Inferring Extinct Reptilian Response to Global Warming: Insights from Modern

Stable Isotope Ratios

Mitchell S. Riegler

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University

in partial fulfillment of the requirements for the degree of

Master of Science

In

Geosciences

Michelle R. Stocker, Chair

Sterling J. Nesbitt

Benjamin C. Gill

Shuhai Xiao

April 20, 2018

Blacksburg, VA

Keywords: lizards, stable isotopes, Anguimorpha, ecology

ii

Inferring Extinct Reptilian Response to Global Warming: Insights from Modern Stable

Isotope Ratios

Mitchell S. Riegler

ABSTRACT

Lizard ecology through time is largely unknown. Understanding ecology is important

because of today’s drastic climate change, but this is not a unique event. Early Cenozoic

hyperthermals were comparable to the perturbations currently experienced by living species.

Understanding ecology through time must acknowledge the dynamic relationship between an

organism and its environment on multiple scales. Ecological inferences can be based on form

equaling function, correlating certain features (e.g. leaf-shaped dentition) with certain

behaviors (e.g. herbivorous diet). Though this applies to specific taxa, there are confounding

examples. Ecology can also be inferred through indirect means, but these are disconnected

from the taxon of interest. Stable isotope geochemistry, however, provides an independent

test. I analyzed stable isotope ratios (δ18O, δ13C) from enamel, providing new data on the

connection between morphology, diet, and environment. I find a trophic separation in δ13C,

and indications of aridity through δ18O. I applied this framework to extinct lizards from an

Early Eocene (Wa4) assemblage, a key time between two major global warming events

(Paleocene-Eocene Thermal Maximum and Early Eocene Climatic Optimum). I identify

xenosaurid and glyptosaurine squamates and alethinophidian snakes. The xenosaurid is one

of the youngest representatives of Restes rugosus, and I provide the first testable hypothesis

of its ecology. These δ18O values corroborate hypotheses of a wet, tropical environment, and

the δ13C values indicate an insectivorous or carnivorous diet for both taxa. My study provides

an independent test of ecology of both extant and fossil lizards, with implications for

differing survivorship throughout the early Cenozoic.

iii

Inferring Extinct Reptilian Response to Global Warming: Insights from Modern Stable

Isotope Ratios

Mitchell S. Riegler

GENERAL AUDIENCE ABSTRACT

We know little about the diet and habitat of lizards. We have a limited knowledge

of these characteristics in living species, but these represents a fraction of the total

number of all lizard species that have ever lived. There are several ways to try to

understand the ecology of an animal. We can observe it directly, we can infer things

about it from comparisons to other living species, or we can make inferences through

indirect proxies. All of these methods have their limitations, however. I am interested in

how lizard ecology changes through geologic time as preserved in the fossil record. This

requires understanding the ecology of extinct lizards. For my thesis, I quantified ecology

using stable isotope ratios in both living and extinct lizard species. Through my analyses,

I was able to differentiate their diets and habitats. My examination of lizard fossils from

~54 million years ago identifies two lizards and one snake, and analyses of the fossil

lizards indicate they were carnivorous or insectivorous and lived in a tropical climate.

These stable isotope analyses not only have the potential to infer diet and habitat, but also

track illegal pet trade and determine if an organism is warm or cold blooded.

iv

For my family, especially my Grandfather, Jerry DeLane:

You instilled a passion for science and supported me every step of the way.

v

TABLE OF CONTENTS

Chapter 1…………………………………………………………………………………...…1

1. Abstract………………………………………………………………………………..2

2. Introduction………………………………………………………………………..….3

3. Material and Methods…………………………………………………………………7

4. Results...……………………………………………………………………………..12

5. Discussion……………………………………………………………………………13

6. Acknowledgments………………...…………………………………………………21

7. References……………………………...……………………………………………22

8. Figures……………………………………………………………………………….33

9. Tables………………………………………………………………………………...43

Chapter 2…………………………………………………………………………………….51

1. Abstract………………………………………………………………………………52

2. Introduction………………………………………………………………………….54

3. Geographic and Geologic Setting……………………………………………………57

4. Methods and Results…………………………………………………………………62

5. Discussion……………………………………………………………………………67

6. Acknowledgments…………………………………………………………………...72

7. References…………………………………………………………………………...72

8. Figures……………………………………………………………………………….84

9. Tables……………………………………………………………………………….102

vi

LIST OF FIGURES

Chapter 1

Figure 1. Tooth comparisons and implantation types between mammals and lizards. Page

33.

Figure 2. δ13C data for five extant lizard species. Page 35.

Figure 3. δ18O data for five extant lizard species. Page 37.

Figure 4. Individual variation amongst all five species. Page 39.

Figure 5. Tof-sims image data for a Savannah monitor tooth. Page 41.

Chapter 2

Figure 1. Geological setting of the Tim’s Confession locality (CM locality #222). Page

84.

Figure 2. Restes rugosus material from the Tim’s Confession locality. Page 86.

Figure 3. Comparison between Restes rugosus (GDB 1) and Xenosaurus grandis (FMNH

211833). Page 88.

Figure 4. GDB 2 maxilla, GDB 6-10 osteoderms, GDB 3 Maxilla. Page 90.

Figure 5. GDB 4 vertebra. Page 92.

Figure 6. Strict consensus tree of Xenosaurus and its relatives, from Bhullar (2011), with

GDB 1 dentary having been added. Page 94.

Figure 7. SEM image and TOF-SIMS map of GDB 3. Page 96.

Figure 8. δ18O data for five extant lizard species and two fossil taxa. Page 98.

Figure 9. δ13C data for five extant lizard species and two fossil taxa. Page 100.

LIST OF TABLES

vii

Chapter 1

Table 1. Taxa name, size, location data, dentition type, average isotope values, and

average standard deviation of each sampled specimen of each of the five species.

Page 43.

Table 2. All isotopic data of all five specimens of all five species. Page 45.

Chapter 2

Table 1. All isotopic data of all five specimens of all five extant species, and both fossil

taxa. Page 102.

viii

ATTRIBUTION

Chapters 1 and 2 were conceived of and designed by MSR and MRS, with input from

BCG and SJN. All data collection and analysis was conducted by MSR with advisement

from MRS and BCG. All figures were made by MSR and all chapters were written by

MSR, with advisement from MRS.

1

Chapter 1

STABLE ISOTOPE RATIOS ACCURATELY DELINEATE TROPHIC STRUCTURE AND

ARIDITY IN EXTANT SQUAMATES: IMPLICATIONS FOR ECOLOGY AND

PALEOBIOLOGY

Mitchell S. Riegler, Department of Geosciences, Virginia Tech, mriegler@vt.edu

2

1. Abstract

Lizard ecology through time is largely unknown. This is more important because of

today’s drastic climate change, but this is not a unique event. Early Cenozoic hyperthermals were

comparable to the perturbations experienced by living species. Understanding ecology thru time

must acknowledge the dynamic relationship between an organism and its environment on

multiple scales. Ecological inferences can be based on form equaling function, correlating certain

features (e.g., leaf-shaped dentition) with certain behaviors (e.g., herbivorous diet). Though this

applies to certain taxa, there are numerous confounding examples. Ecology can also be inferred

through indirect means, but these are disconnected from the taxon of interest. Stable isotope

geochemistry, however, provides an independent test. I analyzed stable isotopic ratios (δ18O,

δ13C) from enamel, providing new data on the connection between morphology, diet, and

environment. Our data indicate a trophic separation in δ13C, with carnivores plotting on the other

end of the spectrum as herbivores, and omnivores plotting variably in between. Results from

δ18O values provide clear indications of wet versus dry environment during the life of the lizard.

Additionally, these δ18O values, which are usually constant (<1 ‰) in endothermic mammals,

are variable (~ 2 ‰) in these ectothermic lizards, supporting the idea that the amount of variation

in δ18O values can serve as a proxy for types of thermoregulation. These analyses can be

extrapolated onto fossil lizards, but because isotopic ratios can be altered depending on the

preservation, additional steps must be taken to ensure the originality of the signal.

3

2. Introduction

Current climate models predict that by the year 2100, Earth’s average temperature will be

4C warmer than it was in 1950 (Hughes, 2000; Solomon et al., 2009). A four-degree increase

over 150 years is likely an order of magnitude faster than any other global warming rate in

Earth’s history. We are only just beginning to understand the impacts of this human driven

global warming event, especially as it comes to humans (e.g., Freely et al., 2004; Rosenzweig et

al., 2008), and these include shifts in crop land, changes in weather and rainfall, and relocation of

coasts and shorelines as only a few of the problems humanity will face if our planet continues to

warm at this current rate. In addition to the significant effects of global warming on humankind,

there will also be a drastic impact on animals. They depend on certain vegetation, various prey or

food items, a familiar climate, and soils or substrates that are conducive to their way of life. All

these elements are key components of an organism’s ecology, and any changes in these elements

usually require at least one of three corresponding responses by the animal. An animal can

change its latitudinal or elevational range, shifting to higher latitudes or elevation to maintain a

certain preferred temperature (Webb et al., 2005; Huey & Tewksbury, 2009; Rage, 2012; Muñoz

& Moritz, 2016). It may also change its behavior, basking in the sun less often or at different

times of day (e.g., Webb et al. 2005). However, if the change is too great or too rapid, or the

animal maintains extremely specific climatic tolerances, some species may be forced to

extinction.

Concrete evidence of how animals respond to global warming may be gathered through

direct observation of the animals today. However, if we are to try and mitigate or prevent any

biological disasters caused by global warming, we need direct observations to predict animal

4

response. Modeling or predicting such response can be challenging, with many factors being

difficult to constrain or parameterize (Huey et al., 2009; Logan et al., 2013). As an alternative to

this method, the rock record preserves what life has already experienced, and we can collect data

on how animals have responded to events in the past.

Paleoecology has been traditionally inferred from the fossil record through examination

of morphology (e.g., Parrish et al., 1987; Narbonne et al., 2014). Paleontologists have long

theorized about the concept that form equals function (Lauder, 1981;. Densmore et al., 1984;

Herrel et al., 2001; Measey et al., 2011; ElShafie, 2014, Melstrom, 2017). That is, the form that

anatomical elements take is intended to serve a specific function. Though there are instances in

which this is true, such as carnassial teeth of mammals being specialized to slicing meat, this is

not always the case. It was long believed that gharials, with long slender snouts could only eat

relatively soft, easy to catch prey like fish (Densmore et al., 1984; Singh, 2015). It was observed

years later that they in fact often eat animals such as turtles (Bezuijen et al., 1997). Similar trends

exist in the teeth of Basiliscus, Enyaliosaurus, and Ctenosaura, all lizards with three simple

cusps. As similar as these teeth are, Ctenosaura and Enyaliosaurus rely mostly on vegetation for

their diets, whereas Basiliscus relies mostly on insects (Montanucci, 1968). To complicate

studies using modern comparisons, the diet of many living lizards is still at times uncertain.

Uromastyx geyri has long been reported to be herbivore, until recent studies noted some eating

insects on a regular basis (Pianka, 1973; Cunningham, 2001).

Instead of relying on morphology alone, newer paleoecological proxies utilize

geochemical analyses of stable isotope ratios (Koch et al, 1995; Cerling et al., 1997, 2003;

Cherel & Hobson, 2005; Passey & Cerling, 2006). When an animal eats or drinks, assimilatory

5

processes occur such that part of those resources make up the animal itself, and certain

ecological factors, like diet or temperature, leave an isotopic signature (Cerling et al., 1997).

Isotopic studies have existed for decades but have largely focused on mammals (Cerling et al.,

1997; Emery et al., 2000; Kelly, 2000; Cerling et al., 2003; Price et al, 2004; Roche et al., 2010;

Ben-David & Flaherty, 2012). Those mammalian studies provide data on paleoenvironments and

biotic response to past events that can be extrapolated onto modern mammals, but given the

broader diversity of life, expansion of this work to different taxa is necessary. Lizards are of

particular interest and represent a logical next step because they are one of the most speciose

terrestrial vertebrates on Earth, and they occupy a wide array of dietary and ecological niches

(Pianka, 1973; Costa et al., 2008; Sinervo et al., 2010). Lizards were also highly diverse

throughout the past 55 million years (e.g., Gauthier, 1982; Bhullar, 2011; Longrich et al., 2012).

