Phylogenetic Character Analysis of Crocodylian Enamel Microstructure and Its Relevance to Biomechanical Performance Jennifer Erin Creech

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

PHYLOGENETIC CHARACTER ANALYSIS OF CROCODYLIAN ENAMEL

MICROSTRUCTURE AND ITS RELEVANCE TO BIOMECHANICAL PERFORMANCE

JENNIFER ERIN CREECH

A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded: Spring Semester, 2004

The members of the Committee approve the thesis of Jennifer Erin Creech defended on March

29th, 2004.

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Gregory M. Erickson Professor Directing Thesis

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Scott J. Steppan Committee Member

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Joseph Travis Committee Member

Approved:

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Timothy S. Moerland, Chairman, Department of Biological Science

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Donald Foss, Dean, College of Arts and Sciences

The Office of Graduate Studies has verified and approved the above named committee members.

This research is dedicated to the memory of my mother

Jessica Irene Buchanan Creech

who passed away during my first year of graduate work.

She taught me to see the world through open eyes; her dedication to her work taught me to

appreciate science, her dedication to her family always made me feel loved.

I wish she could be here to see what I have accomplished by following in her footsteps.

iii

ACKNOWLEDGEMENTS

I want to thank my family for believing in me. Whenever I was in doubt they were sure to know just what to say to get me back on track. Throughout my time at Florida State University there were people who were vital in helping me complete my research. I would like to thank Greg Erickson for always involving me in his research and his input and help with my own research as well as his support during my mother's death. I also would like to thank the rest of my committee; Scott Steppan and Joe Travis for their help and input throughout my research. Jill Holliday's constant support, advice, and encouragement throughout my undergraduate and graduate education were always appreciated. Thank you Jill for getting me interested in research. All of my specimens were the result of the help of Tony Hunter, the St. Augustine Alligator Farm and Zoological Park in St. Augustine, Florida, and the Gladys Porter Zoo in Brownsville, Texas. Many thanks to Kim Riddle and John Ekman from the Florida State University Biological Science Imaging Resource for all of their assistance with my samples and scanning electron microscopy. I am grateful to Sunny Hwang for an endless supply of enthusiasm and an open mind. Michelle Stuckey and the members of EERDG contributed helpful input on my Natural history presentation. And many thanks to James Albright, Sara Tso, and Stacey Halpern for reading so many drafts of my manuscript and making many valuable comments that have improved the final product immeasurably.

TABLE OF CONTENTS

List of Tables ...... vii List of Figures...... viii Abstract ...... ix

INTRODUCTION ...... 1

1. THE EVOLUTION OF PRISMLESS ENAMEL MICROSTRUCTURE WITHIN THE CROCODYLIA...... 9

Introduction...... 9 Materials and Methods...... 12 Specimen selection Element selection Specimen preparation Scanning electron microscope examination Character assignments Statistical analyses Results...... 15 Discussion...... 17 Conclusions...... 19

2. MICRO-SCALE EFFECTS ON THE MECHANICAL PROPERTIES OF PRISMLESS TOOTH ENAMEL AS A RESULT OF MUSEUM PREPARATION TECHNIQUES...... 27

Introduction...... 27 Materials and Methods...... 29 Specimen selection Element selection Specimen preservation methods Specimen preparation for nanoindentation testing Specimen preparation for scratch testing Material testing Statistical analyses Results...... 34 Discussion...... 34

v Conclusions ...... 36

3. ENAMEL MICROSTRUCTURAL VARIATION AND BIOMECHANICAL PROPERTIES TESTING: AN INTEGRATIVE APPROACH ...... 45

Prismless enamel microstructure evolution within the Crocodylia ...... 45 Micro-scale effects on the mechanical properties of prismless tooth enamel ...... 46

APPENDIX A...... 47

APPENDIX B ...... 49

REFERENCES ...... 50

BIOGRAPHICAL SKETCH ...... 59

LIST OF TABLES

1. Species examined within the Crocodylia listed in alphabetical order...... 20

2. Summary of grinding and polishing steps ...... 21

3. Consistency indices (CI) and rescaled consistency indices (RC) for all microstructural characters described in Appendix A...... 22

4. Test parameters assigned to the nanoindentation test procedure...... 37

5. The mean values ± SD of the Vicker's hardness (Hv) and Young's modulus (E) of the enamel 40µm from the enamel edge for the three treatments...... 38

6. Two-level nested ANOVA on Vicker's hardness (Hv)...... 39

7. Two-level nested ANOVA on Young's modulus (E)...... 40

vii

LIST OF FIGURES

1. Cross-section through the tooth crown (A) and longitudinal section through the jaw and tooth (B) of a crocodylian showing the major components of the tooth...... 8

2. Diagram of a caniniform tooth...... 23

3. Three separate preparations from different orientations are necessary to characterize the 3-dimensional structure of crocodylian tooth enamel: longitudinal (A), cross- sectional (B), and tangential (C)...... 24

4. Cutting diagram of caniniform tooth for microstructural examination using SEM (A)...... 25

5. Microstructural character variation mapped onto the robust crocodylian phylogeny (Gatsey et al. 2004)...... 26

6. Cutting diagram of teeth used in biomechanical properties study...... 41

7. Cross-section of a crocodylian tooth...... 42

8. Mean hardness (Vickers) values by treatment technique...... 43

9. Mean Young's modulus (GPa) by treatment technique...... 44

viii

ABSTRACT

Tooth enamel microstructure has been shown to vary among mammals. Such variation has a major bearing upon whole-tooth biomechanical function and may reflect gross-level phylogenetic signal. Although variation is substantial within reptilian lineages, comprehensive standardized sampling has not been done from which a similar understanding can be garnered. In the present study I sampled caniniform teeth from the 23 extant species of Crocodylians. The 3-dimensional enamel microstructure was characterized by examining the tooth enamel in longitudinal, transverse, and tangential views using scanning electron microscopy. The microstructural characters were subsequently mapped onto a robust tree (Gatesy et al. 2004) and the ancestral character states were reconstructed using parsimony. The results of this study showed no correlations between individual microstructural characters and phylogeny or with diet. There is a range and variety of combinations of enamel microstructural variation between species; however there were no distinct sets of characters correlated with each other or with the phylogeny. The distribution of the microstructural enamel characters was extremely random, indicating a great deal of lability in the formation of the enamel within Crocodylia. The microstructural arrangement of apatite crystals has been posited to affect wear- resistance, crack-propagation, and cusp-sharpness in mammals. It seems reasonable that differential crystalline arrangement in non-prismatic enamel may have a similar biomechanical function. In order to make correlations between microstructural variation and the biomechanical properties of enamel it is vital to first determine the effects, if any, of preservation technique on biomechanical properties. Biomechanical testing was conducted using nanoindentation in order to determine the effect of traditional museum preparation techniques (drying or storage in ethanol after fixation in formaldehyde) on the biomechanical properties of prismless enamel. The results of this study indicate that storage methods can affect the biomechanical properties of

ix tooth enamel. Desiccation significantly increases the biomechanical properties (Hv and E) of tooth enamel. Fixation in formalin and subsequent short-term storage in ethanol prior to preparation and testing does not significantly alter Hv nor E values, therefore these specimens may be used in conjunction with fresh specimens in future studies.

INTRODUCTION

Teeth are an important source of information about an animal, whether extant or extinct. Overall gross-tooth morphology is a strong indicator of an organism's diet. While it isn’t possible to determine the specific diet of an organism by examining its dentition, a general estimate can be produced (Butler 1983). By examining the overall dentition it can be determined whether an animal is herbivorous, carnivorous, or omnivorous. Teeth can also be used to determine the approximate age of an organism. Tooth eruption and replacement in addition to subsequent wear are indicators of age in masticating animals such that teeth with high degrees of wear are generally from older animals (Morris 1978, Russell 1996). The statement “Never look a gift horse in the mouth” comes directly from this fact. In addition to tooth wear, the deposition of dentine and cementum are also strong indicators of age in ungulates (Klein and Cruz-Uribe 1983), deer (Mitchell 1967), and some marine mammals such as elephant seals (Laws 1953, Klevezal 1996). Stable isotope analyses of tooth enamel have been used to assess aspects of the animal’s environment (MacFadden 1998) and diet, such as consumption of C3 versus C4 plants (Wang et al. 1994, Quade et al. 1995, Cerling and Sharp 1996, Kohn et al. 1996, Zazzo et al. 2002). In addition to correlations with age and diet, gross tooth morphology has also provided many traditional phylogenetic characters (Wood 1959, Payne 1985, Novacek 1992, O'Leary and Geisler 1999). Because of their durability, in some extreme cases teeth make up the primary source of phylogenetic information for fossil taxa (Butler 1983, Hillson 1986, 1996). Teeth consist of two main elements: a crown, which is the portion of the tooth above the gumline, and a root, which serves as the portion of the tooth attached to the jaw. At a gross morphological level, teeth are composed of three mineralized tissues: enamel, dentin, and cementum (Figure 1). Cementum functions mostly as an attachment tissue. Cementum may also come up the sides of the tooth and “pour” onto the tooth surface during formation to produce additional wear surface on grinding teeth in organisms like horses. The main body of the tooth is

