 55:1118-1137.

Boyer DM, Evans AR, Jernvall J. 2010. Evidence of dietary differentiation among Late Paleocene-Early Eocene Plesiadapids (Mammalia, Primates). Am J Phys Anthropol 142:194-210.

Bunn JM, Ungar PS. 2009. Dental topography and diets of four Old World monkey species. Am J Primatol 71:466-477.

Butler PM. 1972. Some functional aspects of molar evolution. Evolution 26:474-483.

Corruccini RD, Henderson AM. 1978. Multivariate dental allometry in primates. Amer J Phys Anthro 48:203-208.

Czaplewski NJ, Morgan GS, McLeod SA. 2007. Chiroptera. In: C. M. Janis, G. F. Gunnell, and M. D. Uhen, editors. Evolution of tertiary mammals of North America, vol. 2. Cambridge: Cambridge University Press. p174-197.

Evans AR, Hunter J, Fortelius M, Sanson GD. 2005. The scaling of tooth sharpness in mammals. Am Zool Fennici 42:603-613.

Fortelius M. 1985. Ungulate cheek teeth: developmental, functional, and evolutionary mechanisms. Acta Zool Fennica 180:1-76.

Fortelius M. 1990a. Problems with using fossil teeth to estimate body sizes of extinct mammals. In: J. Damuth and B. J. MacFadden, editors. Body size in mammalian paleobiology: estimation and biological implications. Cambridge: Cambridge University Press. p207-228.

Fortelius M. 1990b. The mammalian dentition: a “tangled” view. Neth J Zool 40:312- 328.

Freeman PW. 1998. Form, function, and evolution in skulls and teeth of bats. Papers in Natural Resources 9:140-156.

Genoways HH, Pedersen SC, Larsen PA, Kwiecinski GG, Huebschman JJ. 2007. Bats of Saint Martin, French West Indies/Sint Maarten, Netherlans Antilles. Mastozoologia Neotropical 14:169-188. 70

Gingerich PD, Smith BH, Rosenberg K. 1982. Allometric scaling in the dentition of primates and prediction of body weight from tooth size in fossils. Am J Phys Anthro 58:81-100.

Gingerich PD, Smith BH. 1984. Allometric scaling in the dentition of primates and insectivores. In: W.L. Jungers, editor. Size and scaling in primate biology. Plenum Publishing Corp. p257-272.

Gould SJ. 1975. On scaling of tooth size in mammals. Amer Zool 15:351-362.

Gutzwiller SG, Hunter HP. In press. Evolution and function of the upper molar talon and its dietary implications in microbats. J Morph.

Hogue AS, ZiaShakeri S. 2010. Molar crests and body mass as dietary indicators in marsupials. Austr J of Zool 58:56-68.

Hunter JP, Jernvall J. 1995. The hypocone as a key innovation in mammalian evolution. Proc Natl Acad Sci 92:10718-10722.

Jernvall J. 1995. Mammalian molar cusp patterns: developmental mechanisms of diversity. Acta Zool Fen 198:1-61.

Jones KE, Purvis A, MacLarnon A, Bininda-Emonds ORP, Simmons NB. 2002. A phylogenetic supertree of the bats (Mammalia: Chiroptera). Biol Rev 77:223-259.

Kay RF. 1975. The functional adaptations of primate molar teeth. Am J Phys Anthrop 43:195-216.

Kleiber M. 1947. Body size and metabolic rate. Physiol Rev 27:511-541.

Koopman, K. F. 1993. Bats. In: D.E. Wilson and D. M. Reeder, editors. Mammalian species of the world. A taxonomic and geographic reference. Washington: Smithsonian Institution Press.

Lucas PW. 2004. Dental function morphology: how teeth work. Cambridge UK: Cambridge University Press.

Nowak RM. 1994. Walker's bats of the world. Baltimore: The John Hopkins University Press.

Osborn HF. 1907. Evolution of mammalian molar teeth. New York: Macmillan.

Pilbeam D, Gould S. 1974. Size and scaling in human evolution. Science 186:892-901.

Popowics TE, Fortelius M. 1997. On the cutting edge: tooth blade sharpness in herbivorous and faunivorous mammals. Ann Zool Fennici 34:73-88. 71

Strait SG. 1997. Tooth use and the physical properties of food. Evol Anthropol 5:199- 211.

Ungar PS. 2014. Dental allometry in mammals: a retrospective. Ann Zool Fennici 51:177-187.

Ungar PS, Kay RF. 1995. The dietary adaptations of European Miocene Catarrhines. Proc Natl Acad Sci 92:5479-5481.

Van Cakenberghe V, Herrel A, Aguirre LF. 2002. Evolutionary relationships between cranial shape and diet in bats (Mammalia: Chiroptera). In: Aerts P, D’Août K, Herrel A, and Van Damme R, editors. Topics in functional and ecological vertebrate morphology. Maastricht: Shaker Publishing. p 205-236.

Winchester JM, Boyer DM, St. Clair EM, Gosselin-Ildari AD, SB Cooke, JA Ledogar. 2014. Dental topography of platyrrhines and prosimians: convergence and contrasts. Am J Phys Anthropol 153:29–44.

Chapter 4. Upper Molar Talon and Tooth Strength

Introduction

Mammalian dental morphology represents a tradeoff between optimal design for food breakdown and the maintenance of sufficient tooth strength to avoid enamel cracking during chewing. Sufficient tooth strength can be attained several ways. Enamel microstructure, such as enamel thickness or enamel prism decussation, can be modified

(Bajaj and Arola, 2009; Lawn et al., 2009). Overall robustness of tooth features, such as aspect ratio and radius of curvature of cusps, can be increased (Lucas, 2004; Qasim et al.,

2005; Freeman and Lemen, 2007). Finally, adding novel structures to the tooth can also help to dissipate stress within the crown (such as the cingulum, an enamel ridge located at the base of the tooth; Lucas et al., 2008; Anderson et al., 2011). The evolution of a novel structure to help dissipate occlusal stress may allow the remainder of the tooth to optimize for food breakdown. Thus, when examining the functional evolution of a novel tooth feature, one must not only consider the potential functions it may perform in food breakdown (Chapter 2 and 3), but also in maintenance of tooth strength. The novel feature considered here is the talon, the elaboration of the distolingual region of the primitive tribosphenic upper molar in therian mammals (Fig. 1).