Understanding the surrounding ecology in connection with lizard response to past global

warming events could help set environmental tolerances and provide a model for how modern

reptiles will respond to current climatic perturbations.

Lizards provide a unique study system from mammals in many ways. First, most

mammalian studies have used teeth from large, herbivorous mammals (Emery et al., 2000;

Cerling et al., 2003; Sponheimer et al., 2003). Though abundant and easy to sample, they often

occupy open grasslands. However, tropical forests represent the most diverse habitats ever seen,

occupied by nearly half of the total species on Earth today (Huey et al., 2009; Bush et al., 2011;

McRae et al., 2017). Yet, tropical animals’ potential responses to global warming remain

alarmingly unclear (Hughes, 2000; Huey et al., 2009; Bush, et al., 2011). Lizards, particularly

species-rich tropical clades, offer valuable data for testing how the tropics respond to climate

6

change. The end-Paleocene (~55 Ma) was a time has been interpreted as having been covered in

tropical forests (Jaramillo et al., 2010), making several fossil beds ideal test sites. Additionally,

lizards are ectothermic, adding a physiological complication to their response to climatic

changes. Lastly, most lizards replace their teeth constantly through their lives. Similar to studies

on mammoth tusk growth rings (Rountrey et al., 2007), each tooth carries a signature reflecting

the surrounding environment near the time it was emplaced. Data from lizard teeth thus may

offer a more highly-resolved image of the environment, possibly with seasonal variation.

The teeth of mammals and reptiles are similar in their development and chemical makeup

of differing concentrations of bioapatite in an enamel outer coating over an internal layer of

dentine (Nanci, 2017). However, extant lizards with known ecological data allows for testing of

whether similar isotopic signatures exist across these groups. In addition, these analyses on

extant organisms allow definition of diet and trophic levels using δ13C. As diet and trophic

position changes, for example, from herbivores to carnivores, isotopic fractionations occur that

shifts δ13C values along a gradient (Cerling et al, 1997, 2003; Cherel & Hobson, 2005).

Therefore, when looking at a range of δ13C values, one would predict herbivorous taxa to have

negative values (more enriched in 12C), than animals from higher trophic levels (e.g., carnivores

or insectivores) that would have less negative values (more enriched in 13C) (Sponheimer et al.,

2003). If the goal is to acquire the original atmospheric signal, a trophic correction must be

applied, and that requires knowledge of the diet of the sample under analysis. Inferring the diet

of mammals is possible by observing their specialized dentition, but inferring diet in lizards,

whose teeth are much more simplistic, can be difficult (Herrel et al., 2001; Melstrom, 2017)

7

(Fig.1). Studying extant lizards with known diets can then allow creation of isotopic trophic

ranges and creates a framework to infer of diet in extinct lizards.

δ18O can be a useful isotopic proxy for temperature and aridity and can be analyzed at the

same time and with the same sample from which δ13C is measured. Part of the δ18O composition

of teeth is dependent on aridity. As water evaporates and an environment dries out, a

fractionation results that leaves the heavier isotope in the remaining body of water (Craig, 1961;

Suarez et al., 2012). When it rains, isotopically light rain lowers the available drinking water’s

signature (Craig, 1961). Therefore, more negative δ18O values indicate high rainfall and/or low

evaporation, whereas more positive values indicate an arid environment.

Here we present an independent test of ecomorphology and habitat use in extant lizards

by applying stable isotope analyses to modern tooth enamel in order to test hypotheses and

observations related to the connection between morphology, diet, and environment. We

hypothesize that like mammals, isotopes values from squamates will serve as a consistent proxy

for diet, rainfall, and potentially other ecological factors (i.e. thermoregulation). We show that

these tests have applications for determining diet and environmental preferences in extinct

animals from their fossil materials.

Institutional Abbreviations

VTPCC – Virginia Tech Paleobiology Comparative Collection, Blacksburg, VA, USA

3. Materials and Methods

8

a. Specimen Selection: In order to include as much ecological and morphological diversity (diet,

tooth shape, tooth implantation) as possible while minimizing destructive sampling, we selected

specimens from existing specimens (housed in Dept. of Geosciences, VTPCC) of recently

deceased Salvator merianae, Chamaeleo senegalensis, Iguana iguana, Varanus exanthematicus

and Uromastyx geyri (Table 1) for analysis. We selected five individuals for each species,

analyzing a mesial and distal tooth position from each maxilla and dentary (approximately eight

sites per individual) when possible. We selected this variety of lizard taxa to account for

differences in diet due to trophic structure as well as potential shifts in trophic placement through

ontogeny (e.g., insectivore as a juvenile and herbivore as an adult), and specific features such as

tooth implantation, replacement pattern, size, and position within the jaw, because all these

features may affect the chemical signatures preserved within the teeth. Tooth implantation type

in a lizard is often either pleurodont (tooth attached to the medial wall of the jaw only) or

acrodont (teeth fused into the jaw) rather than socketed (i.e., thecodont) (Fig 1). In addition,

some lizards have the ability to replace teeth through the lifespan of an individual

(polyphyodonty) (Zaher & Rieppel, 1999). This is common in taxa with pleurodont implantation,

whereas acrodont dentition is rarely replaced (Cooper et al., 1970). The isotopic impact of age or

amount of wear is unknown, so selecting lizards that display a variety of conditions, especially

acrodont lizards, was important. Lizards also display occasional heterodonty (multiple tooth

shapes in one animal) (Dessem, 1985). Selecting a taxon that displays such dentition allows

determination of differences in isotopic signature between tooth forms within a single jaw.

Occasionally in lizards, conical teeth with or without serrations are present in carnivorous taxa,

while bulbous ‘molariform’ teeth occur in taxa that feed on gastropods (e.g., caiman lizard)

9

(Cooper & Habegger, 2001). The most complex lizard teeth usually occur in herbivorous taxa

where the teeth are ‘leaf-shaped’ with multiple distal cusps (Rand et al., 1990). However, there

are consistent exceptions to all of these, which make inferring diet from morphology alone

difficult. Isotopic analyses act as a proxy for diet that is quantifiable and independent of

morphology.

Based on known ecology, we predict that Iguana iguana will have more negative

compositions for δ13C and δ18O, based on their tropical environment and herbivorous diet.

Chamaeleo senegalensis are purely insectivorous (Measey et al., 2011), whereas Uromastyx

geyri is both insectivorous and herbivorous (Cunningham, 2001), and each should have more

less negative δ13C values. With respect to δ18O, we predict that Chamaeleo senegalensis will plot

in a similar position to the iguana, whereas the desert dwelling Uromastyx geyri should have less

negative or even positive compositions relative to the other species. Because they also live in

wet, tropical environments, Salvator merianae will plot in a similar δ18O position to the iguana

and chameleon, but as an omnivore it should fall between the δ13C values of the other lizards.

Varanus exanthematicus would be expected to have negative δ18O values but should have the

least negative δ13C, based on their higher trophic position.

b. Gas Source Isotope Ratio Mass Spectrometry (GS-IRMS): We sampled each of the eight

teeth from the larger squamates (Salvator merianae and Varanus exanthematicus) twice, for a

total of 16 samples per individual (Tables 1 & 2). Using a dental-tipped Dremel tool, we

powdered the outermost layer of the tooth, collecting all enamel for the first sample. The lack of

precision of this tool undoubtedly collected some dentine as well, but we are reasonably

10

confident the second sample collected only dentine. If dentine has a different isotopic signature

than enamel, the comparison samples with differing ratios of enamel vs dentine would identify

this. The smaller lizards (Iguana iguana, Uromastyx geyri, Chamaeleo senegalensis) were

sampled once per selected tooth position, for a total of approximately 8 samples, again powdered

with a Dremel tool. The δ13C and δ18O contents were analyzed on a MultiFlowGeo headspace

sampler attached to an Isoprime 100 IRMS. In order to generate enough CO2 to analyze

approximately 5 mg of powdered enamel was required. In some cases, this required multiple

teeth per sample to obtain sufficient powder. Samples were placed in vials sealed with rubber

septums, flushed with helium, and acidified with phosphoric acid in order to liberate CO2.

Samples were reacted for at least 4 hours at 70C to allow for the carbonate to react fully,

producing CO2 gas. This gas was then analyzed for its carbon and oxygen isotope values, which

are reported in the standard δ-notation relative to the Vienna Pee Dee Belemnite (V-PDB)

standard and calibrated to this scale using the international standards IAEA-CO-1 (marble; δ13C

= +2.492‰, δ18O = −2.4‰), IAEA-CO-9 (BaCO3; δ13C = −47.321‰, δ18O = −15.6‰) and

NBS18 (calcite, δ13C = −5.014‰, δ18O = −23.2‰). Reproducibility (1) for single analysis of

the samples was better than ±0.07‰ for δ13C and better than ±0.3‰ for δ18O.

c. Time-of-Flight Secondary Ionization Mass Spectrometry (TOF-SIMS): An additional

objective of this project was to attempt other analytical techniques to facilitate the analysis of

lizard teeth. Enamel, the hardest material in a vertebrate’s body (Kohn et al., 1999; Chenery et

al., 2012), has the best chance of preserving an original chemical signal. Dentine is much more

porous and less dense than enamel. Therefore, it is not ideal for isotopic analysis because it prone

11

to diagenetic alteration (Kohn et al., 1999). Any attempts to apply this method to fossil

specimens should use enamel-only samples if possible. Accordingly, determining approximate

thickness of the enamel was essential. In addition, lizard teeth (millimeter scale or less in

apicobasal length) are generally much smaller than mammal teeth (Fig. 1). We utilized TOF-

SIMS to analyze at a micron-scale.

In order to prep the samples for TOF-SIMS analyses, a dental tip attachment on a Dremel

tool was used to create a flat, smooth surface, starting from the tip of the tooth and grinding

towards the base. A diamond-coated circular blade was then used to polish the surface as much

as possible. Then, using a Leica TIC 020 ion mill located at The University of Texas at Austin,

the surface was ablated using an ion beam. Each tooth was left in the ion mill for approximately

five hours. The final product was a glass like polish that was more ideal for TOF-SIMS analysis

than hand preparing. Once milled, the samples were placed inside an SEM vacuum chamber

overnight, allowing the samples to outgas and reduce the vacuum time in the TOF-SIMS

chamber.

An ION-TOF TOF-SIMS.5 was used with a pulsed (18 ns, 10 kHz) analysis ion beam

consisting of Bi3 + clusters at 30-kV ion energy, which was raster-scanned over areas that

typically varied between 100 × 100 μm2 and 500 × 500 μm2, depending on the quality (i.e.,

corrugation and conductivity) of the sample surface. The polyatomic sputtering was selected to

further enhance the signal. To reduce the sputtering-induced sample charging, a constant energy

(21 eV) electron beam was shot on the sample during the data acquisition. All detected

secondary ions had negative polarity and an average mass resolution of ∼1–2,000 (m/δm).

12

4. Results

a. δ18O: The δ18O values split the five species into two groups (Figure 3). The average values of

the Varanus exanthematicus (-3.33‰), Chamaeleo senegalensis (-1.4‰), Iguana iguana (-

0.41‰), and Salvator merianae (-2.49‰) specimens are all low value negative numbers.

Uromastyx geyri plots in stark contrast, plotting with an average value of +8.74‰. The standard

deviation in these values was also high, with the average standard deviation being ±1.5‰. All

groups had a standard deviation above one except for Chamaeleo senegalensis, which was 0.5‰.

Variation was lower in general at the individual level. Chamaeleo senegalensis again had

the consistently lowest standard deviation, ranging from ±0.18 to 0.54‰. All other species had at

least one specimen with a standard deviation above ±1‰, with Iguana iguana and Salvator

merianae having individuals above ±2‰ (Table 1, Fig. 4). It should be noted that the Iguana

iguana and Salvator merianae specimens were the largest animals, and we were able to acquire

the most sample per individual tooth instead of averaging the sample across several teeth.

b. δ13C: The δ13C average values for all species plotted at more than a half per mil difference or

higher (Figure 2). Salvator merianae plotted the most negative at -15.6‰, followed by Iguana

iguana at -15.0‰, then Chamaeleo senegalensis at -13.7‰, Uromastyx geyri at -11.65‰, and

Varanus exanthematicus at -7.7‰. Standard deviations were high, with the lowest (Chamaeleo

senegalensis) being ±1.02‰ and the highest (Salvator merianae) at ±2.3‰1 (Table 1, Fig. 4).

