Page 1
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
Page 2
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.
Page 3
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.
Page 4
iv
For my family, especially my Grandfather, Jerry DeLane:
You instilled a passion for science and supported me every step of the way.
Page 5
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
Page 6
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
Page 7
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.
Page 8
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.
Page 9
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, [email protected]
Page 10
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.
Page 11
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
Page 12
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
Page 13
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
Page 14
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)
Page 15
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
Page 16
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)
Page 17
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
Page 18
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
Page 19
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).
Page 20
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‰.
Page 21
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 &
Page 22
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.
Page 23
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
Page 24
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.
Page 25
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
Page 26
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
Page 27
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
Page 28
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.
Page 29
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
Page 30
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.
7. References
Anderson, C.V. and Van Heygen, E. 2013 On the sympatry of three Trioceros species in a
tropical upland forest in Cameroon. SALAMANDRA. 49, 215-218.
Anemone RL, Dawson MR, Beard KC. 2012 The Early Eocene Rodent Tuscahomys
(Cylindrodontidae) from the Great Divide Basin, Wyoming: Phylogeny, Biogeography,
and Paleoecology. Annals of Carnegie Museum. 80, 187–205.
Ben-David, M. and Flaherty, E.A. 2012 Stable isotopes in mammalian research: a beginner's
guide. Journal of Mammalogy. 93, 312-328.
Bezuijen, M.R., Hartoyo, P., Elliot, M. and Baker, B. 1997 Second report on the ecology of the
False Gharial (Tomistoma schlegelii) in Sumatera. Wildlife Management International
Pty. Ltd.
Bhullar, B.S. 2011 The power and utility of morphological characters in systematics: a fully
resolved phylogeny of Xenosaurus and its fossil relatives (Squamata:
Anguimorpha). Bulletin of the Museum of Comparative Zoology. 160, 65-181.
Burgos-Rodríguez, J.A., Avilés-Rodríguez, K.J. and Kolbe, J.J. 2016 Effects of invasive Green
Iguana iguana (Iguana iguana) on seed germination and seed dispersal potential in
southeastern Puerto Rico. Biological Invasions. 18, 2775-2782.
Page 31
23
Bush, M., Flenley, J. and Gosling, W. eds. 2011 Tropical rainforest responses to climatic change.
Springer Science & Business Media.
Cerling, T.E., Harris, J.M., Ambrose, S.H., Leakey, M.G. and Solounias, N. 1997 Dietary and
environmental reconstruction with stable isotope analyses of herbivore tooth enamel from
the Miocene locality of Fort Ternan, Kenya. Journal of Human Evolution. 33, 635-650.
Cerling, T.E., Harris, J.M. and Passey, B.H. 2003 Diets of East African Bovidae based on stable
isotope analysis. Journal of Mammalogy. 84, 456-470.
Chenery, C.A., Pashley, V., Lamb, A.L., Sloane, H.J. and Evans, J.A. 2012 The oxygen isotope
relationship between the phosphate and structural carbonate fractions of human
bioapatite. Rapid Communications in Mass Spectrometry. 26, 309-319.
Cherel, Y., & Hobson, K. A. 2005 Stable isotopes, beaks and predators: a new tool to study the
trophic ecology of cephalopods, including giant and colossal squids. Proceedings of the
Royal Society of London B: Biological Sciences. 272, 1601-1607.
Colleary, C., Dolocan, A., Gardner, J., Singh, S., Wuttke, M., Rabenstein, R., Habersetzer, J.,
Schaal, S., Feseha, M., Clemens, M. and Jacobs, B.F. 2015 Chemical, experimental, and
morphological evidence for diagenetically altered melanin in exceptionally preserved
fossils. Proceedings of the National Academy of Sciences. 112, 12592-12597.
Cooper, J.S., Poole, D.F.G. and Lawson, R., 1970 The dentition of agamid lizards with special
reference to tooth replacement. Journal of Zoology. 162, 85-98.
Cooper Jr, W.E. and Habegger, J.J. 2001 Responses by juvenile savannah monitor lizards
(Varanus exanthematicus) to chemical cues from animal prey, plants palatable to
herbivores, and conspecifics. Journal of Herpetology. 35, 618-624.
Page 32
24
Costa, G.C., Colli, G.R. and Constantino, R., 2008 Can lizard richness be driven by termite
diversity? Insights from the Brazilian Cerrado. Canadian Journal of Zoology. 86, 1-9.
Craig, H., 1961 Isotopic variations in meteoric waters. Science. 133, 1702-1703.
Cunningham, P.L. 2001 Notes on the diet, survival rate, and burrow specifics of Uromastyx
aegyptius. Asiatic Herpetological Research. 9, 30-33.
Cunningham, P., 2000 Daily activity pattern and diet of a population of the Spinytailed Lizard,
Uromastyx aegyptius microlepis, during summer in the United Arab Emirates. Zoology in
the Middle East. 21, 37-46.
DeConto, R.M., Galeotti, S., Pagani, M., Tracy, D., Schaefer, K., Zhang, T., Pollard, D. and
Beerling, D.J. 2012 Past extreme warming events linked to massive carbon release from
thawing permafrost. Nature. 484, 87.
Densmore III, L.D. and Dessauer, H.C. 1984 Low levels of protein divergence detected between
Gavialis and Tomistoma: Evidence for crocodilian monophyly?. Comparative
Biochemistry and Physiology Part B: Comparative Biochemistry. 77, 715-720.