Talons vary greatly in shape and size in therians, ranging from a small cingulum, to a large projecting shelf, to a fully-formed hypocone cusp (Hunter and Jernvall, 1995;

Jernvall, 1995; Freeman, 1998; Czaplewski et al., 2008). The remaining primitive portion of the upper molar is considered the trigon (Fig. 1). Talons have evolved convergently over 20 times in therian mammals, and the hypocone cusp is considered by many to be a key innovation allowing for the evolution of derived herbivory (Hunter and Jernvall,

1995; Jernvall, 1995; Hunter, 1998; Butler, 2001; Crompton et al., 2010). However, the function of this region prior to its exaptation in derived herbivores is not fully understood. A better understanding of talon function, from its origin through its expansion, will help us to explore its adaptive significance in therian mammals.

Previous research has focused on the function of the talon during food breakdown, including its role in the crushing and shearing of food and maintenance of occlusal area with increasing body size (Ch. 2 and 3; Butler, 1972; Hunter and Jernvall,

1995; Jernvall, 1995; Butler, 2001; Gutzwiller and Hunter, in press). The evolution of a fully cuspal hypocone is thought to have squared off the upper molar, performing a crushing function brought about by the reduction of the paraconid cusp of the lower molar (Butler 1972; Hunter and Jernvall 1995; Jernvall, 1995; Butler 2001). With the reduction of the paraconid, the hypocone can crush against the entoconid of the anterior lower molar and the metaconid and protoconid of the posterior lower molar (Butler 1972;

Hunter and Jernvall 1995; Jernvall, 1995; Butler 2001). The results presented in Chapter

2 support this notion: the presence of the talon significantly increases the crushing function of the tribosphenic molar (quantified using occlusal relief; Gutzwiller and

Hunter, in press). This crushing function of the talon is consistent across dietary categories (Chapter 2) and across talon sizes (Chapter 3).

However in certain cases, it is unlikely that talon morphology is strictly tied to crushing function. For example, in mammals where the lower molars retain a well- developed paraconid, smaller talons that are lower on the crown probably do not occlude with the lower molars. Results from Ch. 3 may support this notion, finding that talons of small faunivorous bats do not scale with body size, perhaps reflecting a lack of participation in food breakdown. Additionally, the evolutionary benefit of a crushing function provided by the talon for a faunivorous mammal (including insectivores and carnivores that primarily rely on shearing for food breakdown) is not clear (Strait, 1997;

Lucas, 2004; Gutzwiller and Hunter, in press). Finally, as observed in Ch. 2, soft food eaters, such as moth specialists, exhibit talons of lower relief than hard food eaters, such as beetle specialists (Gutzwiller and Hunter, in press). This difference does not translate to a change in the overall crushing function of the whole molar, however, suggesting that a factor other than crushing function may be resulting in this observed difference between hard and soft food eaters (Gutzwiller and Hunter, in press).

These cases suggest that alternative functions of the talon need to be considered.

The difference in the relief of the talons of hard and soft food specialists may point to a potential tooth strength function of the talon. One large difference between chewing hard and soft food is the manner in which stress is distributed within the enamel of the molar

(Qasim et al., 2006; Qasim et al., 2007; Chai et al., 2009). Soft food is defined as a food item that easily deforms, whereas hard food is resistant to deformation (Strait, 1997).

During the breakdown of soft food, the food deforms easily and spreads over the entire tooth surface. This results in occlusal pressure being applied to the entire tooth surface

75 that is facing the food. Experimental loading of real teeth and tooth models has shown that the greatest stress in the enamel that results from this pressure is concentrated at the base of the tooth, leading to crack propagation up the side of the tooth and enamel chipping (Qasim et al., 2007; Chai et al., 2009). Alternatively during the chewing of hard foods, the food does not deform over the entire tooth surface and instead occlusal pressure is concentrated at the location of initial food contact (the cusps tips). The greatest stress is concentrated at these points of food contact, resulting in cracks forming perpendicular to the enamel-dentine junction under the load points (Qasim et al., 2006).

Novel tooth features that dissipate enamel stress and reduce the likelihood of cracking may reflect these differences in hard and soft food chewing (Lucas, 2004; Freeman and

Lemen, 2007; Anderson et al., 2011). Anderson et al. (2011) found that the addition of a cingulum to the base of a simple cone-shaped tooth can increase the strength of the tooth and reduce the likelihood of cracking. Under soft food simulations, both small and large cingula reduced the strain (directly related to stress and tooth cracking) observed in the tooth. Under hard food simulations, only large cingula reduce enamel strain, with smaller cingula actually increasing enamel strain. Anderson et al. (2011) explained the reduced strain caused by large cingula in hard food simulations may result because the cingulum extends more of the enamel bulk (and thus strain distribution) further from the main body of the tooth, where the highest strains are occurring. Overall, the cingulum provided the most consistent benefit during the chewing of soft food, because a cingulum located at the base of the enamel cap dissipates the high stresses that are typical for this area during soft food chewing.

The talon may be providing a similar tooth strength benefit to the tribosphenic upper molar. Indeed, small talons are often considered cingula. The differences between the occlusal relief of the talons of hard and soft food specialists observed in Chapter 2 may support this notion. While occlusal relief is often used to define the shearing and crushing capability of a surface (as in Chapter 2 and 3; M’Kirera and Ungar, 2003; Bunn et al., 2011), it has also been used to define the overall height of molar cusps (see Evans,

2013). A taller cusp will result in a higher relief than a shorter cusp of similar shape. The higher relief of the talons observed in hard food eaters could reflect a tall talon that is more likely to participate in food contact (Gutzwiller and Hunter, in press). During the chewing of hard foods, this additional point of food contact would increase enamel bulk, allow for another point of load concentration on the food, and help dissipate stress across the tooth. On the other hand, the low relief of the talon of soft food eaters likely reflects a short and flat talon. A talon of this shape would help dissipate the stress concentrated at the base of the enamel cap during soft food chewing. While the increased enamel bulk provided by the talon may be beneficial to tooth strength in most cases, the fine scale differences in therian talon shape and size may ultimately correlate with the chewing of hard or soft foods.

The present study examines the potential for the talon to have evolved as a mechanism to increase tooth strength, using a structural modeling technique originating in engineering, Finite Element Analysis (FEA). FEA simulates the physical response

(here focusing on maximum Von Mises stress) from the loading of 2- and 3-dimensional computer models, in order to estimate the potential for failure of the structure. In

Biology, FEA has been used to study how anatomical features, such as skulls and teeth, respond to high loading situations including chewing (e.g., Dumont et al., 2005;

Richmond et al., 2005; Anderson et al., 2011; Dumont et al., 2011). In these studies, the maximum stress observed in the model represents increased likelihood of failure, because stress is directly proportional to failure (i.e. tooth cracking in this case; Dumont et al.,

2009). The failure of a structure occurs when the resulting stress exceeds the stress that the structure can withstand (Dumont et al., 2009). Thus, in FEA, this failure is theoretically determined based on the extent of maximum stress and cannot be directly observed. This modeling technique was used to address the following hypotheses:

Hypotheses

H1: Highest stress will be observed near the cusp tip during hard food chewing (following previous observations of enamel strain and cracking; Qasim et al.,

2007; Anderson et al., 2011).