At the individual scale, variation was much lower. Salvator merianae, for example, had

an average deviation of around ±0.5‰. Typical standard deviations were below ±0.3‰ for other

species, with several being below ±0.1‰.

13

c. TOF-SIMS: The ion-mill that was available to prepare the samples ablates at a temperature

too high for extant, organic material, causing the samples to burn or combust. The extant tooth

was instead hand-milled and analyzed to produce an elemental map (Fig. 5). The elemental map

was normalized to CA+2, to show the difference in apatite concentrations in enamel versus

dentine. The result is an approximation of the thickness of enamel, and the location of the

dentine-enamel contact.

5. Discussion

a. Diet and Trophic Levels Can be Inferred from δ13C: Carbon isotopes in this study were

selected largely for their ability to separate out trophic position (Cerling et al., 1997). Higher

trophic level in mammals is associated with a more 13C enriched enamel (Cerling et al., 2003).

We found this trend within lizards as well (Figure 2). Other than Salvator merianae, Iguana

iguana, the most herbivorous (=lowest trophic level) of the species samples, has the most 12C-

enriched compositions of all other species. The insectivorous (higher trophic level) Chamaeleo

senegalensis have more 13C enriched enamel, followed by the herbivorous and insectivorous

Uromastyx geyri. Lastly, the carnivorous Varanus exanthematicus have the most 13C enriched

enamel.

The only organisms that go partially against our predictions were the omnivorous

Salvator merianae. Natively from Argentina, Salvator merianae sampled here are all from an

invasive population located throughout southern Florida. Their native diet is highly omnivorous,

including arthropods and small vertebrates, fungi, fruits, and eggs (Dessem, 1985; Hobbie &

14

Boyce, 2010). Though many of the tegu data have δ13C values intermediate between herbivorous

and carnivorous lizards, in line with our predictions for an omnivore, some of our Salvator

merianae have more 12C-enriched compositions than the herbivores sampled here. This is likely

a result of their varied diet. Both fungi and egg albumen can have very negative 13C

compositions (-20 to -28‰) (Hobbie & Boyce, 2010; O’Connell & Hedges, 2017). Further, as

with other invasive species, Salvator merianae may not adhere to the same diet as a native

population and individually preferences and availability of food items will be reflected in their

diet (Cerling et al., 2003). Many of these invasive populations are close to urban environments

(e.g., Pernas et al., 2012). It is likely these animals are eating trash or human leftovers, resulting

in large variations in the carbon isotopic composition of their teeth.

Though the Iguana iguana specimens have 13C compositions in the range we expected

(very negative), there is a bimodal distribution. A very tight grouping of points occurs at -17‰,

whereas most of the individuals grouped around -14‰. The split is likely due to the life histories

of the particular organism sampled. Those that plot around -17‰ are from the pet trade

population, whereas those that plot near -14‰ are from a wild population (Tables 1 & 2).

Traditional pet store iguana food comes in pellet form and represents a mix of vegetables and

fruits. Iguana food products from companies such as © 2018 Zilla, © 2018 Exo Terra, or © 2018

Nature Zone Bites for Iguanas include a variety of C3 plants that a wild iguana in the wild would

not eat (Watkins et al., 2017). These include soy, berries, beets, and oats. The interpretation of

this bimodal pattern is that the wild caught individuals were adhering to an herbivorous diet of

tropical C4 plants, whereas the captive individuals were being fed pellets that contain both C3

and C4 plant material.

15

The Chamaeleo senegalensis specimens also plot in line with our predictions. The

insectivorous Chamaeleo senegalensis has compositions centered around -14 ‰. These lizards

represent a wild caught population, feeding on tropical insects that predominately feed on

tropical, C4 vegetation. Uromastyx geyri from Saharan Africa has a mixed diet. Traditionally

they feed on desert plants, including cactus and desert flowers. In times of high heat and dryness,

they become more dependent on the local insect population (Cunningham, 2000). Had they been

purely herbivorous, they still likely would have more 13C-enriched compositions as compared to

the Iguana iguana since they feed on C4 plants. These C4 plants are about 14‰ more positive

than C3 plants (Watkins et al., 2017). With the addition of an occasional insect, also feeding on

C3 plants, it is not surprising that many of the Uromastyx geyri fall between the tropical

herbivorous iguana and the insectivorous chameleon.

The last species, Varanus exanthematicus, our most carnivorous lizard group in the study,

has the most 13C-enriched compositions, around -8‰. These lizards do not adhere to a strict diet,

but instead feed on small vertebrates, mollusks, and insects. There is debate as to how much true

meat these animals eat (Sprackland, 2012), but they will eat mice and amphibians in captivity

(Cooper et al., 2001). Their variable diet may again explain the variation we see in these values,

as some populations tend to favor insects over any vertebrates. Age is also a factor, as these

Varanus exanthematicus, when young, will eat a larger number of scorpions and amphibians

(Cooper et al., 2001).

The large variation between individuals of each species likely speaks to the uniqueness of

the environment each animal’s lives. Variation in vegetation type (C3 or C4) or differing dietary

preferences of the diet of the lower trophic prey could explain the intra-species variation. In

16

addition, because few of these taxa have a strict diet, this variation may be a product of differing

ratios of food sources. Whether a monitor eats more snails or small vertebrates would impact

there respiratory δ13C. The only group to eat exclusively one food item are the Chamaeleo

senegalensis, which are strict insectivores and correspondingly, their variation in δ13C is the

lowest.

b. Aridity and Thermoregulation can be Inferred from δ18O: Oxygen in this study was

selected as a proxy for aridity. More positive δ18O values indicates a more arid environment,

whereas more negative δ18O values indicate a more wet and in the case of the organisms

analyzed here, tropical environment. Our data show a bimodal distribution of δ18O values, with

four of our five species (the Chamaeleo senegalensis, Salvator merianae, Iguana iguana, and

Varanus exanthematicus) plotting close to 0‰, and one species plotting around +10‰ (Fig. 3).

The four species plotting around 0‰ live in wet, tropical environments. The Chamaeleo

senegalensis individuals are native to the southeast region of Africa, occupying such places as

the tropics of Mozambique (Anderson & Heygen, 2013). Salvator merianae, invasive to Florida,

occupies the tropics and marshes there (Pernas et al., 2012). Iguana iguana is native to South and

Central America and occupy the trees of tropical forests (Burgos-Rodriguez et al, 2016). Some of

these animals are captive-bred, and terrariums for Iguana iguana are recommended to contain

water and a moist environment. In keeping with the trend, Varanus exanthematicus is from the

sub-Saharan tropics of Togo (Collection data of U. Florida). All these lizards are in areas with

high rainfall, and all plot accordingly.

17

The one species that is significantly different is Uromastyx geyri, which plots in a much

more positive composition (+10‰). These lizards from Saharan Africa (Harris et al., 2007) and

occupy a habitat that is arid, with low rain fall and high evaporation. Therefore the δ18O values

are in agreement with their ecology.

Though mammalian δ18O values are usually consistent, with intra-tooth variation below

1‰ (Luz & Kolodny, 1985), the lizard δ18O values have interspecies ranges of 3-9‰. δ18O

values are dependent on the temperature at which the tooth formed and the isotopic composition

ingested water, and in the case of mammals, which are endothermic, that temperature will be

largely constant. This is true in other endotherms as well, such as birds, which show narrow δ18O

ranges (Harrell et al., 2016). Ectotherms like lizards, on the other hand, are dependent largely on

their surrounding temperature. Behavioral changes, such as when, where, and if to bask give

them some control over their temperature (Muñoz et al., 2016; Muñoz & Moritz, 2016), but

things like weather and seasons have ultimate control. Variation in δ18O values has been

observed in other ectotherms (fish, turtles) as well (Harrell et al., 2016). As such, δ18O not only

serves as a proxy for aridity, but also for modes of thermoregulation. The larger the range in

values, the more dependent on external temperature the animal must be.

c. Tooth Structure, Composition and Implications for Micro-Isotopic Analyses: The value of

micro-analytical analyses like SIMS cannot be understated (Hoskin, 1998; Passey & Cerling,

2006; Colleary et al., 2015). Whereas the remaining analyses relied on less precise IRMS, they

were performed on extant lizards. Fossils, whose dentine should be excluded from analyses

because of the likelihood of alteration (Kohn et al., 1999), have much less enamel to sample per

18

tooth and are rare. It is important to know where the enamel/ dentine contact is in a tooth and

how thick the enamel is, to prevent any dentine contamination. Using the elemental mapping

abilities of TOF-SIMS, we generated a Ca+ normalized map, to show the contrast between the

amount of apatite in dentine and enamel (Fig. 5). The white outer ridge represents the enamel

portion of the tooth and illustrates how thin the enamel is (~5 microns).

In addition to SIMS, laser ablation inductive-coupled plasma mass spectrometry (LA-

ICP-MS) allows for sampling of teeth that that require a sample area of a few tens of microns

(Passey & Cerling, 2006) and would ideal for sampling lizard teeth. For example, it would likely

require more teeth than one specimen of certain small species (e.g., Anolis carolinensis) has in its

body to acquire enough powder for one sample for GS-IRMS. SIMS and LA-ICP-MS requires a

much smaller scale and broadens the number of species available for isotopic analyses.

Generating an elemental map and sampling at the micron scale will be required to avoid dentine

contamination (Riegler et al., Ch.2)

d. Applications of Stable Isotopes for Conservation Biology: Whereas δ18O and δ13C are two

of the most commonly studied isotopic species for animal ecology, there are others that can serve

as ecological proxies. Strontium isotopes (87Sr/ 86Sr) leave a unique signature depending on the

geographic location (e.g., Ben-Davis & Flaherty, 2012). As such, analyzing these ratios in fossil

specimens has been useful for inferring migration patterns and biogeographic distributions

(Hoppe et al., 1999; Hodell et al., 2004; Price et al., 2004). Using mammoth tusks, which grow

throughout an animal’s life, strontium recorded migration patterns (Hoppe et al., 1999).

However, in the case of lizards, such a proxy could also be useful for living species. It is now

19

known that a large portion of lizards shown in zoos or sold as pets were harvested from the wild,

often illegally (Duckworth et al., 2012). This is especially true in countries like Indonesia (Lyons

& Natusch, 2011); one study found that the annual number of Tokay geckos taken from

Indonesia exceeded one million (Nijman et al., 2012). This represents a major threat to species

longevity, but evidence of those actions is difficult to acquire. Measuring strontium isotope ratios

of teeth and bone in pet and zoo lizards will allow determination of which animals were captive

bred or were taken from the wild, and if so from where they were collected. These analyses can

even be performed on living lizards by collecting teeth that have fallen out of the jaws of newly

acquired specimens.

e. Implications for the Fossil Record and Paleoecology: Global warming is not new to Earth

or to life. Fifty-five million years ago, Earth experienced a major global warming event, the

Paleocene-Eocene Thermal Maximum (PETM) (e.g., Zachos et al., 2001; Zachos et al., 2008;

Deconto, 2012). The PETM is the most recent major global warming event in Earth’s history.

Similar to the current climate change, the PETM was driven by a massive greenhouse gas influx

(CO2), resulting in a 5˚C increase in global temperature over the course of a few thousand years

(Zachos et al. 2001; Rohl et al., 2007; Kraus et al., 2007). Though not as rapid as this current

event, the PETM represents a window into biotic response to geologically rapid (~5,000 years)

climate change and can serve as a lower bound to animal response today. This is especially

useful for understand extinct lizards and their paleoecology.

Paleoecology has traditionally been inferred from the fossil record through examination

of morphology (e.g., Parrish et al., 1987; Narbonne et al., 2014). Analyzing the local biota and

20

making inferences about preferred habitat can also be used to interpret ecology (e.g., Anemone

et al., 2012). These are often circular in nature, relying on ecological assumptions to infer

ecology. More direct analyses like leaf margin analysis or depositional interpretations can also

serve as an ecological proxy for a given area (e.g., Wilf, 1997; Pomar, 2001; Greenwood et al.,

2004). But these do not apply to a specific organism, instead representing an average over an

area. Additionally, an animal may change its behavior within an environment, changing its

ecology independent of the signal preserved in surrounding proxies (Muñoz et al., 2016; Muñoz

& Moritz, 2016). Data from the actual fossils that are separate of the morphology such as stable

isotopes allow independent tests of those ecomorphological hypotheses that are quantitative and

are unique to an individual.