Dessem, D., 1985 Ontogenetic changes in the dentition and diet of Tupinambis (Lacertilia:
Teiidae). Copeia. 1985, 245-247.
Duckworth, J.W., Batters, G., Belant, J.L., Bennett, E.L., Brunner, J., Burton, J., Challender,
D.W.S., Cowling, V., Duplaix, N., Harris, J.D. and Hedges, S. 2012 Why South-east Asia
should be the world’s priority for averting imminent species extinctions, and a call to join
a developing cross-institutional programme to tackle this urgent issue. SAPI EN. S.
Surveys and Perspectives Integrating Environment and Society, (5.2).
Page 33
25
ElShafie, S. 2014 Body size and species richness changes in Glyptosaurinae (Squamata:
Anguidae) through climatic transitions of the North American Cenozoic. Unpublished
M.S. thesis, University of Nebraska, Lincoln, Nebraska, 97.
Emery, K., L. Wright, and H. Schwarcz. 2000 Isotopic analysis of ancient deer bone: Biotic
stability in collapse period Maya land-use. J. Archaeological Society. 27, 537-550.
Feely, R.A., Sabine, C.L., Lee, K., Berelson, W., Kleypas, J., Fabry, V.J. and Millero, F.J. 2004
Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science. 305, 362-
366.
Gauthier, J.A. 1982 Fossil xenosaurid and anguid lizards from the early Eocene Wasatch
Formation, southeast Wyoming, and a revision of the Anguioidea. Rocky Mountain
Geology. 21, 7-54.
Greenwood, D.R., Wilf, P., Wing, S.L. and Christophel, D.C. 2004 Paleotemperature estimation
using leaf-margin analysis: is Australia different?. Palaios. 19, 129-142.
Harrell, T.L., Pérez-Huerta, A. and Suarez, C.A. 2016 Endothermic mosasaurs? Possible
thermoregulation of Late Cretaceous mosasaurs (Reptilia, Squamata) indicated by stable
oxygen isotopes in fossil bioapatite in comparison with coeval marine fish and pelagic
seabirds. Palaeontology. 59, 351-363.
Harris, D.J., Vaconcelos, R. and Brito, J.C. 2007 Genetic variation within African spiny-tailed
lizards (Agamidae: Uromastyx) estimated using mitochondrial DNA
sequences. Amphibia-Reptilia. 28, 1-6.
Page 34
26
Herrel, A., Damme, R.V., Vanhooydonck, B. and Vree, F.D. 2001 The implications of bite
performance for diet in two species of lacertid lizards. Canadian Journal of Zoology. 79,
662-670.
Hobbie, E.A. and Boyce, C.K. 2010 Carbon sources for the Palaeozoic giant fungus Prototaxites
inferred from modern analogues. Proceedings of the Royal Society of London B:
Biological Sciences, p.rspb20100201.
Hodell, D.A., Quinn, R.L., Brenner, M. and Kamenov, G. 2004 Spatial variation of strontium
isotopes (87Sr/86Sr) in the Maya region: a tool for tracking ancient human
migration. Journal of Archaeological Science. 31, 585-601.
Hoppe, K.A., Koch, P.L., Carlson, R.W. and Webb, S.D. 1999 Tracking mammoths and
mastodons: reconstruction of migratory behavior using strontium isotope
ratios. Geology. 27, 439-442.
Hoskin, P. 1998 Minor and trace element analysis of natural zircon (ZrSiO4) by SIMS and laser
ablation ICPMS: a consideration and comparison of two broadly competitive
techniques. Journal of Trace Microprobe Tech. 16, 301-326.
Hughes, L. 2000 Biological consequences of global warming: is the signal already
apparent?. Trends in Ecology & Evolution. 15, 56-61.
Huey, R.B., Deutsch, C.A., Tewksbury, J.J., Vitt, L.J., Hertz, P.E., Pérez, H.J.Á. and Garland, T.
2009 Why tropical forest lizards are vulnerable to climate warming. Proceedings of the
Royal Society of London B: Biological Sciences. 276, 1939-1948.
Huey, R.B. and Tewksbury, J.J. 2009 Can behavior douse the fire of climate
warming?. Proceedings of the National Academy of Sciences. 106, 3647-3648.
Page 35
27
Jaramillo, C., Ochoa, D., Contreras, L., Pagani, M., Carvajal-Ortiz, H., Pratt, L.M., Krishnan, S.,
Cardona, A., Romero, M., Quiroz, L. and Rodriguez, G. 2010 Effects of rapid global
warming at the Paleocene-Eocene boundary on neotropical vegetation. Science. 330, 957-
961.
Kelly, J.F. 2000 Stable isotopes of carbon and nitrogen in the study of avian and mammalian
trophic ecology. Canadian Journal of Zoology. 78, 1-27.
Koch, P.L., Zachos, J.C. and Dettman, D.L. 1995 Stable isotope stratigraphy and
paleoclimatology of the Paleogene Bighorn Basin (Wyoming, USA). Palaeogeography,
Palaeoclimatology, Palaeoecology. 115, 61-89.
Kohn, M., M. Schoeninger, and W. Barker 1999 Altered states: effects of diagenesis on fossil
tooth chemistry. Geochim. et Cosmochim. Acta. 63, 2737-2747.
Kraus, M.J. and Riggins, S. 2007 Transient drying during the Paleocene–Eocene Thermal
Maximum (PETM): analysis of paleosols in the Bighorn Basin,
Wyoming. Palaeogeography, Palaeoclimatology, Palaeoecology. 245, 444-461.