H2: Highest stress will be observed near the base of the occlusal surface during soft food chewing (following previous observations of enamel strain and cracking;

Qasim et al., 2007; Chai et al., 2009; Anderson et al., 2011).

H3: The presence of the talon will reduce maximum enamel stress, increasing tooth strength and reducing the likelihood of tooth cracking (following the function of the cingulum proposed by Lucas et al., 2008 and Anderson et al., 2011).

H4: Talon size will differentially affect tooth strength, dependent on food hardness. Talons of all sizes will reduce maximum stress in soft food chewing (Anderson et al., 2011). Only large talons will reduce maximum stress in hard food chewing

(Anderson et al, 2011).

In this research, FEA was used to examine if the presence of talons of a variety sizes contribute to increased tooth strength during hard and/or soft food loading regimes, using both theoretical and living-species models. Two common talon shapes (cusp vs. shelf) were also examined as potential factors influencing stress values and distribution.

This research was addressed using microbats (suborder Microchiroptera) as a representative therian clade, due to the diversity in talon shape and size observed in this suborder (for examples, see Fig. 5). The ultimate goal of this research is to examine the adaptive and exaptive implications of the evolution of the talon in therian mammals.

Materials and Methods

Specimen Selection

Specimens were borrowed from The Ohio State University, Museum of

Biological Diversity (OSUM), the University of Michigan, Museum of Zoology

(UMMZ), and the Carnegie Museum of Natural History (CMNH). Individuals were chosen for minimal wear on the upper left first molar (M1). One individual, Pipistrellus subflavus OSUM 1047, was selected to represent the primitive tribosphenic upper molar

(lacking a talon) and to act as a baseline to which a variety of theoretical talons would be manually added (see below). Pipistrellus is a generalist insectivore, eating both hard and

79 soft insects (animaldiversity.org). Additionally, four other individuals of extant species were selected to act as real-life comparisons to the baseline tribosphenic. Eptesicus fuscus, OSUM 1829, is a hard insect specialist with a small cuspal talon

(animaldiversity.org). Chrotopterus auritus, UMMZ 124358, is a carnivore with a large talon shelf (animaldiversity.org). Tadarida brasiliensis, OSUM 1744, is a hard insect specialist with a large talon shelf and small talon cusp. Sturnira lilium, CMNH 42862, is a soft fruit specialist, with a medium sized talon cusp and a trigon with a more rounded and flatter topography than the primitive tribosphenic molar.

Micro-computed Tomography Scanning and 3D Rendering

The skull of each specimen was scanned using a micro-computed tomography scanner (microCT; Inveon, Siemens) at the Wright Center of Innovation in Biomedical

Imaging (The Ohio State University). Scans were performed at 100 kV voltage, 200 mA current, and an effective pixel size of 19.4 µm. The resulting digital volumes were imported into Amira 5.6 visualization software (Visualization Sciences Group) for three- dimensional rendering.

In Amira, the upper left M1 of the Pipistrellus baseline and four extant comparisons were segmented, surface smoothed in order to remove artifacts of the scanning process, and exported as a *.stl surface file. The pulp cavity was modeled as empty space (due to its low density and lack of effect on the stress dynamics of the rest of the tooth; Anderson et al., 2011). Unfortunately, due to the scanning resolution constraints on these miniscule teeth, the enamel and dentin were not modeled individually. However, this simplification is unlikely to affect the results, because enamel

80 thickness does not vary to a great degree in microbats (Dumont, 1995). Additionally, because the focus of this project was to examine how the shape and size of the talon affects tooth strength, assuming that all models have a similar ratio of enamel to dentin reduces potential confounding factors.

Each file was then imported into Geomagic Studio 2014 (geomagic.com) for additional smoothing and cleaning, in order to create a model more representative of the real tooth. Using the Pipistrellus model as a baseline tribosphenic tooth, six theoretical models possessing talons were created in Geomagic. Theoretical talons were manually modeled and added to the Pipistrellus rendering, varying according to shape (cusp or shelf) and size (small, medium, and large). Thus, a total of 11 models (the Pipistrellus baseline model, four extant species models, and six theoretical models) were created. For each model, a volume mesh was created as a *.bdf file in Numeca Hexpress

(www.numeca.com) for subsequent Finite Element Analysis.

Convergence Analysis

A convergence analysis was performed to determine the appropriate level of model resolution. This analysis was performed through repeated FEAs of one model with increasing resolution and examination of resulting contour maps (as described below).

Finite Element Analyses

Finite Element Analyses were performed in Strand7 (strand7.com). Each tooth model was assigned a Young’s Modulus of 50 GPa and Poisson’s Ratio of 0.29. These values represent averages between typical enamel and dentin values (Waters, 1980;

Anderson et al., 2011). Although I attempted to assign these physical properties

81 according to values observed in real teeth, the exact values do not affect the comparability of my models. Since all of the models assume homogenous construction, and differences in shape are of primary interest, the models are comparable to each other as long as they are assigned the same physical properties. Each model was restrained

(anchored) at the roots, as a simplification of tooth attachment in the socket (Fig. 13). All analyses were scaled so that 1 MPa of pressure was applied to the surface in the direction of the occlusal vector (Fig. 13). The occlusal vector represents the Phase I power stroke of the chewing cycle (Phase II is thought to be minimal in bats; Crompton and Hiiemae,

1969). The occlusal vector was defined as the direction perpendicular to the model in functional view (where the Phase I wear facets are out of view; Butler, 1972; Wood and

Clemens, 2001; Polly, 2005; Rook et al., 2010). Keeping occlusal pressure constant ensured that the force-to-area ratio was maintained across models of varying sizes and that the resulting stress values were comparable (see Dumont, 2009). Hard and soft food chewing simulations were performed similarly to Anderson et al. (2011).

