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USING STABLE CARBON ISOTOPE, MICROWEAR, AND MESOWEAR
ANALYSES TO DETERMINE THE PALEODIETS OF NEOGENE UNGULATES
AND THE PRESENCE OF C4 OR C3 GRASSES IN NORTHERN AND CENTRAL
JONATHAN M. HOFFMAN
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
Jonathan M. Hoffman
I would like to thank my advisor, Dr. Jonathan Bloch for his guidance on this
proj ect. I would also like to thank my committee members: Dr. David Hodell, Dr.
Richard Hulbert, and Dr. Andrew Zimmerman. Their suggestions and advice are greatly
appreciated. In addition to their helpful advice, Dr. Bloch, Dr. Hulbert, and Dr.
Zimmerman all aided in collecting fossil samples. I would like to thank Art Poyer and
Jeremy Green for their help in collecting samples. I am especially appreciative of the
Englehard Corporation and Dave Mihalik for allowing me to collect fossils from their
mines and being incredibly helpful at those sites.
I would like to thank Dr. Penny Higgins for her assistance in learning the
sampling techniques for stable isotope analysis of fossil teeth and the chemical protocol
for preparing those samples for analysis. I am greatly indebted to Dr. Jason Curtis for
running my samples on the mass spectrometer and for all of his advice.
My fellow graduate students aided me with discussion and advice. I am very
appreciative of P.J. Moore, Warren Grice, Jane Gustayson, and Derrick Newkirk for
listening and providing support. Finally, I would like to thank my family, who has always
supported me and encouraged me to pursue my passions.
TABLE OF CONTENTS
ACKNOWLEDGMENT S ............. ...... .............. iii...
LIST OF TABLES ............. ...... ..............vi...
LI ST OF FIGURE S .............. .................... vii
AB STRAC T ................ .............. ix
1 INTRODUCTION ................. ...............1.......... ......
2 FIELD AREA AND FAUNA ................. ...............17........... ...
3 MESOWEAR METHOD .............. ...............29....
Material s and Method s .............. ...............3 1....
Re sults............. ...... ._ ...............32...
4 MICROWEAR METHOD .............. ...............40....
Materials and Methods .............. ...............41....
Re sults............. ...... ._ ...............44...
5 STABLE ISOTOPE ANALYSIS .............. ...............48....
Back ground ............. ...... ._ ...............48...
Materials and Methods .............. ...............54....
Re sults............. ...... ._ ...............57...
6 DI SCUS SSION ............. ...... .__ ...............67..
7 CONCLUSIONS .............. ...............80....
A MESOWEAR VALUES LISTED BY SAMPLE................ ...............82
B SERIAL STABLE ISOTOPE VALUES .............. ...............86....
LIST OF REFERENCES ................. ...............88................
BIOGRAPHICAL SKETCH ................. ...............102......... ......
LIST OF TABLES
2-1 Biochronological ranges of the Willacoochee Creek Fauna. ............. ........._......23
3-1 Systematic and morphological information about the taxa used in the mesowear
analy si s. .............. ...............3 5....
3-2 Observed percentages of mesowear attributes (% high and % low refer to the
percentage of specimens with high or low occlusal relief and % sharp, % round,
and % blunt refer to the percentage of specimens with sharp, round or blunt cusp
shape). ............. ...............36.....
3-3 Dietary classifications based on hierarchical cluster analyses of mesowear
attributes from Table 3-2 ................. ...............36........... ...
4-1 Individual microwear counts for 3 specimens ofAcritohippus isonesus and 7
specimens of Neohipparion trampa~sense. ............. ...............46.....
4-2 Calculated average microwear values and percentages for Acritohippus isonesus
and Neohipparion trampasense.............. ...............4
4-3 Isotopic data for equids from 4 central Florida sites. ............... ...................4
5-1 List of the 24 specimens analyzed for stable carbon and oxygen isotope analysis..60
5-2 Bulk stable carbon and oxygen isotope values for Willacoochee Creek Fauna.......61
5-3 Descriptive statistics of the 613C and 68 values for 8 herbivores from the
Willacoochee Creek Fauna. ........... ....._.._ ...............62...
6-1 Isotopic data for equids from 4 central Florida sites. ............... ...................7
A-1 Abbreviations: UF ID = catalogue number in Florida Museum of Natural
History collections,............... ..............8
B-1 Abbreviations: ELC = Englehard La Camelia Mine, MGF = Milwhite Gunn
Farm Mine, and LC2 = La Camelia 2 Mine. .............. ...............86....
LIST OF FIGURES
2-1 Index map of middle Miocene localities in northern Florida. ........._._... ........._.....22
2-2 Composite section of the Torreya Formation and the location of the
Willacoochee Creek Fauna within the Dogtown Member. .............. ...................24
2-3 Correlated stratigraphic sections of the Englehard La Camelia and Milwhite
Gunn Farm Mines............... ...............25.
2-4 Fresh cut at the newest site, the Crescent Lake Mine, in Decatur County,
G eor gia. .............. ...............26....
2-5 Stratigraphic and temporal distribution of the 3 fossil sites studied in the
mesowear analysis, as well as a fourth (Moss Acres) ................ ..........__........27
2-6 Tooth positions. .............. ...............28....
3-1 Dendrogram illustrating the hierarchical cluster analysis of the 27 'typical'
grazers, browsers, and mixed feeders from Fortelius and Solounias (2000). ..........34
3-2 Examples of typical mesowear attributes ................. ...............35...............
3-3 Dendrograms illustrating the hierarchical cluster analyses of 6 studied taxa
amongst the 27 'typical' grazers, browsers, and mixed feeders from Fortelius
and Solounias (2000)............... ...............37.
5-1 Schematic of isotopic fractionation between atmospheric carbon and C3 plants,
as well as fractionation between C3 plants and ruminant herbivores. ......................59
5-2 Plot of bulk 813C VS. 6180 values for the 8 herbivores from the Willacoochee
Creek Fauna. ........... ..... ..._. ...............63.....
5-3 613C ValUeS for serial samples of two Aphelops specimens. ................ ................64
5-4 6 O0 values for serial samples of two Aphelops specimens. ................ ................64
5-5 613C ValUeS for serial samples of two M~erychippus primus specimens. ................65
5-6 6 O0 values for serial samples of two M~erychippus primus specimens. ................65
5-7 613C ValUeS for serial samples of two Acriohippus isonesus specimens. .................66
5-8 68 O values for serial sampling of two Acritohippus isonesus specimens. ..............66
6-1 Plot of microwear index versus 613C ValUeS. ................ ............... .............78
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
USING STABLE CARBON ISOTOPE, MICROWEAR, AND MESOWEAR
ANALYSES TO DETERMINE THE PALEODIETS OF NEOGENE UNGULATES
AND THE PRESENCE OF C4 OR C3 GRASSES IN NORTHERN AND CENTRAL
Jonathan M. Hoffman
Chair: Jonathan I. Bloch
Major Department: Geology
Traditionally, hypsodont (high-crowned) teeth in North American ungulates
(hoofed mammals) were thought to have coevolved with grasses during the middle
Miocene. Isotopic evidence has demonstrated that tropical C4 graSses were not dominant
and therefore not abundant enough to be responsible for this adaptive radiation. It has
been proposed that high-altitude C3 graSses were extensive throughout the Great Plains
and were the dietary driving force behind the grazing adaptations. This study will test
this hypothesis to see if it applies to the middle Miocene of the Southeastern United
States. The 613C ValUeS from 24 specimens of 8 ungulate taxa from the Willacoochee
Creek Fauna, an assemblage of middle Miocene mammals from northern Florida and
southern Georgia, are presented here to determine if there is a significant C4 graSS
component in mammalian paleodiets. The 613C ValUeS indicate that all 8 taxa were
consuming C3 plant material, either browse or grass. Furthermore, microwear analyses
conducted on 3 specimens of the most hypsodont taxon indicate that the mammal was
eating grass. Combined with the 613C data, this study concludes that C3 graSSCS WeTO
present in the middle Miocene of northern Florida and at least one hypsodont mammal
was consuming them. This evidence supports a C3 graSs hypothesis of hypsodont
radiations. Also, this study combines the mesowear paleodietary analysis with previously
published isotopic data to 5 equid populations from 3 sites in central Florida, ranging in
age from ~9.5 Ma to ~1.5 Ma, to trace the possible influence of C3 graSses on ungulate
diets. C3 graSses were the primary food source for these horses until approximately 7
Ma. After that, the horses fed on a mixed diet of C3 and C4 graSses until about 1.5 Ma. At
that point in Florida, the abundance of C3 graSses had diminished and the grazers
primarily fed on C4 graSSCS.
Today, over 25% of North American natural biomes are nonforest (Webb, 1977).
The modern vegetation of North America is a sharp contrast to vegetation at the
beginning of the Cenozoic, when nearly all of North America was covered with forests
(Webb, 1977). Traditionally, paleontologists have believed that, beginning in the
Paleocene, parts of North America underwent a stepwise progression from forest to
savanna to grassland biomes (Webb, 1977). For most of the Paleocene, North America
was dominated by evergreen forests and cypress swamps (Wolfe, 1985; Wing and
Tiffney, 1987). It has been proposed that grasses originated amongst this vegetation in
the early Paleocene, although no direct evidence of grasses has been found in sediments
of that age (Linder, 1986; Crepet and Feldman, 1991). Nearly all the mammals in the
Paleocene belong to four orders: the Multiuberculata, Insectivora, Primates, and
'Condylarthra' (Webb and Opdyke, 1995). These mammals were small to medium in size
and were arboreal or scansorial (Webb and Opdyke, 1995). The multituberculate
Ptilodus, for example, possesses morphological adaptations consistent with those of a
modern tree squirrel, suggestive of an arboreal habit (Jenkins and Krause, 1983).
The grassland transitional sequence began meekly, during the late Paleocene with
the appearance of possible protosavannas in scattered open-country areas that constitute
breaks in the forest coverage (Webb, 1977). In the late Paleocene Crazy Mountain Field
of Montana, Simpson (1937) found that 90% of the fossils collected from floodplain
sediments consisted of carnivores and archaic ungulates such as perptychids,
phenacodontids, and arctocyonids. In the same area, taxa collected from swampy
woodland deposits were typically smaller, arboreal mammals (Simpson, 1937). A similar
pattern is apparent in late Paleocene and early Eocene sediments of the Rocky Mountain
intermontane basins. The late Paleocene Fort Union Formation from that region consists
of gray sediments indicative of swampy woodlands bounded by alluvial plains (Van
Houten, 1945; Bown, 1980). Small arboreal mammals have been collected from these
sediments, corroborating the environmental interpretation of woodlands (Van Houten,
1945). The early Eocene Willwood Formation, located in the same area, exhibits red-
banded flood plain sediments and ungulates suited for open-country habitation (Van
Houten, 1945; Bown, 1980; Hickey, 1980; Wing, 1980). Van Houten (1945) concluded
that the late Paleocene Rocky Mountain woodlands had, by the early Eocene, developed
flood plain savannas that existed in breaks in the forest.
A number of subtle modifications are evident in probable open-country taxa during
the late Paleocene and early Eocene. The condylarth M~eniscotherium exhibits molarized
premolars, molar crescents, and some cursorial limb elongation (Gazin, 1965), attributes
that improved the mastication of coarser vegetation and open-country locomotion. In the
Torrej onian North American Land Mammal Age (NALMA) of the middle Paleocene,
three orders of larger Asian immigrants arrived in North America: the Pantodonta,
Taeniodonta, and Dinocerata (Webb and Opdyke, 1995). Titanoides, a late Paleocene
pantodont, bears digging forelimbs that would have excavated coarse roots (Webb, 1977).
Taeniodonts, large clawed opossum-like root grabbers, were the first mammals to
develop hypsodont (high-crowned) teeth (Patterson, 1949). Crested molars and molarized
premolars are also seen at this time in the uintatheres (Order Dinocerata), which bear
"hornlike protuberances" indicative of herding behavior seen in open-country ungulates
Global climate fluctuated during the Paleocene. Deciduous tree populations began
increasing at about 63 Ma, indicative of cooling climate (Rose, 1981). This cooling trend
began near the boundary of the Torrej onian and Tiffanian and continued through the
Tiffanian, accompanied by the disappearance of small mammals and an increased
presence of larger mammals (Webb and Opdyke, 1995). The Tiffanian cooling trend was
followed by the initial appearance of open-country habitats that coincides with a global
warming trend at the end of the Tiffanian (Koch, Zachos, and Gingerich, 1992). This
warming trend began in the middle Paleocene (about 59 Ma) and peaked and ended with
the early Eocene Climatic Optimum (EECO, 52-50 Ma) (Zachos, Pagani, Sloan, Thomas,
and Billups, 2001). At northern latitudes, the peak mean annual temperature in the early
Eocene was between 250C and 300C, 150C to 200C warmer than today (Novacek, 1999).
The warming trend and EECO are marked by a 1.5%o decrease in 68 O values from
benthic foraminifera, the lowest such 68 O values in the Cenozoic (Zachos et al., 2001).
This global warmth had a maj or impact on both North American floral and faunal
communities. Between 55 and 53 Ma, the number of macrofloral species doubled in the
Paleocene/Eocene of the Bighorn Basin (Wing, Alroy, and Hickey, 1995). Early Eocene
fossil leaves exhibit "drip-tips" and smooth margins indicative of a tropical/subtropical
climate (Wolfe, 1978; Prothero, 1994). The evergreen forests of the Paleocene had given
way to early Eocene subtropical forests, while still maintaining some open-country
enclaves (MacGinitie, 1974; Rose, 1981; Bown and Krause, 1981, 1987).