Lauder, G.V. 1981 Form and function: structural analysis in evolutionary
morphology. Paleobiology. 7, 430-442.
Logan, M.L., Huynh, R.K., Precious, R.A. and Calsbeek, R.G. 2013 The impact of climate
change measured at relevant spatial scales: new hope for tropical lizards. Global Change
Biology. 19, 3093-3102.
Longrich, N.R., Bhullar, B.A.S., and Gauthier, J.A. 2012 Mass extinction of lizards and snakes at
the Cretaceous–Paleogene boundary. Proceedings of the National Academy of
Sciences. 109, 21396-21401.
Page 36
28
Luz, B. and Kolodny, Y. 1985 Oxygen isotope variations in phosphate of biogenic apatites, IV.
Mammal teeth and bones. Earth and Planetary Science Letters. 75, 29-36.
Lyons, J.A. and Natusch, D.J. 2011 Wildlife laundering through breeding farms: illegal harvest,
population declines and a means of regulating the trade of green pythons (Morelia
viridis) from Indonesia. Biological Conservation. 144, 3073-3081.
McRae, L., Deinet, S. and Freeman, R. 2017 The diversity-weighted living planet index:
controlling for taxonomic bias in a global biodiversity indicator. PloS One. 12,
p.e0169156.
Measey, G.J., Rebelo, A.D., Herrel, A., Vanhooydonck, B. and Tolley, K.A. 2011 Diet,
morphology and performance in two chameleon morphs: do harder bites equate with
harder prey?. Journal of Zoology. 285, 247-255.
Melstrom, K.M. 2017 The relationship between diet and tooth complexity in living dentigerous
saurians. Journal of Morphology. 278, 500-522.
Muñoz, M.M., Langham, G.M., Brandley, M.C., Rosauer, D.F., Williams, S.E. and Moritz, C.
2016 Basking behavior predicts the evolution of heat tolerance in Australian rainforest
lizards. Evolution. 70, 2537-2549.
Muñoz, M.M. and Moritz, C. 2016 Adaptation to a changing world: evolutionary resilience to
climate change. How Evolution Shapes Our Lives: Essays on Biology and Society. 238.
Nanci, A., 2017. Ten Cate's Oral Histology-E-Book: Development, Structure, and Function.
Elsevier Health Sciences.
Page 37
29
Narbonne, G.M., Laflamme, M., Trusler, P.W., Dalrymple, R.W. and Greentree, C. 2014 Deep-
water Ediacaran fossils from northwestern Canada: taphonomy, ecology, and
evolution. Journal of Paleontology. 88, 207-223.
O'Connell, T.C. and Hedges, R.E.M. 2017 Chicken and egg: testing the carbon isotopic effects of
carnivory and herbivory. Archaeometry. 59, 302-315.
Parrish, J.M., Parrish, J.T., Hutchison, J.H. and Spicer, R.A. 1987 Late Cretaceous vertebrate
fossils from the North Slope of Alaska and implications for dinosaur ecology. Palaios.
377-389.
Passey, B. and T. Cerling. 2006 In situ stable isotope analysis (δ13C, δ18O) of very small teeth
using laser ablation GC/IRMS. Chemical Geology. 235, 238-249.
Pernas, T., Giardina, D.J., McKinley, A., Parns, A. and Mazzotti, F.J. 2012 First observations of
nesting by the Argentine black and white tegu, Tupinambis merianae, in south
Florida. Southeastern Naturalist. 11, 765-770.
Pianka, E.R. 1973 The structure of lizard communities. Annual Review of Ecology and
Systematics. 4, 53-74.
Pomar, L. 2001 Ecological control of sedimentary accommodation: evolution from a carbonate
ramp to rimmed shelf, Upper Miocene, Balearic Islands. Palaeogeography,
Palaeoclimatology, Palaeoecology. 175, 249-272.
Price, T.D., Knipper, C., Grupe, G. and Smrcka, V. 2004 Strontium isotopes and prehistoric
human migration: the Bell Beaker period in central Europe. European Journal of
Archaeology. 7, 9-40.
Page 38
30
Prince, P.A. 1980 The food and feeding ecology of grey‐headed albatross Diomedea
chrysostoma and black‐browed albatross D. melanophris. Ibis. 122, 476-488.
Rage, J.C. 2012 Amphibians and squamates in the Eocene of Europe: what do they tell
us?. Palaeobiodiversity and Palaeoenvironments. 92, 445-457.
Rand, A.S., Dugan, B.A., Monteza, H. and Vianda, D. 1990 The diet of a generalized folivore:
Iguana iguana in Panama. Journal of Herpetology. 24, 211-214.
Roche D., Segalen L., Balan E. and Delattre S 2010 Preservation assessment of Miocene-
Pliocene tooth enamel from Tugen Hills (Kenyan Rift Valley) through FTIR, chemical
and stable-isotope analyses. Journal of Archaeological Science. 37, 1690–1699.
Röhl, U., Westerhold, T., Bralower, T.J. and Zachos, J.C. 2007 On the duration of the Paleocene-
Eocene thermal maximum (PETM). Geochemistry, Geophysics, Geosystems. 8.
Rountrey, A.N., Fisher, D.C., Vartanyan, S. and Fox, D.L. 2007 Carbon and nitrogen isotope
analyses of a juvenile woolly mammoth tusk: evidence of weaning. Quaternary
International. 169, 166-173.