Hard Food Simulation

During the chewing of hard food, the food does not deform over the tooth surface and instead pressure is applied only at the cusp tips (Fig. 13a). In these simulations, the talon only participated in loading if it could be assumed to realistically come into initial contact with the food (occlusion). For the extant species, talon occlusion was determined based on personal observations of the height of the lower paraconid cusp in the following way. All extant species were modeled without the talon occluding, due to a lack of the reduction of the paraconid (personal observation). Note, Freeman (1998)

82 proposes an alternate occlusal relationship in the carnivorous Macroderma, which possesses morphology very similar to Chrotoperus, in which an enlarged lower protoconid occludes in the basin-like talon. However, in order to keep the occlusal assumptions consistent in this analysis (in which pressure is only applied to cusp tips during hard food chewing), Chrotopterus was modeled without the talon occluding. For all theoretical models, except the large cuspal talon, the talon was assumed to be too small and low to participate in initial contact with hard food. As such, only the theoretical large cuspal talon (i.e. a hypocone) participated in loading during hard food simulations.

Soft Food Simulation

During the chewing of soft food, the food deforms over the entire occlusal surface of the tooth. Thus, in these simulations, pressure was applied to the entire occlusal surface (i.e. the surface visible in occlusal view; no underhanging regions of the tooth; Fig. 13b). Since the food deforms over the entire surface, all talons participated in occlusion in soft food loading.

Effect of Added Surface Area versus Remodeling

With the addition of a novel structure to a tooth, two theoretical possibilities exist: the novel structure could add surface area to the tooth or the tooth could be “remodeled” to keep the same surface area. The scaling of all pressures to have the same force-to-surface area ratio (as described above) assumes either that remodeling is taking place (surface area is held constant) or that force can be assumed to increase in proportion with surface area. This scaling is appropriate when comparing different species (and is presented as such for the extant species models), where one can assume

83 that bite force increases with increasing body size (Aguirre et al., 2002). However for my theoretical models, it would be interesting to examine the effect of the increased surface area provided by the talon, in addition to remodeling. For my analyses, surface area refers to the area of pressure application. In my theoretical models, this area is different between models only when the talon is occluding. Thus, to examine the effect of increased surface area of the talon versus remodeling, I performed two separate analyses for those simulations where the talon was occluding (i.e. large cuspal hard food simulation and the soft food simulations; see Table 6 for those models included). In first analysis, the resulting stress values were scaled to reflect the increase in surface area due to the talon. In this way, the effect of the increased surface area of the talon could be directly observed. In the second analysis (presented as “remodeled” in the results below), each model was assumed to be remodeled, where the force-to-surface area ratio was held constant across models (essentially removing the effect of increased surface area of the talon and instead purely examining differences in shape).

Following all FEAs, maximum Von Mises stress of the occlusal surface (max. stress) was measured for each model in hard and soft chewing simulations in Strand7.

The max. stress for each model was compared to the baseline tribosphenic model, resulting in a relative max. stress (%). Because max. stress is proportional to the likelihood of failure, it can act as a predictor tooth cracking. As such, the exact max. stress values are not important (see Anderson et al., 2011). Instead, relative stress values allow for a comparison of the relative risk of tooth cracking. A relative max. stress less than 100% represents a decrease in max. stress when compared to the baseline

84 tribosphenic. A relative max. stress greater than 100% represents an increase in max. stress when compared to the baseline tribosphenic. Comparing relative max. stress across models allows for an assessment of the relative risk of cracking for models of varying morphology under hard and soft food loading conditions. Additionally, contour maps of the stress distribution across the occlusal surface were used to qualitatively examine how the presence of a talon of varying shape and size affects the amplitude and location of max. stress in the tooth crown.

Results

Convergence Analysis

Varying model resolution changed the resulting contour maps very little. As such, a medium level of resolution was determined to be adequate to capture shape differences between the models and produce reliable stress results. The models consisted of 83,158 to

182,929 tetrahedral brick elements. This variability ultimately depended on the complexity needed to model the pulp cavity and is unlikely to affect the results.

Theoretical Models

Hard Food Simulation

Under hard food loading conditions, the max. stress is consistently near the tip of the metacone cusp, at the edge of force loading (Fig. 14a-g). This result is consistent with the location of maximum strains at the cusp tip during hard food loading observed by Anderson et al. (2011). The presence of the talon has variable effects on the max. stress, with some talons increasing and some decreasing relative max. stress (Table

6). The small cuspal talon and the medium talon shelf perform the worst, both increasing max. stress in the model (with the small cusp model increasing max. stress by 4.5% when compared to the baseline tribosphenic, and the medium talon shelf increasing max. stress by 4.2%). Even though the max. stress at the metacone increases in these two models, the stress contours suggest a slight decrease in stress on the protocone and paracone cusps

(Fig. 14c,d). However, given that the location of max. stress is the location most likely to crack, a small cusp or medium basin may increase the likelihood of enamel cracking during hard food chewing. Alternatively, medium and large cuspal talons decrease max. stress to the greatest degree (with the medium cusp model reducing max. stress by 24.8%, and the large cusp model reducing max. stress by 21.1%).

Soft Food Simulation

Under soft food loading conditions, the stress is more evenly distributed across the occlusal surface than in hard food simulations (although the magnitude of this stress is greater than in hard food simulations; see scale in Fig. 15). The max. stress is consistently located in the trigon groove and at the base of the metacone cusp, contrary to my prediction that the max. stress would be located at the base of the occlusal area (Fig.

15a-g). For all shapes and sizes, talon presence lowers the max. stress in the model (with a medium cuspal talon performing best with a reduction of 13.8% ; Table 6).

Effect of Added Surface Area vs. Remodeling

All models that are “remodeled” (to keep surface area constant and examine differences in shape only) decrease max. stress when compared to the baseline tribosphenic (with the large cuspal hard food talon performing best at a 16.2% reduction;

Table 6). However, most of these models do not perform as well as their counterparts in which the talon has been allowed to expand in surface area without this remodeling. In other words, stress declines if the addition of the talon increases the surface area of force application.

Extant Models

Hard Food Simulation

In the hard food simulations, all four extant species models show max. stress values located at the edge of force loading near the cusp tip (primarily metacone and protocone), similar to the baseline tribosphenic model (Fig. 14a,h-k). Max. stress values in these extant models are all lower than the baseline tribosphenic, but to varying degrees (Table 7). Eptesicus fuscus, with a small cuspal talon, has a very similar max. stress to the baseline tribosphenic (1.4% reduction). On the other hand, Sturnira lilium, with a medium cuspal talon and derived trigon, performs best with a 39.0% reduction in max. stress when compared to the baseline tribosphenic.

Soft Food Simulation

In the soft food simulations of extant species, the max. stress is located at the base of the metacone, similar to some of the theoretical models (Fig. 15a,h-k). In all models, except Sturnira lilium, max. stress is increased in the extant models compared to the baseline tribosphenic (with the largest increase being 74.8% in Eptesicus fuscus;

Table 7). However, under soft loading conditions Sturnira lilium significantly reduces max. stress by 56.7%, when compared to the baseline tribosphenic.

