STABLE ISOTOPE PALEOECOLOGY OF AN EARLY MIOCENE EQUID (PARAHIPPUS LEONENSIS) FROM THE THOMAS FARM SITE, GILCHRIST

COUNTY, FLORIDA

By

SEAN MICHAEL MORAN

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2014

© 2014 Sean Michael Moran

To Mom, whose constant bravery and strength during her fight against cancer has been a constant source of inspiration

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ACKNOWLEDGMENTS

First and foremost, I would like to thank my advisor, Bruce MacFadden, for

providing superb guidance of the project along the way. He always pushed me to look at

things in a different light when the data appeared utterly uninteresting. I also thank my

committee, Jonathan Bloch and Andrea Dutton, who provided much needed, valuable

input over the last couple of years. The Geological Society of America, National Science

Foundation (CSBR 1203222, PIRE 0966884), University of Florida Department of

Geological Sciences, and the Southwest Florida Fossil Club provided funding

throughout the duration of this project and for that I am grateful. For those listed below,

who provided assistance, whether lending an ear to listen to various ideas, providing

useful suggestions, critiquing the final product, or assisting with sampling and sample

selection: Elliot Arnold, Jason Bourque, Mark Brenner, Jason Curtis, Amanda Friend,

Pam Haines, Richard Hulbert, Michal Kowalewski, Carly Manz, Ellen Martin, Candace

McCaffery, Paul Morse, Irv Quitmyer, Dave Steadman, Chanika Symister, Julia Tejada,

Lane Wallet, Evan Whiting, Aaron Wood, and my family. This is far from an inclusive list

and I am truly appreciative for the many other people who have provided support.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 10

CHAPTER

1 INTRODUCTION .................................................................................................... 12

Background ............................................................................................................. 12 The Thomas Farm Site ........................................................................................... 13 An Important Miocene Equid ................................................................................... 15 Stable Isotopes ....................................................................................................... 16 A Review of δ13C .................................................................................................... 18 A Review of δ18O .................................................................................................... 19 Research Questions ............................................................................................... 20

2 MATERIALS AND METHODS ................................................................................ 24

Sample Selection .................................................................................................... 24 Isotope Sample Collection ...................................................................................... 25 Sample Pretreatment .............................................................................................. 25 Sample Analysis ..................................................................................................... 26

3 RESULTS ............................................................................................................... 28

δ13C: Corrections .................................................................................................... 28 δ18O: Conversions and Corrections ........................................................................ 28 δ13C: Results ........................................................................................................... 30 δ18Ocarbonate: Results ................................................................................................ 31 δ18Ow: Results ......................................................................................................... 32 Diagenesis .............................................................................................................. 33

4 DISCUSSION ......................................................................................................... 44

No Evidence for C4 Feeding in Parahippus leonensis ............................................. 44 Patterns of Weaning ............................................................................................... 45 Paleoclimatic Interpretations of Thomas Farm ........................................................ 48 No Evidence for Seasonality of Birthing in P. leonensis ......................................... 51

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Mineralization Patterns ........................................................................................... 52

5 CONCLUSIONS ..................................................................................................... 55

APPENDIX

A ISOTOPIC DATA .................................................................................................... 57

LIST OF REFERENCES ............................................................................................... 61

BIOGRAPHICAL SKETCH ............................................................................................ 70

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LIST OF TABLES

Table page 3-1 δ18Ow (V-SMOW) estimates for the four known equations. ................................ 34

3-2 δ13C (V-PDB) data for each of the six sample locations ..................................... 34

3-3 δ18Ocarbonate (V-PDB) data for each of the six sample locations .......................... 34

3-4 δ18Ow (V-SMOW) data for each of the six sample locations ............................... 35

A-1 All isotope data collected from the study ............................................................ 58

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LIST OF FIGURES

Figure page 1-1 Map of Florida showing the location of the Thomas Farm locality and other

data used in the study. ....................................................................................... 22

1-2 Diagram showing δ13C fractionation in vegetation and herbivores. All values given are in reference to V-PDB ......................................................................... 23

2-1 UF 258802 .......................................................................................................... 27

3-1 Plot showing the linear relationship between δ18Ophosphate and δ18Ow ................. 36

3-2 Box and whisker plot showing the variation in δ18Ow estimates between equations 3-3 to 3-6 ............................................................................................ 37

3-3 Bivariate plot of estimates of δ18Ow and δ13C ...................................................... 38

3-4 Box and whisker plot of δ13C by sample location ................................................ 39

3-5 Plot of δ13C values of each sample position compared with a resampled distribution .......................................................................................................... 40

3-6 Plot of mean δ13C against Δδ13Capex-base. ............................................................ 41

3-7 Histogram showing the distribution of Δδ13Capex-base values. ............................... 42

3-8 Box and whisker plot for estimated δ18Ow values. .............................................. 43

4-1 Graph of δ18O values for north-central Florida. ................................................... 54

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LIST OF ABBREVIATIONS

E/P Ratio of evaporation to precipitation

FGS Specimens collected by the Florida Geological Survey, now in collections of the Florida Museum of Natural History Vertebrate Paleontology division

FLMNH Florida Museum of Natural History

M1 Upper first molar

m1 Lower first molar

M2 Upper second molar

m2 Lower second molar

m3 Lower third molar

p2 Lower second premolar

UF Denotes specimens housed in the collections of the Florida Museum of Natural History at the University of Florida

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

STABLE ISOTOPE PALEOECOLOGY OF AN EARLY MIOCENE EQUID

(PARAHIPPUS LEONENSIS) FROM THE THOMAS FARM SITE, GILCHRIST COUNTY, FLORIDA

By

Sean Michael Moran

August 2014

Chair: Bruce MacFadden Major: Geology

The importance of the early Miocene equid, Parahippus leonensis, in the

evolution of modern grazing horses of the subfamily Equinae has long been recognized.

Several important characteristics suggest an increase in grass consumption in P.

leonensis compared to earlier equids, including an increase in molar crown height,

presence of cement, increased wear rates, and a dental microwear pattern with an

abundance of fine scratches. I used carbon and oxygen stable isotope compositions of

a large sample (n=74) of P. leonensis tooth enamel, extracted from along the molar

tooth row to control for ontogenetic effects. The sampled specimens were recovered

from a single early Miocene locality (Thomas Farm, Gilchrist County, Florida) to better

document the paleoecology of this potentially transitional taxon.

Results from stable carbon isotope analyses reveal an increasing δ13C trend

through ontogeny. This is interpreted as a nursing signal and influenced mean δ13C in

four of the six sample locations examined. Mean values of the adult δ13C signals in the

base samples of m2s and m3s indicate a diet of C3 with either substantial incorporation

of water-stressed C3 plants or a very minor C4 component. Evidence of a diet based on

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water-stressed vegetation is corroborated by estimates the δ18O value of water ingested

by P. leonensis. These values are significantly higher than modern precipitation and

river water. They more closely resemble δ18O of modern lake water in north-central

Florida. The indication of a high evaporation to precipitation ratio indicates a drier

paleoclimate than was previously recognized for Thomas Farm.

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CHAPTER 1 INTRODUCTION

Background

Stable isotope ratios have been heavily utilized in vertebrate paleontology to

elucidate a range of paleoecological variables such as diet (e.g., MacFadden et al.,

1999), paleoehabitat (e.g., Nunez et al., 2010), paleoclimate (e.g., Passey et al., 2002),

and migration (e.g., Hoppe et al., 1999). Of particular interest to this study are stable

carbon (δ13C) and oxygen (δ18O) isotope ratios, which are most reliably sampled from

tooth enamel hydroxylapatite (Ca10(PO4,CO3)6(OH)2) due to the high crystal density,

large crystal size, and low organic content in enamel (Koch et al., 1997; Lee-Thorp and

van der Merwe, 1987; Wang and Cerling, 1994; Zazzo et al., 2004). Other sources such

as bone, dentine, and cementum are more easily diagenetically altered, a process that

compromises the original isotopic signal (Lee-Thorp and van der Merwe, 1987; Quade

et al., 1992; Bocherens et al., 1996). Because of these sampling restrictions, the

number of available specimens for isotopic analyses can be limited, making studies of

intrataxonomic variation difficult. Here I use a large sample (n=74) of isotopic samples

from molar enamel of the early Miocene horse, Parahippus leonensis, collected from the

Thomas Farm site in Gilchrist County, Florida, to address questions pertaining to the

paleoecology and local paleoclimate.

Thomas Farm is widely regarded as one of the most important early Miocene

vertebrate fossil localities in the eastern United States due to the high abundance and

diversity of fossils collected from the site. Three equid taxa co-occur at Thomas Farm.

P. leonensis is by far the most abundant of these horses and comprises approximately

70% of all macrovertebrate specimens collected from Thomas Farm since excavation

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began in 1931 (Hulbert, 1984). This has led to an uncommonly large sample of fossil

teeth from a specific source that can be used for stable isotope analyses. Furthermore,

P. leonensis is one of the first horse taxa to show an increase in molar crown height,

deposition of cement, and more complex enamel hypothesized to impede the faster

wear rates associated with grazing (Simpson, 1932; White, 1942; Forsten, 1975;

Schlaikjer, 1937; Downs, 1956; Hulbert and MacFadden, 1991). It has thus been

recognized as the ancestral taxon to the Equinae subfamily (Hulbert and

MacFadden,1991; Maguire and Stigall, 2008). This, in addition to preliminary dental

microwear (Solounias and Semprebon, 2002) and mesowear (Mihlbachler et al., 2011)

analyses, has led to the hypothesis that P. leonensis was incorporating some amount of

grass into its diet. However, there has been much debate regarding the timing and

pattern in the rise and spread of C4 grasses and the origination of grasslands in North

America (e.g., Edwards et al., 2010). Documenting δ13C values of P. leonensis tooth

enamel extends the available data spatially and temporally and has the potential to

better constrain our understanding of the spread of C4 grasses.

