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
4 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 120322 2 , 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 th ose listed below, who provided assistance, whether l ending 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, Mic hal 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 t he many other people who have provided support .
5 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 13C .................................................................................................... 18 A Review of 18O .................................................................................................... 19 Research Questions ............................................................................................... 20 2 MATERIALS AND M ETHODS ................................................................................ 24 Sample Selection .................................................................................................... 24 Isotope Sample Collection ...................................................................................... 25 Sample P retreatment .............................................................................................. 25 Sample Analysis ..................................................................................................... 26 3 RESULTS ............................................................................................................... 28 13C: Corrections .................................................................................................... 28 18O: Conversions and Corrections ........................................................................ 28 13C: Results ........................................................................................................... 30 18Ocarbonate: Results ................................................................................................ 31 18Ow: 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
6 Mineralization Patterns ........................................................................................... 52 5 CONCLUSIONS ..................................................................................................... 55 APPENDIX A ISOTOPIC DATA .................................................................................................... 57 LIST OF REFERENCES ............................................................................................... 61 BIOGRAPHICAL SKETCH ............................................................................................ 70
7 LIST OF TABLES Table page 3 1 18Ow (V SMOW) estimates for the four known equations . ................................ 34 3 2 13C (VPDB) data for each of the six sample locations ..................................... 34 3 3 18Oca rbonate (V PDB) data for each of the six sample locations .......................... 34 3 4 18Ow (V SMOW) data for each of the six s ample locations ............................... 35 A 1 A ll isotope data collected from the study ............................................................ 58
8 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 13C fractionation in vegetation and herbivores. All values given are in reference to V PDB ......................................................................... 23 2 1 UF 258802 .......................................................................................................... 27 3 1 18Ophosphate 18Ow ................. 36 3 2 18Ow estimates between equations 33 to 36 ............................................................................................ 37 3 3 18Ow 13C ...................................................... 38 3 4 13C by sample location ................................................ 39 3 5 13C values of each sample position compared with a resampled distribution .......................................................................................................... 40 3 6 1313Capex base. ............................................................ 41 3 7 13Capex base values. ............................... 42 3 8 18Ow values. .............................................. 43 4 1 18O values for northcentral Florida. ................................................... 54
9 LIST OF ABBREVIATIONS E/P Ratio of evaporation to precipitation FGS Specimens collected by the Florida Geological Survey, now in collectio ns 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 premol ar UF D enotes specimens housed in the collections of the Florida Museum of Natural History at the University of Florida
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Re quirements for the Degree of Master of Science STABLE ISOTOPE PALEOECOLOG Y 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 13C trend through ontogeny. This i s interpreted as a nursing signal and influenced mean 13C in four of the six sample locations examined. Mean values of the adult 13C signals in the base samples of m2s and m3s indicate a diet of C3 with either substanti al incorporation of water stressed C3 plants or a very minor C4 component. Evidence of a diet based on
11 water stressed vegetation is corroborated by estim ates the 18O value of water ingested by P. leonensis . These values are significantly higher than modern precipitation and river water. They more closely resemble 18O of modern lake water in northcentral Florida. The indication of a high evaporation to precipitation ratio indicates a drier paleoclimate than was previously recognized for Thomas Farm.
12 CHAPTER 1 INTRODUCTION Background Stable isotope ratios have been heavily utilized in vertebrate paleontology to elucidate a range of paleoecological variabl es 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 par ticular interest to this study are stable carbon 13C) and oxygen 18O) isotope ratios , which are m ost 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; LeeThorp 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 ( LeeThorp 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 i ntrataxonomic 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 q uestions pertaining to the paleoecology and local paleoclimate. Thomas Farm is widely regarded as one of the most i mportant early Miocene vertebrate fossil localities in the eastern United States due to the high abundance and diversity of fossil s collected from the site. Three equid taxa cooccur 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
13 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; Schla ikjer, 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, i n addition to preliminary dental microwe ar ( Solounias and Semprebon, 2002 ) and mesowear (Mihlbachler et al., 2011) analyses, has le d 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 t he rise and spread of C4 grasses and the origination of grasslands in North America (e.g., Edwards et al., 2010) . Documenting 13C values of P. leonensis tooth enamel extends the available data spatially and temporally and has the potential to better const rain our understanding of the spread of C4 grasses. The Thomas Farm S ite 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 11 ) . Vertebr ate 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 1 8 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
14 (1984) interpreted Thomas Farm as a woodland environment (i.e., mostly forested with some open patches). The validity of Hulbertâ€™s reconstruction, which was based on P. leonensis social structure, h as been questioned by Oâ€™Sullivan (2005) who suggested Thomas Farm was more open than previous ly recognized. F irst 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 , a s 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; Rom er, 1948; Puri and Vernon, 1964), a stream fed sinkhole system ( Bader, 1956), a water filled sinkhole (Simpson, 1932, Estes, 1963; Pratt, 1990), and a sinkholestream cave complex (Olsen, 1959, 1962; Auffenberg, 1963a, 1963b). The most recent and compl ex 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 Crysta l River and Suwannee f ormation s. 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 .
15 Perhaps one of the most important aspects of Thomas Farm to this study is the proposed rapid deposition of the site. Pratt (1990) hypot hesized 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) ex trapolation 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 ev olving taxa such as e quids and h eteromyid s. 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; Oâ€™Sullivan, 2005). If th is 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 E quid 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 brows er 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 ( Oâ€™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
16 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 g razing beginning in the early Miocene and seen as a bridge between the two ecotypes (e.g., Hulbert and MacFadden, 1991; MacFadden, 2005) . P reliminary dental microwear analyses show an abundance of fine scratches on the occlusal surface of Parahippus sp. m olars (Solounias and Semprebon, 2002). Scratches have been interpreted as an endmember 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 largescale 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 trans itional paleopopulation. Stable I sotopes 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 condi tions ( see Clementz , 2012 for a review). Stable oxygen isotopes ( 18O ) have been used as proxies to investigate past climatic conditions ( e.g., Nunez et al., 2010; Higgins and MacFadden, 2004; Koch et al.,
17 1989). Strontium isotope ratios (87Sr/86Sr) have b een used to consider migrat ory patterns in extinct organisms, such as mammoths and mastodons (e.g., Hoppe et al., 1999). Stable nitrogen ratios ( 1 5N) have been used to infer trophic levels of organisms (e.g, Bocherens et al., 1994) . 13C, stable carbon ratios, h as 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; MacF adden et al., 1999) . The stable isotope ratios of carbon, 13C, and oxygen, 18O 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 18O of standard mean ocean water ( i.e., V SMOW) 13C of belemnites from the PeeDee Formation in South Carolina (i.e., V PDB) ). The formula for calculating delta values is as follows: = R R R 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 val ues 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 13C Over the past few decades , ratios of stable carbon isotopes 13C) have become a valuable proxy in reconstructing ancient diet (e.g., Wang et al., 1994; MacFadden and Cerling 1996) and paleoe colog y (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 us i ng the HatchSlack cycle . Alternatively, C3 plants, which comprise approximately 90% of modern plants such as most trees, shrubs, and highlatitude grasses, employ the Calvin cycle. The HatchSlack cycle, in comparison with the Calvin cycle, supports pref erential uptake of the heavier isotope, 13C, into the plant tissue 13C values (Farquahar et al., 1989) 13C for C4 plants range from 15 to 11 , with a mean of 13 , and C3 plants range from 32 to 24 , wit h 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 13C values (v an 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 13C values (Farquhar et al., 1989; Cerling et al., 2004). The foundation for reconstructing 13C values in C4 and C3 plants and subsequent ingestion by the organism .