Discussion

Maximum stress in hard and soft food chewing

The location of maximum Von Mises stress under hard food loading is consistent with H1. Generally, max. stress is located at the cusp tips, especially on the metacone, at the basal edge of force loading. This result is similar to previous studies that found maximum strains and highest likelihood of cracking located at the cusp tips, under the point of force loading (Qasim et al., 2006; Anderson et al., 2011). Any difference between these results and previous work is likely due to the area of force loading. In previous studies, the area of force application was very small, whereas in this study, the force was applied to the entire cusp tip. This larger area of force loading results in max. stresses being located at the edge rather than the center of force loading.

The location of maximum Von Mises stress under soft food loading, however, is not consistent with H2. H2 stated that under soft food loading, max. stress would be located at the base of the occlusal surface. However, in this study max. stress was primarily located at the trigon groove and at the base of the metacone. The difference is likely due to the way that soft food chewing has been modeled in previous work compared to this research. Previous research has primarily modeled teeth as single cones and found that maximum stresses/strains occur at the base of the cone in soft food loading (Qasim et al., 2007; Anderson et al., 2011). The present research however, has modeled a tooth with complex morphology consisting of several cusps, crests, and basins.

Finding max. stresses in the trigon groove and at the base of the cusps was unexpected, but not contrary to previous work. This may ultimately suggest that each cusp is acting

88 somewhat independently in the distribution of stress, as opposed to the tooth acting as an integrated unit as initially expected. Overall, the distribution of maximum stresses in both hard and soft food simulations is consistent with previous experiments on real and model teeth, lending validity to my modeling technique (Qasim et al., 2006; Qasim et al., 2007;

Chai et al., 2009).

Effect of the talon on tooth strength in theoretical models

Presence of the talon does reduce maximum occlusal stress in some cases, increasing tooth strength and supporting H3. Since the amplitude and location of maximum stress is associated with an increased risk of tooth failure, any decrease in max. stress would reflect increased tooth strength. Talon presence increases tooth strength during soft food chewing and sometimes during hard food chewing, dependent upon talon shape and size (discussed further below). In these cases, the talon may have evolved as a novel structure adapted to maintain tooth strength in the face of the high impact of chewing.

There is some evidence to support H4, that talon size differentially affects tooth strength. These results are dependent on food hardness and also talon shape. Under hard food loading simulations, medium and large cuspal talons most successfully reduce max. stress, whereas smaller talons generally do not perform well. This result is consistent with previous work that found that large cingula are able to reduce maximum strains during hard food chewing, whereas small cingula of either shape actually increase maximum strains (Anderson et al., 2011). Because the location of max. stress in hard food simulations is centered at the cusp tips, smaller talons may simply be too far from this

89 location to influence stress dissipation. However, the increased bulk provided by a large talon cusp may help to better dissipate stresses within the occlusal surface. Note also that the large cuspal talon occludes in these hard food simulations, suggesting that it may act as another point of pressure application, additionally helping to spread force out across a greater area and improve stress dissipation. These results suggest that larger talons provide a greater benefit to hard food eaters than do smaller talons.

Under soft food loading, talon shape and size did not affect the degree of stress reduction, further supporting H4. Talons of all shapes and sizes decreased max. stress and improved tooth strength, consistent with previous findings (Anderson et al., 2011). As with the hard food simulations, it is likely that the talon provides this tooth strength benefit during soft food chewing by increasing enamel bulk and the area of force application, helping to dissipate stress within the occlusal surface.

Because the theoretical models were built from the same baseline tribosphenic molar, I was able to keep all aspects of the tooth (except the talon) the same and thus examine separately (1) the talon’s effect on shape only (remodeling) and (2) the potential benefit of increased surface area of the talon. Examining increased surface area assumed that my theoretical models represent closely related species that all share the same body size (and thus bite force), but vary in talon development. When the talon is involved in food contact (as a large cusp in hard food eating or as any shape or size in soft food eating), the increased surface area of pressure application allows for better distribution of force and thus better dissipation of stress across the tooth (as seen above). However, when these teeth are “remodeled”, we are purely examining how shape affects tooth

90 strength. Interestingly, even when these theoretical teeth are remodeled, such that the surface area is kept the same, the talon still provides a benefit (although not to the same degree). These results suggest that both the increased surface area of the talon and the effect that it has on overall molar shape contribute to its role in increasing tooth strength.

This result is contrary to previous research that found that the cingulum did not provide a benefit if the tooth was remodeled and only reduced enamel strains if it was assumed to increase surface area (Anderson et al., 2011).

Effect of the talon on tooth strength in extant species

The amplitude and distribution of occlusal stress are generally similar between my theoretical and extant species models, suggesting that these models are comparable.

However, given that the molars of the extant species vary in ways other than just talon development (e.g. trigon morphology) and that factors such as enamel thickness were not included in my analyses, these models must be interpreted in a holistic view, as follows.

In general, my extant species models (except Sturnira) perform better during the hard food chewing simulations than the soft food chewing simulations. Given that

Eptesicus and Tadarida are hard insect specialists, this observation makes sense.

Chrotopterus, as a carnivore, may also encounter hard food items, including exoskeleton and bone. The molars of these species have likely evolved as a tradeoff between ideal shearing function for the breakdown of tough insect cuticle or tissue material and the maintenance of tooth strength in the face of hard food chewing. The talon in these species is low in relief, being either a rounded cusp or a flat shelf, suggesting that it probably contributes little to shearing function. Instead, the talon may provide a benefit to tooth

91 strength in the hard food specialists. However, it is important to note that changes in the trigon are likely contributing to tooth strength as well. For example, Tadarida and

Chrotopterus have similar talons, yet Chrotopterus was able to reduce relative maximum occlusal stress by 26.1%, whereas Tadarida only reduced relative maximum stress by

18.0%. This difference is likely due to the overall robustness of the trigon in

Chrotopterus, further reducing occlusal stress.