The Thomas Farm Site

The Thomas Farm site located in Gilchrist County, Florida represents one of the

most abundant and diverse early Miocene vertebrate assemblages in North America

(Figure 1-1). Vertebrate biochronology, specifically the collective presence of

Phoberocyon, Leptarctus, Floridaceras, and Metatomarctus, indicates a Hemingfordian

North America Land Mammal Age (NALMA) for the site, estimated to be 18 Ma (Tedford

and Frailey 1976; Tedford et al., 1987; MacFadden, 2001; Hulbert, 2001; MacFadden et

al., 2014). Pratt (1990) used faunal evidence to interpret Thomas Farm as being

deposited in a wooded area with a subtropical to tropical paleoclimate, while Hulbert

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(1984) interpreted Thomas Farm as a woodland environment (i.e., mostly forested with

some open patches). The validity of Hulbert’s reconstruction, which was based on P.

leonensis social structure, has been questioned by O’Sullivan (2005) who suggested

Thomas Farm was more open than previously recognized.

First discovered in 1931 (Simpson, 1932), the site has produced tens of

thousands of vertebrate fossils during its continued excavation. Unlike similar vertebrate

localities in Florida, Thomas Farm preserves a large size range of fossils as both

excavation and screenwashing are viable approaches of fossil collection adding more

specimens to an already large collection (Pratt, 1990). These factors have provided an

overwhelming amount of Miocene vertebrate fossil material available for study, as

documented by the >20,000 individual catalogued fossil of Parahippus leonensis in the

collection.

Hypotheses on the formation of Thomas Farm have proven to be rife with debate

and have ranged from the cutting and filling of a fluvial system (White, 1942; Romer,

1948; Puri and Vernon, 1964), a stream-fed sinkhole system (Bader, 1956), a water-

filled sinkhole (Simpson, 1932, Estes, 1963; Pratt, 1990), and a sinkhole-stream-cave

complex (Olsen, 1959, 1962; Auffenberg, 1963a, 1963b). The most recent and complex

study by Pratt (1990) concluded that taphonomic, taxonomic, and geologic evidence

pointed to a 30 m deep by 35 m diameter sinkhole formed by the collapse of limestone

in the Crystal River and Suwannee formations. Preferred orientations of long bones

suggest some aquatic influence in the deposition of Thomas Farm, but the relative lack

of fish fossils and water worn specimens suggest it was of minor significance and

possibly seasonal.

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Perhaps one of the most important aspects of Thomas Farm to this study is the

proposed rapid deposition of the site. Pratt (1990) hypothesized that the entire deposit

may have been formed on the scale of 1,000 years. She supported this hypothesis with

1) comparison to the infill of modern sinkhole analogs (Laury, 1980); 2) extrapolation of

the deposition of clay-sand couplets that she interpreted to represent at most an annual

cycle; and 3) the negligible amount of evolution observed in specimens recovered from

the upper and lower limits of the site, particularly in rapidly evolving taxa such as equids

and heteromyids. With this limited time scale of deposition at Thomas Farm in mind,

multiple studies have successfully investigated the population dynamics of several

Thomas Farm taxa due to the short timescale in which it formed (Hulbert, 1984;

O’Sullivan, 2005). If this hypothesis of rapid deposition is supported by future study, it

provides an unusually abrupt snapshot of ancient paleoecology and an opportunity to

study about as near to a true population as appears in the vertebrate fossil record.

An Important Miocene Equid

Parahippus leonensis is one of three equids known from Thomas Farm and is

the most abundant large bodied mammal from the site (Hulbert, 1984; Pratt, 1990).

However, it was first described from the Florida panhandle a number of years before the

discovery of the Thomas Farm site (Sellards, 1916). Anchitherium clarencei is the

largest and rarest of the three and considered to be a browser with brachyodont

dentition (MacFadden, 2001). The smallest, Archaeohippus blackbergi, also has short-

crowned molars with molar wear rates indicative of a browser, but is more common at

Thomas Farm than A. clarencei (O’Sullivan, 2005). The molar morphology of P.

leonensis, however, has been described as mesodont or incipiently hypsodont

(Stromberg, 2006; Hulbert, 1984; Hulbert and MacFadden, 1991). This important

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feature of P. leonensis, as well as more complex enamel and cementation, has often

been highlighted in the equid transition from browsing prior to the early Miocene to

grazing beginning in the early Miocene and seen as a bridge between the two ecotypes

(e.g., Hulbert and MacFadden, 1991; MacFadden, 2005).

Preliminary dental microwear analyses show an abundance of fine scratches on

the occlusal surface of Parahippus sp. molars (Solounias and Semprebon, 2002).

Scratches have been interpreted as an end-member for grazing while pitting is

interpreted as being indicative of browsing (see Teaford, 1988 for a review). Because

the abnormally fine scratches observed in Parahippus sp. differ from the coarser pattern

of modern grazers, Soulonias and Semprebon (2002) suggested a C3 grazing or mixed

feeder diet for Parahippus sp., though support for finer scratching in C3 grazers versus

C4 grazers has not been further substantiated. In a large-scale population study of P.

leonensis mandibles from Thomas Farm, Hulbert (1984) also noted that the molar wear

rate in P. leonensis was consistent with a mixed feeding diet and that a lack of

clustering into discrete tooth eruption and wear-based age classes indicated no

seasonal birthing. For these reasons, namely a considerable sample size from Thomas

Farm and its importance in equid evolution, Parahippus leonensis provides a unique

opportunity to better understand isotopic variation in a transitional paleopopulation.

Stable Isotopes

Stable isotope ratios of numerous elements (e.g., carbon, oxygen, strontium,

nitrogen) have been employed in vertebrate paleontology as proxies to elucidate many

hypotheses pertaining to natural processes and conditions (see Clementz, 2012 for a

review). Stable oxygen isotopes (δ18O) have been used as proxies to investigate past

climatic conditions (e.g., Nunez et al., 2010; Higgins and MacFadden, 2004; Koch et al.,

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1989). Strontium isotope ratios (87Sr/86Sr) have been used to consider migratory

patterns in extinct organisms, such as mammoths and mastodons (e.g., Hoppe et al.,

1999). Stable nitrogen ratios (δ15N) have been used to infer trophic levels of organisms

(e.g, Bocherens et al., 1994). δ13C, stable carbon ratios, has perhaps been the most

utilized stable isotope tool in vertebrate paleontology and has been shown to be a

reliable indicator of herbivorous diet between C3 plants and C4 grasses (i.e., browsers

and grazers) (e.g., MacFadden and Cerling, 1996; MacFadden et al., 1999). The stable

isotope ratios of carbon, δ13C, and oxygen, δ18O are of particular interest to this study.

In general, stable isotope values are calculated by measuring the ratio of the two

most abundant isotopes of an element and comparing this to a laboratory standard

(e.g., δ18O of standard mean ocean water (i.e., V-SMOW), δ13C of belemnites from the

PeeDee Formation in South Carolina (i.e., V-PDB)). The formula for calculating delta

values is as follows:

δ =Rsample − Rstandard

Rstandard× 1000

(1-1)

where R is the ratio of the abundance of the heavier isotope to the lighter isotope (e.g.,

18O/16O, 13C/12C). Because meaningful isotopic variations are often very small, the

values are usually reported as per mil (‰).

It should be noted that due to the nature of enamel mineralization in most

mammals, equids included, it is not possible with current protocols and techniques to

identify a daily signal of isotopic values. Instead, the isotopic value at a given point on a

tooth is averaged over the span of weeks or longer and is further attenuated by bulk

sampling (Passey and Cerling, 2002). Therefore the data presented in this study

18

represent an average signal for each sample with the extreme values unlikely to be

detected through the current methods.

A Review of δ13C

Over the past few decades, ratios of stable carbon isotopes (δ13C) have become

a valuable proxy in reconstructing ancient diet (e.g., Wang et al., 1994; MacFadden and

Cerling 1996) and paleoecology (e.g., MacFadden and Higgins, 2004; Nunez et al.,

2010), as well as a variety of other past processes and conditions. These interpretations

are made possible due to the differing fractionation of carbon isotopes in plants

employing varying photosynthetic pathways. C4 plants, including many tropical and

temperate grasses, photosynthesize using the Hatch-Slack cycle. Alternatively, C3

plants, which comprise approximately 90% of modern plants such as most trees,

shrubs, and high-latitude grasses, employ the Calvin cycle. The Hatch-Slack cycle, in

comparison with the Calvin cycle, supports preferential uptake of the heavier isotope,

13C, into the plant tissue and has subsequently higher δ13C values (Farquahar et al.,

1989). Therefore, the δ13C for C4 plants range from -15‰ to -11‰, with a mean of -

13‰, and C3 plants range from -32‰ to -24‰ , with a mean of -27‰ (Dienes, 1980;

Farquahar et al., 1989; Boutton, 1991). The large range of values present in C3 plants is

important as plants found in closed canopy environments frequently have lower δ13C

values (van der Merwe and Medina, 1989; Cerling and Harris, 1999; Cerling et al.,

2004), while those that occur in more water-stressed, open habitats exhibit higher δ13C

values (Farquhar et al., 1989; Cerling et al., 2004). The foundation for reconstructing

paleodiet is thus formed around the different δ13C values in C4 and C3 plants and

subsequent ingestion by the organism.