19 The ingestion of the vegetation imparts 13C 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 1313*dietenamel) 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). dietenamel of 14.1 as has been shown for equids and other largebodied mammals. Therefore, a horse ingesting a pure C3 13C of 13C signal in the enamel hydroxylapatite of 13.9 ; and a horse ingesting a pure C4 diet with a mean 13C of 11 would have a 13C value of 3.1 (Figure 12) . A Review of 18O 18O values of organism sâ€™ body water have been shown to correlate directly with the 18O of ingested water which is in turn related to the 18O of local precipitation (Longinelli , 1984; Luz et al., 1984; Luz and Kolodny, 1985; Bryant and Froelich, 1995; Bryant et al., 1 996a ; 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) . 18O recorded in the biological apatite can be informative as 18O values of precipitation ( 18Omw) 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 18Omw values, whereas a relative depletion in 18O causes lower 18Omw in colder months (McCrea, 1950; Bryant et al., 1996a ; Fricke and Oâ€™Neil, 1996; Feranec and MacFadden, 2000). Additionally, in times of significant precipitation
20 and temperatures greater than 20C the â€œamount effectâ€ can also be a major factor in 18Omw 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 18O values. This phenomenon leads to a pattern of marked decrease in 18Omw due to the preferential depletion of 18O over 16O (Higgins and MacFadden, 2004). Increasing altitude, latitude, and distance from the coast can cause 18O to decrease , but often are only pertinent in comparing modern 18O to past 18O when these factors have shifted significantly during the relevant time span (D ansgaard, 1964; Rozanski et al, 1993) . These compounding factors often m ake 18O more difficult to interpret than 13C with regard to vertebrate paleontology. Research Q uestions The goal of this study is to investigate the stable isotope paleoecology of an important, transitional taxon. P reliminary data (e.g., Soulonias and Se mprebon, 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 paleoec ological reconstruction than has been previously possible. Additionally, m any 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 , 1318O values which are useful in reconstructing aspects of past climatic conditions. Because enamel mineralization of the
21 equid mol ars encompasses several years ( 18O sampled from different locations on cheek teeth has previously be en interpr eted as a re cord of seasonal variability in stable oxygen isotope values (e.g., Nunez et al., 2010) . 18O values can be compared to modern values, in this case from modern horse 18O in meteoric water ( 18Omw) based on latitude, longitude, and elevation ( http://www.waterisotopes.org ; see Bowen and Wilkinson, 2002; Bowen and Revenaugh, 2003) as well as 18O 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: 13C 1. Is there evidence for the presence of C4 grazing in P. leonensis ? 2. 13C 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 ? 18O 4. Did P. leonensis b irth seasonally? 5. How 18Omw from the Miocene of Thomas Farm differ from that observed today? What does this mean about differing climatic conditions during the Hemingfordian and modernday Flori da?
22 Figure 11. Ma p 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 113C 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 ve getation 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 T homas 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 ( o ne 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 m ineralization is conserved from modern Equus to P . leonensis , which has been supported by Hulbert (1984) and Kurten (1953) , sampling m 1s, m 2s, and m 3s records the isotopic signal for the entire range of enamel mineralization (Hoppe et al. , 2004). To the gr eatest extent possible, m 3s were chosen from the same side of mandible to reduce the chances that a single individual was sampled twice. T his was not always possible, due to limitations imposed by the large, but not infinite sample. Furthermore, isolated m 1s and m 2s cannot be distinguished. Therefore, m 1 s and m 2s 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 m 1 and m 2 were present i n the same associated dentition, only one of the teeth was sampled to maximize the number of indiv iduals sampled. Because there was a limited number of associated dentitions that included m 1s or m 2s that were only slightly worn, samples from m 1 and m 2 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 s everal 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 cement um or underlying dentine (Figure 21 ) . 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 micr ocentrifuge 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 al lowed 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 s amples were analyzed at the Department of Geological Sciences Light Stable Isotope Mass Spec trometer 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 FinneganMAT 252 isotope ratio mass spectrometer for 13C and 18Ocarbonate 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 18Ocarbonate and 0.04 for 13C .