Rosenzweig, C., Karoly, D., Vicarelli, M., Neofotis, P., Wu, Q., Casassa, G., Menzel, A., Root,
T.L., Estrella, N., Seguin, B. and Tryjanowski, P. 2008 Attributing physical and
biological impacts to anthropogenic climate change. Nature. 453, 353.
Seimon, T.A., Seimon, A., Daszak, P., Halloy, S.R., Schloegel, L.M., Aguilar, C.A., Sowell, P.,
Hyatt, A.D., Konecky, B. and E SIMMONS, J.O.H.N. 2007 Upward range extension of
Andean anurans and chytridiomycosis to extreme elevations in response to tropical
deglaciation. Global Change Biology. 13, 288-299.
Page 39
31
Sinervo, B., Mendez-De-La-Cruz, F., Miles, D.B., Heulin, B., Bastiaans, E., Villagrán-Santa
Cruz, M., Lara-Resendiz, R., Martínez-Méndez, N., Calderón-Espinosa, M.L., Meza-
Lázaro, R.N. and Gadsden, H. 2010 Erosion of lizard diversity by climate change and
altered thermal niches. Science. 328, 894-899.
Singh, A. 2015 Save the Gharials. ScienceIndia, 33-35.
Solomon, S., Plattner, G.K., Knutti, R. and Friedlingstein, P. 2009 Irreversible climate change
due to carbon dioxide emissions. Proceedings of the national academy of sciences. 106,
1704-1709.
Sponheimer, M., Lee-Thorp, J.A., DeRuiter, D.J., Smith, J.M., Van Der Merwe, N.J., Reed, K.,
Grant, C.C., Ayliffe, L.K., Robinson, T.F., Heidelberger, C. and Marcus, W. 2003 Diets
of southern African Bovidae: stable isotope evidence. Journal of Mammalogy. 84, 471-
479.
Sprackland, R.G. 2012 Savannah and Grassland Varanus exanthematicus: From the Experts at
Advanced Vivarium Systems. i5 Publishing.
Suarez, C.A., González, L.A., Ludvigson, G.A., Cifelli, R.L. and Tremain, E. 2012 Water
utilization of the Cretaceous Mussentuchit Member local vertebrate fauna, Cedar
Mountain Formation, Utah, USA: using oxygen isotopic composition of
phosphate. Palaeogeography, Palaeoclimatology, Palaeoecology. 313, 78-92.
Warchol, G.L. 2004 The transnational illegal wildlife trade. Criminal justice studies. 17, 57-73.
Watkins, J.K., Blatt, S.H., Bradbury, C.A., Alanko, G.A., Kohn, M.J., Lytle, M.L., Taylor, J.,
Lacroix, D., Nieves-Colón, M.A., Stone, A.C. and Butt, D.P. 2017 Determining the
Page 40
32
population affinity of an unprovenienced human skull for repatriation. Journal of
Archaeological Science: Reports. 12, 384-394.
Webb, J.K. and Whiting, M.J. 2005 Why don't small snakes bask? Juvenile broad-headed snakes
trade thermal benefits for safety. Oikos. 110, 515-522.
Wilf, P. 1997 When are leaves good thermometers? A new case for leaf margin
analysis. Paleobiology. 23, 373-390.
Wheatley, P.V., Peckham, H., Newsome, S.D. and Koch, P.L. 2012 Estimating marine resource
use by the American crocodile Crocodylus acutus in southern Florida, USA. Marine
Ecology Progress Series. 447, 211-229.
Zachos, J., Pagani, M., Sloan, L., Thomas, E. and Billups, K. 2001 Trends, rhythms, and
aberrations in global climate 65 Ma to present. Science. 292, 686-693.
Zachos, J.C., Shackleton, N.J., Revenaugh, J.S., Pälike, H. and Flower, B.P. 2001 Climate
response to orbital forcing across the Oligocene-Miocene boundary. Science. 292, 274-
278.
Zachos, J.C., Dickens, G.R. and Zeebe, R.E. 2008 An early Cenozoic perspective on greenhouse
warming and carbon-cycle dynamics. Nature. 451, 279.
Zaher, H. and Rieppel, O. 1999 Tooth implantation and replacement in squamates, with special
reference to mosasaur lizards and snakes. American Museum Novitates; no. 3271.
Page 41
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.
Page 43
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.
Page 44
36
δ13C (‰), VPDB
Page 45
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.
Page 47
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.
Page 49
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.
Page 51
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.
Page 53
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.
Page 54
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
Page 55
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
Page 56
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
Page 57
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
Page 58
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
Page 59
51
std total 1.74 1.02
Page 60
52
Chapter 2
DIVERSITY AND TROPHIC STRUCTURE OF AN EARLY EOCENE HERPETOFAUNA
FROM WYOMING
Mitchell S. Riegler, Department of Geosciences, Virginia Tech, [email protected]
Page 61
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.
Page 62
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
Page 63
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
Page 64
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
Page 65
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
Page 66
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.
Page 67
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
Page 68
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
Page 69
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
Page 70
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
Page 71
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
Page 72
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
Page 73
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
Page 74
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
Page 75
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.
Page 76
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
Page 77
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.,
Page 78
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
Page 79
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
Page 80
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.
7. References
Alvarez, L.W., Alvarez, W., Asaro, F. and Michel, H.V. 1980 Extraterrestrial cause for the
Cretaceous-Tertiary extinction. Science. 208, 1095-1108.