1 composed of dentin, which is much less brittle than enamel and functions as the main tooth support. Dentin is composed of a calcified organic matrix that is less mineralized than enamel. Although not as wear-resistant as enamel, dentin is tougher and can absorb more force before propagating cracks, and therefore serves as a shock absorber for the enamel layer (Hillson 1986). Enamel is the hardest tissue in the body due to its high degree of mineralization and forms a thin, hard layer on the outermost surface of the tooth’s crown. The composition of enamel is integral in determining the basic biomechanical properties of the tooth. Enamel is composed predominantly (97% by weight) of inorganic crystals of hydroxyapatite, a complex crystal of calcium phosphate (Misra 1984), combined with organic proteins and lipids in a non-collagenous matrix (Thewlis 1939, Carlson 1990). The high mineral content of enamel makes it resistant to wear, but extremely brittle and hence relatively vulnerable to cracking. In mammalian enamel the apatite crystals are arranged to form prisms, which are continuous bundles of crystals surrounded by a proteinaceous sheath that extends through at least 75% of the enamel layer from the enamel dentin junction (EDJ) to the outer enamel surface (Sander 1999). In contrast to what is found in mammals, "reptilian" (non-mammalian amniotes excluding birds) enamel is non-prismatic. The only exception is two species of Uromastyx lizards (Cooper and Poole 1973, Sander 1997). The microstructural arrangement of apatite crystals has been posited to affect both wear resistance (Carlson 1990, Maas 1991) and crack propagation (Rensberger and Pfretzschner 1992, Rensberger 1997) in mammals. In prismatic enamel cracks must travel around the prisms, which lessens the depth to which a crack can be driven (Currey and Abeysekera 2003). Additionally, prism arrangements and the resulting differences in resistance to abrasion have been shown to be determinants of wear patterns and consequently regulators of cusp sharpness (Rensberger and Koenigswald 1980, Maas 1993). It seems reasonable that differential crystalline arrangement in non-prismatic enamel may have a similar biomechanical function. It is necessary to understand the historical context of enamel microstructure studies in order to understand the current state of the field of enamel microstructural analysis as well as determine the best place to focus future study efforts. With the advent of magnifying lenses scientists such as Leeuwenhoeck began to notice that tooth enamel was a complex substance with distinct dentine and enamel layers as well dentine tubules (Leeuwenhoeck 1677). It was almost two hundred years later that major improvements in the field of transmission light

2 microscopy allowed scientists to see more detail. In 1850 John Tomes recorded enamel fibers that formed layers in the rodent enamel, but his observations included the caveat that it took a highly trained eye to discriminate the arrangement of these “fibers” (Tomes 1850). While prismatic structure can be seen with a light microscope, it is insufficient to completely examine the fine structure of enamel due to limited resolving power (Thewlis 1939). The advent of polarizing light microscopy contributed to a better understanding of enamel microstructure by greatly enhancing the visibility of Hunter-Schreger bands in prismatic tooth enamel. Hunter-Schreger bands show up as alternating light and dark bands under polarized light and are an effect of prism layering and arrangement (Schmidt and Keil 1971). Because non-prismatic enamel does not exhibit Hunter-Schreger bands and light microscopy cannot clearly resolve apatite crystals, non-prismatic enamel was considered a non-structured amalgam and there was little interest in examining variation within non-mammalian amniotes. The development of Scanning Electron Microscopy in the 1960s allowed examination of specimens with greater resolution at higher magnifications than was possible with light microscopy techniques, making possible a shift from the study of thin sections to work using ground and etched specimens. It isn’t surprising that the previous interest in mammalian enamel and the evolution and function of enamel prisms led to many studies within this group. Mammalian trophic diversification as facilitated by the differences in tooth shape (Butler 1983, Carlson 1990, Hillson 1986) and tooth enamel microstructure (Rensberger and Koenigswald 1980, Koenigswald et al. 1987, Koenigswald and Clemens 1992, Rensberger 1997, Wood and Stern 1997, Wood et al. 1999) has been studied for many years. However, the problem still remained that the reigning view of reptilian enamel is that there is no structure. The limitations of light microscopy had given everyone the impression that there was nothing worth investigating in reptilian enamel, so even with the advent of SEM, investigations into how tooth morphology is produced and preserved in organisms with non-prismatic enamel were severely lacking. The only exceptions to this pattern were the studies by Sander at the University of Bonn (Sander 1997, 1999). To date his survey of reptile enamel is the most comprehensive review of non-prismatic enamel. In order to generate an overview of reptilian tooth enamel diversity and complexity, Sander sampled 43 taxa representing a total of 15 orders and 31 families. He also modified the hierarchy produced by Von Koenigswald and Clemens (1992) so that non-prismatic

3 enamel could be described using terminology consistent to that used to describe mammalian prismatic enamel (Sander 1997, 1999). The extensive survey and characterization of the non-prismatic arrangement of apatite crystals seen in reptiles and early mammals performed by Sander (1997) dispelled the misconception that reptilian enamel is a non-structured amalgam of inorganic and organic components. Discernable microstructural variation was shown, but due to small sample sizes at the species level and lack of standardization of viewing planes, there was no diagnostic representation of differences at a phylogenetic level. As a result of the broadness of his focus there was no way to determine whether the variation present among reptiles was driven by phylogeny or other factors. A single sample from a single species within a family doesn’t say much about the family as a whole. In order to determine if variation is instead correlated with diet or environmental differences studies must be more focused and take into account the phylogeny of the organisms being studied. Previous studies (Harvey and Pagel 1991) have shown that results can be confounded if character correlations are determined without incorporating phylogenetic history. Although gross-tooth characters are an important component of many existing phylogenies, microstructural differences between species and higher order groups have been almost completely disregarded as a source of phylogenetic information. Initial attempts to derive phylogenetic signal from tooth microstructure have, with a few exceptions (Rensberger and Koenigswald 1980, Dalquest et al. 1989, Martin 1994), been unsuccessful. One cause for the lack of phylogenetic signal in previous studies may be that differences are not easily discernable at the taxonomic level at which specimens were compared (species level). Von Koeningswald and Clemens (1992) found that in Mammalia the schmelzmuster level is diagnostic of the family and genus, while the less complex enamel type may only be diagnostic of an order because there are lots of enamel types, prisms, and crystallite arrangements shared by closely related taxa. For non-mammalian species however, there is a gross lack of comprehensive reviews of microstructural data within a phylogenetic framework and the vast majority of reptilian species are yet to be investigated, particularly within a phylogenetic framework. My research contrasts with of Sander’s (1997) approach by attempting to provide background knowledge of interspecies variation within a single clade. This serves to provide a more comprehensive understanding of interspecies variation and variation within reptilian clades. Additionally the

4 examination of the microstructural characteristics of tooth enamel within Crocodylia may shed light onto the relationship between Gavialis gangeticus and Tomistoma schlegelii. There has been a great deal of controversy about the placement of Gavialis, whether as the most basal taxon of the group (morphological tree, Brochu 1997) or as the sister to Tomistoma with subsequent placement basal as sister to the Crocodylidae (molecular tree, Gatesy et al. 2003, Harshman et al. 2003). A comprehensive understanding of tooth function requires a sound understanding of how individual components function and their resulting contribution to the whole. In organic materials it is not merely the structural components but their subsequent arrangement within the material that yields overall tissue property (Ashby et al. 1995). There is no clear understanding of how the diversification of reptilian enamel microstructure facilitated changes in diet, size (both of tooth and animal), and tooth use (in crushing and prey capture). A clear understanding of the diversification of enamel structures and their biomechanical properties will clarify these changes. The first step in this process is to characterize variation in enamel microstructure. Once enamel microstructural variation has been characterized the next step is to evaluate the biomechanical properties conveyed by differing crystallite arrangements. Tooth properties such as wear resistance and crack resistance are determined by gross tooth shape (and resulting force distribution) as well as the intrinsic material properties of the individual tissues that form the tooth. Teeth must withstand both wear and breakage. Though tooth wear is a complex process, the simplified model for determining wear rate (W) includes hardness (H) and a situation specific constant (K) where W=K/H. In this situation higher hardness values are associated with greater wear resistance (Currey and Abeysekera 2003). Hardness is defined as the resistance of a given material against plastic deformation (Lee et al. 2003). Crack resistance, an equally important property, is partially determined by the stiffness of the enamel (measured as the modulus of elasticity). Young's modulus (E) is the ratio of stress to corresponding strain below the yielding point of a material, and dictates the amount of deformation that will occur upon loading (Bowen and Rodiques 1962, Mahoney et al. 2000). The elastic limit, or yielding point, is the maximum stress a material can sustain and still return to its original form. Materials are elastic in relation to an applied stress if the strain completely disappears after the force is removed (elastic recovery).

5 Hardness is generally determined by indentation testing. The large scale of early indentation tests made them inappropriate or problematical for use in determining enamel properties (Waters 1980). Similarly, traditional tests to determine elastic modulus relied on large-scale flexural and tensile tests on notched samples (Rasmussen and Patchin 1984, El Mowafy and Watts 1986, Lin and Douglas 1994). These forms of testing are appropriate for determining average biomechanical properties, but are lack the precision to deduce values at specific locations within thin layers such as enamel (Fong et al. 2000). This is particularly important since the modulus and hardness values of enamel decrease as you move from the enamel surface towards the EDJ (Meredith et al. 1996, Fong et al. 2000, Angker et al. 2003). The development of nano-scale indentation techniques, in addition to a new method developed by Oliver and Pharr (1992) whereby indentation load-displacement curves are analyzed to determine H and E, have made it possible to examine the biomechanical properties of precise locations within thin layers (Lee et al. 2003) such as enamel (Kinney et al. 1996). During indentation testing the indenter tip is driven into the individual test sites by applying an increasing normal load at a fixed rate. The position of the indenter relative to the sample surface is precisely monitored with a differential capacitive sensor during each loading/unloading cycle, and the applied load value is plotted with respect to the corresponding position of the indenter (Gong et al. 2003). In addition to increased accuracy of test position in relation to the EDJ, nanoindentation testing requires a lower normal load than microindentation (Finke et al. 2001), which decreases the chance that total failure of the material will occur. Nanoindentation tests may be performed with a dedicated nanoindentor machine or with a modified atomic force microscope (AFM) (Habelitz et al. 2001). Though many researchers have utilized modified AFMs (Kinney et al. 1996, Finke et al. 2001, Habelitz et al. 2001, Habelitz et al. 2002, Marshall et al. 2003), a specifically designed nanoindentation machine is preferred for several reasons. AFM nano-indentation measurements often suffer from a significant measurement error of between 20–50% due to a lack of accurate knowledge of the cantilever spring-constant and, therefore, in the actual load transfered to the sample upon indentation. AFM tests also have limitated loading capacity with a maximum load of around 0.2 mN while mN-level loads are often called for in the tests of hard materials (Fong et al. 2000).