The data shown here illustrate the potential utility of δ13C and δ18O as proxies for the

paleoecology of extinct lizards. Diet is a huge part of an organism’s ecology (Prince, 1980).

Understanding diet using δ13C can help constrain at which trophic levels lizards struggle during

events like the PETM. Examination of the entire lizard fossil record, a proxy of this nature can

be useful in more accurately determining diet, allowing us to better understand niche

partitioning. Understanding the environment in which lizards lived during such events as the

PETM, and understanding the temperature and rainfall changes they experienced, can help

understand how lizards handled past climate change (Kraus & Riggins, 2007). Variation in δ18O

has the ability to show the approximate amount of temperature change these lizards are

experiencing. Collecting data on what temperature ranges PETM lizards experienced and were

able to tolerate in a variety of environments can help model what lizards might be able to

tolerate today.

21

In addition, a quantified proxy for thermoregulation as demonstrated here with δ18O

could be useful in determining the evolutionary history of endothermy. Endothermy has a large

role in the behavior and life history of an animal (Muñoz et al., 2016), but this aspect of an

organism’s biology is difficult to infer from the fossil record (Harrel et al., 2016). Though there

are several types of thermoregulation that may mirror endothermy’s isotopic signature (e.g.,

gigantothermy), quantifying the amount of variation in a specie’s δ18O values can allow

inferences on ectothermic versus some form of endothermy.

Although the PETM is a period often studied for its similarities to the present rate of

climate change (Deconto et al., 2012), using the proxies outlined here could help with

understanding how lizards responded to any of the many events in Cenozoic. The PETM is one

of several hyperthermal events at the beginning of the Cenozoic (e.g., Zachos et al., 2001;

Zachos et al., 2008). Additionally, these proxies could also be useful in understanding how

organisms responded to any of the ice ages in the Cenozoic (Seimon et al., 2007). The ability to

infer and quantify trophic structure (δ13C), temperature ranges, aridity, and thermoregulation

(δ18O) in lizard fossil taxa will allow for new data on an important and underrepresented group.

6. Acknowledgments

This work was completed as part of an MS thesis by MSR, and comments by committee

members Shuhai Xiao and Sterling Nesbitt, along with the VT Paleobiology and Geobiology

Research Group and Martha Muñoz, greatly increased the quality of this manuscript. We thank

Coleman Sheehy, Sterling Nesbitt, and Alan Resetar for access to specimens in their care and for

permission for destructive sampling. We thank the Geological Society of America and Virginia

22

Tech for providing funding for analytical work to MSR and BCG and the Department of

Geosciences at Virginia Tech for providing funding to MRS for specimen procurement. Andrei

Dolocan and Caitlin Colleary provided skilled assistance using the TOF-SIMS at UT Austin.

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33

8. Figures

Figure 1. Tooth comparisons and implantation types between mammals and lizards. A. Panther

chameleon (Fucifer pardalis) skull, illustrating acrodont and homodont dentition; B. Green

iguana (Iguana iguana) right lower jaw, illustrating pleurodont and homodont dentition; C.

Black and white tegu (Salvator merianae) lower jaw, illustrating pleurodont and heterodont

dentition; D. Elk lower jaw, illustrating complex, thecodont dentition; E. Savannah monitor

(Varanus exanthematicus), illustrating pleurodont and homodont dentition; F. Uromastyx geyri,

illustrating acrodont and homodont dentition. For lizard specimens, anterior is to the left,

mammal specimen anterior is to the right.

34

35

Figure 2. δ13C data for five extant lizard species. Red and blue arrows on the x-axis indicate

predicted plot locations for data based trophic level. Blue diamond = tegu, orange square =

Uromastyx, green plus = chameleon, purple triangle = monitor, teal x = green iguana.

36

δ13C (‰), VPDB

37

Figure 3. δ18O data for five extant lizard species. Red and blue arrows on the y-axis indicate

predicted plot locations for data based on aridity. Blue diamond = tegu, orange square =

Uromastyx, green plus = chameleon, purple triangle = monitor, teal x = green iguana.

38

39

Figure 4. Individual variation amongst all five species. δ18O plots on the y-axis, δ13C plots along

the x-axis. Majority of isotopic variation in both δ18O and δ13C is between individuals of a

species, with data from a single individual often plotting in the same region.

40

41

Figure 5. Tof-sims image data for a Savannah monitor tooth. Image shows occlusal view of a

dentary tooth that has had its crown milled to a flat surface. Image shows CA+, normalized. The

white indicates higher concentrations of CA+. Scale bar = 10 microns.

42

43

9. Tables

Table 1. Taxa name, size, location data, dentition type, average isotope values, and average

standard deviation of each sampled specimen of each of the five species included in this analysis.

44

45

Table 2. All isotopic data of all five specimens of all five species included in this analysis.

Boxed data sets represent averages and standard deviations. Data point names are same as what

were used for analysis. Each species occupies three columns, with isotopic data for each

specimen contained to the right in the same row.

46

Tegu δ13C δ18O Uromastyx δ13C δ18O Chameleon δ13C δ18O

Tegu1-BLM -16.58 -4.17 1 1

Tegu1-BLM -16.93 -4.10 Uro-1 TR -9.09 10.38 Cham.-1 T -14.19 -1.76

Tegu1-BLC -17.09 -4.40 TL -9.35 10.63 Chameleon 1 - BR -14.59 -1.49

Uromastyx 1 -

Tegu1-BLC -16.35 -4.67 BR -11.73 10.90 BL -14.50 -1.55

Tegu1-BRM -17.02 -4.57 BL -11.53 10.23 Cham-2 BR -13.67 -1.08

Tegu1-BRM -16.78 -5.51 aver -10.43 10.54 BL -13.86 -1.65

Tegu1-BRC -16.78 -4.29 std 1.08 0.23 aver -14.16 -1.51

Tegu1-BRC -16.34 -5.25 2 std 0.36 0.23

Tegu1-TLM -16.76 -4.08 Uro.-3 TR -9.68 9.29 2

Tegu1-TLM -16.56 -5.33 Uro.-5 T -9.12 9.21 Cham.-2TR -12.09 -2.56

Tegu1-TLC -16.21 -4.77 TR -11.09 9.85 TL -12.10 -1.98

Tegu1-TLC -15.90 -6.19 TL -11.34 9.20 Cham2.-BRA -12.55 -1.86

47

Tegu1-TRM -16.74 -4.13 aver -10.31 9.39 DL -12.45 -2.48

Tegu1-TRM -17.04 -4.31 std 0.93 0.27 aver -12.30 -2.22

Tegu1-TRC -16.40 -4.36 3 std 0.20 0.31

Tegu1-TRC -16.35 -4.67 Uro-3 TR -13.16 9.81 3

std 0.33 0.58 TL -13.35 9.30 TL -14.26 -2.93

aver -16.61 -4.68 URO 3-BR -13.31 8.82 Cham 3-DR -14.61 -1.63

2 BL -13.30 8.27 BL -14.38 -2.54

Tegu 2 - BLM -12.99 -3.99 aver -13.28 9.05 aver -14.43 -2.28

BLM -11.24 -4.65 std 0.07 0.57 std 0.14 0.54

BLC -11.58 -5.35 4 4

BLC -10.09 -5.29 Uro-4 B -13.65 6.40 Cham.-4 T -15.43 -2.39

BRM -10.77 -4.61 T -13.76 8.37 Cham.-4 BL -15.40 -1.97

BRM -8.76 -5.86 aver -13.71 7.39 Cham 4 - BRB -14.68 -2.04

BRC -11.93 -4.64 std 0.05 0.98 aver -15.17 -2.13

BRC -9.96 -4.87 5 std 0.35 0.18

std 1.23 0.54 Uro-5 TR -10.96 12.27

aver -10.92 -4.91 TL -11.63 11.25 5

3 URO 5-BR -11.98 7.23

Tegu3-BRM -17.92 -3.00 BL -11.63 7.63 Cham 5-BR -13.59 -1.24

Tegu3-BRC -17.52 -3.34 aver -11.55 9.59 BL -13.62 -2.51

Tegu3-TLM -17.44 -3.11 std 0.37 2.20 aver -13.60 -1.87

Tegu3-TLC -17.67 -3.43 average total -11.65 9.39 std 0.01 0.63

Tegu3-TRM -17.72 -3.51 std total 1.53 1.43 average total -13.85 -1.95

Tegu3-TRC -17.80 -5.18 std total 1.02 0.50

LLM -17.13 2.30

LLM -16.91 -0.04

LLM -17.59 -0.79

LTP -17.56 -3.40

LTP -16.57 -1.11

48

LLM LLM std aver

4 Tegu 4 - TRC TRC TLM TLM TLC TLC Tegu 4 - BRM BRM BRC BRC BLM BLM BLC BLC TRM TRM std aver

5 Tegu-5 BRB BRB BRF BRF BLB BLB

-16.88 0.71

-16.74 -2.62

0.42 2.01

-17.34 -2.04

-13.41 -1.98

-13.14 -2.44

-13.41 -2.54

-12.58 -2.46

-13.34 -2.14

-12.98 -2.64

-13.18 -2.06

-13.44 -2.45

-13.23 -2.33

-13.34 -3.33

-14.05 -1.90

-13.76 -3.02

-13.48 -3.28

-13.18 -3.10

-13.19 -2.76

-13.04 -2.35

0.32 0.43

-13.30 -2.55

-17.99 -2.66

-17.29 -2.50

-17.69 -2.77

-17.23 -2.78

-18.21 -2.54

-17.19 -2.41

49

Tegu-4-BRB BRB BRF BRF BLB BLB BLF BLF TRB TRB TRF TRF TLB TLB TLF TLF std aver average total std total

-17.36 0.15

-16.65 -0.05

-17.63 0.35

-16.68 -0.33

-17.52 -0.13

-16.46 -1.09

-17.25 -0.69

-16.69 -0.19

-17.40 -1.47

-16.81 -1.75

-17.72 -1.01

-16.99 -1.30

-17.90 -1.97

-16.88 -2.26

-17.90 -0.95

-16.74 -1.60

0.49 1.01

-17.28 -1.36

-15.62 -2.82

2.31 1.76

Monitor δ13C δ18O Iguana δ13C δ18O

1 1

BRF -8.96 -2.08 Iguana1-BLB -13.41 -3.15

TRB -8.69 -4.24 Iguana 1 - BLF -13.79 -2.08

TRF -9.27 -3.59 BRB -14.87 -2.32

TFB -9.27 -1.98 BRF -14.17 -2.03

TLB -8.95 -4.35 TRB -14.01 -2.37

TLF -9.14 -4.02 TLB -13.03 -4.33

50

aver -9.05 -3.38 aver -13.88 -2.71

std 0.20 0.98 std 0.58 0.81

2 2

Vara-2 BR -5.57 -3.23 Iguana-2 BL -16.63 0.66

BL -5.42 -3.52 Igu 2-BR -17.14 0.72

TR -5.55 -3.12 aver -16.89 0.69

TL -5.75 -3.28 std 0.25 0.03

aver -5.57 -3.29 3

std 0.12 0.15 Iguana-3 BL -16.78 -0.67

3 T -16.70 0.22

Vara-3 BR -7.06 -2.07 aver -16.74 -0.23

BL -6.93 -2.02 std 0.04 0.45

aver -6.99 -2.05 4

std 0.07 0.02 Iguana-4 T -16.37 1.63

4 Igu.4-BRB -16.89 1.27

Vara-4 BR -6.45 -2.92 BL -16.79 0.95

BL -6.36 -2.70 aver -16.68 1.28

TR -5.25 -3.28 std 0.22 0.28

TL -6.43 -2.61 5

aver -6.12 -2.88 Iguana-5 TR -14.21 1.75

std 0.51 0.26 TL -14.07 1.61

5 Iguana 5 -BRB -14.20 2.16

Vara-5 BR -9.69 -1.26 BRF -14.12 2.62

BL -8.70 -1.28 BLB -14.18 1.18

TR -10.42 -1.20 BLF -14.08 1.97

TL -10.30 -0.94 aver -14.14 1.88

aver -9.78 -1.17 std 0.06 0.45

std 0.68 0.14 average total -15.02 -0.01

average total -7.71 -2.68 std total 1.37 2.02

51

std total 1.74 1.02

52

Chapter 2

DIVERSITY AND TROPHIC STRUCTURE OF AN EARLY EOCENE HERPETOFAUNA

FROM WYOMING

Mitchell S. Riegler, Department of Geosciences, Virginia Tech, mriegler@vt.edu

53

1. Abstract

The dawn of the Eocene (55 Ma) occurs in the middle of a drastic change in global

temperature during an event known as the Paleocene-Eocene Thermal Maximum (PETM). This

global warming event shifted temperatures by approximately 6°C and culminated in one final spike

in temperatures at about 52 Ma, the Early Eocene Climatic Optimum (EECO). The Wasatch