The beginning of the Clarkforkian (latest Paleocene), at about 56 Ma, is marked by
the first maj or immigration episode of Asian mammals capable of exploiting the new
North American open-country niches (Webb and Opdyke, 1995; Lofgren, Lillegraven,
Clemens, Gingerich, and Williamson, 2004). The newly arrived Asian orders included
the Tillodontia and Rodentia, as well as the pantodont family Coryphodontidae (Rose,
1981; Krause and Maas, 1990). Many of these mammals had adapted to grazing lifestyles
in Asia. For example, Coryphodon, one of the Asian pantodonts that arrived in North
America in the late Paleocene, is believed to have already been a hippo-like amphibious
grazer that, based on canine grooves, rooted for food (Simons, 1960). In all, nine genera
immigrated from Asia to North America in the Clarkforkian (Stucky, 1990).
The Clarkforkian immigration episode continued into the Wasatchian, in the
earliest Eocene (Webb and Opdyke, 1995). The Clarkforkian-Wasatchian boundary
correlates with the Paleocene-Eocene boundary at about 55 Ma (Gingerich, 2001). The
early Wasatchian was subj ect to the largest mammal immigration wave in the North
American fossil record. At this time, European mammals crossed the North Atlantic over
the Thulean land bridge (Webb and Opdyke, 1995). This immigration episode included
the first North American appearances of order Perissodactyla (Hyracotherium), order
Artiodactyla (Diacodexis), the creodont family Hyaenodontidae, and the primate families
Adapidae and Omomyidae (Rose, 1981). Direct evidence of grass during the late
Paleocene and early Eocene is rare, but there are some fossil grasses that indicate the
presence of protosavannas: the oldest North American grass macrofossil, early Eocene in
age, comes from the Paleocene/ Eocene Wilcox Formation of western Tennessee (Crepet
and Feldman, 1991).
The morphological subtleties visible during the late Paleocene/early Eocene gave
way, in the middle and late Eocene, to more pronounced adaptations and shifts in both
floral and faunal communities due to the changing global climate. In the middle Eocene,
the global climate began cooling again and becoming drier, as evident from a 3.0%o
increase in benthic foraminifera 618O values over a 17 million year span, from 50 Ma to
33 Ma (Savin, 1977; Zachos et al., 2001). The increase in 618O values in the middle
Eocene (50-48 Ma) resulted entirely from a decrease in deep-sea temperature from about
120C to about 4.50C (Zachos et al., 2001). Subsequent ice sheet growth began by the late
Eocene (34 Ma) and was responsible for further 618O enrichment (Miller and Katz, 1987;
Zachos, Stott, and Lohmann, 1994; Zachos et al., 2001). It was also during the middle
Eocene that the first signs of seasonal aridity, such as evaporites and oxidized redbeds,
appear in the Rocky Mountain region (Webb and Opdyke, 1995).
As aridity and cooling increased in the middle Eocene, there was a maj or faunal
turnover in the subtropical forests. By the end of the Duchesnean, 80% of the terrestrial
mammal genera present in the Uintan (the previous age) had become extinct (Savage and
Russell, 1983; Stucky, 1990). The gradual disappearance of tropical forests in North
America prompted the decline of arboreal creatures such as primates (Webb, 1977). The
adapids and paromoyids, the last of the North American primates, disappeared at the end
of the Duchesnean (Prothero, 1994). The orders Condylarthra, Tillodontia, Dinocerata,
and Taeniodonta also disappeared in the late Duchesnean (~40 Ma), while many new
Asian immigrants arrived, most notably the eubrontotheres such as Duchesneodus
(Webb, 1977; Emry, 1981; Krishtalka et al., 1987; Prothero, 1994).
By the late Eocene, the cooler and drier climate allowed for the first true savannas
to dominate the midcontinent (Webb, 1977; Wing and Tiffney, 1987). Savannas, as
defined by Sears (1969), are any biomes that are subtropical open-country plains with
some trees, which includes areas such as thorn scrub and open deciduous forests, but do
not include open steppe or grasslands. The savannas that appeared in the late Eocene
were savanna woodlands, typified by the presence of the Leguminosae, Sapindaceae, and
Anacardiaceae floral families, similar to those of the modern Chihuahua region of
Mexico (Webb, 1977). As the global cooling continued, tropical flora and subtropical
forests retreated south of the Rocky Mountains (Leopold and MacGinitie, 1972).
Members of the grass family Poaceae are also present in the late Eocene.
Representatives of the subfamily Pooideae, from Tribes Stipeae ("e.g.", Stipa florissanti)
and Phalarideae, appear in the Florissant floral assemblage of Colorado, at about 34 Ma
(MacGinitie, 1953; Stebbins, 1981). The presence of these two tribes, endmembers of
two separate evolutionary lineages, suggests that the Pooideae was well-differentiated by
the end of the Eocene (Stebbins, 1981).
The new savanna woodland habitats were exploited by a number of adapting North
American mammals, as well as by late Eocene Asian immigrants already adapted to
savanna habitats. The combined autochthounous and invasive taxa, which followed the
Duchesnean faunal turnover, are termed the "White River chronofauna" (Emry, 1981).
Chronofaunas are assemblages of species that remain compositionally stable over a
significant amount of time (Olson, 1952). The "White River chronofauna" consists of an
increase in herbivore genera and species from the late Eocene through the Oligocene,
beginning around 40 Ma (Krishtalka et al., 1987; Webb and Opdyke, 1995). The number
of identified browsing mammalian genera rose from 8 in the Duchesnean to about 40
after the Duchesnean (Stucky, 1990). Likewise, the overall number of mammalian
herbivore species rose from less than 40 to about 90 during the Eocene-Oligocene
transition (Savage and Russell, 1983). The higher species numbers were maintained
throughout the Oligocene (Webb, 1989). Numerous modern mammalian families make
their first appearance in North America during the late Eocene, including: soricid
insectivores; sciurid, castorid, cricetid, and heteromyid rodents; leporid lagomorphs;
canid and mustelid carnivores; camlids; tyassuids; and rhinocerotids (Webb and Opdyke,
By the middle Oligocene, the "White River chronofauna" had become the first
North American chronofauna to exhibit significant diversity of hypsodont herbivores,
from rodents to ungulates (Gregory, 1971; Webb and Opdyke, 1995). These hypsodont
taxa include: leporids, castorids, comyids, rhinocerotids, hypertragulids, oromerycids,
and oreodonts (Webb, 1977). The oromerycid M~ontanatylopus, for example, has molars
significantly more hypsodont than its brachydont sister taxa (Prothero, 1986). The newest
Asian taxa, introduced through the connection of the North American and Asian
continents (McKenna, 1972), included a suite of selenodont (crescent-toothed)
artiodactyls already adapted for savanna feeding (Webb, 1977). Included in this group are
the families Camelidae, Hypertragulidae, Leptomerycidae, and Agriochoeridae (Webb,
1977). The newly introduced taxa flourished in the fresh savanna environments. One
taxon native to North America, Hyopsodus, acquired more lophodont (crest-toothed)
molars to chew coarser food (Gazin, 1968). Native rodents, such as the protoptychids and
cylindrodontids, developed open-country locomotive adaptations as well as dentitions
suited for coarser foods (Wood, 1962; Black and Dawson, 1966; Galbreath, 1969;
Wahlert, 1973). Larger herbivores existed in two general groups: semiamphibious
streamdwellers such as amynodont rhinos and long-limbed, cursorial taxa that roamed the
interfluves, such as equids (M~esohippus) and selenodont artiodactyls (Wall, 1982).
These adaptations, along with the diversification of many of these cursorial and
hypsodont taxa, mark the dominance of the woodland savannas that had become
widespread in the Oligocene (Webb, 1977). It is also likely that the "White River
chronofauna" established a positive feedback loop with the savanna biomes:
Large herbivores preferred feeding in more open woodlands; in turn, expansion of
open formations facilitated evolution of mixed-feeding herbivores. (p., 192, Webb
and Opdyke, 1995)
Additionally, there was a negative correlation between browsers and the spread of open-
country habitats (Webb and Opdyke, 1995). By the late Oligocene, browsers such as
titanotheres had disappeared as woodland savannas continued to spread (Webb and
The expansion of woodland savannas is supported not only by hypsodont radiations
but other lines of both floral and faunal evidence. Aquatic reptiles in the Rocky Mountain
area underwent severe population decreases as a result of aridity and seasonality during
the late Eocene and Oligocene (Hutchinson, 1982). The late Eocene Florissant Flora of
Colorado records a decrease in the percentage of entire-margined leaves, indicating a
drop in mean annual temperature from 100C to ~12.50C (MacGinitie, 1962; Wolfe,
1985). Additionally, pedological studies on the Brule Formation of South Dakota (~33
Ma, early Orellan) and the Upper John Day Formation in central Oregon (~30 Ma,
earliest Arikareenan) suggest the presence of desert bunch grasslands during the
early/middle Oligocene (Retallack, 1997; Retallack, 2001; Retallack, 2004).
Grass, however, was still relatively rare in the fossil record of the Eocene and
Oligocene (Frederickson, 1981; Webb and Opdyke, 1995; Jacobs, Kingston, and Jacobs,
1999). There are two plausible explanations for this rarity. The first is that woody shrubs
dominated the landscape prior to the profusion of grasses (Huber, 1982). The second
possibility is that grasses suffer from a taphonomic bias, preventing the preservation of
grasses despite their actual abundance (Webb and Opdyke, 1995). Grasses are known to
be abundant elsewhere during the middle Eocene, including Australia (Truswell and
Harris, 1982) and Europe (Litke, 1968).
Immediately following the Eocene-Oligocene transition, the Drake and Tasmania
Passages opened (at ~31 and 32 Ma, respectively), increasing ocean circulation and
proliferating the global cooling trend from the Eocene (Lawyer and Gahagan, 2003). This
also allowed the establishment and preservation of permanent Antarctic ice sheets
(Hambrey, Ehrmann, and Larsen, 1991). The decrease in global temperature continued
until the late Oligocene (26 to 27 Ma), when another warming trend began (Miller,
Wright, and Fairbanks, 1991; Wright, Miller, and Fairbanks, 1992). This warming trend
reduced Antarctic ice sheet volume until the middle Miocene, ~15 Ma (Miller et al.,
1991; Wright et al., 1992). There were some short glaciation events interspersed
throughout this approximately 12 million year interval (Wright and Miller, 1993). This
warming reached its zenith during the middle Miocene Climatic Optimum (MCO), from
17-15 Ma, which is evident from decreased 68"O values (Vincent, Killingley, and Berger,
1985; Flower and Kennett, 1995). Oceanic and atmospheric cooling and ice sheet growth
followed the MCO, marked by about a 1%o increase in foraminifera 618O values from
14.0 to 13.8 Ma (Flower and Kennett, 1995).
There are two hypotheses for the mechanism that drove the middle Miocene
climate variability: greenhouse gases and oceanic circulation (Zachos et al., 1994). From
16.5 to 13.5 Ma, overlapping the MCO, benthic foraminifera 613C ValUeS WeTO eleVated
as high as 2.2%o (Vincent and Berger, 1985). This event, termed the Monterey Excursion,
possibly resulted from the draw down of atmospheric pCO2 through organic carbon
burial in marginal marine sediments (Vincent and Berger, 1985). It has been proposed
that the Monterey Excursion drove middle Miocene climate variability, although there is
a 2.5 million year lag between the onset of the proposed pCO2 draw down (16.5 Ma) and
the atmospheric cooling indicated by the 618O increase at 14 Ma (Vincent and Berger,
1985; Hodell and Woodruff, 1994). Atmospheric CO2 leVOIS and global climate may have
remained high during this lag due to the outgassing of CO2 fTOm the Columbia River
Flood Basalt from 17 to 14.5 Ma (Hodell and Woodruff, 1994). However, other
researchers have suggested that atmospheric CO2 leVOIS were low from 17 Ma to 14 Ma
(Pagani, Freeman, and Arthur, 1999; Flower, 1999; Royer et al., 2001). The middle
Miocene climate variability, therefore, may have resulted from the opening and closing of
tectonic gateways that altered oceanic circulation (Woodruff and Savin, 1989, 1991;
The first immigration episode of the Miocene occurred at approximately the same
time as the beginning of the MCO, from 18-17 Ma in the middle Hemingfordian (Webb
and Opdyke, 1995). These Asian immigrants include comyid rodents, the
biostratigraphically important cricetid rodent Copemys, and the first true cats in the New
World such as Pseudaeherus (Webb and Opdyke, 1995). The immigration also included
four "megaherbivores": the rhinocerotids Teleocera~s and Aphelops and the proboscideans
M~ioma~stodon and Gomphotherium (Webb and Opdyke, 1995). It is assumed that these
"megaherbivores" modified the savanna landscape much like modern elephants,
however, there is no direct evidence to support this hypothesis (Owen-Smith, 1988).
These immigrants comprise part of the "Sheep Creek chronofauna" of the early and
middle Miocene. The "Sheep Creek chronofauna" is also important because it chronicles
the grazing advancement of horses, with the transition of the browsing/mixed feeding
Parahippus to the grazing M~erychippus (Hulbert and MacFadden, 1991).