Anemone, R.L. and Dirks, W. 2009 An anachronistic mammal fauna from the Paleocene Fort
Union Formation (Great Divide Basin, Wyoming, USA). Geological Acta. 7, 113-124.
Page 81
73
Anemone, R.L., Dawson, M.R., and Beard, K.C. 2012 The Early Eocene Rodent Tuscahomys
(Cylindrodontidae) from the Great Divide Basin, Wyoming: Phylogeny, Biogeography,
and Paleoecology. Annals of Carnegie Museum. 80, 187-205.
Bartels, W.S. 1983 A transitional Paleocene-Eocene reptile fauna from the Bighorn Basin,
Wyoming. Herpetologica. 39, 359-374.
Beard, K.C. 2008 The oldest North American primate and mammalian biogeography during the
Paleocene–Eocene Thermal Maximum. Proceedings of the National Academy of
Sciences. 105, 3815-3818.
Ben-David, M. and Flaherty, E.A. 2012 Stable isotopes in mammalian research: a beginner's
guide. Journal of Mammalogy. 93, 312-328.
Bhullar, B.S. 2011 The power and utility of morphological characters in systematics: a fully
resolved phylogeny of Xenosaurus and its fossil relatives (Squamata:
Anguimorpha). Bulletin of the Museum of Comparative Zoology. 160, 65-181.
Bommersbach, B. 2014 Predictive Modeling in the Search for Vertebrate Fossils: Geographic
Object Based Image Analysis (GEOBIA) in the Eocene of Wyoming. Unpublished
Master’s Thesis, Western Michigan University, Kalamazoo, Michigan. 131 pp.
Bowen, G.J., Maibauer, B.J., Kraus, M.J., Röhl, U., Westerhold, T., Steimke, A., Gingerich,
P.D., Wing, S.L., and Clyde, W.C. 2015 Two massive, rapid releases of carbon during
the onset of the Palaeocene–Eocene thermal maximum. Nature Geoscience. 8, 44.
Bush, M., Flenley, J. and Gosling, W. eds. 2011 Tropical Rainforest Responses to Climatic
Change. Springer Science & Business Media.
Butler, P.M. 2003 Ontogenetic aspects of dental evolution. International Journal of
Developmental Biology. 39, 25-34.
Page 82
74
Cerling, T.E., Harris, J.M., Ambrose, S.H., Leakey, M.G., and Solounias, N. 1997 Dietary and
environmental reconstruction with stable isotope analyses of herbivore tooth enamel from
the Miocene locality of Fort Ternan, Kenya. Journal of Human Evolution. 33, 635-650.
Cerling, T.E., Harris, J.M., and Passey, B.H. 2003 Diets of East African Bovidae based on stable
isotope analysis. Journal of Mammalogy. 84, 456-470.
Chenery, C.A., Pashley, V., Lamb, A.L., Sloane, H.J., and Evans, J.A. 2012 The oxygen isotope
relationship between the phosphate and structural carbonate fractions of human
bioapatite. Rapid Communications in Mass Spectrometry. 26, 309-319.
Cherel, Y. and Hobson, K.A. 2005 Stable isotopes, beaks and predators: a new tool to study the
trophic ecology of cephalopods, including giant and colossal squids. Proceedings of the
Royal Society of London B: Biological Sciences. 272, 1601-1607.
Christin, P.A. and Osborne, C.P. 2014. The evolutionary ecology of C4 plants. New
Phytologist. 204, 765-781.
Cohen, A., Coe, A., and Kemp, D. 2007 The Late Palaeocene–Early Eocene and Toarcian (Early
Jurassic) carbon isotope excursions: a comparison of their time scales, associated
environmental changes, causes and consequences. Journal of Geological Society. 164,
1093-1108.
Colleary, C., Dolocan, A., Gardner, J., Singh, S., Wuttke, M., Rabenstein, R., Habersetzer, J.,
Schaal, S., Feseha, M., Clemens, M., and Jacobs, B.F. 2015 Chemical, experimental, and
morphological evidence for diagenetically altered melanin in exceptionally preserved
fossils. Proceedings of the National Academy of Sciences. 112, 12592-12597.
Conrad, J.L., Ast, J.C., Montanari, S., and Norell, M.A. 2011 A combined evidence phylogenetic
analysis of Anguimorpha (Reptilia: Squamata). Cladistics. 27, 230-277.
Page 83
75
Cope, E.D. 1900 The crocodilians, squamates, and snakes of North America. Annual Report of
the United States National Museum, Smithsonian. 155-1294
Costa, G.C., Colli, G.R., and Constantino, R. 2008 Can lizard richness be driven by termite
diversity? Insights from the Brazilian Cerrado. Canadian Journal of Zoology. 86, 1-9.
Craig, H., 1961. Isotopic variations in meteoric waters. Science. 133, 1702-1703.
Cunningham, P.L., 2001 Notes on the diet, survival rate, and burrow specifics of Uromastyx
aegyptius. Asiatic Herpetological Research. 9, 30-33.
DeConto, R.M., Galeotti, S., Pagani, M., Tracy, D., Schaefer, K., Zhang, T., Pollard, D., and
Beerling, D.J. 2012 Past extreme warming events linked to massive carbon release from
thawing permafrost. Nature. 484, 87.
Dessem, D. 1985 Ontogenetic changes in the dentition and diet of Tupinambis (Lacertilia:
Teiidae). Copeia. 1985, 245-247.
Duellman, J.H., Hallermann, J., Huey, R.B., and Kienle, M.S. 2000 How many reptile species?.