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The ingestion of the vegetation imparts the δ13C signal onto the organism, albeit

with further fractionation between ingestion and the incorporation of carbon into tissue,

in the case of this study, the crystalline enamel hydroxylapatite. This enrichment factor

from the δ13C of ingested vegetation to the δ13C of enamel hydroxylapatite (ε*diet-enamel)

in modern horses has been shown to be 14.1‰ (Cerling and Harris, 1999), though this

is taxon specific and is thought to vary slightly due to variation in methane production

during digestion (Passey et al., 2005). For this study, I assume a ε*diet-enamel of 14.1‰ as

has been shown for equids and other large-bodied mammals. Therefore, a horse

ingesting a pure C3 diet with a mean δ13C of -28‰ would have a δ13C signal in the

enamel hydroxylapatite of -13.9‰; and a horse ingesting a pure C4 diet with a mean

δ13C of -11‰ would have a δ13C value of 3.1‰ (Figure 1-2).

A Review of δ18O

δ18O values of organisms’ body water have been shown to correlate directly with

the δ18O of ingested water which is in turn related to the δ18O of local precipitation

(Longinelli, 1984; Luz et al., 1984; Luz and Kolodny, 1985; Bryant and Froelich, 1995;

Bryant et al., 1996a; Kohn, 1996). These values are then imparted onto biological

apatite, both in structural carbonate and phosphate, which mineralize in isotopic

equilibrium with body water (Longinelli, 1984; Luz et al., 1984; Iacumin et al., 1996).

δ18O recorded in the biological apatite can be informative as δ18O values of precipitation

(δ18Omw) vary seasonally due to two major factors: temperature and amount of

precipitation (Dansgaard, 1964). In warmer months, relative enrichment of 18O in

precipitation causes higher δ18Omw values, whereas a relative depletion in 18O causes

lower δ18Omw in colder months (McCrea, 1950; Bryant et al., 1996a; Fricke and O’Neil,

1996; Feranec and MacFadden, 2000). Additionally, in times of significant precipitation

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and temperatures greater than 20ºC the “amount effect” can also be a major factor in

δ18Omw values (Dansgaard, 1964; Rozanski et al., 1993; Bard et al., 2002; Straight et

al., 2004). When meteoric water is precipitated the heavier isotope, 18O, preferentially

falls leading to a depletion of 18O in cloud water and, thus, lower δ18O values. This

phenomenon leads to a pattern of marked decrease in δ18Omw due to the preferential

depletion of 18O over 16O (Higgins and MacFadden, 2004). Increasing altitude, latitude,

and distance from the coast can cause δ18O to decrease, but often are only pertinent in

comparing modern δ18O to past δ18O when these factors have shifted significantly

during the relevant time span (Dansgaard, 1964; Rozanski et al, 1993). These

compounding factors often make δ18O more difficult to interpret than δ13C with regard to

vertebrate paleontology.

Research Questions

The goal of this study is to investigate the stable isotope paleoecology of an

important, transitional taxon. Preliminary data (e.g., Soulonias and Semprebon, 2002;

Hulbert, 1984) indicate Parahippus leonensis may have been incorporating grass into its

diet well before grasslands are hypothesized to have evolved. This study helps to

further specify the dietary niche occupied by P. leonensis which could lead to more

specific paleoecological reconstruction than has been previously possible. Additionally,

many studies utilizing isotopes fail to incorporate a large sample size to take

intrataxonomic variation into account. The nature of the Thomas Farm collection permits

investigation into a large paleopopulation that is not often possible in other situations.

Though the main goal is to focus on stable carbon isotope values in P. leonensis,

the technique used to analyze δ13C also provides δ18O values which are useful in

reconstructing aspects of past climatic conditions. Because enamel mineralization of the

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equid molars encompasses several years (Hoppe et al., 2004), variation in δ18O

sampled from different locations on cheek teeth has previously been interpreted as a

record of seasonal variability in stable oxygen isotope values (e.g., Nunez et al., 2010).

The δ18O values can be compared to modern values, in this case from modern horse

teeth and the distribution of δ18O in meteoric water (δ18Omw) based on latitude,

longitude, and elevation (http://www.waterisotopes.org; see Bowen and Wilkinson,

2002; Bowen and Revenaugh, 2003) as well as δ18O values from local bodies of water

(e.g., Katz et al., 1999; Arnold et al, in press), to draw inferences about past climatic

conditions. Specifically, this study looks to address the following:

Research questions associated with δ13C

1. Is there evidence for the presence of C4 grazing in P. leonensis?

2. Can δ13C values elucidate the timing of weaning in P. leonensis based on samples taken from teeth representing ontogeny?

3. Is dietary variation evident within the Thomas Farm paleopopulation of P. leonensis?

Research questions associated with δ18O

4. Did P. leonensis birth seasonally?

5. How does δ18Omw from the Miocene of Thomas Farm differ from that observed today? What does this mean about differing climatic conditions during the Hemingfordian and modern-day Florida?

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Figure 1-1. Map of Florida showing the location of the Thomas Farm locality and other

data used in the study. The inset shows the area of enlargement. Thomas Farm is denoted by the star. Newnans Lake is the lake illustrated just east of Gainesville and the springs sampled along the Santa Fe River and Suwannee River by Katz et al. (1999) are marked by blue dots.

23

Figure 1-2. Diagram showing δ13C fractionation in vegetation and herbivores. All values

given are in reference to V-PDB. The values on upper portion of the chart reflect actual values of plant tissue with the left being C4 grasses and the right being C3 vegetation with water-stressed and closed canopy components (e.g., Dienes, 1980; Farquahar et al., 1989; Cerling et al., 2004). The bottom portion shows what the isotopic signal of tooth enamel carbonate in large herbivorous mammals feeding on that particular vegetation would be, assuming an isotopic enrichment of approximately 14‰ (Cerling and Harris, 1999).

24

CHAPTER 2 MATERIALS AND METHODS

Sample Selection

A total of 79 samples (with five of those samples removed from statistical

analyses as outliers) from Thomas Farm Parahippus leonensis lower cheek teeth were

sampled for stable carbon and oxygen isotope composition of enamel carbonate. In

addition, several samples were taken from two modern specimens of Equus caballus

from the FLMNH Mammalogy collections (one from Lake City, FL and one from

Gainesville, FL) for isotopic comparison. The study design was intended to sample as

many individuals as possible while still maintaining ontogenetic control of the stable

isotopic ratios. Assuming the pattern of enamel mineralization is conserved from

modern Equus to P. leonensis, which has been supported by Hulbert (1984) and Kurten

(1953), sampling m1s, m2s, and m3s records the isotopic signal for the entire range of

enamel mineralization (Hoppe et al., 2004). To the greatest extent possible, m3s were

chosen from the same side of mandible to reduce the chances that a single individual

was sampled twice. This was not always possible, due to limitations imposed by the

large, but not infinite sample. Furthermore, isolated m1s and m2s cannot be

distinguished. Therefore, m1s and m2s were only sampled when it was possible to

distinguish the tooth position of each (i.e., when teeth were associated or still within the

mandible). In the case where both the m1 and m2 were present in the same associated

dentition, only one of the teeth was sampled to maximize the number of individuals

sampled. Because there was a limited number of associated dentitions that included

m1s or m2s that were only slightly worn, samples from m1 and m2 were not restricted to

a given side of mandible (i.e., left or right). This presents the possibility, though unlikely,

25

due to the sheer number of Parahippus leonensis specimens collected from Thomas

Farm, that several of the samples analyzed may represent the same individual.

Isotope Sample Collection

The teeth were first cleaned of cementum using a Foredom drill to avoid

contamination of the enamel hydroxylapatite sample, as dentine and cementum are

more easily altered by diagenesis (Quade et al., 1992; Wang and Cerling, 1994). More

than 5 mg of enamel per sample were then taken with the hand drill in a line at the apex

(i.e., towards the crown) and base (i.e., towards the root) of each tooth with care to

avoid contamination of the sample by surrounding cementum or underlying dentine

(Figure 2-1). Two samples were taken from each tooth in order to maintain a record of

isotopic change through ontogeny. The teeth were drilled on weighing paper and the

enamel powder was transferred to labeled 1.5 mL graduated microcentrifuge vials.

Sample Pretreatment

The powdered enamel samples were put through a standard hydrogen peroxide

and acetic acid pretreatment regimen to remove organics and secondary carbonates,

respectively (e.g., Higgins and MacFadden, 2004). 1.0 mL of 30% H2O2 was added to

each sample and shaken to thoroughly mix the sample with hydrogen peroxide.

Because gas buildup can force the lid of the vials open, the vials were propped open to

allow gas release. After allowing the samples and hydrogen peroxide to react overnight,

the enamel samples were checked to see if any reaction was ongoing. If the samples

showed continued reaction, they were rinsed three times with distilled water and 1.0 mL

of hydrogen peroxide was again added to the samples, shaken, and allowed to sit

overnight. If the reaction had stopped, the samples were centrifuged for 5 to 10 minutes

at 10,000 rpm and the H2O2 was siphoned off using a pipette. In order to avoid cross-

26

contamination of the samples, the pipette tips were discarded after each sample.

Samples were then rinsed with 1.0 mL of distilled water, shaken, and centrifuged. The

water was then siphoned off, again discarding pipette tips after each sample, and the

rinse was repeated twice more. After the third rinse was completed, 1.0 mL of 0.1 N

acetic acid was introduced to the samples and allowed to react overnight, though not for

more than 24 hours. The samples were then centrifuged, the acetic acid siphoned off,

and the samples were rinsed with distilled water three times. The samples were then

allowed to air dry overnight.

Sample Analysis

Isotopic samples were analyzed at the Department of Geological Sciences Light

Stable Isotope Mass Spectrometer Lab at the University of Florida. Powdered enamel

samples were loaded into a Kiel III preparation device in glass vials and reacted with

phosphoric acid to liberate CO2 from the carbonate. The gas was then analyzed in a

Finnegan-MAT 252 isotope ratio mass spectrometer for δ13C and δ18Ocarbonate in

reference to the NBS-19 standard and then converted to V-PDB. Analytical precision for

each run varied slightly and is described more thoroughly in Appendix A, but was less

than ±0.08 for δ18Ocarbonate and ±0.04 for δ13C.