Treeless grassland prairies were initially thought to have become widespread in the
Great Plains at the beginning of the Barstovian Land Mammal Age (middle Miocene,
~15.8 Ma), replacing the steppe savannas (Kowalevsky, 1872; Webb, 1983). This timing
would correlate with the end of the middle Miocene climatic optimum. This
interpretation for grassland expansion was based largely on the prevalence of mammals
with grazing adaptations, especially horses, and has been viewed as a classic example of
coevolution. Mesodont horses such as Parahippus gave way to hypsodont horses, like
M~erychippus and Hipparion, that also exhibited increased enamel folding and cement
deposition on the cheek teeth (Webb, 1977). These adaptations, which improved the
grinding surface of the tooth, were also featured in camels, pronghorns, oreodonts,
rhinoceroses (diceratherine and teleoceratine), and at least four genera of gomphotheriid
proboscideans (Webb, 1977). The diversification of these taxa is directly correlated to the
degree of hypsodonty; the higher-crowned lineages experienced greater radiations
(Webb, 1977). The succession of grazers, starting at the beginning of the Barstovian
(15.8 Ma) and lasting through the end of the Clarendonian (8.8 Ma), has been termed the
'Clarendonian chronofauna' (Webb, 1983). It has also been suggested that this faunal
succession (and therefore the appearance of grasses) began during the beginning of the
late Hemingfordian at 17.5 Ma (Theodor, Janis, and Broekhuizen, 1998).
In addition to dental adaptations for grazing, many ungulates acquired elongated
limb modifications for open-country locomotion (Webb, 1977). Hypsodont horses, for
example, developed digital springing ligaments at about the same time that they
developed grazing dentitions (Camp and Smith, 1942). Rodents also adapted to savanna
habitats in the Miocene. Their teeth became higher-crowned (Rensberger, 1973) and their
limbs adapted for burrowing habits (Webb, 1977). Mylagaulids (Fagan, 1960),
heteromyids (Lindsay, 1972), geomyoids (Rensberger, 1971), and ochotonid rabbits
(Green, 1972) all diversified into an array of hypsodont burrowers. The abundance of
needlegrass taxa, specifically Stipidium and Berriochloa, in the High Plains region (Elias,
1942) seemingly confirmed the dominance of grasslands in the Great Plains during the
The traditional view of grassland evolution was modified by a study of the late
Miocene (12-13 Ma) Kilgore Flora of Nebraska. This floral assemblage depicted an
environment consisting of savannas with mesic and open grassy forests, but lacking any
open prairies (MacGinitie, 1962). Faunal evidence corroborates this claim. The presence
of arboreal rodents, primates, insectivores, brachydont browsers and mixed feeders
suggests that the Great Plains was still a woodland savanna with riparian forests through
the late Miocene (Gregory, 1971). Grasses, therefore, were abundant through the late
Miocene, but grasslands had yet to dominate the landscape. It was not until the Late
Hemphillian (Early Pliocene, ~5 Ma), that treeless steppe grasslands swept across the
Great Plains. The vertebrate evidence for this consists of the absence of arboreal and
browsing taxa, a limited diversity of grazing taxa, and an overall lower diversity of all
vertebrate taxa (Gregory, 1971).
The traditional grassland story has been further modified, in more recent years,
from multiple types of studies. Paleosol studies indicate the presence of short sod
grasslands in the Great Plains region in the early Miocene, ~19 Ma, and tall sod
grasslands by the late Miocene, ~7 Ma (Retallack, 1997; Retallack, 2001). Paleosol
studies also indicate the presence of sod grasslands in the Hemingfordian (early Miocene,
~ 19 Ma) in central Oregon (Retallack, 2004). Phytoliths, the silica granules found in
many plants, have also been used to identify savanna environments. Morphological
studies on phytolith assemblages from northwestern Nebraska indicate that open-habitat
grasses were present in savanna and woodland environments by the early Miocene
(Stroimberg, 2002; Stroimberg, 2004).
The most extreme revisions have come from stable isotope studies. Stable carbon
isotopes from paleosols and fossil tooth enamel from various regions reveal a global
increase in the C4 biomass during the late Miocene and early Pliocene (7-5 Ma) (Quade
et al., 1992; Cerling, Wang, and Quade, 1993; Wang, Cerling, and MacFadden, 1994;
MacFadden and Cerling, 1996; Cerling et al., 1997b). This observation was based
primarily on the bioapatite of the fossil teeth, which reflect the C3 C4 plant constituents of
the animal's diet through 813C ratios. It was proposed that the increase in C4 biomass was
due to lowered concentrations of CO2 in the atmosphere, which would have caused the
less efficient C3 plants to diminish and the more efficient C4 plants (mainly tropical
grasses) to flourish (Ehleringer, Sage, Flanagan, and Pearcy, 1991; Cerling et al., 1993;
Cerling et al., 1997b). This theory has been countered by Morgan, Kingston and Marino
(1994), who claim that there was no expansion of the C4 biomass and no connection
between the atmosphere and any changes in the C3/ C4 biomass. Rather, Morgan and
colleagues suggest that the apparent increase in C4 COnSumption by mammalian
herbivores is the result of faunal immigration or in situ speciation, not a change in the
floral populations. Also, the proposed decrease in atmospheric CO2 COncentrations has
been challenged due to a lack of direct evidence of a change in the partial pressure of
CO2 (Pagani et al., 1999).
The isotopic evidence modifies the grassland story in two ways: (1) It pushes the
dominance of C4 graSslands back as early as 7 Ma and, more importantly, (2) Denies a
significant C4 preSence before 7 Ma, casting doubt on the idea that grassy savannas
preceded the steppe environment in the Great Plains. Initially, the isotopic and faunal
evidence provided an interesting dilemma. The faunal morphological changes suggested
a strong presence of grass, presumably C4, in the Great Plains at 15.8 Ma (Webb, 1983)
or 17.5 Ma (Theodor et al., 1998). However, a major global increase in C4 biomass is not
documented in the paleodiets until 7 Ma (Cerling et al., 1993). This indicates an apparent
change in faunal morphology that began 8.8 to 10.5 million years before the ungulates
began eating C4 graSses, and therefore long before the dominance of the grasses believed
to have caused the adaptations (Fox and Koch, 2003).
More recent isotopic evidence has addressed this discrepancy by pushing the
appearance of C4 graSses back prior to the original 15.8 Ma age based on faunal
morphology. Fox and Koch (2003) looked at paleosol stable isotopes from the Great
Plains region of North America and suggested that the C4 biomass first appeared no later
than the early Miocene (about 23 Ma), but did not become dominant until the late
Miocene (as evident from the biomass shift seen at 7 Ma). Based on the paleosol and
faunal data, Fox and Koch (2003) suggested that typical Great Plains habitats in the late
Miocene consisted of C3 trees and shrubs over a light carpet of grass.
The apparent temporal gap between grazer morphology and C4 dominance could
then be the result of the expansion of cool climate C3 graSses (Wang et al., 1994; Fox and
Koch, 2003). Expansive C3 graSses in the middle Miocene could have been responsible
for the ungulate grazing adaptations, since C4 graSses were still too relatively low in
abundance to cause such expansive adaptive radiations (Fox and Koch, 2003). The idea
of C3 graSses driving hypsodonty radiations denotes a paradigm shift in how paleodiets
are assessed through stable isotopes. Initially, 613C ValUeS that reflected C3 diets were
typically ascribed to browse material, such as shrubbery and leaves. C3-COnSumers were
then categorized as "browsers" while C4 COnSumers were categorized as "grazers." This
was due, in large part, to the assumption that paleoenvironments are analogous to modern
environments. This assumption has recently been reevaluated. Wang et al (1994) first
suggested that a unique grassland environment composed of low-latitude C3 graSSCS
could have existed in the middle Miocene due to lower concentrations of atmospheric
CO2. JaniS, Damuth, and Theodor (2002) concluded that early Miocene ungulates are
unlike any ungulates from modern grassland and forest environments and, therefore,
represent a paleoenvironment unlike any seen today. This interpretation has been
supported by Fox and Koch (2003), who proposed the hypothesis of expansive C3 graSSCS
in woodland environments. Woodland savannas with C3 graSses would then constitute a
unique environment with no modern analogues.
In order to further assess this claim, and to evaluate the spread of C4 graSses across
North America, this study focuses on the middle Miocene to early Pleistocene (15-5 Ma)
of northern/central Florida and southern Georgia and seeks the presence of grasses in the
diets of ungulates. Of particular interest is the Willacoochee Creek Fauna, an early
Barstovian (~15 Ma) faunal assemblage that is mostly composed of possible early grazers
or mixed feeders ungulatess bearing mesodont to hypsodont teeth). This faunal
community existed at a pivotal point in the evolutionary history of mammals; when open-
habitat morphologies rapidly expanded. This study utilizes three methods of paleodiet
analysis: stable carbon isotope analysis, mesowear analysis, and microwear analysis. The
combination of stable isotopes and mesowear or microwear makes it possible to either
substantiate or refute the presence of C4 graSses in the paleodiets of middle Miocene
ungulates, as well as address alternative driving mechanisms for the adaptation of
hypsodonty. The objectives of this study are (1) Characterize the dietary habits of the
community of herbivores from the Willacoochee Creek Fauna, (2) Determine the
presence of any grasses, C3 Of C4, in nOrthern Florida during the middle Miocene, and (3)
Determine the presence of any grasses, C3 Of C4, in Florida leading up to the carbon
biomass shift at ~7 Ma.
FIELD AREA AND FAUNA
The taxa in this study were excavated from sediments from seven sites in Florida
and Georgia. Three of the sites are located in northern Gadsen County, Florida: the
Englehard La Camelia Mine, the Milwhite Gunn Farm Mine, and the La Camelia 2 Mine
(Figure 2-1). Sediment samples were collected from the Englehard La Camelia and
Milwhite Gunn Mines in the late 1980's by field crews from the Florida Museum of
Natural History (FLMNH) and the University of Florida Department of Geological
Sciences (Bryant, 1991). Vertebrate fossils were also collected from these sites. Most of
the fossils were recovered from spoil piles, but these piles were positively associated with
the Dogtown Member of the Torreya Formation (Bryant, 1991). These fossils comprise
the Willacoochee Creek Fauna (WCF) assemblage, an early Barstovian (middle Miocene)
assemblage of mammals, birds, amphibians, and reptiles found in the Dogtown Member
(Bryant, 1991). The early Barstovian designation of the assemblage is based on the
presence of Copemys, Perognathus, Rakomeryx, and Ticholeptus, as well as the
overlapping age ranges of several other mammals (Bryant, 1991; Table 2-1). The
beginning of the Barstovian is defined by the appearance of Copemys and the early
Barstovian is characterized by the appearance of Perognathus, Rakomeryx, and
Ticholeptus (Tedford et al., 1987). The WCF bears mammals with known age ranges that
begin in the early Barstovian, restricting the age of the WCF to an upper limit of early
Barstovian (Bryant 1991). The overlapping age ranges of other mammals, such as those
of the Merychippine horses, support this age correlation. The absolute age of the early
Barstovian is between about 16.6 to 14.4 Ma (Tedford et al., 1987).
The Torreya Formation is part of the Hawthorn Group and it is the only part of that
group that is present in the eastern Florida panhandle (Scott, 1988; Huddlestun, 1988).
The formation is siliciclastic with scattered carbonate and phosphate deposits and can be
found throughout the eastern Florida panhandle and into southern Georgia (Bryant,
1991). Bryant (1991) described the Dogtown Member of the Torreya Formation as
"largely clay, with varying amounts of sand and dolomite, and is primarily present in
Gadsen County, Florida, and adjacent Decatur County, Georgia" (Figure 2-2). The
Englehard La Camelia Mine was designated as the type section of the Dogtown Member
(Bryant, 1991). Vertebrate fossils are found in the sand and sand/clay layers (Figure 2-3).
As mentioned earlier, the Milwhite Gunn Farm Mine also belongs to the Dogtown
Member, but it represents a unique lithology. It consists of well-indurated, weathering-
resistant, carbonate-cemented sandstone (Bryant, 1991). Bryant (1991) suggested that the
outcrop at the Milwhite Gunn Farm Mine represented a "subaerial exposure surface, but
no pedogenic horizonation is preserved." The Milwhite Gunn Farm Mine and Englehard
La Camelia Mine layers are contemporaneous, based on the in situ presence of the
rodents Copemys and Perognathus at both sites (Bryant, 1991).
There are two new sites, the La Camelia 2 and Crescent Lake Mines. The La
Camelia 2 Mine is located in northern Gadsen County, Florida, near the original
Englehard La Camelia Mine site. Over 2,000 pounds of sediment, as well as some
vertebrate fossils, were collected from La Camelia Mine 2 by an FLMNH/UF Geology
Department field crew in May, 2004. Like the previous sites, the sediment samples and
fossils were collected from spoil piles at this site. These piles, however, were positively
associated with a single unit at the site. This unit consists of interfingering sand and clay
layers and has been correlated with the Dogtown Member and aged as early Barstovian.
The unit correlation is based on lithology and the Barstovian age, which is derived from
the overlapping presence of Aphelops sp., which first appears in the Late
Hemingfordian, and Acritohippus isonesus (Figure 2-2).
The second new site, the Crescent Lake Mine, is located 15 miles north of the
Florida-Georgia Border in Decatur County. Vertebrate fossils and sediment were
collected from this site by FLMNH Hield crews in May, 2005. Unlike the other sites, these
samples were collected in situ along a fresh cut at the mine (Figure 2-4). The sediments at
the Crescent Lake Mine constitute a clayey sand layer representing terrestrial and marine
environments. Marine depositional environments are evident by the presence of abundant
invertebrate fossils, such as gastropods, bivalves, and echinoids (sea urchins), as well as
garfish scales. Based on the lithology of the unit and mammal assemblage, this site
correlates with the Dogtown Member of the Torreya Formation and an early Barstovian
age. The age is based on the presence of the brachydont Anchitherium clarencei and the
mesodont M\~erychippus primus, two horses with biochronological ranges in the WCF that
extended, and terminated, in the early Barstovian (Bryant, 1991; Figure 2-2).
Additionally, the hypsodont horse Acritohippus isonesus and the rhino Aphelops have
overlapping ranges in the Barstovian (Bryant, 1991).