Formation in Wyoming spans the Paleogene, covering the entirety of this climatic transition and

providing insight on its effects on biodiversity and ecology. We describe the fossil reptile assemblage

from the Early Eocene (Wasatchian) Tim’s Confession locality (CM locality #222) in order to shed

light on the herpetofauna during a major global warming event. This locality includes anguimorph

squamates (xenosaurids and glyptosaurines) and alethinophidian snakes. The xenosaurid, represented

by at least two dentaries, is one of the youngest representatives of this clade, helping better

understand the biogeographic and chronologic distribution of a relatively cryptic lineage. In addition

to osteoderms, glyptosaurine anguimorphs are identified based on cranial material, including

maxillae that preserve wide, knob-shaped teeth and pronounced dermal scales on their lateral

surfaces. Trophic structure, as well as other ecological parameters, is poorly understood in fossil

lizards but may provide key data for understanding response to ecological change. Using

geochemical proxies, we were able to quantify certain parts of the ecology in the two lizards

described here. Specimens of these two lizard taxa were subjected to time-of-flight secondary ion

mass spectrometry (TOF-SIMS) and isotope ratio mass spectrometry (GS-IRMS) to test for stable

isotope proxies of diet and aridity. Though it appears the δ18O values were diagenetically altered, the

δ13C values appear original and indicate a higher trophic position (insectivore or carnivore) for both

lizard taxa. Our findings support previous ecomorphological hypotheses attempting to infer diet yet

illustrate the importance of an ecological test independent of morphology.

54

2. Introduction

The Cenozoic (~65 Ma to the present) begins directly after one of the most famous events

in geologic history, the bolide impact that formed the Chicxulub crater and resulted in the

extinction of most dinosaur taxa (Alvarez et al., 1980). Whereas this event marked the end of the

Mesozoic and the beginning of the Cenozoic, it did not mark the end of environmental

perturbations. The early Cenozoic would encompass several hyperthermal events (large

upswings in global temperature) that would each last for millions of years. The earliest and most

heavily-studied of these events is the Paleocene-Eocene Thermal Maximum (PETM) (e.g.

McInerney & Wing, 2011). The PETM was a global warming event ~55 Ma induced by the

release of greenhouse gases, which resulted in a 5C increase in global temperatures (Zachos et

al., 2001; Cohen & Kemp, 2007; Rohl et al., 2007; Kraus et al., 2007; Zachos et al., 2008;

Deconto, 2012; Bowen et al., 2015). The PETM, also known as the Eocene Thermal Maximum 1

(ETM 1), was followed by several smaller events, namely the ETM 2, which were all greenhouse

gas-driven global warming events (Sluijs et al., 2009). Those hyperthermal events likely

culminated to a peak in temperatures during the Early-Eocene Climatic Optimum (EECO)

approximately 52 Ma (Seimon et al., 2007; Zachos et al. 2008; Woodburne et al., 2009).

Changes in an environment at the scale of the PETM or EECO almost always result a

biotic response from the animals in that environment. Most species are accustomed to a narrow

range of environmental parameters including temperature, food sources, rainfall (e.g. Pianka,

1973). When these parameters change, vertebartes are faced with the challenge of adapting to

these changes or going extinct (Webb et al., 2005; Huey & Tewksbury, 2009; Rage, 2012;

Muñoz & Moritz, 2016). In the case of the PETM, the impact and response of animals to those

events have been well studied in mammals (e.g. Beard, 2008; Woodburne et al., 2009). Those

55

ecologically focused studies noted increases in extinction and migrations, and an ever-increasing

understanding of mammalian response to global warming is being formed (e.g. Smith et al.,

2006; Gingerich, 2006; Woodburne et al., 2009). Those studies also developed new proxies to

infer ecology, namely by analyzing stable isotope values in fossil materials that preserve the

original ecology (Koch et al., 1995; Rountrey & Vartanyan, 2007). Such studies have been done

on mammals for decades to infer ecological parameters including diet, migration, and rainfall

(e.g. Cerling et al., 1997; Emery et al., 2000; Kelly, 2000; Cerling et al., 2003; Price et al, 2004;

Roche et al., 2010; Ben-David & Flaherty, 2012; Wheatley et al., 2012). Having high resolution

data on the response of mammals to historic climate change could be especially useful for our

current global warming event (Hughes, 2000). Though modeling floral and faunal response to

climate change can be difficult, several studies have already illustrated the predictive power of

studying animal response to past events (e.g. Kelly, 2000; Cerling et al., 2003; Sponheimer et al.,

2003 Ben-Davis & Flaherty, 2012).

In the case of the PETM, EECO, or current global warming, all major groups animals are

having to adapt to climate change in some way. Additionally, those paleoecological studies

require geochemical sampling that has traditionally been easiest done on larger organisms.

Unfortunately, this then excludes the smaller, tropical organisms (Sponheimer, 2003; Cerling,

2003; Rountrey et al., 2007). The tropics are key spots of biological richness, being called

‘museums’ for their ability to house taxa with greater than average longevity (Lu et al., 2018).

Ectothermic organisms such as squamates are dependent on their surroundings for setting their

internal temperature, and in the case of tropical species, that temperature can be quite constant

(Huey et al., 2009; Lu et al., 2018). When surrounding temperatures vary too far from what a

lizard can tolerate, it can impact energy production, development, and the ability to hunt (Webb

56

& Whiting, 2005; Muñoz et al., 2016; Muñoz & Moritz, 2016). Understanding which taxa can

handle certain temperature ranges can help predict the taxa that are most at risk as temperatures

rise.

Today mammals have approximately 4500+ species (Duellman et al., 2009). Squamates

meanwhile have an approximated 6000+ species (Duellman et al., 2000). This trend exists

throughout the last ~60 Ma, yet a large number of the fossils described for the Cenozoic are

predominately mammalian (e.g. Woodburne et al., 2009; Anemone et al., 2009; 2012; Ben-Davis

& Flaherty, 2012). Our understanding of lizard response to climate change during the Cenozoic

is correspondingly poor (Gauthier, 1982; Huey et al., 2009; Sinervo et al., 2010; Rage, 2012).

Lizards are also informative because of their tendency to occupy tropical climates (Huey et al.,

2009; Bush & Gosling, 2011). In an attempt to expand our ecological data of tropical non-

mammalian taxa during the events of the early Cenozoic, this study analyzes a squamate

assemblage from the early Eocene of Wyoming. We illustrate the feasibility of isotopic analyses

for independent testing of ecomorphology in Cenozoic lizards, and expand our understanding of

squamate ecology at a time of climactic variability.

Institution Abbreviations-

FMNH - Field Museum of Natural History, Chicago, Illinois

GDB - Great Divide Basin group, University of North Carolina Greensboro, Greensboro, North

Carolina

PU - Princeton University, at Yale Peabody Museum, New Haven, Connecticut

UCMP - University of California Museum of Paleontology, Berkeley, California

YPM - Yale Peabody Museum, New Haven, Connecticut

57

CM - Carnegie Museum, Pittsburgh, Pennsylvania

3. Geographic and Geologic Setting

The fossils described here were collected from the Great Divide Basin in Sweetwater

County, Wyoming (Fig. 1), within the Wasatch Formation. This unit is part of the lower Eocene

(~55 Ma) (Savage, 1975, Gauthier, 1982; Woodburne, 2009). In some geologic maps, this site is

mapped as Quaternary (Pipiringos, 1962), but it is in fact an Eocene sandstone (Bommersbach,

2014). It is primarily composed of fluvial and paludal rocks, and intertongues throughout its

vertical section with the lacustrine Green River Formation (Pipiringos, 1955). The squamate

specimens we describe here are from the Tim’s Confession locality (CM locality #222) of the

Wasatch Formation, a highly fossiliferous unit containing several groups of mammals, including

condylarths, perissodactyls, artiodactyls, adapiform primates, euprimates, and creodonts (e.g.

Gauthier, 1982; Anemone et al., 2009, 2012; Gunnell, 2012; Bommersbach, 2014). Based on the

presence of those mammals, namely the omomyids, the Wasatch Formation has been described

as being largely tropical (Anemone et al., 2012). Additionally, Tim’s Confession is dated at Wa4

(~54 Ma) in the Early Eocene (Woodburne et al., 2009; Anemone et al., 2012). This age is

significant because it places the Tim’s Confession locality chronologically between the PETM

(~55 Ma) and the peak of the EECO (~52 Ma) (Woodburne et al., 2009; Anemone et al., 2012).

SYSTEMATIC PALEONTOLOGY

REPTILIA Laurenti, 1768

SQUAMATA Oppel, 1811

ANGUIMORPHA Fürbringer, 1900

58

XENOSAURINAE Cope, 1900

RESTES RUGOSUS Gauthier, 1982

Synonyms: Exostinus rugosus, Gilmore, 1942.

Holotype: PU 14559, Gilmore, 1942, partial right maxilla.

Referred Specimens: GDB 1, left dentary; GDB 5, left dentary.

Locality: Tim’s Confession locality (CM-220), Wasatch Formation, Sweetwater County,

Wyoming (Bommersbach, 2014). Specific locality information is available upon request.

Age: Early Eocene (Wa4, ~54 Ma) (Anemone et al., 2012)

Description and Rationale for Taxonomic Assignment: The holotype specimen of Restes

rugosus is a fragmentary maxilla (Gilmore, 1942; PU 14559). Gauthier (1982) described and

referred a more complete specimen, comprising several cranial elements (maxilla with associated

dentaries) (YPM 14640) (Fig. 2E), that was also collected by Gilmore (Gauthier, 1982). From

that more complete specimen (YPM 14640), Gauthier (1982) described only the frontals and

rediagnosed the taxon based on that element. Subsequently, in his phylogenetic analysis of

anguimorph lizards, Bhullar (2011) included all elements from YPM 14640 in his phylogenetic

analysis, including the dentaries, which are used for comparison here.

59

GDB 1 consists of a nearly complete left dentary missing the angular and surangular

articulation facets. The lateroventral surface of the dentary, which would normally wrap under

the Meckel’s canal, is broken and missing, and the anterior most tip of the dentary is absent. In

total, the dentary is about 10mm in length. All of the posterior tooth positions are preserved, with

the majority of the teeth still in place (Fig. 2). In order to identify GDB 1, we compared it with

the description by Bhullar (2011) and coded in into his character-taxon matrix. The splenial

extends ¾ the length of the dentary, as indicated by the point at which the Meckel’s canal is only

open ventrally (Fig. 3A), and this feature places GDB 1 within Anguimorpha (Gauthier,

2012:375(3)). The Meckel’s canal being open ventrally towards the anterior end of the dentary

(Fig. 3B) places it within Xenosaurinae (Gauthier, 2012:371(1)). In addition, the recurved mesial

teeth and blunt posterior teeth are indicative of Xenosaurinae (Fig. 3). Incipient bicuspid

posterior teeth were considered diagnostic of Restes rugosus by Gilmore (1942), and this is

observed in GDB 1. Additionally, the deep and long groove anterior to the coronoid facet was

found as an autapomorphy of Restes rugosus by Bhullar (2011).