27 Figure 21. 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 13C: Corrections Arens et al. (2000 13CCO2 accounts for over 98% of the 13C in vegetation. Ther e fore, a correction of 0.5 was added to all 1313CCO2 between the prei ndustrial average and the Hemingfordian age of Thomas Farm (Tipple et al., 2010). 18O: Conversions and C orrections 1818O of modern 18Omw) in northcentral Florida, several conver sions were needed. S amples were first converted from the standard V PDB to VSMOW using the equation (Hoefs, 1997; Criss, 1999): 18 O carbonate (V 18 O carbonate (V PDB) + 30.86 ( 3 1) Iacumin et al. (1996) showed a strong relationship (r2= 0 .98) in modern mammals 1818Ocarbonate) and that analyzed from enamel phosphate 18Ophosphate). Assuming this relationship holds true for isotopic samples from the fossil record, it can be used to calculate 18O from the carbonate phase to the phosphate phase using the equation: 18 O phosphate (V 18 O carbonate (V SMOW) 8.5 ( 3 2) 18Ophosphate and the 18O of ingested water 18Ow) in mo dern 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 18Ophosphate 18Ow estimates for each location: 18 O phosphate (V 18 O w (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 (r218Ow from each of the locations: 18 O phosphate (V 18 O w (V SMOW) ( 3 4) Delgado Huertas et al. (1995) 18Ophosphate data of E. caballus 18Ow estimates, to the dataset of Sanchez Chillon et al. (1994) to derive the following equation (r2= 0.90): 18 O phosphate (V SMOW) = 22. 2 9 + 0.7 2 18 O w (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 ta xonomically diverse equid samples , was used for this study : 18 O w (V 18 O phosphate (V SMOW) â€“ 22.60) / 0.71 ( 3 6) Table 3 1 18Ow estimates based on which of the above four relationships are used. Because most precipi tation is ultimately derived from seawater, one final 18Ow to that measured from P. leonensis . 18Osw would have been more enriched in 16O relative to V SMOW
30 due to the absence of polar ice s 18Osw must be inferred for the evaporative source of precipitation (Zachos et al., 2001) . To ensure equivalent 18Osw 18Ow, 1 was added to the calculated 18Ow of the Miocene samples to account for this 18Osw was approximately 1 lower than modern seawater (Lear et al. , 2007; Billups and Schrag, 2003). 13C: R esults 13C collected from Parahippus leonensis tooth enamel carbonate provi de values ranging from 13.85 to 9.43 with a mean of 11.70 (V PDB) (Table 32; Figure 33) . A n 13C for each sample position indicates significant differences exist among sample positions (p<0.001) (Figure 3 4) . I empl oyed a resampling technique to further elucidate the differences indicated by the ANOVA. 13C values were pooled and randomly attributed to a sample location while keeping the sample size of each sample location constant. The randomized data were compi led and means for each sample location calculated iteratively over 10,000 runs. Figure 3 5 shows the resulting graph of the randomized data with m eans of the resampled means for each sample location, actual means, and 95% confidence intervals plotted. All red points fall outside (i.e., m 1 apex, m 2 base, and m 3 base) of the 95% confidence envelope of the resampled distribution. Therefore, the null hypothesis that the data indicated in 13C 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 onetailed 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 ther e is no significant directionality. All but 13C values from the apex to the base with the mean increase being 0.87. An ANOVA shows significant differences due to tooth location in 13Capex base (p =0.003) 13C (p=0.04) . The nonparametric Kruskal Wallis test, however, shows only significance in 13Capex base (p =0.006) and not mean 13C (p=0.06) , though the calculated pvalues differ only slightly from the ANOVA . Figure 3 6 13Capex base) o 13C from apex to base) plotted by mean 13C values. The first tooth to erupt (i.e., m1) shows a more 13C 13C signature than do that the latter erupting teeth (i.e., m2 and m3) 13Capex base. The multivariate, two sample Hotellingâ€™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. 18Ocarbonate: R esults 18Ocarbonate 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 33) . An ANOVA indicates no significant differences present for samples collected at dif ferent sample locations (p=0.20). The mean difference between the apex and base 18Oapex 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 ( O nesample t test,
32 p=0.2291) in the directionality of the differences with 15 specimens showing decreases 18O from the apex to the base and 20 specimens showing increases. An ANOVA fails to reject the null hypothesis that there are no significant dif ferences in 18Oapex base (p=0.07) or mean (p=0.73) between tooth positions. 18Ow: R esults 18Osw and converting to estimated 18Ow (i.e., equations 3 1, 3 2, and 3 6 ), values range from 3.99 to 3.23 (V SMO W) with a mean of 1.45 (Table 34; Figures 33; 38) . Specimens show absolute 18Oapex base variation between 0.08 to 5.55, however only four specimens show 18Oapex base greater than 2.5. The mean magnitude of change between apex and base samples is 1.46 18Ow calculated from the other relationships (i.e., equations 3 3, 3 4, 3 5 ) have means ranging from 1.03 to 2.20. 1818Omw) 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 measu18O of river water to the Thomas Farm locality were taken from various springs in the Suwannee River Basin (Katz et al. 1999). Un fortunately the data provide 18O as all data w ere collec ted in July or August of 1997 and 1998. Nevertheless, the data generally reflect 18Omw with all values from the Santa Fe River springs, Lafayette County springs, Lower Suwannee springs, and Suwannee County springs fall ing between 3.94 and 3.03 These values are consistent with the summer values estimated by the Online Isotopes 18Omw ranges from 3.8 to 3.1. Arnold e t al .
33 (in press ) report 18O 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 18Omw and 18O 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 18Ow values reflective of 18Omw, between 4.8 and 4 .3 , the Lake City specimen, while the Gainesville specimen showed a range in estimated 18Ow from 9.7 to 5.1. Diag enesis 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 13C values ( 5.23 and 5.77) (C. Manz, pers. comm.). The corresponding 18Ocarbonate values ( 1.22 and 0.08) of these samples were lower than most of the samples collected here. This indicates that 18O values at Thomas Farm would likely decrease, rather than increase, when affected by diagenesis with the lower 18O values of local precipitation being the main driver. Because the 18Ow 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 33 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 13C, rather than homogenization of 13C values from different sample locations , further supports a lack of diagenetic impact on the samples.
34 Table 318Ow ( V SMOW) estimates (per mil) for the four known equations. Bryant et al., 1994 is equation 33. S anchez Chillon et al., 1994 is equation 34. Delgado Huertas et al., 1995 is equation 35. All data refer to the relationship calculated from the combined data of the three above references (equation 36). Equation Mean Maximum Minimum Range S D 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 32 . 13C (VPDB) data (per mil) for each of the six sample locations. Sample Location n Mean Maximum Minimum Range S D m 1 Apex 11 12.85 11.92 13.85 1.93 0.56 m 2 Apex 11 12.00 11.20 12.62 1.42 0.41 m 3 Apex 15 11.76 10.85 13.23 2.38 0.79 m 1 Base 11 11.44 10.42 12.51 2.09 0.74 m 2 Base 10 11.14 10.21 11.86 1.65 0.57 m 3 Base 16 11.19 9.43 12.63 3.20 0.86 Total 7 4 11.70 9.43 13.85 4.42 0.88 Table 33 18Ocarbonate (V PDB) data (per mil) for each of the six sample locations. Sample Locati on n Mean Maximum Minimum Range Std m 1 Apex 1 1 1. 46 2.95 0.51 2.44 0.85 m 2 Apex 1 1 1.34 2.70 0.14 2.56 0.80 m 3 Apex 1 5 1.10 2.47 1.49 3.96 1.15 m 1 Base 11 0.50 2.12 2.12 4.24 1.21 m 2 Base 1 0 1.48 2.56 0.00 2.56 0.70 m 3 Base 16 1.14 2.90 0.91 3.81 0.97 Total 7 4 1. 17 2.95 2.12 5.07 0.9 9
35 Table 34 18Ow ( V SMOW) data (per mil) for each of the six sample locations. 18O of ingested water were calculated using equation 3 6. Sample Location n M ean M aximum M inimum R ange SD m 1 Apex 11 1.87 3.99 0.51 3.48 1.20 m 2 Apex 11 1.70 3.64 0.01 3.65 1.13 m 3 Apex 15 1.36 3.31 2.33 5.64 1.64 m 1 Base 11 0.51 2.81 3.23 6.0 4 1.73 m 2 Base 10 1.90 3.43 0.20 3.6 3 0.99 m 3 Base 16 1.42 3.92 1.50 5.4 2 1.39 Total 7 4 1.45 3.99 3.23 7.22 1.41
36 Figure 31. Plot showing the linear relationship 18Ophosphate 18Ow. All four equations show similar relationship with increased disagreement with 18Ophosphate 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 32. 18Ow estimates between equations 33 to 36. The values on the y axis are in relation to V SMOW and are in per mil.