The two sites discovered in the 1980's (Englehard La Camelia and Milwhite Gunn
Mines) have both been analyzed for strontium-isotopic and paleomagnetic dating. Four
reliable sSr/86Sr age estimates place the Dogtown Member between 16.6 + 1.0 and 14.7
+1.5 Ma (Bryan, MacFadden, and Mueller, 1992). The Dogtown Member is entirely of
reversed polarity, and, with biochronologic data, the unit correlates with Chron C5B-R
(Bryant et al., 1992). This correlation narrows the age of the Dogtown Member, and
therefore the WCF, to between 16.2 and 15.3 Ma (Bryant et al., 1992). This range has
been restricted further, to between 15.9 and 15.3 Ma, based on the Hemingfordian-
Barstovian boundary (Woodburne, Tedford, and Swisher, 1990). Bryant et al. (1992)
also determined that the Dogtown Member is time-transgressive, with sediments getting
younger to the north.
In addition to the middle Miocene sites, this study looks at younger sediments to
trace the presence of grasses in Florida. The three younger sites are all located in central
Florida. Ages for two of these sites are determined by biochronology. The Love Bone
Bed Site is late Miocene and designated as late Clarendonian in age (~9.5 Ma) and the
Upper Bone Valley Formation is early Pliocene and late Hemphillian (~4.5 Ma) (Figure
2-5; Hulbert, 1992; Morgan, 1994). The accuracy of these ages is within about f 0.5 Ma
(MacFadden and Cerling, 1996). The third site is the Leisey Shell Pit, which has been
dated at approximately 1.5 Ma, based on biochronological, sSr/86Sr, and paleomagnetic
data (Webb et al., 1989). The accuracy of this age is about f 0. 1 Ma and places the
Liesey Shell Pit in the early Pleistocene with an early Irvingtonian age (Figure 2-5).
These three sites were chosen for this study because stable carbon isotope analyses have
been previously conducted on large populations of fossil horses from these sites
(MacFadden and Cerling, 1996). These large populations are also ideal for mesowear
The specimens analyzed in this study span approximately 14 million years.
Specimens from the Englehard La Camelia, Milwhite Gunn Farm, and La Camelia 2
Mines are representatives of the WCF from the Dogtown Member of the Torreya
Formation and are of early Barstovian (about 15.9 to 15.3 Ma) age (Bryant, 1991).
Before any analyses could be conducted, it was necessary to identify all of the
specimens. Despite the previous description of the WCF by Bryant (1991), many of the
collected fossils were still unidentified and not catalogued. Additionally, all of the fossils
from the new site needed to be identified and catalogued. These fossils, which are all
teeth, were identified by referencing published literature as well as identified specimens
from the FLMNH collections. The vast maj ority of unidentified specimens were horse
teeth. In addition to identifying the taxon, the tooth position was determined for each
tooth by comparing the widths of the parastyle and mesostyle and assessing the angle of
the mesostyle and plane of occlusal surface (Figure 2-6). Typically, the parastyle is wider
than the mesostyle in horse molars and vice versa for horse premolars (Bode, 1931).
More posterior-leaning mesostyle angles and shallower angles of the occlusal plane are
indicative of molars, while more anteriorly-leaning mesostyles and more pronounced
angles of the occlusal plane are apparent in premolars (Bode, 193 1). Identification of the
tooth position is important in this study since paleodiet analyses often use specific teeth
as a standard for paleodiet assessment.
Figure 2-1. Index map of middle Miocene localities in northern Florida. 1 = Milwhite
Gunn Farm Mine, 2 = Englehard La Camelia Mine and La Camelia 2 Mine.
All of the Willacoochee Creek Fauna taxa were collected from the Dogtown
Member of the Torreya Formation. (Modified with permission from Bryant,
DO GTOWNY MB1
Table 2-1. Biochronological ranges of the Willacoochee Creek Fauna. Solid lines
indicate known ranges and asterisks indicate range extensions. Note that all of
the ranges overlap in the early Barstovian. Also, Ticholeptus hypsodus,
Bouromeryx of. parvus, and Rakomeryxr sp. all occur only in the early
Barstovian. These ranges denote an early Barstovian age for the Dogtown
Member of the Torreya Formation. (Modified with permission from Bryant,
TAXON HEMINGFORDIAN BARSTOVIAN CLARENDONIAN
EARLY LATE EARLY LATE EARLY LATE
cf. Protospermophilus sp.
Perognathus cf. minutus
yoc" cf. proterva
Bouromeryx cf. parvus
Anchitherium clarencel ****
KNOWN BIOCHRONOLOGICAL RANGE
wwwwwww BIOCHRONOLOGICAL RANGE EXTENSION
Sclay/sand [sand clay E3cement
Figure 2-2. Composite section of the Torreya Formation and the location of the
Willacoochee Creek Fauna within the Dogtown Member. Note that the
Dogtown member is composed mostly of clayey sand and some clay.
(Modified with permission from Bryant, 1991)
**B VERTEBRATE FOSSIL HORIZON
Figure 2-3. Correlated stratigraphic sections of the Englehard La Camelia and Milwhite
Gunn Farm Mines. Note that the Willacoochee Creek Fauna are found in
layers composed of clayey sand, sand, and sand with carbonate cement.
(Modified with permission from Bryant, 1991)
r -r --
Figre2-. reh utat henees ste te resent~ LaeMie i eatrCony
Georga. A)Crosssectin vie of snd an cla laye~' J~ris aov hefoslieru
layer. B) Bottom of th cut, where the fossiiferos caye ad sepoe.I
the oreroun iswhee teresrialverebrte fssis wee fund whie mrin
invertebrate were- fon lehr ntect
MOSS ACRES HEMPIIL.I.JAN
LIOVE BONE BED -i
Figure 2-5. Stratigraphic and temporal distribution of the 3 fossil sites studied in the
mesowear analysis, as well as a fourth (Moss Acres) discussed later.
Neohipparion trampa~sense, Cormohipparion plicatile, and Cormohipparion
ingenuum are from the Love Bone Bed, Nannippus aztecus is from the Upper
Bone Valley Formation, and Equus "leidyi" is from the Leisey Shell Pits.
(Modified with permission from MacFadden and Cerling, 1996)
Figur 2-6 Tot o'in. )Temdr hrl a ih h nlso emssye
p)~tremola a n pe or ~ (b. oe h dferecsin aasyen
Figre -.mesh os~tylidths and the mdiferencres in ange oft the panges of occlussayle
srandoc.sa (Modfied with permissionh fritom. Bodre, itos 1931)per
The mesowear method was devised by Fortelius and Solounias (2000) as a quick
and inexpensive process of determining the lifelong diet of a taxon. They determined
that, for extant mammals, broad conclusions on the diets, such as a grazer or browser
classification, can be deduced from the shape of the buccal cusps (either the paracone or
metacone) and the relative difference in height between the tip of the cusps and the
intercusp valley. The robustness of this method was confirmed by blind test studies that
revealed that there are no statistical differences in the scoring of attributes between
individual researchers (Kaiser et al., 2000).
The attributes that are evaluated, termed cusp shape and occlusal relief, were
originally only applied to the upper second molar (M2) and require at least 20 specimens
to obtain a reliable classification. Fortelius and Solounias (2000) used cusp shape and
occlusal relief to establish a "typical" set of 27 extant browsers, grazers, and mixed
feeders. This set was illustrated in a hierarchical cluster analysis (Figure 3-1), which
shows clusters of mammals that had similar wear (attrition- or abrasion-dominated). The
classification of each cluster ("e.g.", grazerr") was confirmed by direct observations
made on the diets of the animals. The mesowear technique can be extended to an extinct
taxon, which can then be included in the hierarchical cluster analysis to determine its
dietary classification. Mesowear analyses require large sample populations (>20), which
can be problematic for some localities, but the method yields an accurate depiction of an
animal's average lifelong diet.
In a study on hipparionine and extant equids, the mesowear method was "extended"
to broaden its application beyond the upper M2, to specific combinations of the upper
third premolar (P3), upper fourth premolar (P4), upper first molar (M1), and the upper
third molar (M3) (Kaiser and Solounias, 2003). This study was conducted with the goal
of expanding the application of the mesowear method beyond ungulate populations with
an abundance of M2s. The "extended" mesowear method, the combination of tooth
positions that yielded results most consistent with the M2 results, was that of the
However, the "extended" method still requires positive identification of the tooth
positions. While this is not problematic when studying associated teeth, it can be difficult
to identify isolated teeth. This is especially true for equids. Bode (1931) showed that it
was possible to identify tooth position of unassociated teeth through the anterior-posterior
tilt of each tooth, but it has been noted that this method is time-consuming and does not
always result in a positive identification (Hulbert, 1987). This problem is often
encountered when trying to distinguish between a P3 and P4. Kaiser and Solounias
(2003) showed that the P3 in hipparionine equids was an unreliable indicator of paleodiet,
causing a shift towards a grazer classification. Additionally, they showed that the P4 is a
reliable indicator, particularly when grouped with molars (as in the "extended"
combination of P4-M3). To ensure correct dietary classifications based on unassociated
teeth, this study will apply the mesowear method only to molars, avoiding dubious
premolars. Previous studies have shown that analyses based on Mis and M2s yield the
same classifications as analyses based on only the M2s (Kaiser and Solounias, 2003;
Materials and Methods
Mesowear analyses were conducted on five equids from the younger central Florida sites:
Neohipparion trampa~sense, Cormohipparion plicatile, and Cormohipparion ingenuum
from the Love Bone Bed Site (~9.5 Ma); Nannippus aztecus from the Upper Bone Valley
Formation (~4.5 Ma), and Equus "leidyi" from the Leisey Shell Pit (~1.5 Ma) (Table 3-
1). Based on dental and post-cranial morphology, Neohipparion trampa~sense is a
probable grazer to mixed feeder while the Cormohipparion species are both expected to
be mixed feeders (MacFadden and Cerling, 1996). The taxon analyzed from the Upper
Bone Valley Formation is Nannippus aztecus, an expected grazer. Finally, Equus
"leidyi," an extremely hypsodont grazer from the Leisey Shell pit, was also analyzed.
Following the techniques described in Fortelius and Solounias (2000), the cheek
teeth of six different taxa were assessed for mesowear analysis, in terms of cusp shape
and occlusal relief (Figure 3-2). The cusp shape rating (sharp, round, or blunt) describes
the shape of the apex of the sharper cusp. The occlusal relief describes the height of the
cusps (high or low) relative to the valley between them. This rating can be quantified by
drawing a line that connects the apices of the two cusps, then measuring the vertical
distance between that line and the center of cusp valley. This value is then divided by the
length of the whole tooth. For equids, values greater than 0. 1 signify high occlusal relief
and values below 0. 1 signify low occlusal relief (Fortelius and Solounias, 2000). To
avoid teeth that were in the early or late stages of wear at time of deposition, only teeth
that are 25% to 75% of the maximum crown height were analyzed. Waterworn teeth were
also excluded from the sample set. When necessary, a magnifying lens was used in rating
As stated in Fortelius and Solounias (2000), the mesowear signal becomes stable
when there are more than 20 teeth, although a reasonable approximation is attained at
about 10 samples. In this study, at least 20 samples were measured for each taxon to
ensure correct classifications. The teeth that were analyzed include positively identified
Mis and M2s, as well as molars that could be either an Ml or an M2. Once the
measurements were recorded, the percentages for each attribute were calculated. Next,
these percentages were examined by hierarchical cluster analyses, using SPSS v. 11.5
software, to determine the dietary classification of each taxon. The variables entered into
the hierarchical cluster analyses are percentage high occlusal relief, percentage sharp
cusp shape, and percentage blunt cusp shape. Complete linkage and normalized
Euclidean distance were used in the analysis, following Fortelius and Solounias (2000).
Each taxon was entered into a separate cluster analysis, to avoid altering the morphology
of the dendrogram and possibly yielding biased clusters. The cluster placement of each
fossil taxon was used to determine its dietary classification. For example, a taxon that is
located within the cluster of 'typical' extant grazers would be classified as a grazer.
The cusp shape and occlusal relief ratings for each sample are listed in the
Appendix A. The calculated percentages of the attribute ratings for each taxon are listed
in Table 3-2. In the hierarchical cluster analyses, all Hyve horses (Neohipparion
trampasense,~rtrtrtrt~t~t~ Cormohipparion plicatile, Cormohipparion ingenuum, Nannippus aztecus,
and Equus "le/Jyr )~~ are placed within the cluster of confirmed extant grazers (Table 3 -3,
Figure 3-3). N. trampasenser~rtrtrtrt~t~t~ clusters closest to Ceratotherium simum (White rhinoceros),
while C. plicatile is closest to Damnaliscus lunatus (topi) and C. ingennum is placed
closest to the subcluster of Alcelaphus buselaphus (hartebeest) and Connochaetes
taurinus (wildebeest). Conversely, N. aztecus forms its own sub cluster with Alcelaphus
buselaphus, placing closer than Connochaetes taurinus. Finally, E. "ledivi" is placed
closest to Bison bison (American plains bison). All of these extant herbivores are typical
0 5 10 15 20 25
Fiur 3-.Dnrga lutaigteheaciclcutraayi fte2 tpcl
grzes brwes n ie edr rm otlu n oona 20)
Brwe brvain r nuprcse rzrabeitosaei oe
cae n ie edrabrvain r pe n lwrcs.Abeitos
A =Acesacs S hnceo odiuD =Dieo ions V
Odciesvriins J kpajhntnG =Grfacneoadls
OH Odcilu hezouD=Dceohnssnaress g ael
grati Gt ael hnsni m vbsnocats o artau
Table 3-1. Systematic and morphological information about the taxa used in the
mesowear analysis. Abbreviations: MSCH = Maximum crown height
measured from the occlusal surface to the base of the crown along the
mesostyle. (N. trampasenser~rtrtrtrt~t~t~ tribe, age, and MSCH data from Hulbert, 1987; C.
plicatile and C. ingenuum tribe, age, an MSCH data from Hulbert, 1988; N.
aztecus tribe, age and MSCH data from Hulbert, 1988 and Hulbert, 1990;
Equus "leidyi" tribe, age, and MSCH data from Hulbert, 1995).