27

Figure 2-1. UF 258802, Parahippus leonensis m3. The image is an example of a

sampled m3. The black ovals indicate the location of samples taken at the apex (top) and base (bottom). Scale bar is 1.0 cm.

28

CHAPTER 3 RESULTS

δ13C: Corrections

Arens et al. (2000) showed atmospheric δ13CCO2 accounts for over 98% of the

total variation of δ13C in vegetation. Therefore, a correction of -0.5‰ was added to all

Thomas Farm δ13C measurements to account for differences in atmospheric δ13CCO2

between the pre-industrial average and the Hemingfordian age of Thomas Farm (Tipple

et al., 2010).

δ18O: Conversions and Corrections

In order to compare δ18O values from the Miocene samples to δ18O of modern

meteoric water (δ18Omw) in north-central Florida, several conversions were needed.

Samples were first converted from the standard V-PDB to V-SMOW using the equation

(Hoefs, 1997; Criss, 1999):

δ18Ocarbonate (V-SMOW) = 1.03086 * δ18Ocarbonate (V-PDB) + 30.86 (3-1)

Iacumin et al. (1996) showed a strong relationship (r2=0.98) in modern mammals

between δ18O analyzed from enamel carbonate (δ18Ocarbonate) and that analyzed from

enamel phosphate (δ18Ophosphate). Assuming this relationship holds true for isotopic

samples from the fossil record, it can be used to calculate δ18O from the carbonate

phase to the phosphate phase using the equation:

δ18Ophosphate (V-SMOW) = 0.98 * δ18Ocarbonate (V-SMOW) - 8.5 (3-2)

Lastly, several authors have investigated the association of δ18Ophosphate and the

δ18O of ingested water (δ18Ow) in modern equids (Bryant et al., 1994; Delgado Huertas

et al., 1995; Sanchez Chillon et al., 1994). Bryant et al. (1994) collected oxygen isotopic

samples from African and North American specimens of E. burchelli, E. zebra, and E.

29

caballus and derived the following equation (r2= 0.69) by relating each δ18Ophosphate

sample to δ18Ow estimates for each location:

δ18Ophosphate (V-SMOW) = 22.90 + 0.6863 * δ18Ow (V-SMOW) (3-3)

Sanchez Chillon et al. (1994) looked at both bone and enamel samples from

three equid species (E. prezewalskii, E. caballus, and E. asinus) collected from various

locations throughout Asia, Europe, Africa, and South America. The following

relationship (r2=0.95) was derived after comparison with δ18Ow from each of the

locations:

δ18Ophosphate (V-SMOW) = 22.04 + 0.7369 * δ18Ow (V-SMOW) (3-4)

Delgado Huertas et al. (1995) added several more δ18Ophosphate data of E.

caballus from Argentina, as well as δ18Ow estimates, to the dataset of Sanchez Chillon

et al. (1994) to derive the following equation (r2= 0.90):

δ18Ophosphate (V-SMOW) = 22.29 + 0.72 * δ18Ow (V-SMOW) (3-5)

While the equations presented in these three papers are similar, they do exhibit

small variations and thus the following equation (r2=0.77), which incorporates the data

from all three papers as well as geographically and taxonomically diverse equid

samples, was used for this study:

δ18Ow (V-SMOW) = (δ18Ophosphate (V-SMOW) – 22.60) / 0.71 (3-6)

Table 3-1 displays the variation of δ18Ow estimates based on which of the above

four relationships are used.

Because most precipitation is ultimately derived from seawater, one final

correction must be made to compare modern δ18Ow to that measured from P. leonensis.

In the early Miocene, δ18Osw would have been more enriched in 16O relative to V-SMOW

30

due to the absence of polar ice sheets and thus a correction for δ18Osw must be inferred

for the evaporative source of precipitation (Zachos et al., 2001). To ensure equivalent

comparison of fractionation between δ18Osw and δ18Ow, 1‰ was added to the calculated

δ18Ow of the Miocene samples to account for this relatively ice free world where δ18Osw

was approximately 1‰ lower than modern seawater (Lear et al., 2007; Billups and

Schrag, 2003).

δ13C: Results

Bulk analyses of δ13C collected from Parahippus leonensis tooth enamel

carbonate provide values ranging from -13.85‰ to -9.43‰ with a mean of -11.70‰ (V-

PDB) (Table 3-2; Figure 3-3). An ANOVA testing for differences in δ13C for each sample

position indicates significant differences exist among sample positions (p<0.001) (Figure

3-4).

I employed a resampling technique to further elucidate the differences indicated

by the ANOVA. All δ13C values were pooled and randomly attributed to a sample

location while keeping the sample size of each sample location constant. The

randomized data were compiled and means for each sample location calculated

iteratively over 10,000 runs. Figure 3-5 shows the resulting graph of the randomized

data with means of the resampled means for each sample location, actual means, and

95% confidence intervals plotted. All red points fall outside (i.e., m1 apex, m2 base, and

m3 base) of the 95% confidence envelope of the resampled distribution. Therefore, the

null hypothesis that the data indicated in red have the same δ13C values of the

hypothesized underlying population can be rejected. This will be further investigated in

the discussion, but is likely due to ontogenetic effects (i.e., nursing and weaning).

31

Intra-tooth variations can also be observed because two samples were analyzed

from each specimen, one at the apex of the tooth and the other at the base. A one-

tailed t-test rejects the null hypotheses (p<0.001) that the true mean of the differences

from apex to base is equal to zero and that there is no significant directionality. All but

four of the specimens show increased δ13C values from the apex to the base with the

mean increase being 0.87‰. An ANOVA shows significant differences due to tooth

location in Δδ13Capex-base (p=0.003) and mean δ13C (p=0.04). The non-parametric

Kruskal-Wallis test, however, shows only significance in Δδ13Capex-base (p=0.006) and not

mean δ13C (p=0.06), though the calculated p-values differ only slightly from the ANOVA.

Figure 3-6 depicts the delta (Δδ13Capex-base) of each specimen (i.e., change in δ13C from

apex to base) plotted by mean δ13C values. The first tooth to erupt (i.e., m1) shows a

more 13C depleted δ13C signature than do that the latter erupting teeth (i.e., m2 and m3)

as well as a generally larger Δδ13Capex-base. The multivariate, two sample Hotelling’s t-

test indicates m1 is significantly different than m2 (p=0.01) and m3 (p<0.001), but that

m3 and m2 do not vary significantly (p=0.26). The significance of these results will be

explained in the discussion.

δ18Ocarbonate: Results

δ18Ocarbonate measured directly from the tooth enamel of Parahippus leonensis

range from 2.95‰ to -2.12‰ with a mean value of 1.17‰ (V-PDB) (Table 3-3). An

ANOVA indicates no significant differences present for samples collected at different

sample locations (p=0.20).

The mean difference between the apex and base (Δδ18Oapex-base) of a specimen

is 1.02‰ with a range in differences between 0.06‰ and 3.90‰. All but two specimens

show differences of less than 2‰. There is no significance (One-sample t-test,

32

p=0.2291) in the directionality of the differences with 15 specimens showing decreases

in δ18O from the apex to the base and 20 specimens showing increases. An ANOVA

fails to reject the null hypothesis that there are no significant differences in Δδ18Oapex-base

(p=0.07) or mean (p=0.73) between tooth positions.

δ18Ow: Results

After accounting for differences in Miocene δ18Osw and converting to estimated

δ18Ow (i.e., equations 3-1, 32, and 3-6), values range from 3.99‰ to -3.23‰ (V-SMOW)

with a mean of 1.45‰ (Table 3-4; Figures 3-3; 3-8). Specimens show absolute

Δδ18Oapex-base variation between 0.08‰ to 5.55‰, however only four specimens show

Δδ18Oapex-base greater than 2.5‰. The mean magnitude of change between apex and

base samples is 1.46‰. Estimates for δ18Ow calculated from the other relationships

(i.e., equations 3-3, 3-4, 3-5) have means ranging from 1.03‰ to 2.20‰.

Estimates for δ18O of precipitation (δ18Omw) for modern day Thomas Farm were

calculated using the Online Isotopes in Precipitation Calculator and ranged annually

from -5.5‰ to -2.9‰ with an annual mean of -4.1‰ (http://www.waterisotopes.org; see

Bowen and Wilkinson, 2002; Bowen and Revenaugh, 2003). The nearest direct

measurements of δ18O of river water to the Thomas Farm locality were taken from

various springs in the Suwannee River Basin (Katz et al. 1999). Unfortunately the data

provide little interpretation for annual or seasonal variation in δ18O as all data were

collected in July or August of 1997 and 1998. Nevertheless, the data generally reflect

δ18Omw with all values from the Santa Fe River springs, Lafayette County springs, Lower

Suwannee springs, and Suwannee County springs falling between -3.94‰ and -3.03‰

These values are consistent with the summer values estimated by the Online Isotopes

in Precipitation Calculator that show δ18Omw ranges from -3.8‰ to -3.1‰. Arnold et al.

33

(in press) report δ18O for Newnans Lake which is located approximately 60 km east of

Thomas Farm, just east of Gainesville. The annual average for Newnans Lake is much

higher than δ18Omw and δ18O measured from spring water in the area at 0.54‰ with a

total range of 2.88‰, but the analyzed water was collected from a highly evaporative

boat canal in Newnans Lake. Of the two modern Equus caballus specimens, one had

estimated δ18Ow values reflective of δ18Omw, between -4.8‰ and -4.3‰, the Lake City

specimen, while the Gainesville specimen showed a range in estimated δ18Ow from -

9.7‰ to -5.1‰.