38 Figure 33. 18Ow 13C. Points are coded by toot h 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 wa 18O influenced by evaporation.
39 Figure 34. 13C 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 13C due to decreased consumption of lipids from the motherâ€™s milk (i.e., weaning).
40 Figure 35. 13C 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 13C population. Sample locations are ordered from mineralization starting earliest in ontogeny to latest.
41 Figure 36. Plot of m 13C 13Capex base. The grey symbols reflect the samples an alyzed and the black dots show the centroids for each tooth posit ion. Notice the increasing means from m1s to m3s as well as the larger 13C 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 37. Histogram showing the 13Capex 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 13Capex base values . The values here show a distribution consistent with an increase in 13C from the apex to base samples in the analyzed teeth.
43 Figure 38. Box and whisker plot for estimated 18Ow values. The overlapping values at each sample position indicate no noticeable differences for estimates 18O 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 F eeding in P arahippus 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 hypot hesis of a mixed feeder 13C 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 significa13C 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 e nrichment of 18O in this sample can be explained by feeding on water stressed C3 plants, which is further corroborated by the 18O values. Though the data collected here do not support the hypothesis of a C4 or mixed C3C4 diet for P. leonensis , it d oes 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 C3C4 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 h ypothesis would be supported by specialized browsers with low crowned teeth, C3 grazers with highcrowned teeth, C3C4 grazers later in the Miocene, and C4 grazers . The second hypothesis states decreased temperatures in the middle to late Miocene led t o 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 decreas ed C3 biomass. This second hypothesis w ould be supported by taxa with three distinct characteristics: low crowned teeth with a C3 signature, highcrowned teeth with an intermediate C3C4 signature, and highcrowned teeth with a C4 signature. In Florida, t his 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 indicatin g somewhat open habitats with P. leonensis grazing on C3 resources . Patterns of W eaning Several studies have identified patterns of weaning in isotopic signatures, particularly of 151318O , from the fossil record ( Bryant et al, 1996 b ; Rountrey
46 et al. 2007). Stable nitrogen isotope ratios are most often analyzed from collagen, but d ue to its high 15N analysis for diet is often restricted to the Pleistocene or younger ( Bocherens et al., 1994). Therefore, in the deep fossil record only 13C , and in some cases 18O , can be utilized for investigation into weaning. The 13C data collected in this study strongly suggests an influence of weaning and feeding on motherâ€™s milk during the mineralization of several of the sample locations. Studies of the stable isotopic signature of modern animals identified an 1315N trend from bir th until the end of weaning (e.g., Hobson and Sease, 1998; Balasse et al., 2001). 15N signal of tissue accreted while an organism is feeding on its motherâ€™s milk is hypothesized to be more negative than after due to the socalled trophic level effect, as the offspring is effectively positioned one trophic stage above its mother (Rountr e y et al., 2007; Hobson and 13C values in juveniles feeding from the motherâ€™s milk than individuals feeding independently from the mother are likely due to the incorporation of relatively more lipids , whic h have a lower 13C 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 13C 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 13C trend in the sampled population as a whole. However, it is worth noting that only 4 of the 13C , all of which were m3s and potentially fully weaned during enamel mineralization. The mean increase in 13C 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) . Fut u r e isotopic studies need to understand the enamel mineralization sequence of the study organi sm and the potential enrichment in 12C during weaning. In this study , the tooth apex samples of the oft 13C 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 13C 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 18O associated with weaning. Bryant et a l. ( 1996 b) used a mass balance model for to identify potential patterns in 18O over the period of weaning. They noted the model showed higher 18O values in m1s than in m2s and m3s for foals born in spring and lower 18O 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 18O may be due to low
48 annual variation in 18O values for Florida or due to drinking sources of highly variable 18O values as discussed below. Seasonality recorded 18O of accreted tissue in fossil organisms has been well documented ( e.g., Fox et al., 2007; Fricke and Oâ€™Neil, 1996) . Serial sampling over the span of enamel mineralization 1818O) signals which can be used as time signatures if the general pattern of enamel 18O variability is high, it is hypothetically possible to identify 18O 13C signatures over the molar tooth row of an individual . The low seasonal variability 18O in Florida makes such analysis of precisely timing enamel mineralization difficult in the Thomas Farm specimens. Paleoclimatic I nterpretations of Thomas Farm 18Ow in Parahippus leonensis were surprisingly high with a mean of 1.2 when compared to m odern estimates for 18Omw a t 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 18O , e vaporative effects 18O 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 18O 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 18Omw 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 18O of 1.5 to 1 with an average of 0.5 (Arnold et al., in press ) . These lake values are much closer to t 18Ow 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 h18Ow 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 neglecti ng river, spring, and precipitated water, 18O signature than measured from Newnans Lake. These data support a drier environment in the Hemingfordian of northcentral 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 aquat ic 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 ac cipitrid birds are present that most closely resemble those found in modernday subSaharan Africa ( D. Steadman and E. Whiting, pers. comm. ). Their presence at Thomas Farm also supports a fairly dry ecosystem. Additionally, 13C 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 M iocene 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 18Ow from P . leonensis . Unfortunately, both specimens were collected with little more than locality data for each and i t is unclear whether the specimens were born and raised in Florida. H owever , the carbon isotopic signals show a mixed C3C4 diet for both specimens, 13C= 9 to 6, which would not be expected from freely grazing horses in Florida. Therefore, both specimens appear highly influenced by anthropogenic f actors . Nevertheless, the Gainesville specimen does show 18Ow expected from es 18Om w 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 La18Ow values range from 5.1 and 9.7 and may indicate of a horse that wintered in Florida, while living els ewhere for the remainder of the year. 18O values from precipitation (~ 5 to 3), river and spring water (~ 4 to 3), and lake water (~ 1.5 to 1) from modernday northcentral Florida, the variation in Parahippus leonensis cannot be used to interpret
51 18Ow 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 18O than in the modern. No E vidence for S easonality of B irthing in P. leonensis Hulbert â€™s (1984) analysis of mandibles from Thomas Farm Parahippus leonensis showed no distinct clustering into discrete ageclasses. 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. Thi s deviates from modern wild Equus which typically gives birth in late spring ( Nu ez et al., 2010 ; Keiper and Houpt , 1984). Though the data in this study cannot answer the questi on of seasonality in death because the isotopic signatures had mineralized before the organism died, it can 18Ow signatures will be affected by the water source from which the organism is drinking, as explained 18O should become cl ear 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 m 1) will mineralize in each individual at the same time of year. Therefore, assuming s 18Ow of each sample location should reflect 18O 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 18O signal than other sample positions mineralizing during different times of the year. 18Ow
52 between sample locations were observed in the data collected. This supports the hypothesis that P. leonens is did not give birth seasonally as modern equids usually do. However, b 18Omw in Florida is low and it appears that P. leonensis was drinking from various water sources, this interpretation is preliminary. Further work s hould 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 caball u s is well known (Hoppe et al, 2004). In moder n horses, t he 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 stoppi ng around 37 months and the m3 at 55 months. This pattern is also observed 1313C 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 13C 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 13C ~0.6 lower than m2 and m3 base samples) even th ough 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 13C in P. leonensis . Most interestingly, it appears the m1 base, or at leas t the m3 apex, and all earlier 13C of the motherâ€™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 i n 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 mol ar 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 18O where 18O can be used as a time signature.