Herpetological Review. 31, 13-15
ElShafie, S. 2014 Body size and species richness changes in Glyptosaurinae (Squamata:
Anguidae) through climatic transitions of the North American Cenozoic. Unpublished
M.S. thesis, University of Nebraska, Lincoln, Nebraska, 97 pp.
Emery, K., Wright, L., and Schwarcz, H. 2000 Isotopic analysis of ancient deer bone: Biotic
stability in collapse period Maya land-use. Journal of Archaeological Society. 27, 537-
550.
Estes, R. 1965 Notes on some Paleocene lizards. Copeia. 1965, 104-106.
Estes, R. and Williams, E.E. 1984 Ontogenetic variation in the molariform teeth of
lizards. Journal of Vertebrate Paleontology. 4, 96-107.
Page 84
76
Evans, S.E. 2008 The skull of lizards and tuatara. Biology of the Reptilia. 20, 1-347.
Fricke, H.C. and Wing, S.L. 2004 Oxygen isotope and paleobotanical estimates of temperature
and δ18O–latitude gradients over North America during the early Eocene. American
Journal of Science. 304, 612-635.
Gauthier, J.A. 1982 Fossil xenosaurid and anguid lizards from the early Eocene Wasatch
Formation, southeast Wyoming, and a revision of the Anguioidea. Rocky Mountain
Geology. 21, 7-54.
Gauthier, J.A., Kearney, M., Maisano, J.A., Rieppel, O. and Behlke, A.D. 2012 Assembling the
squamate tree of life: perspectives from the phenotype and the fossil record. Bulletin of
the Peabody Museum of Natural History. 53, 3-308.
Gilmore, C.W. 1942 Paleocene faunas of the Polecat Bench Formation, Park County, Wyoming
Part II. Squamates. Proceedings of the American Philosophical Society. 159-167.
Gingerich, P.D. 2006 Environment and evolution through the Paleocene–Eocene thermal
maximum. Trends in Ecology & Evolution. 21, 246-253.
Goloboff, P., Farris, S. and Nixon, K. 2001 TNT (Tree analysis using New Technology).
Greenwood, D.R., Wilf, P., Wing, S.L., and Christophel, D.C. 2004 Paleotemperature estimation
using leaf-margin analysis: is Australia different?. Palaios. 19, 129-142.
Gunnell, G.F. ed. 2012 Eocene Biodiversity: Unusual Occurrences and Rarely Sampled
Habitats. 18. Springer Science & Business Media, 458.
Harrell, T.L., Pérez-Huerta, A., and Suarez, C.A. 2016 Endothermic mosasaurs? Possible
thermoregulation of Late Cretaceous mosasaurs (Reptilia, Squamata) indicated by stable
oxygen isotopes in fossil bioapatite in comparison with coeval marine fish and pelagic
seabirds. Palaeontology. 59, 351-363.
Page 85
77
Head, J.J. 2002 Snake paleontology of the Siwalik Group (Miocene of Pakistan): correlation of a
rich fossil record to environmental histories. Unpublished Ph.D. dissertation, Southern
Methodist University, Dallas, Texas, 290 pp.
Herrel, A., Damme, R.V., Vanhooydonck, B., and Vree, F.D. 2001 The implications of bite
performance for diet in two species of lacertid lizards. Canadian Journal of Zoology. 79,
662-670.
Hoskin, P. 1998 Minor and trace element analysis of natural zircon (ZrSiO4) by SIMS and laser
ablation ICPMS: a consideration and comparison of two broadly competitive
techniques. Journal of Trace Microprobe Tech. 16, 301-326.
Hughes, L. 2000 Biological consequences of global warming: is the signal already
apparent?. Trends in Ecology & Evolution. 15, 56-61.
Huey, R.B., Deutsch, C.A., Tewksbury, J.J., Vitt, L.J., Hertz, P.E., Pérez, H.J.Á., and Garland, T.
2009 Why tropical forest lizards are vulnerable to climate warming. Proceedings of the
Royal Society of London B: Biological Sciences. 276, 1939-1948.
Huey, R.B. and Tewksbury, J.J. 2009 Can behavior douse the fire of climate
warming?. Proceedings of the National Academy of Sciences. 106, 3647-3648.
Kelly, J.F. 2000 Stable isotopes of carbon and nitrogen in the study of avian and mammalian
trophic ecology. Canadian Journal of Zoology. 78, 1-27.
Koch, P.L., Zachos, J.C., and Dettman, D.L. 1995 Stable isotope stratigraphy and
paleoclimatology of the Paleogene Bighorn Basin (Wyoming, USA). Palaeogeography,
Palaeoclimatology, Palaeoecology. 115, 61-89.
Kohn, M., Schoeninger, M., and Barker, W. 1999 Altered states: effects of diagenesis on fossil
tooth chemistry. Geochimcal et Cosmochimcal Acta. 63, 2737-2747.
Page 86
78
Kraus, M.J. and Riggins, S. 2007 Transient drying during the Paleocene–Eocene Thermal
Maximum (PETM): analysis of paleosols in the Bighorn Basin,
Wyoming. Palaeogeography, Palaeoclimatology, Palaeoecology. 245, 444-461.
Lu, L.M., Mao, L.F., Yang, T., Ye, J.F., Liu, B., Li, H.L., Sun, M., Miller, J.T., Mathews, S., Hu,
H.H. and Niu, Y.T. 2018 Evolutionary history of the angiosperm flora of
China. Nature, 554, 234.