During the soft food simulations, all extant species (excluding Sturnira) performed worse compared to the generalist Pipistrellus. Again, this observation makes sense given the natural diet of these species. Eptesicus and Tadarida are more likely to encounter hard food items than soft food items. Thus their overall molar shape has evolved in response to this selective pressure. Under soft food loading in these species, a very high stress is located at the base of the metacone. This stress concentration is probably due to the shape of the metacone, being generally taller than the other cusps and having a more angled shape (concave below the cusp). This shape is likely ideal for the precise occlusion required for insectivory. However, this shape is not designed for the maintenance of tooth strength in the face of soft food, where highest stresses are located at the base of the cusp. Sturnira, on the other hand, performs very well under soft food loading. This makes sense given that Sturnira is a soft fruit specialist and likely has molars adapted for the breakdown of soft food. The presence of the talon may contribute to the high tooth strength in Sturnira. However, the overall robustness of the trigon makes this molar very resistant to enamel cracking. Ultimately, since frugivore molars are overall more robust, having low occlusal relief ideal for chewing fruit pulp, they may

92 experience less of a tradeoff between tooth function and strength than insectivores and carnivores do. Thus in frugivores such as Sturnira, teasing apart the primary role of the talon (as a crushing surface or a tooth strength mechanism) is difficult.

Conclusions

The therian mammal upper molar talon originated convergently as a small cingulum or metaconule, and has since diversified into a variety of shapes (including shelves, basins, and fully-formed cusps) and sizes (reaching 25% of the molar area in

Ardops nichollsi, for example; personal observation; Butler, 1972; Hunter and Jernvall,

1995; Jernvall, 1995). The research presented here helps us understand the potential functions that the talon performs in these teeth and allows us to further explore the talon as an adaptive feature in the evolution of the therian tribosphenic complex.

In many cases, the presence of the talon does increase the strength of the tribosphenic molar. For soft food specialists, a small, medium, or large talon of either a cusp or shelf shape decreases occlusal stress, reducing the likelihood of tooth cracking.

This benefit is not only due to the increased area of force distribution provided by the talon, but also the added enamel bulk allowing for better dissipation of stress within the tooth crown. During the chewing of soft food, the talon’s location at the distolingual base of the crown is ideal for reducing the maximum stresses at the base of the metacone. For hard food specialists, larger cuspal talons provide the greatest benefit to tooth strength, whereas, in some cases, smaller talons may actually increase occlusal stress. During the chewing of hard foods, large talons provide sufficient enamel bulk to reduce maximum stresses located further away near the tips of the other cusps. Additionally, if a talon

93 becomes large enough to occlude with the lower molars during the chewing of hard food

(such as a fully-formed hypocone cusp), the additional point of force application helps reduce the stress within the rest of the tooth. These results may help explain why hard food specialists tend to have taller (higher relief) talons than soft food specialists, as observed in Chapter 2. In both cases, the talon is optimized for increasing tooth strength.

This research illuminates how talons can act as a mechanism to increase tooth strength, depending upon the selective pressures of a hard or soft diet.

Understanding the role of the talon in the maintenance of tooth strength can help us to examine the adaptive and/or exaptive implications of the origin and elaboration of the talon throughout therian evolution. Since the talon originated as small cingulum or cusp, it was unlikely to contribute greatly to the food breakdown function of the molar.

Instead, this research suggests that small talons could have originated as an adaptation for increased tooth strength during the chewing of soft foods (soft insects, for instance).

Cooptation of the talon for other diets and functions would suggest that a small talon represents an exaptation as well. Additional modifications to the talon (increase in size or change in shape) would then represent subsequent adaptations for other diets and functions. For example, the presence of a crushing-shaped talon in faunivores is unlikely to provide a food breakdown benefit and instead may be explained by the potential for a taller talon to provide additional tooth strength during the chewing of hard foods.

Although a talon in faunivores may represent a tradeoff between optimal shearing function and tooth strength, the increased strength it provides may allow the trigon to further optimize for food breakdown efficiency (precise occlusion and shearing

94 capability). Additionally, the increased crushing provided by the talon in derived frugivores could represent an additional adaptation of the talon to a novel function and diet. Ultimately, the evolution of the talon is likely characterized by the origin of a novel adaptive structure, exaptation of that novel structure for new functions, followed by a series of further adaptations optimized for changing diets and food breakdown functions.

This work exploring the potential adaptation of the talon for increased tooth strength has allowed us to better understand how the origin of the talon, on the one hand, and its expansion, on the other, may have come about in therian mammals.

Figures

Figure 13. FEA Design Pressure and restraint application for (A) hard food simulations where pressure is applied only at the cusp tips and (B) soft food simulations where pressure is applied across the entire occlusal surface. The purple shaded regions and arrows show the location and direction of pressure application. The black arrows show the areas of model restraint.

Figure 14. Hard Food Contour Maps Contour maps of maximum Von Mises stress under hard food loading for (A) the baseline tribosphenic, Pipistrellus subflavus; theoretical models: (B) small shelf, (C) small cusp, (D) medium shelf, (E) medium cusp, (F) large shelf, (G) large cusp; extant models: (H) Eptesicus fuscus, (I) Sturnira lilium, (J) Tadarida brasiliensis, (K) Chrotopterus auritus. Abbreviations: (Me), metacone; (Pa), paracone; (Pr) protocone. 97

Figure 15. Soft Food Contour Maps Contour maps of maximum Von Mises stress under soft food loading for (A) the baseline tribosphenic, Pipistrellus subflavus; theoretical models: (B) small shelf, (C) small cusp, (D) medium shelf, (E) medium cusp, (F) large shelf, (G) large cusp; extant models: (H) Eptesicus fuscus, (I) Sturnira lilium, (J) Tadarida brasiliensis, (K) Chrotopterus auritus. Abbreviations: (Trg), trigon groove. 98

Tables

Table 6. Theoretical Model Max Stresses

Maximum Von Mises Stress for Theoretical Models Rel. Max Remodeled Load Rel. Max Remodeled Model Stress Max Stress Type Stress (%) Max Stress (MPa) (MPa) (%) Hard Baseline tribosphenic 4.83 - - - Hard Small shelf 4.42 91.51% - - Hard Medium shelf 5.03 104.19% - - Hard Large shelf 4.23 87.58% - - Hard Small cusp 5.04 104.45% - - Hard Medium cusp 3.63 75.22% - - Hard Large cusp 3.81 78.88% 4.05 83.82% Soft Baseline tribosphenic 14.50 - 14.50 - Soft Small shelf 12.96 89.38% 12.80 88.28% Soft Medium shelf 12.82 88.41% 12.92 89.10% Soft Large shelf 12.86 88.69% 13.18 90.90% Soft Small cusp 12.63 87.10% 12.80 88.28% Soft Medium cusp 12.50 86.21% 12.61 86.97% Soft Large cusp 13.04 89.93% 13.69 94.41%

Table 7. Extant Model Max Stresses

Maximum Von Mises Stress for Extant Models Load Type Model Max Stress (MPa) Rel. Max Stress (%) Hard Baseline tribosphenic 4.83 - Hard Sturnira lilium 2.95 60.99% Hard Tadarida brasiliensis 3.96 82.01% Hard Chrotopterus auritus 3.57 73.90% Hard Eptesicus fuscus 4.76 98.62% Soft Baseline tribosphenic 14.50 - Soft Sturnira lilium 6.28 43.30% Soft Tadarida brasiliensis 20.15 138.94% Soft Chrotopterus auritus 16.91 116.60% Soft Eptesicus fuscus 25.35 174.84%

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Literature Cited

Aguirre LF, Herrel A, van Damme R, Matthysen E. 2002. Ecomorphological analysis of tropical niche partitioning in a tropical savannah bat community. Proc Biol Sci 269:1271-1278.