Diagenesis

Several factors lead to the assumption that diagenesis did not affect the

values collected in this study. First, two previously analyzed samples from Thomas

Farm P. leonensis teeth were interpreted to have been contaminated by altered dentine

and/or cementum due to unusually high δ13C values (-5.23‰ and -5.77‰) (C. Manz,

pers. comm.). The corresponding δ18Ocarbonate values (-1.22‰ and -0.08‰) of these

samples were lower than most of the samples collected here. This indicates that δ18O

values at Thomas Farm would likely decrease, rather than increase, when affected by

diagenesis with the lower δ18O values of local precipitation being the main driver.

Because the δ18Ow values collected here are higher than any other comparative water

source, it is unlikely diagenesis had a major impact on the sampled enamel.

Furthermore, Figure 3-3 does not exhibit a noticeable inverted J-curve as would be

expected if the samples were affected by meteoric diagenesis (e.g., Lohmann, 1988).

Lastly, the preservation of the expected ontogenetic increase in δ13C, rather than

homogenization of δ13C values from different sample locations, further supports a lack

of diagenetic impact on the samples.

34

Table 3-1. δ18Ow (V-SMOW) estimates (per mil) for the four known equations. Bryant et al., 1994 is equation 3-3. Sanchez Chillon et al., 1994 is equation 3-4. Delgado Huertas et al., 1995 is equation 3-5. All data refer to the relationship calculated from the combined data of the three above references (equation 3-6). Equation Mean Maximum Minimum Range SD Bryant et al., 1994 1.03 3.66 -3.81 7.47 1.46 Sanchez Chillon et al., 1994 2.20 4.64 -2.31 6.95 1.36 Delgado Huertas et al., 1995 1.88 4.38 -2.74 7.12 1.90 All data 1.45 3.99 -3.23 7.22 1.41

Table 3-2. δ13C (V-PDB) data (per mil) for each of the six sample locations. Sample Location

n Mean Maximum Minimum Range SD

m1 Apex 11 -12.85 -11.92 -13.85 1.93 0.56 m2 Apex 11 -12.00 -11.20 -12.62 1.42 0.41 m3 Apex 15 -11.76 -10.85 -13.23 2.38 0.79 m1 Base 11 -11.44 -10.42 -12.51 2.09 0.74 m2 Base 10 -11.14 -10.21 -11.86 1.65 0.57 m3 Base 16 -11.19 -9.43 -12.63 3.20 0.86 Total 74 -11.70 -9.43 -13.85 4.42 0.88

Table 3-3. δ18Ocarbonate (V-PDB) data (per mil) for each of the six sample locations. Sample Location

n Mean Maximum Minimum Range Std

m1 Apex 11 1.46 2.95 0.51 2.44 0.85 m2 Apex 11 1.34 2.70 0.14 2.56 0.80 m3 Apex 15 1.10 2.47 -1.49 3.96 1.15 m1 Base 11 0.50 2.12 -2.12 4.24 1.21 m2 Base 10 1.48 2.56 0.00 2.56 0.70 m3 Base 16 1.14 2.90 -0.91 3.81 0.97 Total 74 1.17 2.95 -2.12 5.07 0.99

35

Table 3-4. δ18Ow (V-SMOW) data (per mil) for each of the six sample locations. Estimates for δ18O of ingested water were calculated using equation 3-6.

Sample Location

n Mean Maximum Minimum Range SD

m1 Apex 11 1.87 3.99 0.51 3.48 1.20 m2 Apex 11 1.70 3.64 -0.01 3.65 1.13 m3 Apex 15 1.36 3.31 -2.33 5.64 1.64 m1 Base 11 0.51 2.81 -3.23 6.04 1.73 m2 Base 10 1.90 3.43 -0.20 3.63 0.99 m3 Base 16 1.42 3.92 -1.50 5.42 1.39 Total 74 1.45 3.99 -3.23 7.22 1.41

36

Figure 3-1. Plot showing the linear relationship between δ18Ophosphate and δ18Ow. All four

equations show similar relationship with increased disagreement with δ18Ophosphate values lower than measured in this study. As noted, the axes are in relation to V-SMOW and the values are per mil.

37

Figure 3-2. Box and whisker plot showing the variation in δ18Ow estimates between

equations 3-3 to 3-6. The values on the y-axis are in relation to V-SMOW and are in per mil.

38

Figure 3-3. Bivariate plot of estimates of δ18Ow and δ13C. Points are coded by tooth

position (shape) and sample location (color). Values higher on the y-axis indicate 1) decreased lipid consumption, 2) incorporation of more water-stressed C3 vegetation, and/or 3) incorporation of more C4 grasses. Higher values on the x-axis indicate water sources with δ18O influenced by evaporation.

39

Figure 3-4. Box and whisker plot of δ13C by sample location. The sample locations are

ordered from earliest to latest in enamel mineralization assuming mineralization in P. leonensis resembles Equus (Hoppe et al., 2004). Note the increasing trend in δ13C due to decreased consumption of lipids from the mother’s milk (i.e., weaning).

40

Figure 3-5. Plot of δ13C values of each sample position compared with a resampled distribution. The plus signs indicate the overall mean of the 10,000 resampled mean with the envelopes indicating 95% confidence intervals of the 10,000 resampled means. The red circles are indicative of actual means of a sample location that plot outside of the 95% confidence intervals, and thus interpreted to be significantly different than the underlying population, and black points are sample location means not significantly different than the underlying δ13C population. Sample locations are ordered from mineralization starting earliest in ontogeny to latest.

41

Figure 3-6. Plot of mean δ13C against Δδ13Capex-base. The grey symbols reflect the

samples analyzed and the black dots show the centroids for each tooth position. Notice the increasing means from m1s to m3s as well as the larger differences in δ13C from apex to base sample for each tooth position. The centroids of the m3 and m1 are significantly different, while the m2s represent a transition from more reliance on nursing (i.e., m1) to less or none (i.e., m3).

42

Figure 3-7. Histogram showing the distribution of Δδ13Capex-base values. The darkest

shading shows m3 values, the moderate shading indicates m2 values, and the lightest represents m1 values. The models on the right show expected distributions for hypotheses of decreasing, increasing, and random Δδ13Capex-

base values. The values here show a distribution consistent with an increase in δ13C from the apex to base samples in the analyzed teeth.

43

Figure 3-8. Box and whisker plot for estimated δ18Ow values. The overlapping values at

each sample position indicate no noticeable differences for estimates of δ18O for ingested water by P. leonensis. This shows there is no evidence for seasonal birthing patterns in P. leonensis.

44

CHAPTER 4 DISCUSSION

No Evidence for C4 Feeding in Parahippus leonensis

Parahippus leonensis has long been considered a important species in the equid

transition from grazing to browsing (Simpson, 1932; Hulbert and MacFadden, 1991;

Maguire and Stigall, 2008). Though certain adaptations, such as hypsodonty, deposition

of cement, and more complex enamel, as well as calculated enamel wear rates

(Hulbert, 1984), support this hypothesis of a mixed feeder, δ13C data presented here for

Thomas Farm specimens indicate C4 grazing was not an integral part of the paleodiet in

P. leonensis. It is unlikely the diet of P. leonensis incorporated enough C4 grasses to

contribute in any significant way to the δ13C signal, as all samples interpreted to

represent the adult signal (i.e., m2 and m3 base samples) average -11.2‰. The

maximum value analyzed in all of the 74 samples, -9.43‰, may indicate some

insignificant portion of C4, but much of the enrichment of 18O in this sample can be

explained by feeding on water-stressed C3 plants, which is further corroborated by the

high δ18O values.

Though the data collected here do not support the hypothesis of a C4 or mixed

C3-C4 diet for P. leonensis, it does not preclude the possibility that P. leonensis

incorporated C3 grasses into its diet. In fact, preliminary dental microwear analyses, as

well as the aforementioned grazing adaptations, seem to support at least some grazing

aspect to its diet. It seems highly unlikely P. leonensis would distinguish between C4

and C3 grasses while later equids show a clear mixed C3-C4 signal with hypothesized

grazing adaptations (MacFadden and Cerling, 1996). Thus, the abundance of C4

45

grasses in the Hemingfordian near Thomas Farm was likely very low, if not completely

absent.

Fox and Koch (2004) developed two hypotheses for the spread of grasslands in

North America. The first hypothesis suggests the decline of wooded habitats due to the

emergence of C3 grasslands after the peak in browser diversity in the middle Miocene

and throughout the remaining duration of the Miocene (e.g., Janis et al., 2000). This

predominantly C3 ecosystem is only replaced by C4 grasses after the end of the

Miocene. Fox and Koch note this hypothesis would be supported by specialized

browsers with low-crowned teeth, C3 grazers with high-crowned teeth, C3-C4 grazers

later in the Miocene, and C4 grazers. The second hypothesis states decreased

temperatures in the middle to late Miocene led to a shorter growing season and

increased competition of resources among browsers. Increased browsing competition

on available vegetation would have acted as an ecological disturbance allowing for C4

grasses to become dominant by the Pliocene due to decreased C3 biomass. This

second hypothesis would be supported by taxa with three distinct characteristics: low-

crowned teeth with a C3 signature, high-crowned teeth with an intermediate C3-C4

signature, and high-crowned teeth with a C4 signature. In Florida, this study provides

support for the first hypothesis with Parahippus leonensis showing adaptations for

grazing (i.e., cementation, increase in crown height over earlier equids, more complex

enamel), but still retaining a distinct C3 dietary signal indicating somewhat open habitats

with P. leonensis grazing on C3 resources.