Taxon Tribe Site and Ag~e Level MSCH Morpholog
Nohipparion trampasense Hipparionini Love Bone Bed, ~9.5 Ma 60 mm Hypsodont
Cormohipparion plicatile Hipparionini Love Bone Bed, ~9.5 Ma 58 mm Hypsodont
Cormohipparion ingenuum Hipparionini Love Bone Bed, ~9.5 Ma 49 mm
Nanppus aztecus Hipparionini Upper Bonie Valleyi Fm., ~4.5 Ma 51 mml Mdrtl
Eus"leidyi" Equinni Liesey Shell Pit, ~1.5 Ma 91.3 mm Extremelyhysdn
siharlp round blunt
Cusp Shrape (C: 5)
Figure 3-2. Examples of typical mesowear attributes. The three types of cusp shape are
sharp, round, and blunt. The types of occlusal relief are high and low. The
dotted lines indicate the height of the occlusal relief, from the bottom of
intercusp valley to the apex of the highest cusp. (Modiefied with permission
from Kaiser and Fortelius, 2003)
Table 3-2. Observed percentages of mesowear attributes (% high and % low refer to the
percentage of specimens with high or low occlusal relief and % sharp, %
round, and % blunt refer to the percentage of specimens with sharp, round or
blunt cusp shape).
% % % % %
Taxon N high low sharp, round blunt
Neohipparion trampasense 49 24.5 75.5 2 57.2 40.8
Cormohipparion plicatile 45 35.6 64.4 8.9 60 31.1
Cormohipparion ingenuum 26 50 50 0 84.6 15.4
Nannippus aztecus 35 54.3 45.7 0 82.9 17.1
Equs leidyi 30 0 100 6.7 20 73.3
Taxon Site Age Classification
Nohipparion trampasense Love Site 9.5 & 0.5 Ma Grazer
Cormohipparion plicatile Love Site 9.5 & 0.5 Ma Grazer
Cormohipparion ingenuum Love Site 9.5 & 0.5 Ma Grazer
Nanppus aztecus Upper Bone Valley Fm. 4.5 & 0.5 Ma Grazer
Equs leidyi Leisey Shell Pit 1.5 & 0.1 Ma Grazer
Table 3-3. Dietary classifications based
attributes from Table 3-2.
on hierarchical cluster analyses of mesowear
Bescalec1 Distance Cluster Combine
0 5 10 15 20 25
Figure 3-3. Dendrograms illustrating the hierarchical cluster analyses of 6 studied taxa
amongst the 27 'typical' grazers, browsers, and mixed feeders from Fortelius
and Solounias (2000). A) Neohipparion trampasenser~rtrtrtrt~t~t~ (Nt). B)
Cormohipparion plicatile (Cp). C) Cormohipparion ingenuum (Ci). D)
NNNNNNNNNNNNNNNNNNNNanipu aztecus (Na). E) Equus "leidyi" (El). Note that all five equids place
within the "Grazers" cluster. Browser abbreviations are in upper case, grazer
abbreviations are in lower case, and mixed feeder abbreviations are in both
upper and lower case. Abbreviations: AA= Alces alces, RS= Rhinoceros
sondaicus, DB= Diceros bicornis, OV= Odocoileus virginianus, OJ= Okapia
johnstoni, GC= Giraffa camnelopardalis, OH= Odocoileus hemionus, DS=
Dicerorhinus sumatrensis, Gg= Galzella granti, Gt= Galzella thomsoni, Om=
Ovibos moschatus, To= Taurotragus oryx, Ts= Tragelaphus scriptus, Cc=
Cervus canadensis, Cs= Capricornis sumatraensis, Me= Aepyceros
melampus, ab= Alcelaphus buselaphus, ct= Connochaetes taurinus, he=
Hippotragus equinus, rr- Redunca redunca, ke= Kobus ellipsipyrmnus, hn=
Hippotragus niger, eb= Equus burchelli, eg= Equus grevyi, dl= Damnaliscus
lunatus, cs= Ceratotherium simum, bb= Bison bison.
Bescalec1 Distance Cluster Combine
0 5 10 15 20 25
+----------- ---- -+- ---- --- -+-- --- ----+-- --- ---- +
Rescaled Distance Cluster Combine
0 5 10 15 2~ 0 5
+----------- ---- -+- ---- --- -+-- --- ----+-- --- ---- +
Becalc1lc Distance Cluster Combine
5 10 15 2 0
Rcscalec1 Distance Cluster Combine
The microwear method is a dietary analysis that quantifies the microscopic wear on
herbivore teeth. The consumption of herbivorous diets leaves microscopic scratches and
pits on the tooth enamel known as microwear. Studies on microwear of extant herbivores
with known diets have revealed a correlation between microwear and diet (Teaford and
Walker, 1984; Grine, 1986; Teaford, 1988; Solounias and Hayek, 1993) One noticeable
trend is that grazers typically have more scratches than browsers while browsers bear
more pits than grazers (Solounias and Semprebon, 2002). These observations have led to
the establishment of a Microwear Index (MI) (MacFadden, Solounias, and Cerling,
1999). For a stated area of enamel ("e.g.", 0.5mm x 0.5mm), the MI is calculated as the
total number of scratches divided by the total number of pits. As a standard, an MI below
1.5 indicates a browsing diet and an MI above 1.5 indicates a grazing diet (MacFadden et
al., 1999). Additional conclusions can be drawn by comparing the analyzed tooth to a
database of the microwear of extant animals with observed known diets. Plots of the
number of pits versus the number of scratches for these extant herbivores reveal distinct
morphospaces indicative of browsing, grazing, and mixed diets (Solounias and
Microwear studies were initially conducted at high magnification using Scanning
Electron Microscopy (SEM) imagery (Grine, 1986). Recently, however, a low-
magnification technique was developed to reduce the time and financial costs of the
microwear method. Analyzing microwear at only 35x magnification has been shown to
reveal the same results as high magnifieation (Solounias and Semprebon, 2002).
Solounias and Semprebon (2002) also created four additional quantifying characters to
attain a more detailed understanding of dietary habits. These characters can be used in
hierarchical cluster analyses to determine which kind of extant herbivores most closely
resemble the fossil taxa in terms of microwear, and therefore diet. However, microwear is
subj ect to the "Last Supper Effect," the limitation of reflecting an individual's last few
meals, rather than the life-history of diet (Grine, 1986). Therefore, microwear analyses
are based on the assumption that the animal's typical diet is reflected by these Einal
Materials and Methods
Microwear analyses were conducted on three samples of the most hypsodont taxon
from the WCF assemblage, Acritohippus isonesus. Microwear analyses were conducted
instead of mesowear because of the small sample size. The standard tooth position for
microwear analysis is the second molar (upper or lower) (Solounias and Semprebon,
2002; Rivals and Deniaux, 2003). No other horses, such as M~erychippus primus or
M~erychippus gunteri, from the WCF were analyzed for microwear because there are not
enough M2s for a statistical analysis. Due to the very limited sample size of the WCF,
only one tooth ofA. isonesus (part of an associated dentition) was positively identified as
a second molar (M2). The other two samples were identified as first or second molars
(M1/2s). Based on the curve of the mesostyle, it appears likely that both of these teeth are
second molars, but it cannot be stated with certainty. Statistical analyses, such as the
unpaired Students t-test and nonparametric Mann-Whitney test, were conducted on the
collected results to determine whether there exist significant differences between the
M1/2s and the one positively identified M2. Microwear analyses were also conducted on
specimens of the hypsodont horse, Neohipparion trampasense,~rtrtrtrt~t~t~ from the Love Bone Bed
site (~9.5 Ma) of north central Florida. These microwear results were used for
comparison to A. isonesus.
This study followed the procedure for microwear analysis outlined in Solounias and
Semprebon (2002). The first step is to clean the occlusal surface of each tooth using 95%
alcohol and cotton swabs. A mold is then made of the tooth using high-precision
polyvinylsiloxane dental impression material. The mold is removed and discarded in
order to remove any remaining debris from the enamel. A second mold is then made.
After the second mold has hardened, it is used to make a clear, high-quality epoxy cast
(using a resin to hardener ratio of 5:1). After 1-2 days, the cast has hardened and is ready
The standard for microwear analysis consists of looking at a 0.4 mm x 0.4 mm area
of the cast under 3 5x magnification (Solounias and Semprebon, 2002). Those dimensions
(0.4mm x 0.4mm) were used because the objective crosshairs had 0.4 mm increments at
that magnification. It should be noted that the only stereomicroscope with crosshairs
available for this study had 50x magnification, not 35x. Additionally, the crosshair
increments had larger spacing, causing the search area to be larger, about 0.5mm x 0.5
mm. The increased magnification and search area result in higher numbers of scratches
and pits than would be found using the standard magnification and search field. While
this has no effect on the calculated ratio of scratches to pits or on the percentages of other
quantitative categories, the increase in average pit and scratch numbers prevents the use
of hierarchical cluster analyses to assess dietary subcategories. However, these
subcategories are not essential for this study, which is only concerned with whether the
taxa were eating grass or browse.
To observe the micowear features, a light source is shone through the cast at a
shallow angle to the occlusal surface. The casts are analyzed in a dark room and
adjustments to the intensity and angle of the light source are often made to best observe
the microwear features. Two 0.5 mm x 0.5 mm areas were assessed for each tooth.
Following Solounias and Semprebon (2002), these areas were located along the shear
facet of the second enamel band on the paracone of each upper tooth. For lower teeth, the
search areas were restricted to the shear facet on the protocone. Pits are defined as
microscopic defects in the enamel that have a length:width ratio < 4, while scratches are
defects that have a ratio > 4 (Grine, 1986; Teaford and Robinson, 1987). These categories
are obvious under low magnification. Pits usually appear as fairly round dots with a
length:width ratio of about 1 or 2, while scratches are always much longer and typically
have length:width ratios much higher than 4. For each tooth, the pit and scratch numbers
from the two search areas are averaged. Pits are further classified as large or small. Small
pits are the most numerous type and are fairly rounded. There are usually a few pits that
are at least twice the diameter of the small pits. These are categorized as large pits and
appear deeper and dark due to less refraction. Scratches are subdivided into fine or
coarse. Fine scratches are the narrowest scratches. They are shallow and often are white
from light reflection. Coarse scratches are often dark and longer than most fine scratches.
It is also noted when a sample bears more than four cross scratches. Cross scratches are
any scratches that run roughly perpendicular to the maj ority of scratches. Finally, the
average microwear index for each taxon is calculated.
The observed counts of microwear features for each specimen are recorded in Table
4-1 and the calculated MI and percentages are presented in Table 4-2. Table 4-1 follows
the format and uses some of the quantitative parameters established by Solounias and
Semprebon (2002). The average number of pits and scratches for each taxon is listed,
followed by the microwear index (the ratio of scratches to pits), and two of the newer
quantitative variables. Since the microscope field used in this study is not the commonly
used size, these variables cannot be utilized in cluster analyses to determine more detailed
similarities to the diets of extant mammals. However, these variables are helpful in
making general inferences. The listed percentages in Table 4-2 for each of these attributes
refer to the percentages of specimens that bear that corresponding attribute.
Unpaired Students t-tests and nonparametric Mann-Whitney tests were used to
examine the concordance of the three A. isonesus specimens. The MI' s of the two
dubiously identified teeth (UF 223080 and UF 223081) were compared to the MI of the
positively identified M2 (UF 223063). The Students t-test and Mann-Whitney p-values
for comparing UF 223080 UF 223063 are 0.6826 and 0.4386, respectively. The Students
t-test and Mann-Whitney p-values for comparing UF 223081 to UF 223063 are 0.2195
and 0. 1213, respectively. For both of the M1/2 specimens, the Mann-Whitney test and
Students t-test yield p-values greater than 0.05 and reveal no significant differences
between the MI's. For each sample of both A. isonesus and N. trampa~sense, the average
number of scratches is almost double the average number of pits. The average MI's for A.
isonesus and N. trampasenser~rtrtrtrt~t~t~ are 1.97 and 1.98, respectively. The MI' s ofA isonesus and
N trampa~sense were compared for statistical differences using an unpaired Students t-test
and nonparametric Mann-Whitney test. The p-values for the Students t-test and Mann-
Whitney test are 0.5707 and 0.8690, respectively. These p-values are greater than 0.05
and show that the MI's of A. isonesus and N. trampa~sense are not statistically distinct.
Additionally, all of the samples for both taxa had scratches that were predominantly fine.
Each sample also had at least four cross scratches.