6 In testing tooth enamel it is necessary to perform repetitive indentations at each individual test site in order to produce accurate H and E values. This is partly because a single measurement at a given location may not penetrate through any enamel matrix loosened by preparation or storage. Several of the tooth enamel and dentin studies utilizing nanoindentation techniques have not taken this into account (Meredith et al. 1996, Cuy et al. 2002, Habelitz et al. 2002). Other studies suffer from a lack of power due to low sample size when only a single tooth specimen was examined (Cuy et al. 2002, Currey and Abeysekera 2003). In addition to the above mentioned issues with previous studies of enamel biomechanical properties, there have not been any statistically significant quantitative comparisons of enamel between species. In many cases only a single representative is tested from each species. It is often difficult to obtain and prepare fresh specimens of several different species within a suitable time scale for a comparative study. Often samples must be collected from several separate field sites and subsequently stored in some manner prior to testing as opposed to being immediately prepared and tested. In many cases it may be necessary to resort to previously collected or museum specimens. Not only can the storage of fresh specimens in saline solutions alter hardness and Young's modulus values of enamel by 10-25%, presumably by demineralization (Habelitz et al. 2002), dehydration of biological materials such as bone can have an equally dramatic effect on biomechanical properties (Rho and Pharr 1999, Hengsberger et al. 2002). It is therefore necessary to examine the effect of the common museum storage preparation techniques on enamel before differences in biomechanical properties can be compared between species (and hopefully correlated to microstructural differences in the enamel).

7 Lingual B A Minor Carina Enamel

Dentin Crown

Major Pulp Cavity Carina Pulp Cavity Cementum Root Enamel Bone Dentin Labial

Figure 1. Cross-section through the tooth crown (A) and longitudinal section through the jaw and tooth (B) of a crocodylian showing the major components of the tooth. The orientation of tooth A is indicated by lingual and labial designations.

CHAPTER 1

THE EVOLUTION OF PRISMLESS ENAMEL MICROSTRUCTURE WITHIN THE CROCODYLIA

Teeth are a significant source of information about an animal. Tooth morphology is a strong indicator of an organism's diet (Butler 1983) and age (Morris 1978, Russell 1996, Klein and Cruz-Uribe 1983, Mitchell 1967, Laws 1953, Klevezal 1996). Additionally, gross-tooth morphology has also provided many traditionally employed phylogenetic characters (Wood 1959, Payne 1985, Novacek 1992, O'Leary and Geisler 1999). At a gross morphological level, teeth are composed of three mineralized tissues: enamel, dentin, and cementum (Figure 1). In mammalian enamel apatite crystals are arranged to form prisms (Sander 1999). With the exception of two species of Uromastyx lizards reptilian tooth enamel is non-prismatic (Cooper and Poole 1973, Sander 1997). Mammalian trophic diversification as facilitated by differences in tooth enamel microstructure (Rensberger and Koenigswald 1980, Koenigswald et al. 1987, Koenigswald and Clemens 1992, Rensberger 1997, Wood and Stern 1997, Wood et al. 1999) has been studied for many years, however for non-mammalian species there is a gross lack of comprehensive reviews of microstructural data within a phylogenetic framework. The primary goal of this study is to characterize the enamel microstructural variation within Crocodylia and determine if there is interspecies variation. Studies by Sander (1997, 1999) and others have been cursory looks at specimens readily available for study. Discernable microstructural variation within reptiles has been shown, but due to small sample sizes at the species level there was no diagnostic representation of differences at a phylogenetic level. Due to the broadness of his focus there was no way to determine whether the variation present among reptiles is driven by phylogeny or other factors. This work focuses on the microstructural variation between species seeks to explain reasons for the microstructural differences. The

9 present research represents the first cohesive investigation of reptilian (non-mammalian amniotes exclusive of birds) within a phylogenetic framework. The subsequent objectives of this study are to (1) determine if the microstructural characteristics are phylogenetically informative and (2) determine if there is a distinct phylogenetic pattern to the microstructural characters. A distinct pattern includes one in which a suite of characters exist within definable clades. This is in contrast to cases where all characters occur in a random distribution throughout Crocodylia. Of equal interest is to (3) determine if there is a suite of characters that appear to be strongly correlated with diet as opposed to phylogeny. This would indicate that structure determines function in the ecological setting and is labile enough to evolve in the same manner multiple times. A strong correlation between microstructure and diet would indicate that there is a correlation between prismless enamel microstructural variation and the biomechanical properties of the tooth enamel. This hypothesis could subsequently be tested by conducting nanoindentation tests on tooth enamel from species with different diets and microstructural characteristics to determine if there is a quantifiable difference in the biomechanical properties of prismless enamel with differing microstructural characteristics. I chose crocodylians in particular for several reasons. The crown group of Crocodylia evolved 80 million years ago, but the major diversification within Crocodylinae was only 5-7 million years ago (Buffetaut 1989). Crocodylia is a relatively unexamined group with only two of the extant species having been previously studied in regards to their tooth enamel microstructure (Sander 1997) with scanning electron microscopy. Complete taxon sampling is key to understanding the overall microstructural variation between species. In some reptilian families there were too many species to feasibly produce a comprehensive examination, while other groups were so small that there was not enough variation in diet and morphology between species. With just 23 extant species it was possible to sample every living species. Crocodylians range in size from the dwarf Paleosuchus species that seldom exceed 1.5 meters (Magnusson 1992) to the largest of all of the crocodylians the salt-water crocodile Crocodylus porosus that has been reported to reach greater than 6.3 meters (Ross and Magnusson 1989). In addition to this four-fold difference in size between crocodylian species there is a dramatic difference in snout shape and resultant feeding regimes. Snout shape varies from the generally broad alligatoroid form to the narrower wedge shaped snout of most crocodiloids to independently

10 derived slender-snouted forms. There are three main feeding strategies among adult crocodylians. Most species employ a generalist strategy, slender-snouted species such as Gavialis and Tomistoma feed exclusively on fish, while the diet of durophagous taxa consists predominantly of snails and mussels (Rao 1994). This variation in diet allows testing of correlations between diet and microstructure. Sander (1997) modified the hierarchy of Koenigswald and Clemens so that nonprismatic enamel could be described with the same basic terminology as prismatic enamel. The most basic level is a description of hydroxyapatite crystallite arrangement. Crystallites may be parallel to each other, or they may be oriented at angles to each other in a continuous or discontinuous manner with zones of convergence and divergence. The next level is the module level. Modules are repeatable volumes of enamel that are delimited by crystallite discontinuities or by zones of changing crystallite orientation. These can consist of columnar units, micro-units that are simply small helical clusters of crystals, or compound units made up of smaller modules. Prisms also fall into the module level. Enamel type is defined by large volumes of enamel, which consist of crystallites or modules of the same type and of similar orientation. There are 4 enamel types possible in non-prismatic enamel: parallel enamel, wavy, micro-unit enamel, and columnar enamel. The subsequent 3-dimensional arrangement of enamel types and major structural discontinuities in one tooth makes up the Schmelzmuster level. Dentition level variation is the highest level in the hierarchy and consists of variation in schmelzmuster throughout the whole tooth. In my study of Crocodylian microstructure I characterized the enamel at the crystallite and module levels, and determined enamel types in addition to examining a few novel characteristics (characters 2-5 in Appendix A). Previous work on mammalian tooth enamel has postulated that there are biomechanical advantages of differing prismatic arrangements (Koenigswald et al. 1987, Rensberger and Koenigswald 1980, Koenigswald 1992, Rensberger and Pfretzschner 1992, Maas 1993, Currey and Abeysekera 2003) and it seems reasonable that there may be similar correlations between the microstructure of nonprismatic enamel and diet. It would therefore seem reasonable to expect to find compound unit enamel and/or incremental lines associated with a durophagous diet. The increased complexity of the arrangements of apaptite crystals in compound unit enamel should slow down crack propagation in comparison to the homogenous arrangement present in the parallel enamel type in a similar manner to that postulated for prismatic enamel. The differences

11 in mineralization that cause incremental lines could protect tooth the tooth from breakage by shearing off only part of the enamel layer when an unexpected force was encountered. Similarly, species that are predominantly fish eaters (piscivorous species) should theoretically encounter lower forces on their teeth due to the decreased amount of bone that would be encountered during feeding. Subsequently it would seem reasonable to expect to find enamel consisting solely of parallel enamel crystallites due to a lack of need for increased wear resistance or crack- suppressing.

Materials and Methods

Specimen selection

Caniniform teeth from adult specimens of each of the 23 extant species (Brochu 2001, Gatesy et al. 2003, 2004) within Crocodylia were examined (Table 1). Shed teeth from each of the 21 extant species of crocodylians on display at the St. Augustine Alligator Farm and Zoological Park (St. Augustine, FL) were collected from the habitat exhibits. A single tooth from Crocodylus porosus was removed from a commercially prepared display skull, while the final specimen, Crocodylus mindorensis, was obtained from the Gladys Porter Zoo (Brownsville, TX).