54 Figure 41. Graph of 18O values for northcentral Florida. The curve represents annual estimates for 18Omw for the latitude, l ongitude, and elevation at Thomas Farm ( http://www.waterisotopes.org ; see Bowen and Wilkinson, 2002; Bowen and Revenaugh, 2003). The lightly shaded bar shows 18O values for springs in the Suwannee River Basin ( Katz et al., 1999) and the darker bar represents 18O 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 gr ey 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 18O values to modern values and differ by 1 due to lower ice cover i n the Miocene.
55 CHAPTER 5 CONCLUSIONS The stable isotopic analysis from the Parahippus leonensis paleopopulation at 1813C data 18Ow 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 b13C 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 support s a previous hypothesis proposed by Hulbert (1984) that P. leonensis did not exhibit seasonal birthing with no evidence of significant differences in 18O relative to sample location. However, this interpretation is preliminary as P. leonensis was likely i 18O signatures and in area where seasonal variability 18Omw 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 13C 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 On 13C 13C 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 interprete13C than carbohydrates from vegetation, in the diet while nursing. This study shows that weaning 13C when analyzed over the e namel 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 acc 13C values may contain some nursing signature, potentially further into enamel mineralization than was originally recognized in some taxa. Because the nursing sign al occurs in sample locations in P. leonensis that are mineralized after nursing i n 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 locat ion. The tooth indicated by ** had previously been measured to have extraordinarily high values of 13C and thus was resampled. However, it was not included in the analysis because the sample location differed from all of the other samples. Analytical prec ision for samples analyzed on different dates are as follows: For 5/30/2013, 1318O 0.084. 13181318O 1318O 0.044. UF 1505 was collected from Gainesville and UF 32653 was collected from Lake City.
58 Table A 1 . A ll isotope data collected from the study. Catalog Number Species Tooth position Sample location Dat e Analyzed 13 C carbonate (V PDB) 18 O carbonate (V PDB) 18 O phosphate (V SMOW) 18 O w (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. leonensi s 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, rig ht 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. leonens is 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 13 C carbonate (V PDB) 18 O carbonate (V PDB) 18 O phosphate (V SMOW) 18 O w (V SMOW) UF 214867 P. leon ensis 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 A pex 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, lef t 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 2.3 3 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. leonens is 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 13 C carbonate (V PDB) 18 O carbonate (V PDB) 18 O phosphate (V SMOW) 18 O w (V SMOW) FGS 7171 P. leonensis m3, left lower Base 9/25/2013 1 1.46 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 1 1004 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 lowe r 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 LIST OF REFERENCES Ambrose, S.H., Norr, L., 1993. Experimental evidence for the relationship of the carbon isotope ratios of whole diet and dietary protein to those of bone collagen and carbonate. In: Lambert, J.B., Groupe, G. (Eds.). Prehistoric Human Bone: Archaeology at the Mo lecular Level. Springer, Berlin, pp. 137. Arens, N.C., Jahren, A.H., Amundson, R., 2000. Can C3 plants faithfully record the carbon isotopi c compositio n of atmospheric carbon dioxide? Paleobiology 16, 137164. Arnold, T.E., Brenner, M., Curtis, J.H., Dutton, A., Baker, S.M., Escobar, J.H., Ortega, C.A., in press. Application of stable isotopes ( 18O) to determine growth patterns of the invasi ve gastropod Pomacea maculata in Florida lakes. Florida Scientist. Auffenberg, W., 1963a. Present problems about the past: biological sciences curriculum study. University of Florida Pamphlet 6, 135. Auffenberg, W., 1963b. The fossil snakes of Florida. Tulane Studies in Zoology 10, 131216. Bader, R.S., 1956. A quantitative study of the Equidae of the Thomas Farm Miocene. Museum of Comparative Zoology Bulletin 115, 4978. Balasse , M., Bocherens, H., Mariotti, A., Ambrose, S.H., 2001. Detection of dietary changes by intratooth carbon and nitrogen isotopic analysis: and experimental study of dentine collagen of cattle ( Bos taurus ). Journal of Archaeological Science 28, 235245. Bard, E., Delaygue, G., Rostek, F., Antonioli, F., Silenzi, S., Schrag, D.P., 200 2. Hydrological conditions over the western Mediterranean basin during the deposition of the cold Sapropel 6 (ca. 175 kyr BP). Earth and Planetary Science Letters 202, 481494. Beck, D.D., 2009. Biology of Gila Montsters and Beaded Lizards. University of C alifornia Press, Berkeley , CA. Bhular, B.A., Smith, K.T., 2008. Helodermatid lizard from the Miocene of Florida, the evolution of the dentary in Helodermatidae, and comments on dentary morphology in Varanoidea. Journal of Herpetology 42, 286302. Billups , K., Schrag, D.P., Application of benthic foraminiferal Mg/Ca ratios to questions of Cenozoic climate change. Earth and Planetary Science Letters 209, 181 195. Bocherens , H., Fizet, M., Mariotti, A., 1994. Diet, physiology and ecology of fossil mammals as i nferred from stable carbon and nitrogen isotope biogeochemistry: implications for Pleistocene bears. Palaeogeography, Palaeoclimatology, Palaeoecology 107, 213225.