The dentition was one of the first described characteristics in the holotype of Restes

rugosus, PU 14559 (Gilmore, 1942). Gilmore (1942) found that the tooth morphology was

diagnostic to this species, and the same morphology is seen in the dentary here. At the anterior

half of the dentary of GDB 1, the teeth are recurved posteriorly and have a conical base. The

absolute number of recurved teeth in the dentary is difficult to ascertain, because the anterior tip

nearest the symphysis is absent. However, in the dentary of Xenosaurus grandis (FMNH

211833) there are 18 teeth. GDB 1 has 16 tooth positions, so it is likely missing only about two.

Therefore, assuming two missing teeth, the total number of recurved more mesial teeth is

approximately 12 in GDB 1. At the posterior end of the dentary, the teeth are no longer recurved

60

but extend dorsally without any distal deflection at the tips. Gilmore (1942) (PU 14559)

observed diagnostic bicuspid crowns on the posterior teeth. We compared the dentition of the

extant Xenosaurus grandis (FMNH 211833) with that of the fossil dentary (GDB 1). Xenosaurus

grandis is described as bicuspid (Estes, 1965), but, as illustrated (Fig. 3), it is weak or absent in

many specimens. This is similar to what is present in GDB 1, which might indicate a worn

surface that was likely bicuspid when freshly erupted.

The lateral surface of GDB 1 is smooth and appears to preserve its original morphology.

Posteriorly, the coronoid articulation surface is preserved along the dorsal surface. This surface

is a deep, extended groove, but does not have a sharp surrounding ridge. This is identical with

the material of Restes rugosus described and characterized by Bhullar (2011) (YPM 14640)

(186(1), 187(0)). Along the lateral face, approximately four circular foramina are present (Fig.

2).

The Meckel’s canal is mostly preserved (Fig. 2B) in GDB 1. In lingual view, the canal is

restricted dorsally by a dental shelf to which the teeth are attached. As the shelf extends

anteriorly, it becomes dorsoventrally taller (Bhullar, 2011:191(1)). Ventrally, the lateroventral

surface of the dentary extends underneath to form the lower surface of the canal. Though much

of that surface is broken, the orientation of the canal and the remaining fragments shows that the

posterior portion of the canal would be exposed lingually, common in squamates (Phrynosoma,

Iguana) (Evans, 2008; Gauthier et al., 2012). As the canal extends anteriorly, the canal becomes

ventrally oriented and is present along the ventral edge of the dentary (comparable to Gauthier et

al., 2012:371(1)). At the point where the canal transitions from a lingual orientation to a ventral

one, the splenial ends. It can no longer articulate with the lateroventral surface becuase the

61

surface no longer extends medially. This occurs at approximately ¾ of the way toward the

anterior end of the dentary (Gauthier et al., 2012:375(3)).

REPTILIA Laurenti, 1768

SQUAMATA Oppel, 1811

ANGUIDAE Gray, 1825

GLYPTOSAURINAE McDowell and Bogert, 1954

PROXESTOPS Gilmore, 1942

Holotype: PU 14565, Gilmore, 1942, partial right maxilla.

Referred Specimens: GDB 2, fragmentary maxilla; GDB 3 fragmentary maxilla; GDB 6-10

assorted osteoderms.

Description and Rationale for Taxonomic Assignment: The osteoderms of Proxestops are

intermediate in size and rugosity to the other glyptosaurine taxa (Smith, 2011). Unlike most

glyptosaurines, which have purely tuberculate osteoderms, Proxestops has a slightly more

vermiculate pattern (Fig. 4e).

A fragmentary maxilla (GDB 2) is identified as Proxestops on the basis of the fused

osteoderms on the lateral surface. Seven teeth are in place, and one alveolus is missing a tooth,

for a total of eight tooth positions within the maxilla. The teeth widen distally through the

toothrow, with the three posterior teeth preserving black enamel caps. These enamel caps

preserve a raised anterior-posterior oriented ridge that forms from grooves within the enamel.

From the inclination of the base, it appears that the tooth is slightly posteriorly recurved.

Laterally, there is a linear row of foramina opening laterally from the facial surface of the

62

maxilla. There are seven present, with variable sizes and shape. From a dorsal view, a strong

palatal process/dental shelf exists that contains a single foramen pointing dorsally (Fig. 4).

REPTILIA Laurenti, 1768

SQUAMATA Oppel, 1811

SERPENTES Linnaeus, 1758

ALETHINOPHIDIA Nopcsa, 1923

Referred Specimens: GDB 4, trunk vertebra

Description and Rationale for Taxonomic Assignment: GDB 4 represents a procoelous

vertebra with obvious zygosphene-zygantrum complexes. The identification of GDB 4 as an

alethinophidian snake is based on the characters of Head (2002), including a sharp hemal keel

that terminates anteriorly to a point just anterior to the condyles, anterior cotyles that are

expanded with delineated margins, and paired and symmetrical subcentral foramina (Fig. 5).

4. Methods and Results

a. Phylogenetic Analyses: The squamate fossil materials analyzed here were identified and

described in part using the matrices of Gauthier et al. (2012) and Bhullar (2011). In order to test

the phylogenetic position and character state distribution of the GDB Restes rugosus material, we

coded GDB 1 as a separate Operational Taxonomic Unit (OTU) in the character-taxon matrix of

Bhullar (2011). Nexus files were generated using Mesquite v.2.01 (Maddison and Maddison,

2008). Phylogenetic analyses were performed using both TNT (Goloboff et al., 2001) and

PAUP* v. 4.0b10 (Swofford, 2002). Analyses were run in accordance with the taxon sampling

63

and parameters set by Bhullar (2011). Default options other than tree bisection and reconnection

(TBR) branch swapping was enabled with 1,000 random addition sequences. Multistate

characters were run as ordered.

There were 14 characters from Bhullar (2011) that were related to the jaw or dentition (1,

184-197). When incorporated, the dentary described here was scored identically to that of the

Restes rugosus specimens scored by Bhullar (2011). The results from our analyses produced two

most parsimonious trees (MPTs) with a tree length of 950 (Fig 6.). We recovered slight

differences with the topology of our trees as compared with the tree reported by Bhullar (2011),

who found only one most parsimonious tree with 875 steps, 75 steps fewer than in our strict

consensus of two trees. The difference in topology relates to the positions of Elgaria

multicarinata and Ophisaurus ventralis. However, the inconsistent tree lengths and differing

relationships of the two mentioned taxa do not impact the relationships in the portion of the tree

in which Restes rugosus is recovered. As such, we generated a strict consensus tree in which the

problematic lineages were consolidated into a Varanoidea + Anguidae lineages. With a Bremer

value of 1, we recover the GDB 1 dentary as the sister taxon to Restes rugosus, confirming our

descriptive conclusions. There was one unambiguous synapomorphy for Restes rugosus, which

was the posterior, rising section of dorsal edge of dentary extending for six or fewer tooth

positions (Bhullar, 2011, 185 (1)).

b. Isotopic Analyses: Biogeographic and temporal distribution is not all that is necessary to

understand biotic response to climate change. Understanding taxon survivorship and response

through an event like the PETM is more significant if we understand the ecological parameters

surrounding the organism. If we can infer the trophic structure of organisms in the PETM, we

64

could then possibly infer which taxa today are more at risk based in their diet. The same is true

for understanding the temperature range that an organism can tolerate. Using stable isotope

proxies tested by Riegler et al. (Ch.1), we performed stable isotope analyses on the two lizard

taxa identified at Tim’s Confession to infer the ecology of lizards living between two global

warming events. In addition, three mammal teeth (Meniscotherium tapiacitum, GDB 11-13) and

the enamel-like ganoine from three gar scales were analyzed to compare isotopic data across taxa

and check to chemical alteration (Fricke & Wing, 2004). We analyzed two isotopic species,

δ18O and δ13C, each serving as proxies for different ecological factors. δ13C was selected in this

study largely for its ability to separate out diet and trophic position (e.g. herbivore versus

carnivore; Cerling et al., 1997; Cerling et al, 2003; Sponheimer et al., 2003, Riegler et al., Ch.1).

δ18O in this study was selected as a proxy for aridity and temperature range (Luz & Kolodny,

1985; Ben-David & Flaherty, 2012; Chenery et al., 2012; Suarez et al., 2013; Harrell et al., 2016;

Riegler et al., Ch. 1).

In order to test diet, trophic position, and aridity, we performed both traditional isotope

ratio mass spectrometry (GS-IRMS) and time-of-flight secondary ion mass spectrometry (TOF-

SIMS) on our squamate specimens. For these analyses, we selected the most fragmentary though

diagnostic material for Restes rugosus and Prosextops in order to minimize the amount of fossil

material sacrificed for destructive sampling. It was important to determine dental tissue location

and thickness for these analyses. Enamel is the strongest material in the body and is least likely

to be altered during fossilization (Kohn & Barker, 1999; Colleary et al., 2015) and has a better

potential to preserve original isotopic signals. To determine that enamel was present, and its

thickness, TOF-SIMS analysis was performed in the Texas Materials Institute facilities at The

University of Texas at Austin. Samples were prepped with a dental tip attachment on a Dremel

65

tool to create a flat, smooth surface, starting from the tip of the tooth and grinding down towards

the base. A diamond-coated circular blade was used to polish the surface as much as possible.

Using a Leica TIC 020 ion mill, the surface was ablated using an ion beam. Each tooth was left

in the ion mill for approximately 5 hours. The final product was a glass-like polish that was more

ideal for TOF-SIMS analysis than hand preparation. Once milled, the samples were placed inside

an SEM vacuum chamber overnight, allowing the samples to outgas and reduce the vacuum time

in the TOF-SIMS chamber.

An ION-TOF TOF-SIMS.5 was used with a pulsed (18 ns, 10 kHz) analysis ion beam

consisting of Bi3 + clusters at 30-kV ion energy, which was raster-scanned over areas that

typically varied between 100 × 100 μm2 and 500 × 500 μm2, depending on the quality (i.e.,

corrugation and conductivity) of the sample surface. The polyatomic sputtering was selected to

further enhance the signal. To reduce the sputtering-induced sample charging, a constant energy

(21 eV) electron beam was shot on the sample during the data acquisition. All detected

secondary ions had negative polarity and an average mass resolution of ∼1–2,000 (m/δm).

Part of what differentiates enamel from dentine is the concentration of bioapatite within

the two materials (Nanci, 2017), and this difference can be identified in the different

concentrations of Ca+ across the tooth cross-sectional surface created with the ion beam. We

were able to detect the enamel-dentine contact, and we determine that the thickness of enamel in

these samples ranged from 0 to 18 microns (Fig. 7). Sampled teeth were then SEM scanned in an

SEM Quanta FEG 600 (Fig. 7) to provide an additional test to determine enamel versus dentine,

because the dentine in the inner portion of the teeth is much more porous than the outer enamel

layer. The outer rim of the tooth, approximately 30 microns, was smooth and lacked any pores.

Interior to that, the tooth was consistently porous, and more irregular. While not as diagnostic as

66

an elemental map, this appears to represent a visual separation of enamel and dentine.

Additionally, one tooth was sputtered (blasted at a high intensity to create a smooth surface) for

8 hours to illustrate the size that is required for consistent SIMS data (Fig. 7).

In order to infer diet and aridity, tooth enamel from both lizard species was then

isotopically analyzed using the GS-IRMS in the Department of Geosciences at Virginia Tech.

The teeth were milled with a dental-tipped Dremel tool, creating the 5mg of powder necessary to

derive enough carbonate for analysis (Riegler et al., Ch. 1). This resulted in one sample for each

species. In some cases, multiple teeth where needed to obtain the 5 mg of powder necessary. The

δ13C and δ18O contents were analyzed on a MultiFlowGeo headspace sampler attached to an

Isoprime 100 GS-IRMS. Samples were placed in rubber septum vials, flushed with helium, and

acidified with phosphoric acid. Samples were then reacted for at least 4 hours at 70C to allow

for the carbonate to react fully, producing CO2 gas. This gas was then analyzed for δ13C and δ18O

contents. Carbon and oxygen isotope values are reported in the standard δ-notation relative to the

Vienna Pee Dee Belemnite (V-PDB) standard and calibrated to this scale using the international

standards IAEA-CO-1 (marble; δ13C = +2.492‰, δ18O = −2.4‰), IAEA-CO-9 (BaCO3; δ13C =

−47.321‰, δ18O = −15.6‰) and NBS18 (calcite, δ13C = −5.014‰, δ18O = −23.2‰).