Patterns of Weaning

Several studies have identified patterns of weaning in isotopic signatures,

particularly of δ15N, δ13C, and δ18O, from the fossil record (Bryant et al, 1996b; Rountrey

46

et al. 2007). Stable nitrogen isotope ratios are most often analyzed from collagen, but

due to its high susceptibility to diagenetic alteration, δ15N analysis for diet is often

restricted to the Pleistocene or younger (Bocherens et al., 1994). Therefore, in the deep

fossil record only δ13C, and in some cases δ18O, can be utilized for investigation into

weaning. The δ13C data collected in this study strongly suggests an influence of

weaning and feeding on mother’s milk during the mineralization of several of the sample

locations.

Studies of the stable isotopic signature of modern animals identified an

increasing δ13C and decreasing δ15N trend from birth until the end of weaning (e.g.,

Hobson and Sease, 1998; Balasse et al., 2001). The δ15N signal of tissue accreted

while an organism is feeding on its mother’s milk is hypothesized to be more negative

than after due to the so-called trophic level effect, as the offspring is effectively

positioned one trophic stage above its mother (Rountrey et al., 2007; Hobson and

Sease, 1998; Balasse et al., 2001). On the other hand, the more negative δ13C values in

juveniles feeding from the mother’s milk than individuals feeding independently from the

mother are likely due to the incorporation of relatively more lipids, which have a lower

δ13C value than the carbohydrates vegetation fed on by browsers or grazers after

weaning (DeNiro and Epstein, 1977; Ambrose and Norr, 1993; Rountrey et al., 2007).

As would be predicted from the aforementioned studies, the data analyzed in this

paper show a clear and statistically significant increasing trend in mean δ13C from

approximately -12.9‰ to -11.2‰ from the m1 apex to the m3 base sample locations,

the earliest and latest in the mineralization sequence, respectively. Because the main

goal of this study is to look at the variation in P. leonensis by analyzing as many

47

individuals from the paleopopulation as possible, the collected data cannot analyze the

significance of a consistent 1.7‰ increase in any given individual. These data only

support an increasing δ13C trend in the sampled population as a whole. However, it is

worth noting that only 4 of the 35 specimens had decreasing δ13C, all of which were

m3s and potentially fully weaned during enamel mineralization. The mean increase in

δ13C of each specimen was just below 1‰, though each tooth position only represents a

portion of the overall enamel mineralization period of a given individual (Hoppe et al.,

2004).

Future isotopic studies need to understand the enamel mineralization sequence

of the study organism and the potential enrichment in 12C during weaning. In this study,

the tooth apex samples of the oft-sampled m3 have a mean δ13C value that is 0.5‰

higher than the base samples and likely incorporate some weaning signal. The base

samples of the m1, m2, and m3 begin to plateau towards what is interpreted as the

adult dietary signal, but even the m1 base sample has a mean δ13C that is

approximately 0.3‰ lower than the m2 and m3 signals.

Interestingly, in the P. leonensis teeth sampled for this study, there does not

appear to be any trend in δ18O associated with weaning. Bryant et al. (1996b) used a

mass balance model for to identify potential patterns in δ18O over the period of weaning.

They noted the model showed higher δ18O values in m1s than in m2s and m3s for foals

born in spring and lower δ18O values in m1s than in m2s and m3s for fall-born foals.

Though the data collected here shows m1s with lower values than m2s or m3s, this is

not statistically significant. The lack of a weaning signal in δ18O may be due to low

48

annual variation in δ18O values for Florida or due to drinking sources of highly variable

δ18O values as discussed below.

Seasonality recorded in δ18O of accreted tissue in fossil organisms has been well

documented (e.g., Fox et al., 2007; Fricke and O’Neil, 1996). Serial sampling over the

span of enamel mineralization can elucidate summer (high δ18O) and winter (low δ18O)

signals which can be used as time signatures if the general pattern of enamel

mineralization is known. Therefore, in areas where seasonal δ18O variability is high, it is

hypothetically possible to identify the precise timing of weaning by examining the δ18O

and δ13C signatures over the molar tooth row of an individual. The low seasonal

variability of δ18O in Florida makes such analysis of precisely timing enamel

mineralization difficult in the Thomas Farm specimens.

Paleoclimatic Interpretations of Thomas Farm

Estimates for δ18Ow in Parahippus leonensis were surprisingly high with a mean

of 1.2‰ when compared to modern estimates for δ18Omw at Thomas Farm which range

from -5.1‰ to -2.9‰ with an annual mean of -4.1‰ (http://www.waterisotopes.org; see

Bowen and Wilkinson, 2002; Bowen and Revenaugh, 2003). This large discrepancy

between the Miocene and modern values was surprising. Assuming there is not a

dramatic change in source water δ18O, evaporative effects, where the δ18O values

increase due to preferential evaporation of the lighter 16O relative to 18O, are the only

factor that can account for this large disparity (Hodell et al., 1991; Brenner et al., 2003).

To further investigate this, I looked at lake and river/spring δ18O values published from

the area surrounding Thomas Farm. Though these data are fairly sparse, values from

springs in the Suwannee River Basin near Thomas Farm tend to reflect the δ18Omw with

summer values from -3‰ to -4‰. Lake values, however, collected from Newnans Lake

49

which is located just 60 km east-southeast of the Thomas Farm locality show a range in

δ18O of -1.5‰ to 1‰ with an average of 0.5‰ (Arnold et al., in press). These lake

values are much closer to the δ18Ow calculated in P. leonensis, though still nearly 1‰

lower. It should also be noted that the lakewater analyzed by Arnold et al. (in press),

was collected from a boat canal that is highly affected by evaporation.

The simplest explanation for these high δ18Ow is the water ingested by the P.

leonensis deposited at Thomas Farm was enriched in 18O due to high evaporation

versus precipitation (high E/P). As it seems unlikely P. leonensis would have ingested

only highly evaporated lake water while neglecting river, spring, and precipitated water,

it is likely the lake water ingested had a higher δ18O signature than measured from

Newnans Lake. These data support a drier environment in the Hemingfordian of north-

central Florida than was previously recognized. Several taxa present at Thomas Farm

also support this paleoclimatic interpretation. Compared to other fossil sites in Florida,

Thomas Farm is relatively depauperate in aquatic testudines and only one rare emydid,

Deirochelys sp., and the tortoise, Hesperotestudo tedwhitei, are known from the site (J.

Bourque, pers. comm.). Additionally, a helodermatid lizard also occurs at Thomas Farm,

which inhabits drier environments today (Bhullar and Smith, 2008; Beck, 2009). Within

the Thomas Farm avifauna, several accipitrid birds are present that most closely

resemble those found in modern-day sub-Saharan Africa (D. Steadman and E. Whiting,

pers. comm.). Their presence at Thomas Farm also supports a fairly dry ecosystem.

Additionally, the δ13C values from the teeth interpreted to be formed after

weaning is fairly high for distinct C3 feeders. This indicates either minor incorporation of

C4 vegetation into the diet or of water-stressed C3 plants. With the supporting evidence

50

from the stable oxygen isotopic data, present fauna, and hypothesized dominance of C3

vegetation over C4 in the Hemingfordian, it appears likely this can be interpreted as

dietary reliance on water-stressed C3 vegetation. This interpretation of the paleoclimate

of Thomas Farm contrasts with the heavily forested, tropical to subtropical interpretation

of Pratt (1990) and suggests a drier and more open early Miocene than previously

proposed.

Two modern horses from the FLMNH Mammalogy collections, one from Lake

City (~40 km northeast of Thomas Farm) and one from Gainesville (~54 km east-

southeast of Thomas Farm), were sampled to compare with calculated δ18Ow from P.

leonensis. Unfortunately, both specimens were collected with little more than locality

data for each and it is unclear whether the specimens were born and raised in Florida.

However, the carbon isotopic signals show a mixed C3-C4 diet for both specimens,

δ13C= -9 to -6‰, which would not be expected from freely grazing horses in Florida.

Therefore, both specimens appear highly influenced by anthropogenic factors.

Nevertheless, the Gainesville specimen does show estimated δ18Ow expected from

estimates of δ18Omw with both samples falling between the -5.4‰ and -2.9‰ estimates

for precipitation in Gainesville (http://www.waterisotopes.org; see Bowen and Wilkinson,

2002; Bowen and Revenaugh, 2003). The Lake City specimen δ18Ow values range from

-5.1‰ and -9.7‰ and may indicate of a horse that wintered in Florida, while living

elsewhere for the remainder of the year.

Because of the wide range of δ18O values from precipitation (~-5‰ to -3‰), river

and spring water (~-4‰ to -3‰), and lake water (~-1.5‰ to 1‰) from modern-day

north-central Florida, the variation in Parahippus leonensis cannot be used to interpret

51

seasonality in Florida for the Hemingfordian. Though the δ18Ow values presented in this

study range from -3‰ to 4‰, this is likely due more to differing water sources for P.

leonensis than to greater seasonal variation in δ18O than in the modern.

No Evidence for Seasonality of Birthing in P. leonensis

Hulbert’s (1984) analysis of mandibles from Thomas Farm Parahippus leonensis

showed no distinct clustering into discrete age-classes. He interpreted this to mean that

births and deaths of P. leonensis did not occur seasonally in the attritional assemblage

that Thomas Farm is thought to have represented. This deviates from modern wild

Equus which typically gives birth in late spring (Nuñez et al., 2010; Keiper and Houpt,

1984). Though the data in this study cannot answer the question of seasonality in death

because the isotopic signatures had mineralized before the organism died, it can

provide further evidence about seasonality in birthing. Although the δ18Ow signatures will

be affected by the water source from which the organism is drinking, as explained

earlier, seasonal differences in δ18O should become clear with a sample size of

sufficient magnitude and the averaging out of the ingestion of different water sources.

Even though the absolute variation may be meaningless, any significant differences

between samples collected at different locations of the tooth row may not be.