Table 4-1. Individual microwear counts for 3 specimens ofdcritohippus isonesus and 7
specimens of Neohipparion trampasense. Each specimen has two areas (A
and B) assessed for microwear. Abbreviations: UF ID = catalogue number in
the Florida Museum of Natural History collections, # x-scratches = number of
# fine # coarse
Taxon flF IE) # pits # scratches # x-scratches scratches scratches
Acritohippus 223063A 22 86 39 83 3
isonesus 223063B 43 96 20 81 15
223080/< 17 58 16 53 5
223080B 37 57 10 53 4
223081/< 83 118 27 110 8
223081B 65 111 40 106 5
Nohipparion 27991A 29 70 15 62 8
tapsense 27991B 42 48 13 45 3
27992;\ 45 59 26 55 6
27992B 22 57 17 52 5
27993/< 11 34 18 28 6
27993B 23 39 5 35 4
32280A 68 103 31 98 5
32280B 42 76 16 67 9
32291/< 25 49 9 48 1
32291B 24 85 25 79 6
36287A 29 58 26 53 5
36287B 30 69 25 58 11
32114A 37 103 17 99 4
32114B 32 60 18 54 6
Table 4-2. Calculated average microwear values and percentages for Acritohippus
isonesus and Neohipparion trampasense.~rtrtrtrt~t~t~ Abbreviations: N = sample size,
Avg. # pits = the average number of pits for the sample population, Avg. #
scratches = the average number of scratches for the sample population, MI =
microwear index, % Fine scratches = the percentage of the sample population
that possess scratches that are predominantly fine, % coarse scratches = the
percentage of the sample population that possess scratches that are
predominantly coarse, % cross scratches = the percentage of the sample
population that exhibits at least 4 cross scratches.
Avg. Avg. % Fine % Coarse % Cross
Taxon N # pits # scratches MI scratches scratches scratches
A. isonesus 3 44.5 87.67 1.97 100 0 100
N.trampasense 7 32.79 65 1.98 100 0 100
Table 4-3. Isotopic data for equids from 4 central Florida sites. Abbreviations: UF ID =
catalogue number in Florida Museum of Natural History collections.
(Modified from MacFadden and Cerling, 1996)
Taxon Site UF ID Material 613 (ooo
9.5 Ma Level, late Clarendonian, late Miocene
Nohipparion trampasense Love Bone Bed 32230 R. P3- 3 -11.6
Cormohipparion plicatile Love Bone Bed 32265 R. P2 -12.6
Cormohipparion plicatile Love Bone Bed 32265 R. P3 -12.5
Cormohipparion plicatile Love Bone Bed 32265 R. P4 -11.0
Cormohipparion plicatile Love Bone Bed 32265 R. M' -12.0
Cormohipparion ingenuum Love Bone Bed 60396 R. P2 -10.8
Cormohipparion ingenuum Love Bone Bed 35979 R. P2 -10.8
7.0 Ma level, "middle" Hemphillian, late Miocene
Nannippus aztecus Moss Acres 69933 R. M3 -7.9
cf. Nannippus Moss Acres None R. p2 -5.3
4.5 Ma level, latest Hemphillian, early Pliocene
Nanppus Upper Bone Valley None L. M -6.0
Nannippus aztecus Upper Bone Valley 63633 L. M2 -9.1
Nannippus aztecus Upper Bone Valley 65796 R. M' -2.4
1.5 Ma level, early Irvingtonian, early Pleistocene
Eq~uus "leidyi Leisey 1A 80047 R. M3 -1.5
Equs leidyi Leisey 1A None R. P2 -3.5
STABLE ISOTOPE ANALYSIS
Plants utilize three different types of metabolic pathways to process and use carbon.
These pathways are: the C4 pathway (or Hatch-Slack cycle), the C3 photosynthetic
pathway (the Calvin cycle), and the CAM (Crassulacean acid metabolism) pathway
(Bender, 1971). The C4-dicarboxylic acid pathway utilizes CO2 through carboxylation of
phosphoenolpyruvate (O'Leary, 1988). The Calvin Cycle, used by C3 plants, uses the
enzyme ribulose biphosphate carboxylase to fix CO2 (O'Leary, 1988). The CAM
pathway also uses ribulose biphosphate carboxylase to take in CO2, but the process is
more similar to what happens in the bundle sheath cells of C4 plants (O'Leary, 1988).
Plants that use the C4 pathway are primarily tropical grasses, but also include some fruits
and vegetables (O'Leary, 1988). C3 plants include trees, shrubs, and cool-climate
grasses, and dicots (O'Leary, 1988). The CAM plants consist primarily of desert
succulents (O'Leary, 1988).
Each plant metabolic pathway results in the isotopic fractionation of carbon (Figure
5-1) as it is taken into the plant cells from CO2 (Bender, 1971). The heavier carbon
isotope, 13C, iS discriminated against to varying degrees dependent on the pathway used,
and the 13 /12C iSotopic ratios of plants decreases relative to atmospheric isotopic ratios
(Bender, 1971). The lighter isotope is preferentially used due to the physical and
chemical properties associated with its mass (O'Leary, 1988). The carbon isotopic ratio
(813C) is represented as the parts per thousand difference between the sample and a
standard, the Peedee Belemnite from South Carolina (Craig, 1957).
C3 plants have an average 613C Value Of -27.1l%o f 2.0%o and a range from about
-3 5%o to -22%o, while C4 plants have an average 613C Value Of -13.1l%o f 1.2%o and have a
more limited range from -14%o to -10%o (O'Leary, 1988). CAM plants have a range of
-10%o to -20%o that distinguishes them from C3 plants, but not C4 plants (O' Leary, 1988).
Since CAM plants are known to be dominant only in xeric habitats (Ehleringer et al.,
1991), they are typically not considered in paleodiet analyses of temperate or subtropical
p al eoenvi ronm ents .
When mammalian herbivores consume plants, the carbon isotopes are fractionated
once again. By determining the fractionation factor for the mammal in question, the
carbon isotopic signature can be used to determine the diet, in terms of C3 and C4
consumption, of the herbivore. Stable carbon isotope analyses were first used in
archaeology to determine the paleodiets of ancient human populations (MacFadden and
Cerling, 1996). These studies analyzed the inorganic apatite in bone, which exists
primarily as hydroxyapatite, or Calo(PO4)6(OH)2 (Hillson, 1986; Wang and Cerling,
1994; MacFadden and Cerling, 1996). Trace amounts of carbon are present in this
mineral when it is commonly altered during skeletal formation, with carbonate (CO3 -2
replacing phosphate to yield Callo(PO4,CO3)6(OH)2 (Hillson, 1986; Newesely, 1989;
McClellan and Kauwenbergh, 1990). Due to the porous nature of bone collagen, the
hydroxyapatite with structural carbonate is extremely susceptible to diagenetic alteration
(Quade et al., 1992). Dental enamel, however, is much more resistant to diagenesis
because it is has very low porosity and is more than 96% inorganic by weight (Quade et
al., 1992; Wang and Cerling, 1994). Additionally, enamel is more than 95%
hydroxyapatite (Hillson, 1986). Multiple studies have found that the biogenic apatite of
enamel does not undergo diagenesis in most depositional situations and therefore retains
primary isotopic values (Quade et al., 1992; Wang and Cerling, 1994).
Cerling and Harris (1999) determined an enrichment factor of +14. 1 f 0.5%o
between the 613C Of the plants consumed and the bioapatite in the enamel of extant
ruminant mammals. This consistent enrichment establishes distinct ranges of isotope
ratios for ruminants that consume C4 Of C3 plants. Grazers (animals that feed primarily
on C4 graSses) fall in a range of 0%o to +4%o while browsers (animals that feed off of
leaves from C3 Shrubs and trees) fall within a range of -21%o to -8%o (Cerling and Harris,
1999). Stable carbon isotope analyses can therefore be conducted on ruminant teeth to
determine diet. This method has been extended to extinct ruminants to determine a
general classification of feeding habit. To do this, paleodietary analyses have adopted the
categories used to describe extant herbivores, established by Hofmann and Stewart
(1972). There are three general categories: concentrate selectors (browsers), bulk and
roughage feeders (grazers), and intermediate feeders (mixed feeders that consume both
browse and grass) (Hofmann and Stewart, 1972). These categories have been subdivided
further to detail the complexity of the feeding strategy. These subdivisions, however, are
rarely used in paleodiet analyses because it is difficult to calculate percentages of plant
type for an extinct animal. It is also important to recognize that paleodiet studies that
utilize stable isotope analyses rely on two assumptions: 1) The metabolic pathways of the
plants behaved similarly in the past as they do today ("i.e.", fractionated to the same
degree), and 2) the metabolic pathways and enrichment factor of ruminant mammals have
been consistent throughout time.
There is another factor that must be corrected when analyzing stable carbon
isotopes in fossils. As stated above, the 613C ValUeS of modern ruminants are derived
from the fractionation of carbon from plant intake, which is fractionated from the fixation
from atmospheric CO2. Atmospheric 613C ValUeS have decreased from approximately
-6.5%o to approximately -8.0%o over the course of the last 200 years, due to the burning
of fossil fuels (Friedli, Loitscher, Oeschger, Siegenthaler, and Stauffer, B, 1986; Marino,
McElroy, Salawitch, and Spaulding, 1992). Due to this change in atmospheric 613C
values, fossil bioapatite values are about 0.5%o to 1.3%o more positive than the values of
modern mammals (Koch, Hoppe, and Webb, 1998). This adjustment shifts the
endmembers on the scale for paleodiet interpretation: a diet strictly consisting of C3
plants would be no more positive than -8.7%o and a pure C4 diet would be no more
negative than -0.5%o (Feranec, 2003). Using these endmembers, a browser (less then 10%
C4 intake) would have a 613C Value leSs than -7.9%o, a grazer (less than 10% C3 intake)
would have a 613C value greater than -1.3%o, and a mixed feeder would have a 613C ValUe
between -7.9%o and -1.3%o (Feranec, 2003).
In addition to the utility of 613C ValUeS, Valuable dietary and environmental
information can also be attained from the 6 O0 values of structural carbonate and
phosphate in enamel. Several studies have determined that the 6 O0 of a mammal's body
water is directly correlated to the 6 O0 of ingested water from drinking water and food,
such as plants (Luz, Kolodny, and Horowitz, 1984; Luz and Kolodny, 1985; Bryant and
Froelich, 1995; Bryant, Froelich, Showers, and Genna, 1996a; Kohn, 1996). Furthermore,
these 618O values are recorded in the structural carbonate and phosphate of mammalian
tooth enamel, which mineralizes in isotopic equilibrium with body water (Longinelli,
1984; Luz et al., 1984). This conclusion has been corroborated by the calculation of a
consistent fractionation factor for oxygen between body water and structural carbonate in
modern equids (Bryant, Koch, Froelich, Showers, and Genna, 1996). Therefore, the 618O
values of mammals can be used to determine the 618O values of their water source.
However, it must first be determined whether that particular mammal obtains most of its
body water from plants or drinking water.
For obligate drinkers, tooth enamel can reflect the 618O values of meteoric water.
The 618O values of precipitation are controlled by temperature (Dansgaard, 1964).
Typically, this link causes the 618O values of meteoric water to be enriched during
periods of warm weather and depleted during periods of cool weather (McCrea, 1950;
Bryant et al., 1996a). The 618O values of meteoric water can therefore be used to interpret
seasonality. As mentioned previously, the 618O values of water sources are reflected in
the carbonate and phosphate of mammalian tooth enamel (Longinelli, 1984; Luz et al.,
1984). However, the metabolic rate of the mammal can influence the 618O values
recorded in the enamel (Bryant and Froelich, 1995; Kohn, 1996; Kohn, Schoeninger, and
Valley, 1998; Zhow and Zheng, 2002). This poses a problem for using small mammals
as environmental indicators, but Bryant and Froelich (1995) note that "because the
proportion of oxygen taken up as liquid water increases while the food requirement
decreases, the proportion of surficial drinking water reflected in [68"O of body water] will
increase with increasing body size." The 618O of surface water will then be reflected in
the enamel phosphate and carbonate of large mammals that weigh more than 1 kg (Bryant
and Froelich, 1995). The use of 618O values from tooth enamel as environmental
indicators has been extended to fossils, where the variation of 68"O values of enamel
phosphate along serially sampled teeth from Miocene horses (Bryant et al., 1996a) and
Holocene bison and sheep (Gadbury, Todd, Jahren, and Amundson, 2000) has been
determined to reflect seasonality. Similar studies have been conducted using the
structural carbonate in tooth enamel (Cerling and Sharp, 1996; Higgins and MacFadden,
2004; MacFadden and Higgins, 2004). Therefore, along the serially sampled tooth of an
obligate drinker, more positive (or enriched) 68xO values will indicate summer and more
negative (or depleted) values will indicate winter (Fricke and O'Neil, 1996; Feranec and
For herbivores that are not obligate drinkers, the 618O of structural carbonate in
fossil teeth reflects the 618O of the water in consumed leaves (Longinelli, 1984; Bryant,
Froehlich, Showers, and Genna, 1996; Koch, 1998). The 618O of water in plants is
influenced by temperature, and humidity (Dongman, Nurnberg, Fiirstel, and Wagener,
1974; Epstein, Thompson, and Yapp, 1977; Sternberg, Mulkey, and Wright, 1989). Due
to this relationship, evapotranspiration causes leaves in forest canopies and dry, open
habitats to have higher 618O values than ground-level leaves in cool, humid forests
(Fiirstel, 1978; Kohn, Schoeninger, and Valley, 1996; Cerling, Harris, Ambrose, Leakey,
and Solounias, 1997a). These values can be enriched by more than 20%o compared to the
68xO of local precipitation (Fiirstel, 1978; Kohn et al., 1996). For non-obligate drinkers,
the 618O of structural carbonate can yield some general information about the mammal's
habitat ("e.g.", arid, open areas versus cool forests), but it does not accurately reflect
Materials and Methods
The assemblage of eight ungulates analyzed ranges from expected browsers
(brachydont dentitions found in the unidentified artiodactyl, oreodont, one rhinocerotid,
and one equid) to expected mixed feeders/grazers (mesodont to hypsodont dentitions
found in three equids and one rhinocerotid). Due to the eruption order of teeth and the
weaning stage of mammals, the tooth positions most appropriate for a post-nursing diet
analysis are the third premolar (P3), fourth premolar (P4), and third molar (M3) (Bryant
et al., 1996a; Fricke and O'Neil, 1996; Hoppe, Stover, Pascoe, and Amundson, 2004).