Element selection

The exact location of collected teeth could not be determined due to the manner of specimen collection. In order to standardize specimens between species, caniniform teeth (teeth from the anterior region of the jaw that are conical in shape with a single main cusp and a carina running along the mesial-distal surface of the tooth) (Hershkovitz 1971, Hillson 1986) (Figure 2) were chosen. All crocodilians have caniniform teeth that are used in the procurement of prey, while molariform teeth are only present in some species. A single tooth from each species can be compared because enamel structure is constant in the same tooth position of different individuals within a species (Sander 1999).

12 Specimen preparation

Each tooth was set in a hard, clear resin (EpoFix resin, Struers Inc., Cleveland, Ohio) and cut to produce three views of the enamel surface. The crystallites that make up enamel are arranged in 3-D space; to characterize how they are arranged it is necessary to look at least three different preparations of the tooth enamel. The three views necessary to accurately characterize the enamel were: tangential at the enamel surface, transverse, and longitudinal (Figure 3). The longitudinal section was cut along the vertical axis of the tooth passing just through the tip of the tooth, the cross section passed through the main body of the crown and the tangential section just broke the surface of the enamel leaving a clean oval-shaped facet of enamel for examination (Figure 4). Each tooth section was then reset in EpoFix resin prior to polishing. Specimens were prepared using a rotary polishing system (Rotoforce, Struers Inc., Cleveland, OH) with the following protocol (Table 2): Grinding 1) The tooth surface was exposed using 320-grit sandpaper at a speed of 300 rpm and a force of 35 N. Water was used to lubricate the disc surface. Specimens were rinsed thoroughly with distilled water between all subsequent steps of grinding and polishing to prevent contamination of the grinding/polishing surfaces by larger particles from the previous step. 2) The exposed surface was sanded with 9µm diamond spray (Struers Inc., Cleveland, OH) on a MD-Largo disc (Struers Inc., Cleveland, OH) at a speed of 150 rpm and a force of 40 N for five minutes. DP-Blue (Struers Inc., Cleveland, OH) at a dosing level of 5 was used to lubricate the disc during this step of preparation. Polishing 1) The exposed surface was polished with 3µm diamond spray (Struers Inc., Cleveland, OH) on a MD-Dac cloth disc (Struers Inc., Cleveland, OH) at a speed of 150 rpm and a force of 40 N for three minutes. DP-Blue at a dosing level of 9 was used to lubricate the disc during this step of preparation. 2) The final polishing was achieved with 0.04µm abrasive in an OP-S suspension (Struers Inc., Cleveland, OH) at a dosing level of 6. A MD-Chem cloth disc (Struers Inc., Cleveland, OH) was used at a speed of 150 rpm and a force of 40 N for one minute.

13 Ultra-sonic Cleaning and Acid Etching 1) Specimens were cleaned ultra-sonically for 20 seconds in an EtOH bath after final polishing. 2) Before acid-etching the surface of the specimens were treated for 10 seconds with a air-polishing device (Prophy-Jet, Dentsply International, York, Pennsylvania) utilizing an air/water/sodium bicarbonate slurry to enhance the microstructural appearance (Boyde 1984, Wood et al. 1999).

3) Specimens were then acid-etched for 20 seconds using 1% H3PO4. Etching was stopped by immersion in running de-ionized water for 60 seconds. 4) Specimens were again cleaned ultra-sonically for 20 seconds with EtOH.

Scanning electron microscope examination

Specimens were sputter-coated with gold-palladium at 20 kV and the three views of the enamel layer of each species were examined using a scanning electron microscope (JSM 840, JEOL USA, Inc., Peabody, MA) at the Florida State University Biological Science Imaging Resource (BSIR).

Character assignments

Species diet was assigned based on the following criteria: a species was considered a generalist if their diet varied highly both within and between locations presumably as a result of individual preference and food availability; piscivorous species fed almost entirely on fish, except in such cases where they were not readily available; while durophagous species incorporated a high degree of hard material in their diet (such as mussels or turtles) whenever such foods were available, as well as having flattened bulbous teeth in the anterior of the mouth (Fittcau 1973, Diefenbach 1979, Godshalk 1982, Huang 1982, Webb et al. 1983, Brisbin et al. 1986, Magnusson et al. 1987, Gurzula and Seijas 1989, Thorbjarnarson 1989, Waitkuwait 1989, Messel et al. 1992, Tucker et al. 1993, Arteaga et al. 1994, Elsey et al. 1994, Platt 1994, Rao 1994, Solmu 1994, Velasco et al. 1994, Tucker et al. 1996). Microstructural characters (Appendix A) were based upon the descriptive terminology of Sander (1999). Upon examination the enamel was characterized and the characters were mapped onto the phylogeny of Gatesy et al. (2004) using McClade 3.07 (Maddison and Maddison 1997).

14 Gatesy's current phylogenetic hypothesis is the most robust tree of extant Crocodylians and uses molecular as well as morphological data. The enamel character matrix is located in Appendix B.

Statistical analyses

The consistency index (CI) and rescaled consistency index (RC) were calculated for each character using parsimony to estimate the ancestral character states. Consistency indexes are a measure of how well an individual character fits onto a given tree. CI is calculated by dividing the minimum possible number of changes by the observed number of steps. If the minimum number of steps is the same as the observed number of steps, then the character will have a consistency index of one. If a character is not completely compatible with a tree its CI value will be smaller. CI can be an overestimate of a character's agreement with the tree (as is the case with autapomorphies) (Wiley et al. 1991). The RC overcomes this problem looking at the product of a character's CI and its retention index (RI). RI is calculated by dividing the (G-S) by (G-M) where G is equal the number of steps necessary to explain the character evolution under the hypothesis of a phylogenetic "bush", while M is equal to the minimum number of steps possible for the given phylogeny, and S is equal to the actual number of steps necessary (Farris 1989). Character correlations were tested using pairwise comparisons of phylogenetically separate pairs (Maddison 2000) in order to determine character associations. Calculations were made in Mesquite (Maddison and Maddison 2004) using the pairwise comparisons package (Maddison 2004). The criterion for choosing pairs of taxa was such that each taxon exhibited different states for both characters being compared (Read and Nee 1995).

Results

All characters were mapped onto the current crocodylian tree (Figure 5). The CI for individual characters ranged from 0.11 to 1.0, while the RC values ranged from 0.0 to 0.1(Table 3). I decided a priori that in order for a character to be considered in support of the phylogeny its RC must be 0.5 or greater. There are four possible enamel types in non-prismatic enamel (Sander 1997): parallel enamel, wavy enamel, micro-unit enamel, and columnar enamel. Because of the way in which enamel is laid down by the ameloblasts from the dentin interface outwards it is possible to have

15 more than one enamel type in successive layers. All four possible non-prismatic enamel types were present in Crocodylia. Seventeen species were composed of a single enamel type; parallel enamel was present in ten species, while seven species were composed entirely of micro-unit enamel. The remaining six species were composed of more than one enamel type. Five species were composed of a layer of parallel enamel as well as a layer of microunit enamel, while a single species (Alligator mississippiensis) was composed of compound unit enamel and columnar enamel. With the exception of layers of different enamel type within a single tooth, the only major Schmelzmuster level organization of enamel types within Crocodylia was the presence of compound unit enamel consisting of columnar and microunit enamel in Alligator missippiensis. Incremental lines (Retzius lines) were present in 16 of the 23 species with scalloping of the lines being present in five species. Incremental lines, a product of the enamel formation process, were widespread in both the Alligatoridae and Crocodylidae. Minor carinae on the surface of the teeth were a result of either a curvature of the enamel dentin junction (EDJ) or solely due to convergences and divergences of apatite crystals in the enamel layer itself. Seven species had minor carinae present from the EDJ, two species had enamel derived minor carina, while six species had both. While enamel derived minor carina were more prevalent in the Crocodylidae, they were also present in Melanosuchus niger, an alligatoroid. There are 13 unique sets of characters and only five character arrangements that are shared by two species, and none shared by more than two species. The pairs of species that share the same microstructural character sets are: Caiman yacare and Paleosuchus palpebrosus, Melanosuchus niger and Crocodylus moreletii, Paleosuchus trigonatus and Crocodylus intermedius, with the final pair consisting of Alligator sinensis and Tomistoma schlegelii. All correlations between individual microstructural characters were nonsignificant (p>0.05) with the exception of two associations with parallel enamel. The presence of parallel enamel was negatively correlated with the presence of microunit enamel (p= 0.008). Additionally, marginal significance for a positive association between parallel enamel and the presence of high numbers of enamel tubules was seen. 37 different sets of comparisons with seven pairs of taxa in each set were tested with p= 0.187 for 24 of the sets, and p= 0.031 for 12 of the sets. The association between parallel enamel and the presence of large numbers of

16 enamel tubules is nonsignificant when the p-values for the 37 sets of comparisons are averaged (p=0.13). All correlations between microstructural characters and diet were nonsignificant (p>0.05).