Anderson PSL, Gill PG, Rayfield EJ. 2011. Modeling the effect of cingula structure on strain patterns and potential fracture in tooth enamel. J Morphol 272:50-65.

Bajaj D, Arola D. 2009. Role of prism decussation on fatigure crack growth and fracture of human enamel. Acta Biomater 5:3045-3056.

Butler PM. 1972. Some functional aspects of molar evolution. Evolution 26:474-483.

Butler PM. 2001. Evolutionary transformations in the mammalian dentition. Mitt Mus Nat Kd Berl Zool 77:167-174.

Chai H, Lee JJ-W, Kwon J-Y, Lucas PW, Lawn BR. 2009. A simple model for enamel fracture from margin cracks. Acta Biomat 5:1663-1667.

Crompton AW, Hiiemae K. 1969. Functional occlusion in tribosphenic molars. Nature 222:678-679.

Crompton AW, Owerkowicz T, Skinner J. 2010. Masticatory motor pattern in the koala (Phascolarctos cinereus): a comparison of jaw movements in marsupial and placental herbivores. J Exp Zool 313A:1-15.

Czaplewski NJ, Morgan GS, McLeod SA. 2007. Chiroptera. In: C. M. Janis, G. F. Gunnell, and M. D. Uhen, editors. Evolution of Tertiary mammals of North America, vol. 2. Cambridge: Cambridge University Press. p174-197.

Dumont ER. 1995. Enamel thickness and dietary adaptation among extant primates and chiropterans. J Mamm 76:1127-1136.

Dumont ER, Davis JL, Grosse IR, Burrows AM. 2011. Finite element analysis of performance in the skulls of marmosets and tamarins. J Anatomy 218:151-162.

Dumont ER, Grosse IR, Slater GJ. 2009. Requirements for comparing the performance of finite element models of biological structures. J Theor Biol 256:96-103.

101

Dumont ER, Piccirillo J, Grosse IR. 2005. Behavior and bone stress in the facial skeletons of bats. The Anatomical Record 283A:319-330.

Evans AR. 2013. Shape descriptors as ecometrics in dental ecology. Hystrix, Ital J Mam 24:133-140.

Freeman PW. 1998. Form, function, and evolution in skulls and teeth of bats. Papers in Natural Resources 9:140-156.

Freeman PW, Lemen CA. 2007. The trade-off between tooth strength and tooth penetration: predicting optimal shape of canine teeth. J Zool 273:273-280.

Gutzwiller SG, Hunter HP. In press. Evolution and function of the upper molar talon and its dietary implications in microbats. J Morph.

Hunter JP. 1998. Key innovations and the ecology of macroevolution. Trends Ecol Evol 13:31-36.

Hunter JP, Jernvall J. 1995. The hypocone as a key innovation in mammalian evolution. Proc Natl Acad Sci 92:10718-10722.

Jernvall J. 1995. Mammalian molar cusp patterns: developmental mechanisms of diversity. Acta Zool Fen 198:1-61.

Lawn BR, Lee JJ-W, Constantino PJ, Lucas PW. 2009. Predicting failure in mammalian enamel. J Mech Behav Biomed Mater 2:33-42.

Lucas PW. 2004. Dental function morphology: how teeth work. Cambridge UK: Cambridge University Press.

Lucas PW, Constantino PJ, Wood BA, Lawn BR. 2008. Dental enamel as a dietary indicator in mammals. Bioessays 30:374-385.

M’Kirera F, Ungar PS. 2003. Occlusal relief changes with molar wear in Pan troglodytes troglodytes and Gorilla gorilla gorilla. Am J Primatol 60:31-41.

Polly PD, Le Comber SC, Burland TM. 2005. On the occlusal fit of tribosphenic molars: are we underestimating species diversity in the Mesozoic? J Mamm Evol 12:283-299.

Qasim T, Bush MB, Hu X, Lawn BR. 2005. Contact damage in brittle coating layers: influence of surface curvature. J Biomed Mater Res B 73:179-185.

Qasim T, Ford C, Bush MB, Hu X, Lawn BR. 2006. Effect of off axis concentrated loading on failure of curved brittle layer structures. J Biomed Mater Res B 76:334- 339.

102

Qasim T, Ford C, Bush MB, Hu X, Malament KA, Lawn BR. 2007. Margin failures in brittle dome structures: relevance to failure of dental crowns. J Biomed Mater Res B 80:78-85.

Richmond BG, Wright BW, Grosse I, Dechow PC, Ross CF, Spenser MA, Strait DS. 2005. Finite element analysis in functional morphology. The Anatomical Record 283A:259-274.

Rook DL, Hunter JP, Pearson DA, Bercovici A. 2010. Lower jaw of the Early Paleocene mammal Alveugena and its interpretation as a transitional fossil. J Paleontol 84: 1217- 1225.

Strait SG. 1997. Tooth use and the physical properties of food. Evol Anthropol 5:199- 211.

Waters NE. 1980. Some mechanical and physical properties of teeth. In: J.F. V. Vincent and J.D. Currey, editors. The mechanical properties of biological materials. Symposia of the Society of Experimental Biology 34. Cambridge UK: Cambridge University Press. pp 99-135.

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Chapter 5. Conclusions: Adaptation and Exaptation

The evolution of the therian talon is marked by numerous convergent origin events of small cingula or metaconules, followed by subsequent expansions into broad- reaching shelves or large hypocone cusps filling in the entire distolingual region of the molar. This dissertation explored three potential functions of the Microchiropteran talon, in order to understand the factors affecting its origin and elaboration in therian mammals.