62 Bocherens, H., Koch, P.L., Mariotti, A., Geraads, D., Jaeger, J.J.,1996. Isotopic biogeochemistry (13C, 18O) of mammalian enamel from African Pleistocene hominid sites. Palaois 11, 3063 18. Boutton, T.W., 1991. Stable carbon isotope ratios of natural minerals: II. Atmospheric, terrestrial, marine and freshwater environments. In: Coleman, D.C., Fry, B. (Eds.), Carbon Isotope Techniques. Academic Press, San Diego, pp. 173195. Bowen, G.J., Revenaugh, J., 2003. Interpolating the isotopic composition of modern meteoric precipitation. Water Resources Research 39, 337348. Bowen, G.J., Wilkinson, B., 18O in meteoric precipitation. Geology 30, 315â€“ 3 18. Brenner, M., Hodell, D.A. , Curtis, J.H. , Rosenmeier, M.F. , Anselmetti, F.S. , Ariztegui , D., 2003. Paleolimnological approaches for inferring past climate change in the Maya region: recent advances and methodological limitations. In : Gomez Pompa, A., Fedick , S. ( Eds .), Lowland Maya Area, Three Millennia at the HumanWi ldland Interface. Haworth Press, Inc., New York, pp. 4576 Bryant, J.D., Froelich, P.N., 1995. A model of oxy gen isotope fractionation in body water of large mammals. Geochimica et Cosmochimica Acta 59, 45234537. Bryant, J.D., Froelich, P.N., Showers, W.J., Genna, B.J., 1996 a . Biologic and climatic signals in the oxygen isotopic composition of EoceneOligocene equid enamel phosphate. Palaeogeography, Palaeoclimatology, Palaeoecology 126, 7589. Bryant, J., Froelich, P., Showers, W., Genna, B., 1996b. A tale of two quarries: biologic and taphonomic signatures in the oxygen isotopic composition of tooth enamel phosphate from modern and Miocene equids. Palaios 11, 397408. Bryant, J.D., Luz, B., Froelich, P.N., 1994. Oxygen isotope compos ition of fossil horse tooth phosphate as a record of continental paleoclimate. Palaeogeography, Palaeoclimatology, Palaeoecology 107, 303316. Ce rling, T.E., Harris, J.M., 1999. Carbon isotope fractionation between diet and bioapatite in ungulate mammals and implications for ecological and paleoecological studies. Oecologia 120, 347363. Cerling, T.E., Hart, J.A., Hart, T.B., 2004. Stable isotope ecology in the Ituri Forest. Oecologia 138, 512. Clementz, M., 2012. New insights from old bones: stable isotope analysis of fossil mammals. Journal of Mammalogy 93, 368380. Criss, R.E., 1999. Principles of Stable Isotope Distribution. O xford University Press, New York, p. 254.
63 Dansgaard, W., 1964. Stable isotopes in precipitation. Tellus 16, 436468. Delgado Huertas, A., Iacumin, P., Stenni, B., Sanchez Chillon, B., Longinelli, A., 1995. Oxygen isotope variations of phosphate in mammal ian bone and tooth enamel. Geochimica et Cosmochimica Acta 59, 42994305. DeNiro, M.J., Epstein, S., 197 7 . Mechanism of carbon isotope fractionation associated with lipid synthesis. Science 197, 261263. Dienes, P., 1980. The isotopic composition of reduced organic carbon. In: Fritz , P ., Fontes , J. C ., (Ed s. ) Handbook of E nvironmental I sotopic Geochemistry 1, Elsevier, New York , pp 329â€“ 406 Downs, T., 1956. The Mascall Fauna from the Miocene of Oregon. University of California Publications in Geological Sciences 31, 199354. Edwards, E.J., Osborne, C.P., Stromberg, C.A.E., Smith, S.A., C4 Grasses Consortium, 2010. The origins of C4 grasslands: integrating evolutionary and ecosystem science. Science 328, 587591. Estes , R., 1963. Early Miocene lizards and salamanders from Florida. Quarterly Journal of the Florida Academy of Sciences 26, 234256. Farquhar, G.D., Ehleringer, J.R., Hubick, K.T., 1989. Carbon isotopic discrimination and photosynthesis. Annual Review Plant Physiology. Plant Molecular Biology 40, 503537. Feranec, R.S., MacFadden, B.J., 2000. Evolution of the grazing niche in Pleistocene mammals from Florida: evidence from stable is otopes. Palaeogeography, Palaeo climatology, Palaeoecology 162, 155169. Forsten, A., 1975. The fossil horses of the Texas Gulf Coastal Plain: A r evision. Pierce Sellards Series of the Texas Memorial Museum 22, 187. Fox , D.L, Koch, P.L., 2004. Carbon and oxygen isotopic variability in Neogene paleosol carbonates: constraints on the evolution of the C4grasslands of the Great Plains, USA. Palaeogeography, Palaeo climatology, Palaeoecology , 207, 305329. Fox , D.L., Fisher, D.C., Vartanyan, S., Tikhonov, A.N., Mol, D., Buigues, B., 2007. Paleoclimatic implications of oxygen isotopic variation in late Pleistocene and Holocene tusk s of Mamm u thus primigenius from northern Eurasia. Quaternary International 169170, 154165. Fricke, H.C., O'Neil, J.R., 1996. Interand intratooth variation in the oxygen isotope composition of mammalian tooth enamel phosphate: implications for palaeocli matological and palaeobiological research. Palaeogeography, Palaeoclimatology, Palaeoecology 126, 9199.