Discussion

The results of this study of the enamel microstructural variation in Crocodylians are inconsistent with correlations between individual microstructural characters and either phylogeny or diet. There is a range of enamel microstructural variation between species with various different combinations of enamel characteristics, however there were no distinct sets of characters that were correlated with each other nor subsequently with the phylogeny. The five non-unique sets of microstructural characters were distributed without regards to the phylogeny. None of the sets were shared by sister taxa, and three of the sets were split across the phylogeny at the split between Alligatoridae and the rest of the extant crocodylians (Melanosuchus niger- Crocodylus moreletii, Paleosuchus trigonatus-Crocodylus intermedius, and Alligator sinensis- Tomistoma schlegelii). Due to the presence of columnar and compound unit enamel in a single species (autapomorphy) the subsequent CI values for these characters are inflated. The calculated RC values for these and all other characters were well below the a priori RC value of 0.5 indicating that no single character corresponded well with the accepted phylogeny. Only one microstructural character had any significant association with any other microstructural character. Though both parallel enamel and microunit enamel can be present in the same tooth in subsequent layers of the enamel, the presence of more than one enamel type may be limited by enamel thickness. The negative correlation between the presence of parallel enamel and the presence of microunit enamel (when parallel enamel is present, microunit is not and vice versa) may be a direct result of enamel layer thickness. The distribution of the microstructural enamel characters within Crocodylia was extremely random, indicating a great deal of lability in the formation of the enamel. This lability may be directly tied to the way in which the enamel layer is formed. Very little is known about how the development and maturation of nonprismatic enamel correlates with microstructural variation (Sander 1999). Current models of enamel formation and subsequent microstructural

17 arrangements (Lester and Koenigswald 1989, Carlson 1990) have proven to be too simplistic, and did not take into account the 3-dimensional natural of the enamel (Sander 1999). In the general model of enamel formation, full-length amelogenin proteins are produced by differentiated ameloblasts from the EDJ outward. The amelogenin is then degraded, producing smaller peptides that provide the skeleton for mineralization and crystal formation. The organic components in the enamel are gradually withdrawn during maturation, and inorganic mineral components deposit into the matrix (Li et al. 2003). Nothing is known about the relationship between the ameloblasts that initially lay down the enamel matrix and the subsequent microstructural arrangements of apatite crystals. It is possible that in nonprismatic enamel a minute change in the ameloblasts could result in differences in microstructural arrangements. This could help account for the findings within Crocodylia. There are several reasons why there may not be a significant correlation between microstructural arrangements in the enamel and differences in diet. The current theory in mammalian research is that prismatic enamel arose in response to evolutionary pressures to increase the wear and crack-resistance of enamel (Koenigswald et al. 1987). It is possible complex apatite arrangements did not arise in correlation with a durophagous diet because crocodylians did not experience the same evolutionary pressures. Unlike mammals who are diphyodont, crocodylians (and most other reptiles as well) are polyphyodont and undergo continuous tooth replacement throughout their lifespan. Crocodylians do not masticate their food, and in conjunction with polyphyodonty and a lower base metabolic rate and subsequent decreased food intake (Pooley 1989) any individual tooth is less likely to experience fatigue and subsequent failure as a result of repetitive loading of forces. Additionally because the teeth of crocodylians are not in occlusion there is less predictability of forces in crocodylians versus the predictability of masticatory forces in mammals. The combination of these properties may have made the evolution of specific microstructural arrangements in response to diet impossible. Despite these differences between mammals and crocodylians it appears that with the lack of correlation between microstructural arrangements and diet in the simpler model of tooth enamel microstructure of crocodylians (as opposed to mammals) that the case for a correlation between prismatic enamel microstructure and increased biomechanical advantage with subsequent expansions of dietary niches in Mammalia has been overstated.

18 Though the interspecies variation within Crocodylia is not correlated with either diet or phylogeny it is still worth determining whether there are biomechanical advantages of differing crystallite arrangements. Almost all of the studies concerning biomechanical properties such as hardness and wear resistance have been qualitative (Rensberger and Koenigswald 1980, Carlson 1990, Maas 1991) and with the advent of new technologies for testing materials at the micro- and nano-scale it is now possible conduct tests of enamel at scales small enough correlate structural variations with mechanical properties.

Conclusions

While there is interspecies tooth enamel microstructural variation in Crocodylia, there are no defined suites of characters that correlate with either the phylogeny or the diet in these animals. There do not appear to be any characters that are diagnostic within this group, nor that can be specifically used as phylogenetically informative characters. Although the microstructural variation within Crocodylia is not diagnostic at the species level further studies within the reptile lineage are necessary to determine if there are taxonomic levels at which microstructure is phylogenetically informative. Despite the differences in evolutionary pressures on tooth enamel evolution between mammals and crocodylians, it must be stated that with the lack of correlation between microstructural arrangements and diet in crocodylians it appears that the case for differential microstructural arrangements in prismatic enamel contributing significantly to biomechanical advantage and subsequent the expansion of dietary niches in mammals has been overstated.

19 Table 1. Species examined within the Crocodylia listed in alphabetical order.

ALLIGATORIDAE CROCODYLIDAE Alligator mississippiensis Crocodylus acutus Alligator sinensis Crocodylus cataphractus Caiman crocodilus Crocodylus intermedius Caiman latirostris Crocodylus johnstoni Caiman yacare Crocodylus mindorensis* Gavialis gangeticus Crocodylus moreletii Melanosuchus niger Crocodylus niloticus Paleosuchus palpebrosus Crocodylus novaeguineae Paleosuchus trigonatus Crocodylus palustris Crocodylus porosus* Crocodylus rhombifer Crocodylus siamensis Osteolaemus tetraspis Tomistoma schlegeli

All specimens were collected from the St. Augustine Alligator Farm and Zoological Park except for those designated by a *. The C. mindorensis specimen was obtained from the Gladys Porter Zoo (Brownsville, TX), while the C. porosus specimen was removed from a commercially prepared display skull from a captive raised animal.

20 Table 2. Summary of grinding and polishing steps.

Step 1 2 3 4

Abrasive Silicon-carbide Diamond Diamond OP-S Suspension

Consumable disc - MD-Largo MD-Dac MD-Chem

Grit/grain size 320 9µm 3µm -

Lubricant Water DP-Blue DP-Blue -

Rotational speed (rpm) 300 150 150 150

Force (N) 35 40 40 40

Time (min) As needed 5 3 1

Specimens were prepared with an automated rotory polishing system (Rotoforce, Struers Inc., Cleveland, OH). All specimens were rinsed with distilled water between each step of the process to prevent contamination of the grinding/polishing surfaces. All consumables are products of Struers Inc. (Cleveland, OH).

21 Table 3. Consistency indices (CI) and rescaled consistency indices (RC) for all microstructural characters described in appendix A.

Character Consistency index (CI) Rescaled consistency index (RC) 1: Incremental lines 0.14 0.0 2: Scalloped incremental lines 0.25 0.0 3: Enamel tubules 0.20 0.10 4: Enamel derived minor carina 0.17 0.05 5: Minor carina present from EDJ 0.13 0.03 6: Compound unit enamel 1.00* 0.0 7: Parallel enamel 0.13 0.0 8: Microunit enamel 0.11 0.02 9: Columnar enamel 1.00* 0.0

It was chosen a priori that a CI of 0.5 or greater was necessary to indicate a character's compatability with the phylogenetic tree by (Gatsey et al. 2004). The consistency index may be an overestimation in cases such as autapomorphies (*) and the rescaled consistency index utilizes the retention index to control for these inflated CI values.

Carina

Figure 2. Diagram of a caniniform tooth. Crocodylian teeth are conical, similar in shape to mammalian canines, thereby the term "caniniform." Caniniform teeth are not true canines because the occur along most of the dental arcade and are not consistant with the mammalian dental formula for identifying tooth form. A single major carina occurs as a ridge on the mesiodistal surface (indicated by an arrow).

23 A B C

Figure 3. Three separate preparations from different orientations are necessary to characterize the 3-dimensional structure of crocodylian tooth enamel: longitudinal (A), cross-sectional (B), and tangential (C). The top row of illustrations indicate the placement of the plane of section through which the tooth must be cut. The bottom row of illustrations show the orientation of the plane when the tooth is viewed from above.

24 1 A B

1 2

Figure 4. Cutting diagram of caniniform tooth for microstructural examination using SEM (A). The initial cut was made to produce a longitudinal section (1). The second cut was made through one of the longitudinal sections to produce a cross-section (2). The tangential section was produced using the top half of the cross-sectioned portion of the tooth (3). Diagram of sections produced from one tooth (B).

25 descriptions). branches indicate the presence ofthemicrostruc while apiscivorousdiet is indicatedbythefine Solid branchesindicatea genera (Gatsey Figure 5.Microstructuralcharactervaria Parallel enamel incremental lines Scalloped Incremental lines Caiman yacare et al. Caiman crocodilus 2004).Thedietofeachspeci Caiman latirostris Melanosuchus niger Paleosuchus palpebrosus Paleosuchus trigonatus Alligator mississippiensis

list diet,thebold-striped bran Alligator sinensis Gavialis gangeticus Enamel tubules (high density) Compound unit enamel Microunit enamel Tomistoma schlegelii tion mapped ontotherobustcrocodylian phylogeny

es isindicatedbythediffere Crocodylus cataphractus -striped branches.The 26

tural characters (seeAppendixA forcharacter Osteolaemus tetraspsis

Crocodylus acutus Crocodylus intermedius Crocodylus moreletii ches indicateadurophagous diet, Crocodylus rhombifer Crocodylus niloticus

Columnar enamel carina presentfromMinor EDJ Enamel derived minor carina Crocodylus novaguineae boxes atthetipsof nt branchesonthetree. Crocodylus mindorensis Crocodylus johnstoni Crocodylus porosus Crocodylus palustris Crocodylus siamensis

CHAPTER 2

MICRO-SCALE EFFECTS ON THE MECHANICAL PROPERTIES OF PRISMLESS TOOTH ENAMEL AS A RESULT OF MUSEUM PREPARATION TECHNIQUES

It is necessary to understand the relationship between the microstructure and biomechanical properties of enamel in order to model the dissipation of stress within a tooth and explain tooth function (Habelitz et al. 2001). Tooth properties such as wear resistance and crack resistance are determined by both gross tooth shape (and resulting force distribution) and the intrinsic material properties of the individual tissues that form the tooth. Though tooth wear is a complex process, the simplified model for determining wear rate (W) includes hardness (H) and a situation specific constant (K) where W=K/H. In this situation higher hardness values are associated with greater wear resistance (Currey and Abeysekera 2003). Hardness is defined as the resistance of a given material against plastic deformation (Lee et al. 2003). Crack resistance, an equally important property, is partially determined by the stiffness of the enamel (measured as the modulus of elasticity). Young's modulus (E) is the ratio of stress to corresponding strain below the yielding point of a material, and dictates the amount of deformation that will occur upon loading (Bowen and Rodiques 1962, Mahoney et al. 2000). The elastic limit, or yielding point, is the maximum stress a material can sustain and still return to its original form. With the growing interest in the microstructural properties (hardness/crack-resistance, Young's modulus/resistance of plastic deformation, and wear/scratch resistance) of biologic structural elements such as bone (Rho and Pharr 1999, Rho et al. 1999, Hoffler et al. 2000, Swadener et al. 2001, Fan et al. 2002, Hengsberger et al. 2002) enamel (Kishen et al. 2000, Finke et al. 2001, Marshall et al. 2001) and dentine (Meredith et al. 1996, Angker et al. 2003) it is of increasing importance to consider the factors that can influence the materials being tested.