The research described in Chapter 2 addressed the long-proposed hypothesis that the talon is an adaptation for increased crushing. This hypothesis was supported, with microbat talons generally being low in occlusal relief, increasing the crushing potential of the tribophenic molar. Whereas the trigon is specialized according to diet (with frugivores being more crushing specialized and faunivores being more shearing specialized), the talon exhibits a crushing-dominated shape across all diets. Increased surface area dedicated to crushing would provide a fitness benefit to a frugivorous bat that relies greatly on crushing for efficient food breakdown. However, the benefit of a low-relief talon to a faunivorous bat that relies on shearing is not fully understood.

Perhaps the increased crushing function provided by the talon allows for a more generalist faunivore diet. Or, perhaps the faunivore talon represents an evolutionary trade-off, with a shape that is not ideal for insect or tissue breakdown but provides some other function and fitness benefit. Although the presence of the talon across all species

104 was found to shift the whole molar function towards increased crushing, one could argue that a smaller talon may not provide the same functional impact as a larger talon. For this reason, it was important to not only examine the evolution of talon shape, but also the evolution of talon size. However, as observed in Chapter 3, talon crushing or shearing specialization is unrelated to talon size. This means that large talons are not more likely to reflect a given crushing or shearing specialization. With most talons being low in relief, it is possible that large talons are specialized for crushing in frugivorous bats.

However, given that a large low-relief talon will not provide a shearing benefit to a faunivorous bat, it is more likely that a large talon provides an alternative benefit in some species.

Chapter 3 allowed for an examination of a potential alternative talon function unrelated to crushing or shearing specialization. This research explored the potential for the talon to act as a more generalized structure, whose increased occlusal area allows for maintenance of metabolic needs at larger body sizes. Overall scaling trends show that both the trigon and talon scale with body size in some cases, suggesting that both regions can be involved in food breakdown. This participation in food breakdown means that the talon does act as part of the tribosphenic chewing complex in certain cases and its size is closely tied to the metabolic needs of increasing body size.

For frugivores, increased talon size could provide multiple benefits. It not only increases occlusal area allowing for larger body sizes, but also increases surface area specialized in crushing. In faunivores, the benefits of the talon likely change depending on the body size of the bat. In large faunivores, talon size scales closely with body size.

105

This suggests that talons are indeed participating in food breakdown in large faunivores.

But, due to low relief, the talon may simply not be specialized in shearing as we might expect. Instead, the talon may play a more generalist role, but still allowing the bat to increase occlusal area in order to maintain food intake at large body size. However, in small faunivores, talon size does not scale as closely with body size and thus the talon is unlikely to play a significant role in food breakdown. In fact, many small faunivores lack a talon entirely. This suggests that for small faunivores the shearing-dominated trigon is likely sufficient to maintain food intake as body size increases. So what function would a small talon that does not contribute to food breakdown perform in a small-bodied faunivorous bat?

The results from Chapter 4 provide a potential explanation. Talon presence was found to increase tooth strength during the chewing of soft food and sometimes hard food, depending on the shape and size of the talon. Larger talons provided reduced stress during the chewing of hard food, likely due to the additional point of occlusal force distribution. Talons of all shapes and sizes reduce occlusal stress during soft food chewing, likely due to the increased enamel bulk and surface area for distribution of occlusal force. It is possible the talons of small faunivorous bats provide a strength benefit during the chewing of hard or soft food. Bats of other diets and size classes may also benefit from the increased strength of the talon, in addition to the food breakdown benefits mentioned above.

Thus, all three functions of the talon – specialization for crushing, increased occlusal area for large body sizes, and increased tooth strength – are plausible depending

106 on the diet and body size of the mammal. An exploration of these potential functions was vital to be able to place the talon back into the context of therian dental evolution and understand its adaptive and exaptive significance.

Given that the talon originated as a small feature in a primitive tribosphenic molar of an insectivorous bat, it is most likely that it was initially adapted to increase tooth strength during the chewing of soft food (such as moths). From this point of origination, talons were able to be coopted into new functions, suggesting that a small talon represents an exaptation for derived functions and diets in microbats. Subsequently, the talon further adapted for specialization in these derived functions and diets, through changes in shape and size. One such adaptation would be a small talon in a derived frugivore. In a frugivore, the small talon could still provide the benefit of tooth strength in the face of soft food. But, in addition, the low-relief shape of the talon would allow for increased surface area dedicated to the crushing of fruit. The expansion of size of the talon could have provided a variety of benefits, representing additional adaptations. A large talon could further increase the crushing potential, and thus food intake efficiency, of a frugivorous mammal. A large talon could also provide a tooth strength benefit, potentially allowing for a dietary shift from soft to hard food. Finally, a large talon may provide a site of increased generalized occlusion, allowing for the evolution of increased body size in faunivores (especially carnivores that represent some of the largest living microbats).

In these ways, the talon represents a complicated, yet impactful, player in the evolution beyond the tribosphenic molar. The talon may be considered an adaptation

107 providing an initial benefit, but also an exaptation for derived diets and functions following the trajectory of therian dietary and body size evolution. The many convergent origins and elaborations of the talon across therians reflect the highly evolvable nature of teeth, responding to initial selective pressures and modifying morphology accordingly as those selective pressures change. Ultimately, the evolutionary legacy of the talon lies not only in its origin and expansion, but also go beyond to morphology no longer classified by the term “talon”. In some derived herbivores, the talon represents a key innovation, eventually allowing for the evolution of further derived shearing ridges and expansion into novel niches. In this way, the talon is not only represented by a diversity of cingula, shelves, and cusps in living mammals, but its evolutionary vestiges remain in many further-derived morphologies as well.

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Comprehensive Literature Cited

Aguirre LF, Herrel A, van Damme R, Matthysen E. 2002. Ecomorphological analysis of tropical niche partitioning in a tropical savannah bat community. Proc Biol Sci 269:1271-1278.

Anderson PSL, Gill PG, Rayfield EJ. 2011. Modeling the effect of cingula structure on strain patterns and potential fracture in tooth enamel. J Morphol 272:50-65.

Bajaj D, Arola D. 2009. Role of prism decussation on fatigure crack growth and fracture of human enamel. Acta Biomater 5:3045-3056.

Boyer DM. 2008. Relief index of second mandibular molars is a correlate of diet among Prosimian primates and other Euarchontan mammals. J Hum Evol 55:1118-1137.

Boyer DM, Evans AR, Jernvall J. 2010. Evidence of dietary differentiation among Late Paleocene-Early Eocene Plesiadapids (Mammalia, Primates). Am J Phys Anthropol 142:194-210.

Bunn JM, Ungar PS. 2009. Dental topography and diets of four Old World monkey species. Am J Primatol 71:466-477.

Butler PM. 1972. Some functional aspects of molar evolution. Evol 26:474-483. 109