64 Higgins, P., MacFadden, B.J., 2004. â€œAmount Effectâ€ recorded in oxygen isotopes of Late Glacial horse ( Equus ) and bison ( Bison ) teeth from the Sonoran and Chihuahuan deserts, southwestern United States. Palaeogeograp hy, Palaeoclimatology, Palaeoecology 206, 337353. Hobson, K.A., Sease, J.L., 1998. Stable isotope analyses of tooth annuli reveal temporal dietary records: an example using Stellar sea lions. Marine Mammal Science 14, 116129. Hodell, D.A., Curtis, J.H., Jones, G.A., Higuera Gundy, A., Brenner, M., Binford, M.W., Dorsey, K.T., 1991. Reconstruction of Caribbean climate change over the past 10,500 years. Nature 352, 790793. Hoefs, J., 1997. Stable Isotope Geochemistry. Springer Verlag, Berlin. Hoppe, K.A., Koch, P.L., Carlson, R.W., Webb, S.D., 1999. Tracking mammoths and mastodons: reconstructing of migratory behavior using strontium isotope ratios. Geology 27, 439442. Hoppe, K.A., Stover, S.M., Pascoe, J.R., Amundson, R., 2004. Tooth and enamel biomineralization in extant horses: implications for isotopic microsampling. Palaeogeography, Palaeoclimatology, Palaeoecology 206, 355365. Hulbert Jr., R.C., 1984. Paleoecology and population dynam ics of the early Miocene (Hemingfordian) horse Parahippus leonensis from the Thomas Farm Site, Florida. Journal of Vertebrate Paleontology 4, 547558. Hulbert Jr., R.C., 2001. The Fossil Vertebrates of Florida. Universit y Press of Florida, Gainesville , FL. Hulbert Jr., R.C., MacFadden, B.J., 1991. Morphological transformation and cladogenesis at the base of the adaptive radiation of Miocene hypsodont horses. American Museum Novitates 3000, 161. Iacumin, P., Boccherini, H., Mariotti, A., Longinelli, A., 1996. Oxygen isotope analyses of coexisting carbonate and phosphate in biogenic apatite: a way to monitor diagenetic alteration of bone phosphate. Earth and P lanetary Science Letters 142, 16. Janis, C.M., Damuth, J., Theodor, J.M., 2000. Miocene ungulates a nd terrestrial primary productivity: where have all the browsers gone? PNAS 97, 78997904. Katz , B.G., Hornsby, H.D., Bohlke, J.F., Mokray, M.F., 1999. Sources and chronology of nitrate contamination in spring waters, Suwannee River Basin, Florida. U.S. Ge ological Survey Water Resources Investigations Report 994252, 154. Keiper , R., Houpt , K., 1984. Reproduction in feral horses: an eight year study. American Journal of Veterinary Research 45, 991995.
65 Koch, P.L, Fisher, D.C., Dettman, D., 1989. Oxygen iso tope variation in the tusks of extinct proboscideans: a measure of season of death and seasonality. Geology 17, 515519. Koch, P.L., Tuross, N., Fogel, M.L., 1997. The effects of sample treatment and diagenesis on the isotopic integrity of carbonate in biogenic hydroxylapatite. Journal of Archaeological Science 24, 417429. Kohn, M.J., 1996. Predicting animal 18O : accounting for diet and physiological adaptation. Geochemica et Cosmochimica Acta 60, 4811â€“ 4829. Kurten, B., 1953. On the variation and populati on dynamics of fossil and recent mammal populations. Acta Zoologica Fennica 76, 1122. Laury , R.L., 1980. Paleoenvironment of a late Quaternary mammothbearing sinkhole deposit, Hot Springs, South Dakota. Geological Society of America Bulletin 91, 465475. Lear , C.H., Elderfield, H., Wilson, P.A., 2000. Cenozoic deepsea temperatures and global ice volumes from Mg/Ca in benthic foraminiferal calcite. Science 287, 269272. LeeThorp, J.A., Van der Merwe, N.J., 1987. Carbon isotope analysis of fossil bone apatite. South African Journal of Science 83, 7174. LeGrande, A.N., Schmidt, G.A., 2006. Global gridded data set of the oxygen isotopic composition in seawater. Geophysical Research Letters 33, 15. Lohman,K.C., 1987. Geochemical patterns of meteoric diagenetic systems and their application to studies of paleokarst. In: James, N.P., Choquette, P.W. (Eds.), Paleokarst. Springer Verlag, New York, pp. 5880. Longinelli, A., 1984. O xygen isotopes in mammal bone phosphate: a new tool for paleohydrological and paleoclimatological research? Geochimica et Cosmochimica Acta 48, 385â€“ 390. Luz, B., Kolodny, Y., 1985. Oxygen isotope variations in phosphate of biogenic apatites, IV. Mammal teeth and bones. Earth and Planetary Science Letters 75, 29â€“ 36. Luz , B., Kolodny, Y. , Horowitz, M., 1984. Fractionation of oxygen isotope variations in phosphate of deer bones. Geochimica et Cosmochimica Acta 54, 16891693. MacFadden, B.J., 2001. Threetoed browsing horse Anchitherium clarencei from the Early Miocene (Hemingfordian) Thomas Farm, Florida. Bulletin of the Florida Museum of Natural History 43, 79 109. MacFadden, B.J., 2005. Fossil horses --evidence for evolution. Science 307, 17281730.
66 MacFadden, B.J., Bloch, J.I., Evans, H., Foster, D.A., Morgan, G.S., Rincon, A., Wood, A.R. , 2014. Temporal calibration and biochronology of the Centenario Fauna, early Miocene of Panama. The Journal of Geology 122, 113135. MacFadden, B.J., Cerling, T.E., 1996. Mammalian herbivore communities, ancient feeding ecology and carbon isotopes: a 10 m illion year sequence from the Neogene of Florida. Journal of Vertebrate Paleontology 16, 103115. MacFadden, B.J., Cerling, T.E., Harris, J.M., Prado, J., 1999. Ancient latitudinal gradients of C3/C4 grasses interpreted from stable isotopes of New World Pleistocene horse ( Equus ) teeth. Global Ecology and Biogeography 8, 137149. MacFadden, B.J., Higgins, P., 2004. Ancient ecology of 15million year old browsing mammals within C3 plant communities from Panama. Oecologia 140, 169182. Maguire, K.C., Stigall, A.L., 2008. Paleobiogeography of Miocene Equidae of North America: a phylogenetic biogeographic analysis of the relative roles of climate, vicariance, and dispersal. Palaeogeography, Palaeoclimatology, Palaeoecology 267, 175184. McCrea, J.M., 1950. On th e isotopic chemistry of carbonates and a paleotemperature scale. Journal of Chemical Physics 18, 849â€“ 857. Mihlbachler, M.C., Rivals, F., Solounias, N., Semprebon, G.M., 2011. Dietary change and evolution of horses in North America. Science 331, 11781 181. Nu ez, C.M.V., Adelman, J.S., Rubenstein , D.I., 2012. Immunocontraception in wild horses ( Equus caballus ) extends reproductive cycling beyond the normal breeding season. PLoS ONE 5, e13635. Nunez, E.E., MacFadden, B.J., Mead, J.I., Baez, A., 2010 . Ancient forests and grasslands in the desert: diet and habitat of Late Pleistocene mammals from Northcentral Sonora, Mexico. Palaeogeography, Palaeoclimatology, Palaeoecology 297, 391400. Olsen , S.J., 1959. Fossil mammals of Florida. Florida State Geological Sur vey Special Publications 6, 175. Olsen , S.J., 1962. The Thomas Farm fossil quarry. Quarterly Journal of the Florida Academy of Sciences 25, 142146. Oâ€™Sullivan, J.A., 2005. Population dynamics of Archaeohippus blackbergi (Mammalia; Equidae) from the Miocene Thomas Farm fossil site of Florida. Bulletin of the Florida Museum of Natural History 45, 449463. Passey, B.H., Cerling, T.E., 2002. Tooth enamel mineralization in ungulates: implications for recovering a primary isotopic time series. Geochimica et Cos mochimica Acta 66, 32253234.