27 When planning any experiment variation both within a sample and between samples must be considered. Though statistical analyses such as ANOVA (one-way with blocking or two- way) can account for much of this variation, it is vital to control for as much possible variation (outside the interest of the question being asked) through experimental planning. This is especially true when testing enamel at the micro-scale. The significant variation in biomechanical properties on a gradient across the enamel layer from the EDJ to the enamel surface (Meredith et al. 1996, Fong et al. 2000, Angker et al. 2003) must be taken into consideration when using nanoindentation due to the small volumes of material involved in each test. A major source of variation that is seldom considered is the way in which samples have been stored prior to preparation. Storage of fresh specimens in saline after preparation and prior to biomechanical testing have a significant effect (>20% reduction in hardness and elastic modulus) on enamel biomechanical properties such as hardness and elastic modulus (Habelitz et al., 2002). Desiccation of biological materials such as bone has an equally significant effect and can increase hardness and elastic modulus values by as much as 20% (Rho and Pharr 1999, Hengsberger et al. 2002). Other studies of bone have shown that embalming increases hardness and modulus values (in tension) and ethanol fixation decreases deflection (Evans 1973). Due to the difficulty and expense of obtaining fresh samples (particularly when investigating rare or distantly indigenous species) it is often necessary for researchers to obtain samples from the preserved collections available in museums. Many of these collections have specimens that have been stored via one of several methods. The most common methods include desiccation, storage in formaldehyde, or fixation in formaldehyde and subsequent storage in ethanol (Kenny Krysco personal communication). It is therefore important to determine the effects of these treatments on the biomechanical properties of the enamel before testing and comparing specimens that may have been preserved by different methods. The object of this study is to determine the effect of museum preparation techniques on the biomechanical properties (Vicker's hardness and Young's modulus) of the nonprismatic enamel of Alligator mississippiensis. A nanoindentation technique was used in order to make an accurate assessment of the enamel at a fixed distance from the enamel surface. This assessment may be important in future evaluations of biomechanical property comparisons between species particularly with regard to differential biomechanical properties resulting from enamel

28 microstructural variation. The information yielded on the amount of difference between hydrated (fresh) and desiccated specimens may be extended to adjust biomechanical results of desiccated enamel to a value for "fresh" specimens in future comparisons. Nanoindentation is the preferred method for determining the hardness (H) and Young's modulus (E) of enamel for several reasons. Traditional tests to determine E depended on large- scale tests of notched samples (Rasmussen and Patchin 1984, El Mowafy and Watts 1986, Lin and Douglas 1994), which were difficult to produce from enamel and determined the average biomechanical properties of the material. The large scale of early indentation tests also made them appropriate for determining average biomechanical properties, but they lacked the precision to deduce values at specific locations within enamel (Fong et al. 2000). Due to the way in which the biomechanical properties of enamel change across the enamel layer these were unsatisfactory methods of testing. In addition to increased accuracy of test position in relation to the EDJ, nanoindentation testing requires a lower normal load than microindentation (Finke et al. 2001), which decreases the chance that total failure of the material will occur.

Materials and Methods

Specimen selection

Prisms located in tooth enamel may influence mechanical properties (Carlson 1990, Maas 1991, Rensberger and Pfretzschner 1992, Rensberger 1997, Currey and Abeysekera 2003), particularly during nano-scale testing. Therefore the nonprismatic tooth enamel present in Alligator mississippiensis is a simplified system for testing the effect of preparation techniques on the mechanical properties of interest (hardness and elastic modulus). Teeth from 5 mature individuals of the species A. mississippiensis were extracted for use in material testing. Specimens were obtained from a nuisance alligator trapper in Leon County, FL. All specimens collected from the field were frozen immediately after collection (and are therefore considered fresh specimens). The organisms had recently been collected from the field and teeth could be extracted prior to further preparation, making the specimens ideal for this experiment. The teeth of A.

29 mississippiensis undergo replacement throughout life, so recently produced; relatively unworn teeth could be used in the testing.

Element selection

A major canine was extracted from the upper jaw of five similarly sized adults. These teeth were chosen because they are the largest caniniform teeth present in the jaw and were easily cut along the transverse plane into 3 regions (Figure 6) with the maximal quantity of tooth enamel surface for the test preparation process.

Specimen preservation methods

The whole-tooth specimens were kept frozen at -20º prior to removal from the jaw. After removal the teeth were wrapped in ectotherm ringer solution (Kiernan 1990) soaked toweling (to prevent freeze drying/freezer burn) and kept frozen at -20º prior to cutting along the transverse plane and application of preservation techniques. All teeth were divided in the same manner. Transverse cuts were made every 3 mm using a precision saw (Isomet 1000, Buehler, Lake Bluff, IL) equipped with a diamond-tipped wafering blade (Allied HP, inc, Rancho Dominguez, California) to create three separate specimens from each tooth. In order to determine the effect of museum preservation techniques on the mechanical properties of the tooth enamel I tested one control ("Fresh") sample and two treated samples from each tooth. Preservation methods are consistent with the preparation methods used at the Florida Museum of Natural History, Gainesville (Krysco personal communication). Preservation methods 1. The control sample was kept frozen at -20º until final test preparation. 2. The first preservation method consisted of drying the specimen. A section was desiccated at 33ºC for three weeks, which is a consistent temperature to the bug- boxes in which museums clean and dry bony elements. (Treatment designated "Dry"). 3. For the second preservation method a tooth section was fixed in formalin for seven days and then moved to 70% ethanol for an additional two weeks. (Treatment designated "EtOH").

30 Specimen preparation for nanoindentation

After the preservation techniques were applied, all specimens were prepared for biomechanical testing. Each tooth section was set in a clear, hard resin (EpoFix resin, Struers Inc., Cleveland, OH). Specimens were then prepared using a rotary polishing system (Rotoforce, Struers Inc., Cleveland, OH) with the following protocol (Table 2): Grinding 1. The transverse-plane of the tooth was exposed using 320-grit sandpaper at a speed of 300 rpm and a force of 35 N. Water was used to lubricate the disc surface during grinding. Specimens were rinsed thoroughly with distilled water between all subsequent steps of grinding and polishing to prevent contamination of the grinding/polishing surfaces by larger particles from the previous step. 2. The exposed surface was sanded with 9µm diamond spray (Struers Inc., Cleveland, OH) on a MD-Largo disc (Struers Inc., Cleveland, OH) at a speed of 150 rpm and a force of 40 N for five minutes. DP-Blue (Struers Inc., Cleveland, OH) at a dosing level of 5 was used to lubricate the disc during this step. Polishing 1. The exposed surface was polished with 3µm diamond spray (Struers Inc., Cleveland, OH) on a MD-Dac cloth disc (Struers Inc., Cleveland, OH) at a speed of 150 rpm and a force of 40 N for three minutes. DP-Blue at a dosing level of 9 was used to lubricate the disc during this step of preparation. 2. The final polishing was achieved with 0.04 µm abrasive in an OP-S suspension (Struers Inc., Cleveland, OH) at a dosing level of 6 on an MD-Chem cloth disc (Struers Inc., Cleveland, OH). The samples were polished for one minute at a speed of 150 rpm and a force of 40 N. Fresh and ethanol-stored specimens were maintained in the hydrated state throughout polishing by immersion in ectotherm ringer's solution and ethanol (respectively) between steps. Immediately after polishing the reverse side of all specimens was ground with 320-grit sandpaper to ensure a level surface (to within 0.1 mm) for testing. Specimens were tested following final polishing and leveling to remove immersion-storage effects on the prepared surface of wet specimens (ethanol-stored and fresh) (Mahoney et al. 2000, Habelitz et al. 2002). All tests were performed at room temperature on room temperature specimens. If specimens

31 could not be tested immediately, the prepared specimens were stored to sustain their treated state prior to testing: desiccated specimens were kept at 33ºC in the same humidity at which they were prepared, while wet specimens were wrapped in toweling that had been dampened with their respective storage liquid (ethanol or ringer's solution) and stored at 2ºC until testing could be performed. Desiccation of wet specimens during testing was minimized by keeping samples wrapped between testing series (Habelitz et al. 2002) since E-values can increase as much as 15% over a 72 hour period as hydrated samples are allowed to dry to ambient conditions (Cuy et al. 2002).