Luz, B. and Kolodny, Y. 1985 Oxygen isotope variations in phosphate of biogenic apatites, IV.
Mammal teeth and bones. Earth and Planetary Science Letters. 75, 29-36.
Maddison, W.P. and Maddison, D.R. 2008 Mesquite: a modular system for evolutionary
analysis. Version 2.0, 2008. URL http://mesquiteproject.org.
McDowell, S.B. and Bogert, C.M. 1954 The systematic position of Lanthanotus and the
affinities of the anguimorphan squamates. Bulletin of the American Museum of Natural
History. 105, article 1.
McInerney, F.A. and Wing, S.L. 2011 The Paleocene-Eocene Thermal Maximum: A
perturbation of carbon cycle, climate, and biosphere with implications for the
future. Annual Review of Earth and Planetary Sciences. 39, 489-516.
Measey, G.J., Rebelo, A.D., Herrel, A., Vanhooydonck, B., and Tolley, K.A. 2011 Diet,
morphology and performance in two chameleon morphs: do harder bites equate with
harder prey?. Journal of Zoology. 285, 247-255.
Melstrom, K.M. 2017 The relationship between diet and tooth complexity in living dentigerous
saurians. Journal of Morphology. 278, 500-522.
Montanucci, R.R. 1968 Comparative dentition in four iguanid lizards. Herpetologica. 24, 305-
315.
Page 87
79
Muñoz, M.M., Langham, G.M., Brandley, M.C., Rosauer, D.F., Williams, S.E. and Moritz, C.
2016 Basking behavior predicts the evolution of heat tolerance in Australian rainforest
lizards. Evolution. 70, 2537-2549.
Muñoz, M.M. and Moritz, C. 2016 Adaptation to a changing world: evolutionary resilience to
climate change. How Evolution Shapes Our Lives: Essays on Biology and Society, 238.
Passey, B. and Cerling, T. 2006 In situ stable isotope analysis (δ13C, δ18O) of very small teeth
using laser ablation GC/IRMS. Chemical Geology. 235, 238-249.
Nanci, A. 2017 Ten Cate's Oral Histology-E-Book: Development, Structure, and Function.
Elsevier Health Sciences.
O'Connell, T.C. and Hedges, R.E.M. 2017 Chicken and egg: testing the carbon isotopic effects of
carnivory and herbivory. Archaeometry. 59, 302-315.
Pianka, E.R. 1973 The structure of lizard communities. Annual Review of Ecology and
Systematics. 4, 53-74.
Pipiringos, G.N. 1955 Tertiary rocks in the central part of the Great Divide Basin, Sweetwater
County, Wyoming. Green River Basin; 10th Annual Field Conference Guidebook.
100-104.
Pipiringos, G.N. 1962 Uranium-bearing coal in the central part of the Great Divide Basin.
Geological Survey Bulletin. 1099A, 1-103.
Pomar, L. 2001 Ecological control of sedimentary accommodation: evolution from a carbonate
ramp to rimmed shelf, Upper Miocene, Balearic Islands. Palaeogeography,
Palaeoclimatology, Palaeoecology. 175, 249-272.
Page 88
80
Price, T.D., Knipper, C., Grupe, G., and Smrcka, V. 2004 Strontium isotopes and prehistoric
human migration: the Bell Beaker period in central Europe. European Journal of
Archaeology. 7, 9-40.
Rage, J.C. and Werner, C. 1999 Mid-Cretaceous (Cenomanian) snakes from Wadi Abu Hashim,
Sudan: the earliest snake assemblage. Paleontology of Africa. 35, 85-110.
Rage, J.C. 2012 Amphibians and squamates in the Eocene of Europe: what do they tell
us?. Palaeobiodiversity and Palaeoenvironments. 92, 445-457.
Riegler, M., Gill, B.C., Stocker, M.R. 2018 Stable isotope ratios accurately delineate trophic
structure and aridity in extant squamates: Implications for ecology and paleobiology.
Thesis Ch.1.
Roche D., Segalen L., Balan E., and Delattre S. 2010 Preservation assessment of Miocene-
Pliocene tooth enamel from Tugen Hills (Kenyan Rift Valley) through FTIR, chemical
and stable-isotope analyses. Journal of Archaeological Science. 37, 1690–1699.
Röhl, U., Westerhold, T., Bralower, T.J., and Zachos, J.C. 2007 On the duration of the
Paleocene-Eocene thermal maximum (PETM). Geochemistry, Geophysics,
Geosystems, 8, 1-13.
Rountrey, A.N., Fisher, D.C., Vartanyan, S. and Fox, D.L. 2007 Carbon and nitrogen isotope
analyses of a juvenile woolly mammoth tusk: evidence of weaning. Quaternary
International. 169, 66-173.
Savage, D.E. 1975 Cenozoic-the primate episode. Contributions to Primatology. 5, 2.
Seimon, T.A., Seimon, A., Daszak, P., Halloy, S.R., Schloegel, L.M., Aguilar, C.A., Sowell, P.,
Hyatt, A.D., Konecky, B., and Simmons, J. 2007 Upward range extension of Andean
Page 89
81
anurans and chytridiomycosis to extreme elevations in response to tropical
deglaciation. Global Change Biology. 13, 288-299.
Sinervo, B., Mendez-De-La-Cruz, F., Miles, D.B., Heulin, B., Bastiaans, E., Villagrán-Santa
Cruz, M., Lara-Resendiz, R., Martínez-Méndez, N., Calderón-Espinosa, M.L., Meza-
Lázaro, R.N. and Gadsden, H. 2010 Erosion of lizard diversity by climate change and
altered thermal niches. Science. 328, 894-899.