For example, in a population that exhibits seasonal birthing, a given sample

location (e.g., the base of m1) will mineralize in each individual at the same time of year.

Therefore, assuming seasonal birthing, the δ18Ow of each sample location should reflect

the annual δ18O signature of ingested water year after year. If this were supported by

the data collected here, a given sample location would be expected to have a

significantly different δ18O signal than other sample positions mineralizing during

different times of the year. As was discussed earlier, no significant differences in δ18Ow

52

between sample locations were observed in the data collected. This supports the

hypothesis that P. leonensis did not give birth seasonally as modern equids usually do.

However, because the seasonal variability of δ18Omw in Florida is low and it appears that

P. leonensis was drinking from various water sources, this interpretation is preliminary.

Further work should investigate this pattern in horses from more northern localities with

greater variability as well as an increased sample size.

Mineralization Patterns

Enamel mineralization patterns in Equus caballus is well known (Hoppe et al,

2004). In modern horses, the m1 apex is first to mineralize shortly after birth with the m2

apex and m3 apex following at approximately 7 and 21 months, respectively. The end of

m1 mineralization (i.e., m1 base sample) occurs at approximately 23 months with the

m2 stopping around 37 months and the m3 at 55 months. This pattern is also observed

in the δ13C data presented here. There is an overall increase in δ13C in the same

pattern that plateaus with the m2 base and m3 base samples. It is debatable whether

the m1 apex is influenced by nursing as its mean δ13C is only 0.2‰ lower than m2 apex

sample and 0.15‰ lower than the m3 apex sample. However, it does seem likely that

the m3 apex sample is influenced by nursing (mean δ13C ~0.6‰ lower than m2 and m3

base samples) even though in Equus it begins mineralizing only two months prior to the

m1 base. Further, more sample intensive work will be needed to resolve where exactly

nursing no longer influences δ13C in P. leonensis.

Most interestingly, it appears the m1 base, or at least the m3 apex, and all earlier

mineralized samples have been influenced by the δ13C of the mother’s milk during

nursing in P. leonensis. In Equus, weaning occurs between about 9 and 15 months. If

the length of enamel mineralization in Equus was conserved in P. leonensis, which is

53

unlikely due to the shorter crown height in P. leonensis, this would place full weaning at

approximately two years. Kurten (1953) estimated full molar eruption of an equid

occurring in the late Hemingfordian slightly after P. leonensis, i.e., Merychippus primus.

The m3 of M. primus was estimated to be fully erupted at three years, with the m2

erupted at two years, and the m1 at one year. The full eruption of the m3 in M. primus is

approximately six months to a year prior to the full eruption of m3 in Equus with the m2

and m3 more or less the same (Hoppe et al., 2004). While the eruptive timing sequence

between Equus and M. primus is likely similar, the time period of enamel mineralization

would likely have been shorter in horses with shorter-crowned teeth, such as P.

leonensis and M. primus. Therefore it is unlikely weaning in P. leonensis occurred a full

year later than in Equus and can perhaps be more easily explained by a decrease in the

length of enamel mineralization over the molar tooth row, with all tooth apices, and

perhaps the entire m1, mineralized prior to the organism being fully weaned. This would

indicate a marked difference from Equus where the m1 continues to mineralize for a full

year after eruption (Hoppe et al., 2004). Further study will need to identify whether these

differences in the weaning signal between P. leonensis and Equus are due to later

weaning, earlier termination in enamel mineralization, or both. As described above, this

may be possible by serially sampling Hemingfordian equids in areas where greater

seasonal variability in δ18O where δ18O can be used as a time signature.

54

Figure 4-1. Graph of δ18O values for north-central Florida. The curve represents annual

estimates for δ18Omw for the latitude, longitude, and elevation at Thomas Farm (http://www.waterisotopes.org; see Bowen and Wilkinson, 2002; Bowen and Revenaugh, 2003). The lightly shaded bar shows δ18O values for springs in the Suwannee River Basin (Katz et al., 1999) and the darker bar represents δ18O values measured from Newnans Lake (Arnold et al., in press). The box and whisker plot and open circles show the range of values estimated in P. leonensis. The Lake City Equus caballus is indicated by grey circles and the Gainesville E. caballus by black. Values that plot higher on the y-axis represent greater influence by evaporation. The secondary y-axis represents equivalent Miocene δ18O values to modern values and differ by 1‰ due to lower ice cover in the Miocene.

55

CHAPTER 5 CONCLUSIONS

The stable isotopic analysis from the Parahippus leonensis paleopopulation at

Thomas Farm reveals several interesting results from both the δ18O and δ13C data

collected. With estimated δ18Ow for P. leonensis being unexpectedly high, with a mean

value of 1.2‰, it is clear that bodies of water surrounding Thomas Farm experienced

high amounts of evaporative depletion of 16O relative to 18O (high E/P). This

interpretation, that Thomas Farm was fairly dry, is supported both by mean δ13C values

of the adult dietary signal around -11.2‰, indicating ingestion of mostly water-stressed

C3 plants or a small portion of C4 grasses, and the presence of avifauna and

herpetofauna often associated with drier climates.

This study supports a previous hypothesis proposed by Hulbert (1984) that P.

leonensis did not exhibit seasonal birthing with no evidence of significant differences in

δ18O relative to sample location. However, this interpretation is preliminary as P.

leonensis was likely ingesting several water sources with varying δ18O signatures and in

area where seasonal variability in δ18Omw is low.

Few samples show any evidence for incorporation of C4 grasses into the diet of

P. leonensis, especially in light of a drier paleoclimatic interpretation where water-

stressed plants can account for much of the lower than expected δ13C from a pure C3

feeder. However, other evidence, such as more hypsodont, cementum-bearing teeth,

preliminary dental microwear analyses, and calculated molar wear rates argue that

grazing was at least partially undertaken by P. leonensis. In conjunction with the data

collected here, it seems more likely C4 grasses had a very low abundance in the

Hemingfordian of Florida and P. leonensis was a C3 mixed feeder.

56

One of the most convincing signals analyzed in the data was the increase of δ13C

over ontogeny. Mean δ13C values show an increase from -12.9‰ to -11.2‰ from the

sample position that first mineralizes to the last enamel to mineralize. The signal is

interpreted to be the due to the relatively higher proportion of lipids, with lower δ13C than

carbohydrates from vegetation, in the diet while nursing. This study shows that weaning

trends can be observed in the deep fossil record from δ13C when analyzed over the

enamel mineralization sequence. The data also show that the nursing signal continues

perhaps throughout the entire m1 mineralization of P. leonensis and certainly the

initiation of enamel mineralization of the m2 and m3. Future studies need to take into

account that δ13C values may contain some nursing signature, potentially further into

enamel mineralization than was originally recognized in some taxa. Because the

nursing signal occurs in sample locations in P. leonensis that are mineralized after

nursing in Equus, it is apparent that either P. leonensis weaned longer than Equus or

enamel mineralization was terminated much sooner following eruption in P. leonensis

than Equus. Further study is needed to indentify which hypothesis is supported.

57

APPENDIX

ISOTOPIC DATA

Table A-1 shows all of the isotopic data collected in the study. Outliers that were

removed are indicated by * due to inferred machine error or because the values plotted

well outside of the values from other values from the same sample location. The tooth

indicated by ** had previously been measured to have extraordinarily high values of

δ13C and thus was resampled. However, it was not included in the analysis because the

sample location differed from all of the other samples. Analytical precision for samples

analyzed on different dates are as follows: For 5/30/2013, δ13C±0.008 and δ18O ±0.084.

For 9/25/2013, δ13C±0.022 and δ18O ±0.037. For 2/28/2014, δ13C±0.031 and δ18O

±0.067. For 3/13/2014, δ13C±0.029 and δ18O ±0.044. UF 1505 was collected from

Gainesville and UF 32653 was collected from Lake City.

58

Table A-1. All isotope data collected from the study. Catalog Number

Species Tooth position Sample location

Date Analyzed

δ13Ccarbonate (V-PDB)

δ18Ocarbonate (V-PDB)

δ18Ophosphate (V-SMOW)

δ18Ow (V-SMOW)