Material testing

Testing apparatus available at the FSU/FAMU engineering facility was used for all biomechanical tests. Nanoindentation was used to find the hardness (Vicker's) and Young's modulus (E) of the enamel. A nano-hardness tester fitted with a Berkovitch diamond tip (CSEM Instruments SA, Neuchâtel, Switzerland) was used to perform the nano-indentation testing. Indentation testing determines modulus and hardness in the following manner: the indenter tip is driven into the individual test sites by applying an increasing normal load at a fixed rate. Once the load reaches a maximum value the load is reduced to 1 mN. This procedure is repeated in order to stabilize the depth measurements and at each stage of the experiment the position of the indenter relative to the sample surface is precisely monitored with a differential capacitive sensor. For each loading/unloading cycle the applied load value is plotted with respect to the corresponding position of the indenter. The CSEM software then automatically calculates quantitative Vicker's hardness (Hv) and modulus (E) values from the resulting load/displacement curves data using the Oliver-Pharr method (Oliver and Pharr 1992). Protocol The tip was cleaned prior to testing and between each specimen test by indenting into a copper puck with a load of 200 mN and a loading/unloading rate of 400 mN/min to remove any specimen residue that may have adhered to the diamond tip. Measurement accuracy (within 2%) of each series of tests was determined by testing the Young's modulus of a fused-quartz standard with a true modulus of 72 GPa before and after testing each sample to determine any thermal drift (Lee et al. 2003). Measurements were examined for precision between testing cycles at each given testing point.

32 The parameters for each nanoindentation are located in Table 4. Each individual test consisted of a single cycle of indentation. Testing cycles were performed in order to stabilize the value of E at each indentation location in the enamel. This is partly because an individual measurement (at a given location) may not penetrate through any enamel matrix loosened by preparation or storage. I determined through previous tests that 20 cycles are enough to ensure stabilization of the modulus results for desiccated specimens while wet specimens may require as many as 40 cycles. The enamel of each specimen was tested at a distance of 40 µm from the enamel surface (Figure 7) to minimize the effect of variations in mineralization on the biomechanical properties. A series of five indentation locations 20 µm apart to minimize proximity affects (Hengsberger et al. 2002) and parallel to the enamel surface were tested in a single run with multiple indentations (20-40) per site. The first and last indentation location values were not included in calculations. Immediately after testing all indentations were examined with a light microscope to evaluate the quality of the indentation. Poor quality indentations may be misplaced or located on a crack or scratch and do not yield reliable results (Mahoney et al. 2000) therefore results from poor quality indentations were not included in the calculations.

Statistical analyses

At a given testing site the 3rd through 7th values whose graphical representation showed a stable penetration value during the pause occurring at maximum penetration were considered to be stabilized measurements and were used to determine the mean Hv and E-values at each testing location. The overall means for hardness (Hv) and elastic modulus (E) were also calculated (Table 5). Two-level mixed-model nested ANOVA's were used to compare means to determine the effect of storage method on Vicker's hardness and Young's modulus. The ANOVA was set up as follows: measurements were made at three locations (n) on the enamel of each specimen with five specimens (samples) (b) in each of three treatments (a) (a= 3, b=5, n=3) (Sokal and Rohlf 2001). The effects in the model were Treatment and Location [Sample]. In order to determine which treatment means were significantly different from each other I performed an all-pairs Tukey-Kramer honestly significant difference (HSD) test for Hv and E with p=0.05 using JMP software (SAS Institute Inc., Cary, NC).

Results

Across all treatments the overall mean for the tooth enamel hardness (Hv) ranged from 275 to 329 (Vicker's), while the mean elastic modulus (E) ranged from 59 to 68 GPa (Table 5). The ANOVA model showed that there was a significant difference between Treatment means for both Hv (Table 6) and E (Table 7) (with p=0.10 and 0.025 respectively), however there was also a significant effect of Sample within each treatment (with p=0.01). Due to unequal variance between treatments (O'Brein [.5] p=0.03) in the hardness test a Welch ANOVA was performed to determine if there was a difference between the treatment means. The Welch ANOVA indicated a statistically significant (p=0.0001) difference between treatments. The Tukey-Kramer HSD tests showed that the mean Hv (Figure 8) was significantly greater for the Dry treatment than for the Fresh and EtOH treatments (an increase of 20%). Similarly the mean E value (Figure 9) was significantly greater (13%) for the Dry treatment. There was no significant difference between the mean values of Hv and E between Fresh and EtOH treatments.

Discussion

The results of the nanoindentation testing show that desiccation of enamel increases mean hardness (Hv) and elastic modulus (E) by 20% and 13% respectively. This is consistent with previous testing on bone (Evans 1973, Rho and Pharr 1999, Hengsberger et al. 2002). The significance of sample on Treatment means is due to the way order in which samples were tested. Samples were tested such that the three treatments were all tested on the same day. Testing was performed over several days; subsequently the last specimen to be tested had higher modulus and Young's modulus values (due to dehydration after preparation). Although enamel is a highly mineralized tissue, in vivo it is present in a consistently moist environment, allowing for complete hydration. The presence of water within a sample reduces electrostatic interactions and acts as a plasticizer, subsequently lowering the elastic modulus and hardness in resin-modified glass ionomer cements (used in dental restoration)

34 (Nicholson et al. 1992, Cattani-Lorente et al. 1999). A decrease in hardness and modulus with increased moisture content has also been shown in other biological materials such as keratin (Collins et al. 1998, Bonser 2002, Taylor et al. 2004) and whole seeds (Bay et al. 1996, Murthy and Bhattacharya 1998). In contrast to the effects of air-drying, hardness and elastic modulus did not differ between ethanol-stored and fresh samples. Though alcohol dehydrated the enamel it appears that the saturation of the sample with ethanol causes the enamel to retain its normal biomechanical properties upon short-term storage (< 1 month). The duration of storage for this experiment was rather short and it is possible that long- term storage of enamel in ethanol and formalin may have an effect on the biomechanical properties of enamel via demineralization or breakdown of inorganic components. Storage in water alone can cause hydrolysis and dissolution of some components and has been shown to affect hardness in some materials (Cattani-Lorente et al. 1999). Additionally, long-term storage in formalin results in dissolution of mineral components of the enamel (Waters 1980), which may alter biomechanical properties as well. A study comparing museum specimens that have been stored for different (long) periods of time may yield discernable differences in biomechanical properties. Previous tests have shown that rehydration of dried bone lowers hardness and modulus to values that approach those of the fresh state (Evans 1973). Enamel has been shown in this study to respond to dehydration and storage in ethanol with biomechanical changes similar to those of bone; therefore, rehydration of enamel prior to testing may be possible for enamel as well. If subsequent testing supports this hypothesis, it may be possible use rehydrated specimens in conjunction with fresh specimens in future biomechanical property tests. This study is meant to be a starting point for studies examining biomechanical advantage of specific crystallite arrangements in reptiles and differential arrangements of prisms in mammals. Fresh samples may be formalin fixed and subsequently stored in ethanol for short periods of time prior to preparation for testing without significantly altering the biomechanical properties (Hv and E). This result has implications in regards to sampling in distant field sites, allowing for a greater period of time between initial collection of specimens and subsequent biomechanical testing. Fresh samples may be collected and immediately deposited into formalin while in the field and stored for as long as a week. Subsequently specimens may be transferred

35 to ethanol for additional storage for as long as 3-4 weeks prior to preparation for biomechanical testing. However, it is important that samples are tested immediately after preparation so that neither dehydration nor dissolution of inorganic material at the test surface occurs.

Conclusions

Desiccation significantly increases the biomechanical properties (Hv and E) of tooth enamel. Fixation in formalin and subsequent short-term storage in ethanol prior to preparation and testing does not significantly alter Hv nor E values, therefore these specimens may be used in conjunction with fresh specimens in future studies.

36 Table 4. Test parameters assigned to the nanoindentation test procedure.

Test Parameters

Indenter type Berkovitch (pyramidal triangular)

Max indentation force (mN) 10

Pause at maximum load (sec) 10

Load/Unload rate (mN/min) 200

Number of cycles per test location 20-40

Number of test locations per matrix cycle 5

Distance of tests from enamel edge (µm) 40

Distance between tests in matrix (µm) 20

37 Table 5. The mean values ± SD of the Vicker's hardness (Hv) and Young's modulus (E) of the enamel 40µm from the enamel edge for the three treatments. n=5.

Treatment Hardness (Vicker's) Elastic modulus (GPa) Fresh 275.6 ± 27.5 60.4 ± 4.5 EtOH 274.9 ± 65.3 59.3 ± 6.8 Dry 329.4 ± 43.4 67.8 ± 4.6

38 Table 6. Two-level nested ANOVA on Vicker's hardness (Hv).

Source of variation DF SS MS Expected MS F Ratio Prob>F

2 2 2 Among Treatment 2 29357 14678 σ +3 σ BA+15 σ A 2.94 0.1 2 2 Among Samples within Treatment 12 59814 4984 σ +3 σ BA 4.048 0.01 Within Location 30 36937 1231 σ2 Total 44 126108

The ANOVA was set up with 3 measurements (Location) of hardness for each of 5 samples within three treatments (a=3, b=5, n=3) with Location[Sample].

39 Table 7. Two-level nested ANOVA on Young's modulus (E).

Source of variation DF SS MS Expected MS F Ratio Prob>F

2 2 2 Among Treatment 2 637.2 318.6 σ +n σ BA+nb σ A 5.686 0.01

2 2 Among Samples within Treatment 12 672.4 56.0 σ +n σ BA 3.149 0.01 Within Location 30 543.9 17.8 σ2 Total 44 1843.5

The ANOVA was set up with 3 measurements (Location) of elastic modulus (E) for each of 5 samples within three treatments (a=3, b=5, n=3) with Location[Sample].

40 3 mm

Figure 6. Cutting diagram of teeth used in biomechanical properties study. Each tooth was repeatedly cut along the transverse plane to produce three slices, each with a height of 3 mm. Each section then underwent a different preparation technique.

41 Enamel Surface Enamel Resin Dentin * * Enamel Dentin Junction

Figure 7. Cross-section of a crocodylian tooth. All biomechanical tests are performed 40µm from the enamel surface as indicated by the *. This controls for biomechanical property variations that occur along a gradient from the enamel surface to the enamel dentin junction (EDJ).