Sluijs, A., Schouten, S., Donders, T.H., Schoon, P.L., Röhl, U., Reichart, G.J., Sangiorgi, F.,
Kim, J.H., Damsté, J.S.S., and Brinkhuis, H. 2009 Warm and wet conditions in the Arctic
region during Eocene Thermal Maximum 2. Nature Geoscience. 2, 777.
Smith, K.T. 2006 Horizontal and vertical aspects of species diversity in the fossil record: alpha,
beta, and the temporal nature of the richness-temperature relation. Unpublished Ph.D.
dissertation, Yale University, New Haven, Connecticut, 606 pp.
Smith, T., Rose, K.D. and Gingerich, P.D. 2006 Rapid Asia–Europe–North America geographic
dispersal of earliest Eocene primate Teilhardina during the Paleocene–Eocene thermal
maximum. Proceedings of the National Academy of Sciences, 103, 11223-11227.
Smith, K.T. 2011 The long-term history of dispersal among lizards in the early Eocene: new
evidence from a microvertebrate assemblage in the Bighorn Basin of Wyoming,
USA. Palaeontology. 54, 1243-1270.
Smith, K.T. and Gauthier, J.A. 2013 Early Eocene squamates of the Wasatch Formation near
Bitter Creek, Wyoming: diversity and paleoenvironment during an interval of global
warming. Bulletin of the Peabody Museum of Natural History. 54, 135-230.
Sponheimer, M., Lee-Thorp, J.A., DeRuiter, D.J., Smith, J.M., Van Der Merwe, N.J., Reed, K.,
Grant, C.C., Ayliffe, L.K., Robinson, T.F., Heidelberger, C., and Marcus, W. 2003 Diets
Page 90
82
of southern African Bovidae: stable isotope evidence. Journal of Mammalogy. 84, 471-
479.
Stocker, M.R. and Kirk, E.C. 2016 The first amphisbaenians from Texas, with notes on other
squamates from the middle Eocene Purple Bench locality. Journal of Vertebrate
Paleontology. 36, p.e1094081.
Suarez, C.A., González, L.A., Ludvigson, G.A., Cifelli, R.L., and Tremain, E. 2012 Water
utilization of the Cretaceous Mussentuchit Member local vertebrate fauna, Cedar
Mountain Formation, Utah, USA: using oxygen isotopic composition of
phosphate. Palaeogeography, Palaeoclimatology, Palaeoecology. 313, 78-92.
Sullivan, R.M. 1991 Paleocene Caudata and Squamata from Gidley and Silberling quarries,
Montana. Journal of Vertebrate Paleontology. 11, 293-297.
Swofford, D.L. 2003 PAUP*: phylogenetic analysis using parsimony, version 4.0b10.
Watkins, J.K., Blatt, S.H., Bradbury, C.A., Alanko, G.A., Kohn, M.J., Lytle, M.L., Taylor, J.,
Lacroix, D., Nieves-Colón, M.A., Stone, A.C., and Butt, D.P. 2017 Determining the
population affinity of an unprovenienced human skull for repatriation. Journal of
Archaeological Science: Reports. 12, 384-394.
Page 91
83
Webb, J.K. and Whiting, M.J. 2005 Why don't small snakes bask? Juvenile broad-headed snakes
trade thermal benefits for safety. Oikos. 110, 515-522.
Wheatley, P.V., Peckham, H., Newsome, S.D. and Koch, P.L. 2012 Estimating marine resource
use by the American crocodile Crocodylus acutus in southern Florida, USA. Marine
Ecology Progress Series. 447, 211-229.
Wilf, P. 1997 When are leaves good thermometers? A new case for leaf margin
analysis. Paleobiology. 23, 373-390.
Woodburne, M.O., Gunnell, G.F., and Stucky, R.K. 2009 Climate directly influences Eocene
mammal faunal dynamics in North America. Proceedings of the National Academy of
Sciences. 106, 13399-13403.
Zachos, J., Pagani, M., Sloan, L., Thomas, E., and Billups, K. 2001 Trends, rhythms, and
aberrations in global climate 65 Ma to present. Science. 292, 686-693.
Zachos, J.C., Shackleton, N.J., Revenaugh, J.S., Pälike, H., and Flower, B.P. 2001 Climate
response to orbital forcing across the Oligocene-Miocene boundary. Science. 292, 274-
278.
Zachos, J.C., Dickens, G.R., and Zeebe, R.E. 2008 An early Cenozoic perspective on greenhouse
warming and carbon-cycle dynamics. Nature. 451, 279.
Zaher, H. and Rieppel, O. 1999 Tooth implantation and replacement in squamates, with special
reference to mosasaur lizards and snakes. American Museum Novitates. no. 3271, 1-20.
Zahradnicek, O., Buchtova, M., Dosedelova, H. and Tucker, A.S. 2014 The development of
complex tooth shape in reptiles. Frontiers in Physiology. 5, 74.
Page 92
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).
Page 94
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.
Page 96
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.
Page 98
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.
Page 100
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.
Page 102
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.
Page 104
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.
Page 106
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.
Page 107
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
Page 108
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.
Page 109
101
δ13C (‰),
VPDB
Page 110
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.
Page 111
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
Page 112
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
Page 113
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
Page 114
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
Page 115
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
Page 116
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
Page 117
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
Page 118
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