UF 60 P. leonensis m2, left lower Apex 9/25/2013 -12.35 2.03 23.80 2.69 UF 60 P. leonensis m2, left lower Base 9/25/2013 -11.18 1.26 23.01 1.58 UF 40020 P. leonensis m2, right lower Apex 9/25/2013 -12.01 1.71 23.47 2.22 UF 40020 P. leonensis m2, right lower Base 9/25/2013 -11.76 0.88 22.63 1.04 UF 44815 P. leonensis m3, left lower Apex 9/25/2013 -13.11 -0.69 21.04 -1.20 UF 44815 P. leonensis m3, left lower Base 9/25/2013 -11.62 0.04 21.78 -0.15 UF 95364 P. leonensis m1, left lower Apex 9/25/2013 -12.88 0.84 22.59 0.98 UF 95364 P. leonensis m1, left lower Base 9/25/2013 -12.16 -0.96 20.78 -1.57 UF 99392 P. leonensis m2, right lower Apex 5/30/2013 -11.84 2.70 24.47 3.63 UF 155373 P. leonensis m3, right lower Apex 2/28/2014 -12.68 2.47 24.24 3.31 UF 155373 P. leonensis m3, right lower Base 2/28/2014 -12.10 1.07 22.82 1.32 UF 157579 P. leonensis m2, right lower Apex 9/25/2013 -11.20 1.14 22.89 1.42 UF 157579 P. leonensis m2, right lower Base 9/25/2013 -10.21 1.59 23.35 2.05 **UF 158266 P. leonensis p2, left lower Base 2/28/2014 -11.53 1.53 23.29 1.97 UF 158290 P. leonensis m3, right lower Apex 3/13/2014 -13.23 -1.49 20.24 -2.33 UF 158290 P. leonensis m3, right lower Base 3/13/2014 -12.63 -0.91 20.82 -1.50 UF 164767 P. leonensis m2, right lower Apex 5/30/2013 -11.80 1.64 23.39 2.12 UF 164767 P. leonensis m2, right lower Base 5/30/2013 -11.44 1.54 23.30 1.98 *UF 172397 P. leonensis m1, right lower Apex 5/30/2013 -9.23 -0.12 21.62 -0.38 *UF 172397 P. leonensis m1, right lower Base 5/30/2013 -9.39 -0.01 21.73 -0.23 UF 176616 P. leonensis m1, left lower Apex 9/25/2013 -13.09 0.54 22.29 0.56 UF 176616 P. leonensis m1, left lower Base 9/25/2013 -10.96 0.60 22.35 0.65 UF 192280 P. leonensis m3, left lower Apex 5/30/2013 -11.70 0.38 22.13 0.33 UF 192280 P. leonensis m3, left lower Base 5/30/2013 -11.75 0.81 22.56 0.94 UF 192310 P. leonensis m3, left lower Apex 9/25/2013 -11.75 1.04 22.79 1.27 UF 192310 P. leonensis m3, left lower Base 9/25/2013 -10.92 2.90 24.68 3.92 UF 201702 P. leonensis m3, left lower Apex 5/30/2013 -10.86 1.98 23.75 2.62 UF 201702 P. leonensis m3, left lower Base 5/30/2013 -11.33 1.53 23.29 1.98 UF 203391 P. leonensis m3, left lower Base 5/30/2013 -11.42 1.45 23.21 1.86 UF 203391 P. leonensis m3, left lower Apex 2/28/2014 -11.06 2.24 24.01 2.98 UF 213777 P. leonensis m3, right lower Apex 9/25/2013 -12.45 2.47 24.24 3.31 UF 213777 P. leonensis m3, right lower Base 9/25/2013 -11.69 1.60 23.36 2.06 UF 214560 P. leonensis m1, right lower Apex 5/30/2013 -12.92 0.78 22.53 0.90 UF 214560 P. leonensis m1, right lower Base 2/28/2014 -12.41 2.12 23.88 2.81 UF 214590 P. leonensis m1, right lower Apex 5/30/2013 -12.73 1.83 23.59 2.39 UF 214590 P. leonensis m1, right lower Base 5/30/2013 -10.42 0.45 22.20 0.43 UF 214867 P. leonensis m2, right lower Apex 5/30/2013 -11.96 0.20 21.95 0.08

59

Table A-1. Continued Catalog Number

Species Tooth position Sample location

Date Analyzed

δ13Ccarbonate (V-PDB)

δ18Ocarbonate (V-PDB)

δ18Ophosphate (V-SMOW)

δ18Ow (V-SMOW)

UF 214867 P. leonensis m2, right lower Base 5/30/2013 -11.72 1.28 23.04 1.62 UF 215280 P. leonensis m3, left lower Apex 5/30/2013 -10.88 0.73 22.48 0.84 UF 215280 P. leonensis m3, left lower Base 5/30/2013 -10.22 0.08 21.82 -0.09 UF 215289 P. leonensis m2, right lower Apex 5/30/2013 -12.22 0.60 22.35 0.65 UF 215289 P. leonensis m2, right lower Base 5/30/2013 -10.42 0.00 21.75 -0.20 UF 215308 P. leonensis m3, left lower Apex 9/25/2013 -11.52 0.48 22.23 0.48 UF 215308 P. leonensis m3, left lower Base 9/25/2013 -11.29 1.89 23.65 2.48 UF 215783 P. leonensis m2, left lower Apex 9/25/2013 -12.13 1.06 22.81 1.30 UF 215783 P. leonensis m2, left lower Base 9/25/2013 -11.85 2.00 23.77 2.64 UF 216291 P. leonensis m1, right lower Apex 5/30/2013 -12.59 2.73 24.50 3.67 UF 216291 P. leonensis m1, right lower Base 5/30/2013 -10.43 0.02 21.76 -0.18 *UF 257391 P. leonensis m2, right lower Apex 5/30/2013 -9.55 1.13 22.88 1.40 UF 257391 P. leonensis m2, right lower Base 5/30/2013 -10.87 2.56 24.33 3.43 UF 257785 P. leonensis m2, left lower Apex 9/25/2013 -11.49 1.76 23.52 2.30 *UF 257785 P. leonensis m2, left lower Base 9/25/2013 -12.35 1.96 23.72 2.58 UF 258694 P. leonensis m1, right lower Apex 5/30/2013 -12.76 2.95 24.73 3.99 UF 258694 P. leonensis m1, right lower Base 5/30/2013 -11.70 1.16 22.91 1.44 UF 258802 P. leonensis m3, left lower Base 5/30/2013 -9.43 2.31 24.08 3.08 UF 259493 P. leonensis m1, right lower Base 5/30/2013 -11.82 -2.12 19.60 -3.22 UF 259493 P. leonensis m1, right lower Apex 2/28/2014 -13.73 1.78 23.54 2.33 UF 259495 P. leonensis m1, left lower Apex 9/25/2013 -13.85 1.69 23.45 2.19 UF 259495 P. leonensis m1, left lower Base 9/25/2013 -12.51 0.16 21.91 0.02 UF 262997 P. leonensis m3, left lower Base 5/30/2013 -9.51 0.98 22.74 1.19 UF 262997 P. leonensis m3, left lower Apex 2/28/2014 -11.23 1.29 23.05 1.63 UF 269846 P. leonensis m1, right lower Apex 9/25/2013 -12.36 0.51 22.25 0.51 UF 269846 P. leonensis m1, right lower Base 2/28/2014 -11.00 1.40 23.16 1.78 UF 270907 P. leonensis m2, right lower Apex 5/30/2013 -12.62 0.14 21.88 -0.01 UF 270907 P. leonensis m2, right lower Base 5/30/2013 -10.78 1.77 23.53 2.31 UF 276773 P. leonensis m3, left lower Apex 9/25/2013 -11.40 1.24 22.99 1.55 UF 276773 P. leonensis m3, left lower Base 9/25/2013 -10.82 2.27 24.04 3.03 FGS 6427 P. leonensis m3, left lower Apex 5/30/2013 -10.85 0.58 22.33 0.62 FGS 6427 P. leonensis m3, left lower Base 5/30/2013 -11.27 0.48 22.23 0.47 FGS 6441 P. leonensis m1, left lower Apex 9/25/2013 -11.92 0.82 22.57 0.96 FGS 6441 P. leonensis m1, left lower Base 9/25/2013 -10.99 1.57 23.33 2.03 FGS 6749 P. leonensis m1, left lower Apex 9/25/2013 -12.54 1.59 23.35 2.06 FGS 6749 P. leonensis m1, left lower Base 9/25/2013 -11.46 1.12 22.88 1.39 FGS 7171 P. leonensis m3, left lower Apex 9/25/2013 -11.80 2.13 23.89 2.82

60

Table A-1. Continued Catalog Number

Species Tooth position Sample location

Date Analyzed

δ13Ccarbonate (V-PDB)

δ18Ocarbonate (V-PDB)

δ18Ophosphate (V-SMOW)

δ18Ow (V-SMOW)

FGS 7171 P. leonensis m3, left lower Base 9/25/2013 -11.46 1.15 22.90 1.43 FGS 7172 P. leonensis m3, left lower Apex 9/25/2013 -11.83 1.65 23.41 2.14 FGS 7172 P. leonensis m3, left lower Base 9/25/2013 -11.52 0.58 22.33 0.62 FGS 11004 P. leonensis m2, left lower Apex 9/25/2013 -12.43 1.80 23.56 2.35 FGS 11004 P. leonensis m2, left lower Base 9/25/2013 -11.18 1.91 23.67 2.51 UF 1505 E. caballus m1, left lower Apex 2/28/2014 -6.01 -3.25 18.46 -5.83 UF 1505 E. caballus m1, left lower Base 2/28/2014 -6.93 -5.96 15.72 -9.69 UF 1505 E. caballus m2, right lower Apex 2/28/2014 -8.91 -4.57 17.13 -7.71 UF 1505 E. caballus m2, right lower Base 2/28/2014 -6.80 -3.87 17.83 -6.71 UF 1505 E. caballus m3, right lower Apex 2/28/2014 -8.91 -2.71 19.01 -5.06 UF 32653 E. caballus M1, left upper Base 2/28/2014 -5.96 -2.17 19.55 -4.29 UF 32653 E. caballus M2, left upper Base 3/13/2014 -7.39 -2.50 19.22 -4.76

61

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BIOGRAPHICAL SKETCH

Sean Moran was born in 1988 in Sewell, NJ. The oldest of five children, he

graduated from Gloucester Catholic High School in Gloucester, NJ, just across the river

from the great city of Philadelphia. Sean spent much of his free time in high school

volunteering at the Academy of Natural Sciences in Philadelphia and traipsing around

Monmouth County, NJ creeks with his father and younger siblings collecting shark teeth

and other Cretaceous fossils. He started his undergraduate career at the College of

William and Mary in 2007. Sean was awarded with a Roy R. Charles Center Honors

Fellowship which culminated in the writing of his honors thesis, “The Paleoecology,

Taphonomy, and Depositional Environment of Vertebrate Microfossil Bonebeds from the

Late Cretaceous Hell Creek Formation in Garfield County, Montana.” He graduated

from William and Mary with honors in geology in 2011. Sean received his M.S. in

geology in 2014. He and his brother, Ryan, plan to celebrate the end of their formal

education by spending six weeks driving to national parks across Colorado, Utah,

Wyoming, Montana, and South Dakota. Plans outside of the immediate future are more

uncertain.