Figure 8. Mean hardness (Vicker's) values by treatment technique. * Statistically significant difference with p=0.05 with an all-pairs Tukey-Kramer HSD test. The center lines of the diamonds are the group means with the 95% CI indicated by the top and bottom of the diamonds. The bold circles are the 95% CI's for each group. The bold circle represents the Dry treatment which is significantly different from the other treatments.

Figure 9. Mean Young's modulus (GPa) by treatment technique. * Statistically significant difference with p=0.05 with an all-pairs Tukey-Kramer test. The center lines of the diamonds are the group means with the 95% CI are indicated by the top and bottom of the diamonds. The bold circles are the 95% CI's for each group. The bold circle represents the Dry treatment which is significantly different from the other treatments.

CHAPTER 3

ENAMEL MICROSTRUCTURAL VARIATION AND BIOMECHANICAL PROPERTIES TESTING: AN INTEGRATIVE APPROACH

Despite the increased interest in comparative studies of tooth enamel (Currey and Abeysekera 2003) quantitative reviews correlating specific microstructural arrangements with biomechanical property differences have not been produced. By integrating studies of microstructural variation (morphology) with tests of biomechanical properties (bioengineering) a better understanding of tooth function can be generated. My research is meant to provide the background information necessary for future integrative studies. In this chapter, I briefly summarize the results from my studies of enamel microstructural variation within the Crocodylia and the effects of sample storage on the biomechanical properties of Alligator tooth enamel.

Prismless enamel microstructure evolution within the Crocodylia (summary of results from chapter 1)

The results of this study do not support correlations between individual microstructural characters and either phylogeny or diet. There is a range of enamel microstructural variation between species, however there were no distinct sets of correlated characters. Pairwise comparisons between microstructural characters as well as between the characters and diet were nonsignificant with exception of a single negative correlation between parallel enamel and microunit enamel. Additionally, with the exception of layers of different enamel type within a single tooth, the only major Schmelzmuster level organization of enamel types within Crocodylia was the presence of compound unit enamel consisting of columnar and microunit enamel in

45 Alligator missippiensis. The distribution of the microstructural enamel characters was extremely random, indicating a great deal of lability in the formation of the enamel within Crocodylia. Despite the differences in evolutionary pressures on tooth enamel evolution between mammals and crocodylians, it must be stated that with the lack of correlation between microstructural arrangements and diet in crocodylians that the case for prismatic enamel microstructure contributing a significant biomechanical advantage and subsequently opening new dietary niches to mammals may have been overstated.

Micro-scale effects on the mechanical properties of prismless tooth enamel as a result of museum preparation techniques (summary of results from chapter 2)

The results of the nanoindentation testing show the method of storage prior to testing may have a significant effect on the subsequent biomechanical properties of the enamel. Desiccation of enamel increases mean hardness (Hv) and elastic modulus (E) by 20% and 13% respectively. This is consistent with studies of correlation between the moisture content of other biological materials. Increases in hardness and modulus with decreased moisture content have been found in bone (Evans 1973, Rho and Pharr 1999, Hengsberger et al. 2002), keratin (Collins et al. 1998, Bonser 2002, Taylor et al. 2004), and whole seeds (Bay et al. 1996, Murthy and Bhattacharya 1998). Short term storage in ethanol (< 1 month) after formalin fixation did not have a significant effect on hardness and elastic modulus values. This result has implications in regards to sampling in distant field sites, allowing for a greater period of time between initial collection of specimens and subsequent biomechanical testing. The duration of storage for this experiment was rather short and it is possible that long- term storage of enamel in ethanol and formalin may have an effect on the biomechanical properties of enamel. The hydrolysis and dissolution of mineral components has been observed after long-term storage in water (Cattani-Lorente 1999) and formalin (Waters 1980), which may alter biomechanical properties as well. A study comparing museum specimens that have been stored for different (long) periods of time may yield discernable differences in biomechanical properties.

APPENDIX A

Microstructural Character Descriptions

Characters 1-9 are based the microstructural descriptions in Sander (1999); character 10 is based on Brochu (2001).

1. Incremental Lines - Incremental lines are caused by differences in mineralization resulting in their enhanced appearance after preparation. They run parallel to the enamel dentin junction (EDJ). (0) Unclearly defined; (1) clearly defined.

2. Scalloped Incremental Lines - When looking at a cross-section the incremental lines appear scalloped in regions of enamel where the EDJ and the enamel surface are both smooth. The scalloped appearance is caused by elongate zones of convergence and divergence with an overall wavelength of ~20 µm. (0) Absence; (1) presence.

3. Enamel Tubules - Seen in the tangential view, enamel tubules appear as small circular openings in the enamel between 2 and 5 µm in diameter connecting the dentin and the enamel. (0) Presence in low numbers (<25/50µm2); (1) presence in high numbers.

4. Enamel Derived Minor Carina - Minor carina are longitudinal ridges in the enamel surface of the tooth. Seen in the cross-sectional view the enamel derived (ED) minor carina are solely a result of crystallite arrangement and the EDJ does not contribute to their development. (0) Absence; (1) presence.

5. Minor Carina Present from EDJ - Seen in the cross-sectional view the longitudinal ridges enamel derived (ED) minor carina are a result of both crystallite arrangement and the shape of the EDJ. (0) Absence; (1) presence.

6. Compound Unit Enamel - Compound unit enamel is composed of units in which modules (microunits or columnar units) are spatially arranged into higher order units. (0) Absence; (1) presence.

7. Parallel Enamel - Apatite crystals are arranged parallel to each other and normal to the EDJ. Incremental lines may occur, but are not always in evidence. (0) Absence; (1) presence.

47 8. Microunit Enamel - Microunit enamel consists of parallel apatite crystals that are grouped in units. These units may be non-parallel to each other and may be grouped into higher order structures. (0) Absence; (1) presence.

9. Columnar Enamel - Columnar enamel consists of columnar divergence units that are parallel to each other. (0) Absence; (1) presence.

10. Snout Type - sensu Brochu (2001): (0) generalized; (1) slender; (2) blunt.

11. Diet - (0) generalist; (1) piscivorous; (2) durophagous.

APPENDIX B

Character Matrix for Crocodylia

This matrix lists all extant species of Crocodylis. Codings for microstructural characters are based on Sander (1999); snout type was base on Brochu (2001). ______

1 2 3 4 5 6 7 8 9 10 11 Alligator mississippiensis 0 - 1 0 0 1 1 0 1 0 2 Alligator sinensis 1 1 1 0 1 0 1 1 0 0 2 Paleosuchus palpebrosus 1 0 0 0 1 0 1 0 0 2 0 Paleosuchus trigonatus 1 0 0 0 0 0 0 1 0 2 0 Caiman crocodilus 1 1 0 0 0 0 1 1 0 0 0 Caiman latirostris 0 - 0 0 0 0 0 1 0 0 2 Caiman yacare 1 0 0 0 1 0 1 0 0 0 0 Melanosuchus niger 0 - 0 1 1 0 1 0 0 0 0 Osteolaemus tetraspis 1 0 0 0 1 0 0 1 0 2 2 Tomistoma schlegelii 1 1 1 0 1 0 1 1 0 1 1 Gavialis gangeticus 1 0 0 0 1 0 0 1 0 1 1 Crocodylus acutus 0 - 0 1 0 0 1 0 0 0 0 Crocodylus cataphractus 0 - 0 0 1 0 1 1 0 1 1 Crocodylus intermedius 1 0 0 0 0 0 0 1 0 1 1 Crocodylus johnstoni 1 0 0 1 1 0 0 1 0 1 1 Crocodylus mindorensis 0 - 0 1 1 0 1 1 0 0 0 Crocodylus moreletii 0 - 1 1 1 0 1 0 0 0 0 Crocodylus niloticus 1 0 0 1 0 0 1 0 0 0 0 Crocodylus novaguineae 1 0 1 1 1 0 1 0 0 0 0 Crocodylus palustris 1 0 1 0 0 0 1 0 0 0 0 Crocodylus porosus 1 0 1 1 1 0 1 0 0 0 0 Crocodylus rhombifer 1 0 1 0 0 0 0 1 0 0 0 Crocodylus siamensis 1 1 1 0 0 0 1 0 0 0 0

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BIOGRAPHICAL SKETCH

Educational Background: 1997-2001 Florida State University, B.S., Cum laude

Research Experience: R.A. for Greg Erickson; Summer 2003, Fall 2003

Teaching experiences: Teaching Assistant, Comparative Vertebrate Anatomy Lab; Spring 2002, Spring 2003, Spring 2004 Teaching Assistant, Prokaryotic Microbiology Lab; Summer 2002, Fall 2002 Teaching Assistant, Biology I Lab; Fall 2001

Informal teaching activities: Florida State University Athletic Department tutor, Biology I for non-majors; Fall 2002 Senior T.A. Biology I; Spring 2001 Senior T.A. Biology II; Fall 2000

Other activities and honors: Golden Key National Honor Society member

Selected abstracts: 1. Creech JE, Erickson GM. “Enamel microstructure of chamaeleonids and the evolution of reptilian enamel prisms” SICB 2002 annual meeting, Anaheim, CA. 2. Creech JE, Erickson GM. “Biomechanical properties of Iguanian enamel and its correlation to the arrangement of apatite-crystals” 62nd Annual Meeting of the Society of Vertebrate Paleontology, Norman, Oklahoma. 3. Creech JE. "Variation in crocodylian enamel microstructure." SICB 2004 annual meeting, New Orleans, LA.

Professional memberships: The Society for Integrative and Comparative Biology The Society of Vertebrate Paleontology