67 Passey, B.H., Cerling, T.E., Perkins, M.E., Voorhies, M.R., Harris, J.M., Tucker, S.T., 2002. Environmental change in the Great Plains: an isotopic record from fossil horses. Journal of Geology 110, 123140. Passey, B.H., Robinson, T.F., Ayliffe, L.K., Cerling, T.E., Sponheimer, M., Dearing, M.D., Roeder, B.L., Ehleringer, J.R., 2005. Carbon isotope fractionation between diet, breath CO2, and bioapatite in different mammals. Journal of Archaeological Science 32, 14591470. Prat t, A.E., 1990. Taphonomy of the large vertebrate fauna from the Thomas Farm locality (Miocene, Hemingfordian), Gilchrist County, Florida. Bulletin of the Florida Museum of Natural History 35, 35130. Puri, H.S., Vernon, R.O., 1964. Summary of the geology of Florida and a guidebook to the classic exposures. Florida State Geological Survey Special Publications 5, 1312. Quade, J., Cerling, T.E., Barry, J.C., Morgan, M.E., Pilbeam, D.R., Chivas, A.R., Lee Thorp, J.A., Van der Merwe, N.J., 1992. A 16Ma record of paleodiet using carbon and oxygen isotopes in fossil teeth from Pakistan. Chemical Geology 94, 183 192. Romer, A.S., 1948. The fossil mammals of Thomas Farm, Gilchrist County, Florida. Quarterly Journal of the Florida Academy of Sciences 10, 111. Rou ntrey , A.N., Fisher, D.C., Vartanyan, S., Fox, D.L., 2007. Carbon and nitrogen isotope analyses of a juvenile woolly mammoth tusk: evidence of weaning. Quaternary International 169170, 166173. Rozanski, K., Aragus Aragus, L., Gonfiantini, R., 1993. Isotopic patters in modern global precipitation. In: Swart, P.K., Lohmann, K.C., McKenzie, J., Savin, S. (Eds.), Climate Change in Continental Isotopic Records. American Geophysical Union Geophysical Monograph, vol. 78. American Geophysical Union, Washington, DC, pp. 1 36. Sanchez Chillon, B., Laberdi, M.T., Leone, G., Bonadonna, F.P., Stenni, B., Longinelli, A., 1 994. Oxygen isotopic composition of fossil equid tooth and bone phosphate: an archive of difficult interpretation. Palaeogeography, Palaeoclimatology, Palaeoecology 107, 317328. Schlaikjer, E.M.,1937. A study of Parahippus wyomingensis and a discussion of the phylogeny of the genus. Bulletin of the Museum of Comparative Zoology 80, 255280. Schmidt , G.A., Bigg, G.R., Rohling, E.J., 1999. Global seaw ater oxygen18 database â€“ v1.21. http://data.giss.nasa.gov/o18data/.
68 Sellards, E.H., 1916. Fossil vertebrates from Florida: a new Miocene fauna; new Pliocene species; the Pleistocene fauna. Annual Report of the Florida State Geological Survey 8, 77119. Simpson, G.G., 1932. Tertiary land mammals from Florida. Bulletin of the American Museum of Natural History 59, 149211. Solounias, N., Semprebon, G., 2002. Advances in the reconstruction of ungulate ecomorphology with application to early fossil equids. American Museum Novitates 3366, 149. Straight, W.H., Barrick, R.E., Eberth, D.A., 2004. Reflections of surface water seasonality and climate in stable oxygen isotopes from tyrannosaurid tooth enamel. Palaeogeography, Palaeoclimatology, Palaeoecology 206, 239256. Stromberg, C.A.E., 2006 . Evolution of hypsodonty in equids: testing a hypothesis of adaptation. Paleobiology 32, 236258. Teaford, M.F., 1988. A review of dental microwear and diet in modern mammals. Scanning Microscopy 2, 11491166. Tedford, R.H., Frailey, D., 1976. Review of some Carnivore (Mammalia) from the Thomas Farm Local Fauna (Hemingfordian: Gilchrist County, Florida). American Museum Novitates 2610, 19 Tedford, R.H., Skinner, M.F., Fields, R.W., Rensberger, J.M., Whistler, D.P., Galusha, T. , Taylor, B.E., Macdonald, J.R., and Webb, S.D., 1987. Faunal succession and biochronology of the Arikareean through Hemphillian interval (late Oligocene through earliest Pliocene epochs) in North America. In: Woodburne, M.O. (Ed.), Cenozoic Mammals of Nor th America: Geochronology and Biostratigraphy. University of California Press, Berkeley, CA, pp. 153210. Tipple, B.J., Meyers, S.R., Pagani, M., 2010. Carbon isotope ratio of Cenozoic CO2: a comparative evaluation of available geochemical proxies. Paleoceanography 25, pa3202. Van der Merwe, N.J., Medina, E., 1989. Photosynthesis and 13C/12C ratios in Amazonian rain forests. Geochemica et Cosmochimica Acta 53, 10911094. Wang, Y., Cerling, T.E., 1994. A model of fossil tooth and bone diagenesis: implications for paleodiet reconstruction from stable isotopes. Palaeogeography, Palaeoclimatology, Palaeoecology 107, 281289. White, T.E., 1942. The lower Miocene mammal fauna of Florida. Bulletin of the Museum of Comparative Zoology 92, 149. Zachos , J.C., Pagani, M., Sloan, L., Thomas, E., Billups, K., 2001. Trends, rhythms, and aberrations in global climate 65 Ma to present. Science 292, 686693.
69 Zazzo, A., Lecuyer, C., Sheppard, S.M.F., Grandjean, P., Mariotti, A., 2004. Diagenesis and the reconstruction of paleoenvironments: a method to restore 18O values of carbonate and phosphate from fossil tooth enamel. Geochemica et Cosmochimica Acta 68, 22452258.
70 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 i n 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, â€œThe Paleoecology, Taphonomy, and Depositional Environment of Vertebrate Microfossil Bonebeds from the Late Cretaceous Hell Creek Formation in Garfield County, Montana.â€ He gra duated from W illiam 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, Wyo ming, Montana, and South Dakota. Plans outside of the immediate future are more uncertain.