Reproducibility (1) for the analysis of the samples and standards were better than ±0.07‰ for

δ13C and better than ±0.3‰ for δ18O.

The data was plotted in combination with those from Riegler et al. (Ch.1), in which five

extant lizard taxa were analyzed for identical stable isotope values (Figs. 8 & 9). The data points

for the two extinct taxa in each instance plot very near each other in both the δ18O and the δ13C

plots (Fig. 8 & 9). Regarding δ18O, the values appear consistent with an original signal. Though

much more negative than modern taxa (~8‰), all four sampled taxa are within ~5‰ of each

67

other. These values also agree with Fricke & Wing (2004), who sampled mammal teeth and gar

scales in the near-by Big Horn Basin. These samples were of similar age and averaged around

(17‰). The inference that can be made from these values are that these organisms were living in

a very wet, tropical environment. The δ13C values also appear to be original to the specimens. An

altered carbon signal would incorporate the surrounding signal in the sediment in which it is

buried (Watkins et al., 2017). That sediment would contain large amounts of plant matter, which

in the early Eocene would mean all C3 plants (Christin & Osborne, 2014), and the isotopic signal

of those plants would be very negative (-24‰) (Watkins et al., 2017). The fact that the value

recovered for the two samples here is actually more positive than most extant taxa indicates an

unaltered sample.

5. Discussion

a. Significance of the Herpetofauna from Tim’s Confession: A vast majority of the materials

identified as Restes rugosus are highly fragmented (YPM VPPU 17144, YPM VPPU 14640, PU

14559). Although GDB 1 is not complete, it likely preserves nearly all tooth positions, and the

three-dimensional structure more so than other Restes rugosus materials. GDB 1 best exemplifies

the heterodont dentition from mesial to distal in a single specimen, and shows the Meckel’s canal

in all angles, and in its entirety. When the dentary was incorporated in the character taxon matrix

of Bhullar (2011), it was recovered as the sister taxon to the other described material of Restes

rugosus (Appendix 1; Fig. 6). The inclusion of more taxa is necessary to verify the effectiveness

of this matrix outside of Xenosaurus, but it appears that the dentary is diagnostic for Restes

rugosus.

68

Tim’s Confession is dated at Wa4, putting it in the Early Eocene (~54 Ma). Whereas

Proxestops and alethinophidian snake elements are common throughout much of the early

Cenozoic, Restes rugosus had long been listed as a Paleocene lineage (Clarkforkian, Tiffanian),

until Gauthier (1984) found and described Eocene specimens (Bartels, 1983; Conrad et al., 2011;

Rieppel, 1980; Sullivan, 1991; Gunnell, 2012). The Restes rugosus material found at the Oh!

Locality by Gauthier has generally been dated to be Wa5 (Gauthier, 1984; Smith, 2006), but

more recent studies have again stated Restes rugosus existed only in the Paleocene (Bhullar,

2011). Additional studies have mentioned finding Restes rugosus as late as Wa7 and the Late

Gardnerbuttean (Brla; ~50 Ma); however, no illustration, description, or rationale was given for

those identifications (Gunnell 2012). Our study is the first to describe and date Restes rugosus

from Wa4 and reaffirms that this species exists beyond the Paleocene, having survived through

the PETM.

b. Morphological Variation in Restes rugosus: Ontogenetic changes to the morphology of an

organism can alter character state interpretations and thus interpreted systematic relationships of

extinct taxa. With respect to Restes rugosus, these effects should be considered especially in

regard to the bicuspid crowns of the posterior teeth (Estes, 1984; Dessem, 1985; Butler, 2003;

Bhullar, 2011. Bhullar (2011) stated that bicuspid teeth are an indication of maturity and should

be scored as such. However, there is likely a wear factor to consider. The extant Xenosaurus

grandis (FMNH 211833) that was examined was from a mature specimen and had several

bicuspid posterior teeth (Fig. 3). However, it had a few teeth that had a flat or knob-like crown,

while still being surrounded by other bicuspid teeth. This likely indicates that this variation in the

number of cusps is a product of wear as the tooth ages. In application, this makes ontogenetic

69

identification difficult in fossils, and we hypothesize that in the dentary here described (GDB 1),

the teeth are worn and are not in an immature specimen.

c. Implications for Squamates in the Early Cenozoic: Proxestops and Restes rugosus are both

found before the PETM (e.g. Gilmore,1942; Smith, 2011). The presence of these taxa at the

Tim’s Confession locality (Wa4) means that they both survived through the PETM. While

alethinophidian snakes are a long-lived clade, extending from the Cretaceous to the present, we

can at least say that this site also supported such large predators (vertebrae length up to ~6 mm)

(Rage & Werner, 1999). However, in comparisons with other herpetofaunal assemblages from

adjacent or similarly-aged localities (Gauthier 1984; Smith, 2006; Stocker & Kirk, 2016), several

prominent taxa appear to be missing (e.g. amphisbaenians, iguanids). This could represent a

sampling bias or actual extirpation from this area as a result of the PETM. Additionally, while

alethinophidian snakes survive to the present, and Proxestops survived to ~50 Ma, there is little

evidence that Restes rugosus existed past the EECO (Gauthier, 1984; Gingerich, 1989; Gunnell,

2012). This is not necessarily in alignment with our understanding of the EECO and the PETM,

with the PETM being a more rapid event, and the EECO being a rather gradual event with few

extinctions (Zachos et al., 2001; Kraus & Riggins, 2007; Zachos et al., 2008). If the EECO did in

fact drive Restes rugosus to extinction, what driver was responsible that had not already

happened in the PETM? Additional isotopic sampling and herpetofaunal analysis is needed to

constrain how the environment and ecology changed before and after the EECO and the PETM.

d. Ecomorphology and ecology in the Early Eocene: Previous inferences of diet in extinct

lizards have been based on tooth morphology and comparisons to modern taxa (e.g. Herrel et al.,

70

2001; Measey et al., 2011; ElShafie, 2014, Melstrom, 2017). While these analyses can be helpful

and utilized the best of what was available, dental morphology in lizards is often shared amongst

different diets. For example, the teeth of Basiliscus, Enyaliosaurus, and Ctenosaura, are all

pleurodont with three simple cusps. As similar as these teeth are, Ctenosaura and Enyaliosaurus

rely mostly on vegetation for their diets, whereas Basiliscus relies mostly on insects

(Montanucci, 1968). To complicate studies using modern comparisons, the diet of many modern

lizards is still at times uncertain. Uromastyx has long been reported as an herbivore, until recent

studies noted some eating insects on a regular basis (Pianka, 1973; Cunningham, 2001). More

recent ecomorphological studies have quantified tooth shape and found more consistent results in

predicting the diet of lizards (Melstrom, 2017). There are still large regions of overlap between

differing diets, and it is clear additional objective data are necessary. Isotopic analyses allow

independent inferences of diet, separate from morphology, that are unique to an individual

specimen (Cherel & Hobson, 2005). Additional inferences using stable isotopes in ecology have

been made based on depositional environment or vegetation, but this only represents an average

over time (Wilf, 1997; Pomar, 2001; Greenwood et al., 2004). Complicating this, animal

behavior may impact an animal’s ecology independent of the surrounding environment. Isotopic

analyses serve as a unique proxy, providing data at the individual scale.

In this study, we performed isotopic analysis on two lizard taxa, Restes rugosus and

Proxestops. In each case, it appears the δ18O values were original (Fig. 8). This data allows for

environmental inferences, and inferences in paleotemperature. Our δ13C values appear to be

original as well. These δ13C values indicate a higher trophic position for both taxa, likely as an

insectivore to occasional carnivore (Fig. 9) (O’Connell & Hedges, 2017). This is in agreement

with other inferences on the diet of Proxestops, which indicated it should likely be an insectivore

71

(Elshafie, 2014). No prior inferences have been made on the diet of Restes rugosus, so this

stands as the first.

An additional proxy that δ18O values can serve that could not be shown in this study but

was illustrated by Riegler et al. (Ch. 1) is for the temperature regulation of the organism. In the

case of an ectotherm, that is generally the temperature surrounding the individual (Muñoz et al.,

2016; Muñoz & Moritz, 2016). Because lizards are polyphyodont (i.e. replace their teeth

constantly throughout their life time), the δ18O value of a tooth will be unique to when it was

emplaced (Zaher & Rieppel, 1999). Variation in δ18O values within a single ectothermic

individual therefore indicates temperature changes an animal experienced during its lifetime.

Understanding what temperature ranges an animal can tolerate is important for understanding

lizard response in the PETM and in the present.

It is worth noting that Proxestops and related glyptosaurines are common throughout

early Cenozoic deposits (e.g. Gauthier, 1982; Smith, 2011; Gunnell, 2012; Stocker & Kirk,

2016). Though this may appear to reduce the significance of that fossil material, it makes the

specimens more informative from a paleoecological standpoint. Geochemical analyses are

generally destructive and are best suited for common or uninformative materials. Proxestops is

not only common, but has robust dentition, with obvious black enamel caps in most instances

(Fig. 4). This makes tooth-bearing specimens of Proxestops ideal candidates for isotopic

analyses, with larger than average enamel samples being available. In addition, they are often

reported, as was the case with this study, as having been found in association with or in the same

locality as many other taxa (e.g. Gauthier, 1982; Smith, 2011; Gunnell, 2012). Many taxa, such

as amphibians, are not yet able to be isotopically analyzed. Others are too rare or missing jaw

elements to be sampled. Analyzing these glyptosaurins found in association with other taxa can

72

allow us to make ecological inferences for a wide range of groups whose ecology is poorly

constrained.

6. Acknowledgments

This work was completed as part of an MS thesis by MSR, and comments by committee

members Ben Gill, Shuhai Xiao and Sterling Nesbitt, along with the VT Paleobiology and

Geobiology Research Group, greatly increased the quality of this manuscript. We thank UNCG

fieldwork crews, Sterling Nesbitt, and Alan Resetar for access to specimens in their care and for

permission for destructive sampling. We thank the Geological Society of America and Virginia

Tech for providing funding to MSR for analytical work, the Department of Geosciences at

Virginia Tech for providing funding to MRS for specimen procurement, and NSF-BCS 1227329

to RA for funding fieldwork in the GDB. All fossils described here were collected by field crews

under the direction of RA on federal land under BLM Permit 287-WY-PA95. We also thank Ben

Gill for his assistance and guidance with GS-IRMS anaylsis. Andrei Dolocan and Caitlin

Colleary provided skilled assistance using the TOF-SIMS at UT Austin. Mario Bronzati’s and

C.T. Griffin’s assistance with phylogenetic analyses greatly strengthened this paper, and

illustration assistance from Alex Bradley was greatly appreciated.

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84

8. Figures

Figure 1. Geological setting of the Tim’s Confession locality (CM locality #222). Left, map of

Wyoming, with blue square covering the majority of the Wasatch Formation, Sweetwater County

(modified from Bommersbach, 2014:Figure 2); Right, stratigraphic column showing Wasatch

Formation and the surrounding beds (modified from Bommersbach, 2014:Figure 3).

85

86

Figure 2. Restes rugosus material from the Tim’s Confession locality. A-D. Left dentary (GDB

1) in; A. Lingual view; B. Line drawing of lingual view; C. Labial view; D. Line drawing of

labial view; E. Dentaries of YPM 14640 (modified from Bhullar (2011)); F. Lingual view of left

dentary (GDB 5), specimen was sampled isotopically. Scale bar = 3 mm for A.-D. & F.; 1 mm

for E.

87

88

Figure 3. Comparison between Restes rugosus (GDB 1) and Xenosaurus grandis (FMNH

211833). A. GDB 1, dentary in lingual view; B. GDB 1, dentary in ventral view; C. GDB 1,

dentary in labial view; D. FMNH 211833, lower jaw in lingual view; E. FMNH 211833, lower

jaw in labial view.

89

90

Figure 4. A. GDB 2 maxilla in labial view; B. Line drawing of GDB 2 maxilla in labial view; C.

GDB 6-10 osteoderms; D. GDB 3 maxilla in labial view, specimen was sampled isotopically.

Scale bar = 1 cm.