Other teeth were analyzed for taxa that lacked any positively identified P3 s, P4s, or M3 s.
These taxa include Anchitherium clarencei, Teleocera~s, and Aphelops. In all, twenty-four
teeth were selected for bulk stable carbon and oxygen isotope analysis of enamel
carbonate (Table 5-1). Bulk samples, taken along the entire height of the tooth, yield an
average stable isotopic ratio for the time during which the enamel mineralized. Six
specimens, two from three different taxa, were also chosen for serial sampling to obtain
isotopic ratios with finer resolution. The taxa chosen were: Aphelops sp., the brachydont,
long-limbed rhinocerotid expected to be an open-country cursorial browser; M~erychippus
primus, a hypsodont horse presumed to be a mixed feeder; and Acritohippus isonesus, the
most hypsodont taxon from the assemblage that is expected to be a grazing horse.
Serial samples of bioapatite were taken from 6 teeth at 5-millimeter intervals along
the length of each tooth. Serial stable isotope analyses were conducted in order to analyze
the variation of diet for an individual. Since a tooth mineralizes from the crown to the
base over the course of a few months to two years (Hillson, 1986), serial samples reveal a
dietary interpretation from a smaller time interval than a bulk sample. However, the
isotopic signals are masked due to the slightly non-perpendicular mineralization of tooth
enamel (Passey and Cerling, 2002). Despite this effect, the serial samples still yield an
isotopic ratio that is averaged over shorter time intervals than a bulk sample and allows
some variation to be discerned (MacFadden and Higgins, 2004). For the purposes of this
study, it is important to show any C4 dietary influences throughout the mineralization
stage of the animal.
The sampling and preparation procedures for stable isotope analysis are comprised
of two main components: 1) physical sampling and 2) chemical processing. The physical
sampling consists of the actual removal of enamel for analysis and the chemical
processing is the procedural treatment of the enamel to remove organic compounds and
all other contaminants that will affect the stable isotope signature. The physical sampling
begins by identifying any enamel flakes on the tooth that appear pliable. The surface of
the flake is cleaned using brushes, and then removed. A drill is then used to remove any
dentine remaining on the inside surface of the flake. It is then ground in a clean mortar
and pestle, and the enamel powder is placed in a labeled microcentrifuge vial. If there are
no flakes apparent, then a part of the tooth must be drilled, preferably where the enamel is
thick. The surface is prepared by cleaning it with carbide dental drill bits and brushes.
All the cementum and soil must be removed from the tooth to avoid contaminating the
sample. Once the surface is cleaned, a Foredom drill is used at low RPM' s to remove
approximately 5 milligrams of pristine enamel. The drill operator must be careful not
drill into the dentine, as dentine has a different 813C Value fTOm enamel and will
contaminate the sample. Bulk samples were taken along the entire height of the tooth,
while serial samples were taken at 5-millimeter increments starting at the base of the
tooth. All the enamel powder is collected on weighing paper and placed into a labeled
The first step in the chemical treatment of the enamel samples is to remove all
organic material that might affect the carbon isotope ratio. To do this, 1 mL of 30%
H202 is added to each sample. Then, the vials are closed and shaken on a Thermolyne
Type 16700 Mixer in order to thoroughly mix the enamel powder with the H202. The
samples are then placed, with the lids off, in a reaction cabinet overnight. After
approximately 24 hours, some of the samples may still be reacting with the H202,
indicating the presence of abundant organic residues. The samples are all treated a
second time with H202 to remove these organic materials.
After the second H202 treatment, the samples are centrifuged and the H202 is
removed. One milliliter of distilled water is added to each vial and then mixed. The
samples are centrifuged, and the water is removed using a pipetter. This rinsing step is
repeated two more times. After the third rinse, 1 mL of 0. 1 M acetic acid is added to the
vials to remove carbonates from the samples. The samples are shaken and left in the
reaction cabinet overnight. The samples are not allowed to react with the acetic acid for
more than 24 hours, since it has been shown that prolonged exposure to acetic acid can
affect the carbon isotope signatures (Lee-Thorpe, Sealy, and van der Merwe, 1989;
Vennemann, Hegner, Cliff, and Benz, 2001).
The next day, the samples are centrifuged and the acetic acid is removed. Distilled
water is used to rinse the samples three more times. After the rinses, 95% methanol is
added to each sample to remove water. Again, the samples are shaken to mix the
methanol with the enamel, and then the methanol is removed. Finally, the samples are
placed in the reaction cabinet to dry overnight.
After the samples have dried, they are analyzed using a VG Prism mass
spectrometer at the Department of Geological Sciences at the University of Florida.
Approximately 1 mg of each sample is placed in a small sample boat and loaded into the
mass spectrometer, accompanied by standards. The bulk samples are standardized to
either the MEme (MacFadden Elephantus maximus enamel, 613C = -10.43%o) or the
NBS-19 (613C = +1.95%o; Coplen, 1996) standard to ensure the precision of the results
and the calibration of the mass spectrometer. All of the serial samples are calibrated to
the NBS-19 standard. All of the measured isotopic values are then calibrated to the
universal V-PDB (Vienna Pee Dee Belemnite). The values are presented in standard
6-notation: 613C Of 6180 = [(Rsample RV-PDB) 1] x 1000, where Rsample IS the measured
13 /12C Of 180/160 ratio of the sample. The analytical precision of samples run with the
MEme standard is f0.05 and f0.07 for 613C and 68 ", respectively. The analytical
precision of samples run with the NBS-19 standard is f0.09 and f0.18 for 613C and 68 ",
respectively. The analytical precision is measured by calculating one standard deviation
of the corrected 813C and 68 values of the standards.
The bulk 813C ValUeS for all the sampled specimens range from -11.39%o to -8.30%o
(Table 5-2, Table 5-3, Figure 5-2). The most positive mean bulk 813C value belongs to
Aphelops sp. and the most negative belongs to Teleocera~s sp., although Teleocera~s has a
sample size of only one. The bulk 68 O values for all the sampled specimens range from
-2.15%o to 2.71%o (Table 5-3, Figure 5-2).
Three taxa were selected for serial sampling: one expected browser with
brachydont dentition (Aphelops sp.) and two expected grazing/mixed feeding horses with
hypsodont dentitions (M\~erychippus primus and Acritohippus isonesus). Individual serial
stable isotope values are presented in Appendix B. The serial stable isotope samples for
the first specimen of Aphelops (UF 1 16827) range from -8.37%o to 11.72%o for carbon
and from -1.99%o to -0.31%o for oxygen. (Table 5-3, Figures 5-3 and 5-4). The second
specimen (UF 104227) has a narrower range of -9.76%o to -10.73%o for carbon and a
much wider and more positive range of 0.71%o to 3.46%o for oxygen (Table 5-3, Figures
5-3 and 5-4). For M\~erychippus primus, the serial 613C ValUeS range from -9.26%o to
-10.54%o (UF 221419) and from -9.39%o to -9.72%o (UF 221427) and the serial 6 O0
values range from 0. 15%o to 1.99%o (UF 221419) and from 1.12%o to 2.83%o (UF
221427) (Table 5-3, Figures 5-5 and 5-6). Acritohippus isonesus exhibits serial 813C
ranges of -9.92%o to -10.66%o (UF 221407) and -10.33%o to -11.96%o (UF 217590) as
well as serial 6 O0 ranges of 0.25%o to 2.56%o (UF 221407) and 0.62%o to 2.37%o (UF
217590) (Table 5-3, Figures 5-7 and 5-8).
-1 9 5%
Figure 5-1. Schematic of isotopic fractionation between atmospheric carbon and C3
plants, as well as fractionation between C3 plants and ruminant herbivores.
613C ValUeS are listed in parentheses. The average 613C Value foT C3 plants is
approximately -27.1%o and the average 613C Value foT C3-COnSuming
ruminants is approximately -13%o. The average isotopic fractionation between
atmospheric carbon and C3 plants is a depletion of about -19.5%o. The isotopic
fractionation between plants and ruminants is an enrichment of approximately
14.1l%o. (Modified with permission from Koch et al., 1992)
Table 5-1. List of the 24 specimens analyzed for stable carbon and oxygen isotope
analysis. Specimens are representatives of the Willacoochee Creek Fauna
from the early Barstovian (middle Miocene) Dogtown Member of the Torreya
Formation. Abbreviations: UF ID = catalogue number in the Florida Museum
of Natural History collections, ELC = Englehard La Camelia Mine, LC2 = La
Camelia 2, and MGF = Milwhite Gunn Farm Mine.
UJF ID Taxon Crown Height Tooth Site
221429 Teleoceras sp. Brachydont R. I ELC
104227 Aphelops sp. Brachydont Tooth fragment MGF
116827 Aphelops sp. Brachydont R. Mx fragment ELC
217565 cf. Aphelops Brachydont L. Px LC2
114723 Anchitherium clarencei Brachydont L. P2 ELC
221402 Anchitherium clarencei Brachydont R. M1 ELC
217590 Acritohippus isonesus Hypsodont L. P4 LC2
217562 Acritohippus isonesus Hypsodont R. M3 LC2
221405 Acritohippus isonesus Hypsodont L. M3 ELC
221407 cf. Acritohippus isonesus Hypsodont R. P3/4 ELC
114721 Merychippus gunteri Mesodont R. M3 ELC
116829 Merychippus gunteri Mesodont L. P3/4 ELC
221416 Merychippus gunteri Mesodont L. P3/4 ELC
221408 Merychippus gunteri Mesodont L. M3 ELC
114976 Merychippus primus Mesodont R. P3/4 ELC
104208 Merychippus primus Mesodont L. P3/4 MGF
221415 Merychippus primus Mesodont R. P3 ELC
221419 Merychippus primus Mesodont L. P4 ELC
221426 Merychippus primus Mesodont R. M3 ELC
221427 Merychippus primus Mesodont R. M3 ELC
221428 Merychippus primus Mesodont R. M3 ELC
221418 Merychippus primus Mesodont R. M3 ELC
116823 Ticholeptus hypsodus Brachydont R. M3 ELC
221434 Artiodactyla Brachydont R. M? ELC
Table 5-2. Bulk stable carbon and oxygen isotope values for Willacoochee Creek Fauna.
Abbreviations: UF ID = catalogue number in Florida Museum of Natural
History collections, Unident. = unidentified tooth position, ELC = Englehard
La Camelia Mine, MGF = Milwhite Gunn Farm Mine, and LC2 = La Camelia
UJF ID Taxon 613C ooo) 6180 (%o) Tooth Site
221429 Teleoceras sp. -11.39 -0.41 R. I ELC
104227 Aphelops sp. -9.48 1.01 Unident. MGF
116827 Aphelops sp. -8.72 -2.15 R. Mx ELC
217565 cf. Aphelops -10.23 0.38 L. Px LC2
114723 Anchitherium clarencei -8.90 2.32 L. P2 ELC
221402 Anchitherium clarencei -10.23 1.45 R. M1 ELC
217590 Acritohippus isonesus -10.87 2.03 L. P4 LC2
217562 Acritohippus isonesus -11.00 -0.35 R. M3 LC2
221405 Acritohippus isonesus -9.67 1.95 L. M3 ELC
221407 cf. Acritohippus isonesus -10.16 1.02 R. P3/4 ELC
114721 Merychippus gunteri -11.09 -0.65 R. M3 ELC
116829 Merychippus gunteri -8.30 -0.16 L. P3/4 ELC
221416 Merychippus gunteri -9.72 1.86 L. P3/4 ELC
221408 Merychippus gunteri -11.19 0.67 L. M3 ELC
114976 Merychippus primus -8.78 1.36 R. P3/4 ELC
104208 Merychippus primus -9.54 -0.82 L. P3/4 MGF
221415 Merychippus primus -8.79 2.10 R. P3 ELC
221419 Merychippus primus -10.00 0.85 L. P4 ELC
221426 Merychippus primus -9.76 1.14 R. M3 ELC
221427 Merychippus primus -8.70 1.03 R. M3 ELC
221428 Merychippus primus -9.73 0.45 R. M3 ELC
221418 Merychippus primus -9.50 0.97 R. M3 ELC
116823 Ticholeptus hypsodus -9.83 0.33 R. M3 ELC
221434 Artiodactyla -10.87 0.66 R. M? ELC
Table 5-3. Descriptive statistics of the 613C and 68 values for 8 herbivores from the
Willacoochee Creek Fauna. Abbreviations: Unident. = unidentified, N =
sample size, SD = standard deviation.
Taxon N 813 o/oo) 6180 (%o)
Mean SD Range Mean SD Range
Anchitherium clarencei 2 -9.57 0.94 -10.23 to -8.90 1.89 0.94 1.45 to 2.32
Merychippus primus 8 -9.35 0.51 -10.39 to -8.70 0.89 0.84 -0.82 to 2.10
Merychippus gunteri 4 -10.08 1.36 -11.19 to -8.30 0.43 1.10 -0.65 to 1.86
Acritohippus isonesus 5 -10.43 0.62 -11.00 to -9.67 1.16 1.11 -0.35 to 2.03
Teleoceras sp. 1 -11.40 ---0.41
Aphelops sp. 3 -9.48 0.76 -10.23 to -8.72 -0.25 1.67 -2.15 to 1.01
Ticholeptus hypsodus 1 -9.83 --0.33
Unident. artiodactyl 1 -10.87 --0.66
Taxon N 813 o/oo) 6180 (%o)
Mean SD Range Mean Range
Aphelops sp. 19 -10.52 0.82 -10.23 to -8.72 0.06 -1.99 to 3.46
Merychippus primus 9 -9.67 0.43 -10.39 to -8.70 1.66 0.15 to 2.90
Acritohippus isonesus 12 -10.66 0.65 -11.19 to -8.30 1.51 0.25 to 2.56
Figure 5-2. Plot of bulk 813C VS. 6180 values for the 8 herbivores from the Willacoochee Creek Fauna. Note that all the herbivores
bear bulk 813C ValUeS below -8.30%o and most specimens cluster between -9.50%o and -10.50%o.