91

92

Figure 5. A. GDB 4 vertebra in anterior view; B. Line drawing of GDB 4 vertebra in anterior

view; C. GDB 4 vertebra in posterior view; D. GDB 4 vertebra in ventral view; E. GDB 4

vertebra in dorsal view. Scale bar = 2 mm.

93

94

Figure 6. Strict consensus tree of Xenosaurus and its relatives, from Bhullar (2011), with GDB 1

dentary having been added. GDB 1, although scored with dentary and dentition characters only

(1, 184-197), was found as a sister taxon to R. rugosus.

95

96

Figure 7. A. SEM image of GDB 3. Square pit in black box represents sample area needed for

SIMS analysis; B. GDB 3, tooth in occlusal view, tooth has been ion-milled to a flat surface; C.

GDB 3, tooth in occlusal view, porous surface represents dentine, solid outer layer represents

enamel; D. GDB 3, tooth in occlusal view (position 1), Ca+ normalized map, illustrating enamel-

dentine boundary; E. GDB 3, tooth in occlusal view (position 2), Ca+ normalized map,

illustrating enamel-dentine contact.

97

98

Figure 8. δ18O data for five extant lizard species and two fossil taxa. Red and blue arrows on the

x-axis indicate predicted plot locations for data based trophic level. Blue diamond = tegu, orange

square = Uromastyx, green plus = chameleon, purple triangle = monitor, teal x = green iguana,

orange line = Proxestops (GDB 3), blue dot = Restes rugosus (GDB 5), green circles = fossil

mammal teeth, brown circles = fossil gar scales. Average for each extant species is signified with

black X.

99

-20.00

-15.00

-10.00

-5.00

0.00

5.00

10.00

15.00

δ18

O (

‰),

VP

DB

δ18O Isotopic Data

Tegu Uromastyx Chameleon Monitor Green Iguana

Glyptosaur R. rugosus Average Fossil Mammal Fossil Gar Scales

100

Figure 9. δ13C data for five extant lizard species and two fossil taxa. Red and blue arrows on the

x-axis indicate predicted plot locations for data based trophic level. Blue diamond = tegu, orange

square = Uromastyx, green plus = chameleon, purple triangle = monitor, teal x = green iguana,

orange line = Proxestops (GDB 3), blue dot = Restes rugosus (GDB 5). Average for each extant

species is signified with black X.

101

δ13C (‰),

VPDB

102

9. Tables

Table 1. All isotopic data of all five specimens of all five extant species, and both fossil taxa.

Boxed data sets represent averages and standard deviations. Data point names are same as what

was used for analysis. Each species occupies three columns, with isotopic data for each specimen

contained to the right in the same row.

103

Tegu

δ13C

δ18O Uromastyx

δ13C

δ18O Chameleon

δ13C δ18O

Monitor

δ13C δ18O

Iguana

δ13C δ18O

Tegu1-BLM

-16.5

8

-4.1

7 1 1 1 1

Tegu1-BLM

-16.9

3

-4.1

0 Uro-1 TR

-9.0

9 10.38 Cham.-1 T

-14.19 -1.76 BRF

-8.9

6 -2.08

Iguana1-BLB

-13.41 -3.15

Tegu1-BLC

-17.0

9

-4.4

0 TL

-9.3

5 10.63

Chameleon 1 - BR

-14.59 -1.49 TRB

-8.6

9 -4.24

Iguana 1 - BLF

-13.79 -2.08

Tegu1-BLC

-16.3

5

-4.6

7 Uromastyx 1 - BR

-11.73

10.90 BL

-14.50 -1.55 TRF

-9.2

7 -3.59 BRB

-14.87 -2.32

Tegu1-BRM

-17.0

2

-4.5

7 BL

-11.53

10.23 Cham-2 BR

-13.67 -1.08 TFB

-9.2

7 -1.98 BRF

-14.17 -2.03

Tegu1-BRM

-16.7

8

-5.5

1 aver

-10.43

10.54 BL

-13.86 -1.65 TLB

-8.9

5 -4.35 TRB

-14.01 -2.37

Tegu1-BRC

-16.7

8

-4.2

9 std 1.0

8 0.2

3 aver

-14.16 -1.51 TLF

-9.1

4 -4.02 TLB

-13.03 -4.33

Tegu1-BRC

-16.3

4

-5.2

5 2 std 0.3

6 0.23 aver

-9.0

5 -3.38 aver

-13.88 -2.71

Tegu1-TLM

-16.7

6

-4.0

8 Uro.-3 TR

-9.6

8 9.2

9 2 std 0.2

0 0.98 std 0.5

8 0.81

Tegu1-TLM

-16.5

6

-5.3

3 Uro.-5 T

-9.1

2 9.2

1 Cham.-2TR

-12.09 -2.56 2 2

Tegu1-TLC

-16.2

1

-4.7

7 TR

-11.09

9.85 TL

-12.10 -1.98

Vara-2 BR

-5.5

7 -3.23

Iguana-2 BL

-16.63 0.66

Tegu1-TLC

-15.9

0

-6.1

9 TL

-11.34

9.20

Cham2.-BRA

-12.55 -1.86 BL

-5.4

2 -3.52

Igu 2-BR

-17.14 0.72

Tegu1-TRM

-16.7

4

-4.1

3 aver

-10.31

9.39 DL

-12.45 -2.48 TR

-5.5

5 -3.12 aver

-16.89 0.69

104

Tegu1-TRM

-17.0

4

-4.3

1 std 0.9

3 0.2

7 aver

-12.30 -2.22 TL

-5.7

5 -3.28 std 0.2

5 0.03

Tegu1-TRC

-16.4

0

-4.3

6 3 std 0.2

0 0.31 aver

-5.5

7 -3.29 3

Tegu1-TRC

-16.3

5

-4.6

7 Uro-3 TR

-13.16

9.81 3 std

0.12 0.15

Iguana-3 BL

-16.78 -0.67

std 0.33 0.5

8 TL

-13.35

9.30 TL

-14.26 -2.93 3 T

-16.70 0.22

aver

-16.6

1

-4.6

8 URO 3-BR

-13.31

8.82 Cham 3-DR

-14.61 -1.63

Vara-3 BR

-7.0

6 -2.07 aver

-16.74 -0.23

2 BL

-13.30

8.27 BL

-14.38 -2.54 BL

-6.9

3 -2.02 std 0.0

4 0.45

Tegu 2 - BLM

-12.9

9

-3.9

9 aver

-13.28

9.05 aver

-14.43 -2.28 aver

-6.9

9 -2.05 4

BLM

-11.2

4

-4.6

5 std 0.0

7 0.5

7 std 0.1

4 0.54 std 0.0

7 0.02

Iguana-4 T

-16.37 1.63

BLC

-11.5

8

-5.3

5 4 4 4

Igu.4-BRB

-16.89 1.27

BLC

-10.0

9

-5.2

9 Uro-4 B

-13.65

6.40 Cham.-4 T

-15.43 -2.39

Vara-4 BR

-6.4

5 -2.92 BL

-16.79 0.95

BRM

-10.7

7

-4.6

1 T

-13.76

8.37 Cham.-4 BL

-15.40 -1.97 BL

-6.3

6 -2.70 aver

-16.68 1.28

BRM -

8.76

-5.8

6 aver

-13.71

7.39

Cham 4 - BRB

-14.68 -2.04 TR

-5.2

5 -3.28 std 0.2

2 0.28

BRC

-11.9

3

-4.6

4 std 0.0

5 0.9

8 aver

-15.17 -2.13 TL

-6.4

3 -2.61 5

BRC -

9.96

-4.8

7 5 std 0.3

5 0.18 aver

-6.1

2 -2.88

Iguana-5 TR

-14.21 1.75

105

std 1.23 0.5

4 Uro-5 TR

-10.96

12.27 std

0.51 0.26 TL

-14.07 1.61

aver

-10.9

2

-4.9

1 TL

-11.63

11.25 5 5

Iguana 5 -BRB

-14.20 2.16

3 URO 5-BR

-11.98

7.23

Vara-5 BR

-9.6

9 -1.26 BRF

-14.12 2.62

Tegu3-BRM

-17.9

2

-3.0

0 BL

-11.63

7.63 Cham 5-BR

-13.59 -1.24 BL

-8.7

0 -1.28 BLB

-14.18 1.18

Tegu3-BRC

-17.5

2

-3.3

4 aver

-11.55

9.59 BL

-13.62 -2.51 TR

-10.42 -1.20 BLF

-14.08 1.97

Tegu3-TLM

-17.4

4

-3.1

1 std 0.3

7 2.2

0 aver

-13.60 -1.87 TL

-10.30 -0.94

aver

-14.14 1.88

Tegu3-TLC

-17.6

7

-3.4

3 average total

-11.65

9.39 std

0.01 0.63 aver

-9.7

8 -1.17 std 0.0

6 0.45

Tegu3-TRM

-17.7

2

-3.5

1 std total 1.5

3 1.4

3 average total

-13.85 -1.95 std

0.68 0.14

average total

-15.02 -0.01

Tegu3-TRC

-17.8

0

-5.1

8 std total 1.0

2 0.50

average total

-7.7

1 -2.68

std total

1.37 2.02

LLM

-17.1

3 2.3

0

std total

1.74 1.02

LLM

-16.9

1

-0.0

4

LLM

-17.5

9

-0.7

9

LTP

-17.5

6

-3.4

0

106

LTP

-16.5

7

-1.1

1

LLM

-16.8

8 0.7

1

LLM

-16.7

4

-2.6

2

std 0.42 2.0

1

aver

-17.3

4

-2.0

4

4

Tegu 4 - TRC

-13.4

1

-1.9

8

TRC

-13.1

4

-2.4

4

TLM

-13.4

1

-2.5

4

TLM

-12.5

8

-2.4

6

TLC

-13.3

4

-2.1

4

TLC

-12.9

8

-2.6

4

Tegu 4 - BRM

-13.1

8

-2.0

6

BRM

-13.4

4

-2.4

5

BRC

-13.2

3

-2.3

3

BRC

-13.3

4

-3.3

3

107

BLM

-14.0

5

-1.9

0

BLM

-13.7

6

-3.0

2

BLC

-13.4

8

-3.2

8

BLC

-13.1

8

-3.1

0

TRM

-13.1

9

-2.7

6

TRM

-13.0

4

-2.3

5

std 0.32 0.4

3

aver

-13.3

0

-2.5

5

5

Tegu-5 BRB

-17.9

9

-2.6

6

BRB

-17.2

9

-2.5

0

BRF

-17.6

9

-2.7

7

BRF

-17.2

3

-2.7

8

BLB

-18.2

1

-2.5

4

BLB

-17.1

9

-2.4

1

Tegu-4-BRB

-17.3

6 0.1

5

108

BRB

-16.6

5

-0.0

5

BRF

-17.6

3 0.3

5

BRF

-16.6

8

-0.3

3

BLB

-17.5

2

-0.1

3

BLB

-16.4

6

-1.0

9

BLF

-17.2

5

-0.6

9

BLF

-16.6

9

-0.1

9

TRB

-17.4

0

-1.4

7

TRB

-16.8

1

-1.7

5

TRF

-17.7

2

-1.0

1

TRF

-16.9

9

-1.3

0

TLB

-17.9

0

-1.9

7

TLB

-16.8

8

-2.2

6

TLF

-17.9

0

-0.9

5

TLF

-16.7

4

-1.6

0

109

std 0.49 1.0

1

aver

-17.2

8

-1.3

6

average total

-15.6

2

-2.8

2

std total 2.31 1.7

6

Fossil Lizrads δ13C δ18O

Fossil Mammals δ13C δ18O Fossil Gar Scales δ13C δ18O

Glypto-1 -7.00 -11.86 0.25 Mammal 1 -7.326999925 -11.35732548 Gar Scale 1 -5.900570892 -15.90973888

Xeno-1 -6.30 -11.81 0.5 Mammal 2 -6.449627643 -10.02735155 Gar Scale 2 -4.878986133 -14.30454816

Mammal 3 -8.10144967 -14.28272153 Gar Scale 3 -5.362621094 -16.31340534

110

Appendix 1.

Character scores for Restes rugosus (GDB 1) from the character matrix of Bhullar (2011); only

dentition and dentary characters (1; 184-197)

Restes rugosus (GDB 1)

2 ? 1 1 0 ? ? ? 1 0 1 0 1 1 0