6 C (%9)~
+ Artiodactyla Acritohippus isonesus + Merychippus primus A Merychippus gunteri
SAnchitherium clarencei + Aphelops x TeloceraIs
cl Ticholeptus hypsoclus
0 5 10 15 20 25 30 35 40 45 50
Position along tooth (mm)
Figure 5-3. F13C ValUeS for serial samples of two Aphelops specimens. Note that all the
values are lower than -8.37 %o. Position along tooth refers to the distance from
the base of the tooth.
-1.50 -o--UF116827 -m-UF104227
0 5 10 15 20 25 30 35 40 45 50
Position Along Tooth (mm)
Figure 5-4. 6 O0 values for serial samples of two Aphelops specimens. Note that both
specimens exhibit variation outside of the range of error. Position along tooth
refers to the distance from the base of the tooth.
-*- UF 221419 -m- UF 221427
Position along tooth (mm)
Figure 5-5. 613C ValUeS for serial samples of two M~erychippus primus specimens. Note
that all the values are lower than -9.26%o. Position along tooth refers to the
distance from the base of the tooth.
O 5 10 15 20
Position along tooth (mm)
Figure 5-6. 618O values for serial samples of two M~erychippus primus specimens. Note
that both specimens exhibit variation outside of the range of error. Position
along tooth refers to the distance from the base of the tooth.
-4-UF 221407 -m-UF 217590
10 15 20
Position along tooth (mm)
Figure 5-7. 613C ValUeS for serial samples of two Acriohippus isonesus specimens. Note
that all the values are lower than -9.92%o. Position along tooth refers to the
distance from the base of the tooth.
5 10 15 20
Position along tooth (mm)
Figure 5-8. 618O values for serial sampling of two Acritohippus isonesus specimens.
Note that both specimens exhibit variation outside of the range of error.
Position along tooth refers to the distance from the base of the tooth.
Of the eight taxa analyzed from the Willacoochee Creek Fauna (WCF), three are
expected to yield significant proportions of browse in their diets: Anchitherium clarencei,
Ticholeptus hypsodus, and Aphelops. A. clarencei is a three-toed horse with low-crowned
cheek teeth and stocky limbs suggestive of a browse diet in a woodland habitat (Janis et
al., 2002). The only positively identified artiodactyl from the WCF, T. hypsodus, has a
mesodont dentition and is expected to be a mixed feeder (Lander, 1998). Aphelops is a
hornless, brachydont aceratherine rhino with long limbs suggestive of a cursorial habit
(Matthew, 1932; Janis, 1982). Aphelops is traditionally described as an open-country
browser, with the extant black rhino (Diceros bicornis) cited as a modern analog
(Matthew, 1932). Furthermore, Janis (1982) described all aceratherine rhinos as browsers
within woodland savannas. There has been some variance on the dietary classification of
Aphelops. Webb (1983) recognized Aphelops and Teleocera~s as grazers from the
'Clarendonian Chronofauna,' the succession of Barstovian-Clarendonian grazers.
However, stable isotopic analyses ofAphelops specimens from multiple Florida localities
(ranging from 9.5 to 4.5 Ma), combined with crown-height data, suggest that Aphelops
was eating C3 browse material before and after the ~7 Ma spread of C4 graSSCS
(MacFadden, 1998). The WCF Aphelops specimens are approximately 6 million years
older than the previously analyzed specimens, but the dental and postcranial
morphologies still suggest a diet of C3 browse.
Teleocera~s is a moderately hypsodont teleoceratine rhino with short limbs. It has
been traditionally interpreted as semi-aquatic grazer, comparable to the extant
hippopotamus, Hippopotamust~~~~~tttt~~~~ amphibious (Scott, 1937; Voorhies, 1981; Webb, 1983;
Prothero, 1992). Matthew (1932) rej ected the hypothesis of a semi-aquatic habitat for
Teleocera~s, and instead suggested that the rhino grazed on grassy plains. Voorhies and
Thomasson (1979) determined that Teleocera~s was a grazer, based on the presence of
grass anthoecia in the oral and body cavities of specimens from the Ashfall Fossil Beds in
Nebraska (10 Ma). They were, however, unable to determine whether that grass
originated from mesic or lacustrine environments. Recent isotopic evidence, however,
has revealed that: 1) Teleocera~s was, based on 68 values, not primarily aquatic; and 2)
Teleocera~s was a mixed feeder, consuming significant portions of C3 graSs prior to the 7
Ma spread of C4 graSses and then shifting to a diet of C4 graSses after 7 Ma (MacFadden,
1998). This interpretation likens Teleocera~s to the extant white rhino (Ceratotheriunt
sinsun), a grazer (MacFadden, 1998). Based on this interpretation of Teleocera~s, the
specimens from the WCF would be expected to have 613C Signatures indicative of a
mixed diet, possibly composed of C3 graSSCS.
The remaining equids from the WCF are all hypsodont three-toed horses. The
hypsodont teeth of 2erychippus primus and M~erychippus gunteri suggest a
grazing/mixed diet for these equids. Janis (1988) points out that, despite fairly hypsodont
cheek teeth, the M~erychippus dentition is more similar to that of extant mixed feeders
than to grazers. Whether M~erychippus was a grazer or mixed feeder, the tooth
morphology suggests that there is at least some grazing component to the animal's diet.
Acritohippus isonesus, the most hypsodont taxon of the WCF, would be expected to be a
Based on average bulk stable carbon isotope values, all eight taxa from the WCF
fell within the range of a C3-dominated diet, between -21%o and -8%o (Cerling and
Harris, 1999). Additionally, only one sample had a 613C Value more positive than -8.7%o
(a M~erychippus gunteri specimen had a value of -8.30O%o), indicating that nearly the entire
WCF community was consuming strictly C3 plants, according to the pre-industrial ranges
of Feranec (2003). Even the most positive value was still more negative than -7.9%o,
indicating that the animal was eating less than 10% C4 graSS.
However, atmospheric 613C ValUeS during the middle Miocene were considerably
higher than during the pre-industrial Holocene (Vincent and Berger, 1985). During the
Monterey Excursion, from 16.5 Ma to 13.5 Ma, 613C ValUeS of benthic foraminifera were
as high as +2.2%o, indicative of higher atmospheric 613C (Zachos et al., 2001). The higher
atmospheric 613C WOuld raise the 613C Of plants, slightly raising the 613C Of the enamel of
mammals that consume the plants. This increase would be no greater than about 1%o.
Taking this enrichment into account, the 613C Signatures of all the samples are well
within a pure C3 diet. This suggests that each ungulate was either grazing on C3 graSSCS Of
browsing, and not consuming any significant amounts of C4 graSs. The serial sampling of
Aphelops sp., M~erychippusprimus, and Acritohippus isonesus, also reveal no significant
C4 fluctuations in diet at any point during the mineralization of the enamel. For both
Aphelops sp. and M\~erychippus primus, the most enriched serial sample was not greater
than -8.7%o, indicating a pure C3 diet. Once again, when the higher atmospheric 613C iS
considered, the serial samples fall well within the range of a pure C3 diet.
The stable carbon isotope analyses support the browser interpretations based on
dental morphologies for A. clarencei, T. hypsodus, and Aphelops. Conversely, the
dentitions and lack of a C4 Signal for Teleoceras and the three hypsodont equids suggest
that they were consuming C3 graSses. Unfortunately, the sample sizes ofM. primus, M~
gunteri, and Teleocera~s are inadequate for further investigation of grass consumption
("e.g.", microwear or mesowear analyses). Likewise, the sample sizes of the suspected
browsers are also insufficient for direct confirmation of a browse-dominated diet. A.
isonesus, however, can be assessed for evidence of a grass diet through microwear
analy si s.
While conducting the microwear analyses, there was some doubt as to whether
two of the A. isonesus molars were M2s. However, since the parametric and
nonparametric statistical tests found no significant differences between the microwear
indices (MI) of each A. isonesus specimen, they are accepted here as all being M2 teeth.
The average microwear index (MI) for A. isonesus is 1.97, well above the 1.5 threshold
for grazers (MacFadden et al., 1999). The MI for N. trampa~sense is 1.98, which is also
significantly higher than 1.5. This supports the mesowear analysis, which classifies N.
trampasenser~rtrtrtrt~t~t~ as a grazer. The MI of A. isonesus is not statistically different from that of
N. trampasense.~rtrtrtrt~t~t~ The relatively high MI of A. isonesus, coupled with the lack of a
significant difference to the MI from the grazing N. trampa~sense, suggests that A.
isonesus was also dominantly a grazer, with no significant browse component. The 613C
data and microwear index indicate that A. isonesus was a C3 grazer (Figure 6-1). This
conclusion is supported by the scratch morphology. The quantified scratches in extant
grazers have indicated that a prevalence of fine scratches are observed in consumers of C3
grasses, while coarse scratches dominate the enamel wear of C4 graZeTS (Solounias and
Semprebon, 2002). For each sample ofA. isonesus, fine scratches composed at least 84%
of the total number of scratches.
Further paleoecological interpretations can be made from the bulk and serial 613C
values of the WCF. In closed canopy forests, CO2 becomes trapped near the forest floor,
elevating the CO2 COncentration near the forest floor and in turn depleting the 613C Of that
air which, in turn, results in the depletion of 13C in plant tissues (Medina and Minchin,
1980; Medina, Montes, Cuevas, and Rokzandic, 1986; van der Merwe and Medina, 1989;
Cerling et al., 1997a). Further depletion of 13C in plant tissues results from the lack of
light reaching the forest floor, caused by the dense cover of the canopy (Medina and
Minchin, 1980; Medina et al., 1986; van der Merwe and Medina, 1989). These factors
cause the understory plants of a closed canopy forest to bear 613C ValUeS as low as -37%o,
a phenomenon known as the "Canopy Effect" (Medina and Minchin, 1980; Medina et al.,
1986; van der Merwe and Medina, 1989). The average 613C Value for these plants is -
30.9%o (Ehleringer, Field, Lin, and Kuo, 1986). Modern C3 plants that grow in open
areas and do not suffer from water stress exhibit a 813C range from -26 to -27%o (Cerling
et al., 1997a).
The range of bulk 813C ValUeS for all the sampled WCF is -11.39%o to -8.3%o,
which, considering analytical and fractionation error, indicates a diet that ranges in
isotopic composition from -25.49%o a 0.59%o to -22.40%o a 0.59%o. An adjustment for
the Monterey Excursion of -1%o changes the range to approximately -26.49%o a 0.59%o to
-23.40%o a 0.59%o. With this adjustment, all of the bulk samples have 613C ValUeS above
the maximum limit (-27%o) for the "Canopy Effect," although the error range of the
Teleocera~s sample extends down to -27.08%o. Even the two brachdyont taxa,
Anchitherium clarencei and Aphelops, have dietary bulk 813C ranges that preclude them
from eating ground-level browse in a closed canopy forest. The serial sampling also does
not support a closed-canopy dietary component. All the serial samples for M. primus,
Aphelops, and A. isonesus indicate diets that have 613C ValUeS above -27%o, except one A.
isonesus sample that yields a dietary value of -27.06%o. The error range of this sample,
along with the higher serial 613C ValUeS for this specimen, suggests that this specimen
was still not eating in a closed-canopy forest. The range of bulk and serial 613C ValUeS
for the WCF reveals that the mammals lived and ate in an open habitat, such as a
woodland savanna or grassland.
Paleoecological interpretations can also be made from the bulk 813C ValUeS of the
WCF. When C3 plants are suffering water stress, they become enriched in 13C (Ehleringer
et al., 1986; Ehleringer and Cooper, 1988; Ehleringer, 1991). During episodes of water
stress, plants close their stomata to conserve water, consequently reducing CO2 intake
and enriching the 613C Of C3 plant tissues as high as -22%o (Ehleringer et al., 1986;
Ehleringer and Cooper, 1988; Ehleringer, 1991). As mentioned above, the calibrated
isotopic range of the WCF diet is -25.49%o a 0.59%o to -22.40%o a 0.59%o. An adjustment
for the Monterey Excursion of -1%o changes the range to approximately -26.49%o &
0.59%o to -23.40%o f 0.59%o. This range suggests that the WCF were feeding off of C3
plants in open areas that periodically experienced water stress. This interpretation is
supported by the serial sampling, which reveals dietary ranges of -24.36%o a 0.59%o to
-25.64%o a 0.59%o for two serially sampled M. primus specimens and -25.02%o a 0.59%o
to -27.06%o a 0.59%o for the two serially sampled A. isonesus specimens. The serially
sampled specimens of Aphelops also support an interpreted diet of water-stressed C3
plants, with significantly variant dietary ranges of -23.47%o a 0.59%o to -26.82%o &
0.59%o. The stable carbon isotopic evidence suggests that the WCF were consuming
water-stressed plants in open arid and seasonal environment, such as open-country plains.
Interpretations about local seasonality can be made from the serial 6 O0 values
from the structural carbonate in the enamel of the WCF. Assuming that equids from the
middle Miocene were obligate drinkers like modern equids, the serial sampling should
reveal a noticeable curve in 68 O values. For M. primus, specimens UF221419 and
UJF221427 display trends that are nearly mirror images of one another. Enamel
mineralization, which begins at the crown, began during the winter for UF 221419, which
is evident from the low 6 O0 values at 15 and 10 mm from the base of the tooth. Enamel
mineralization, which takes about 1.5 to 2.8 years for cheek teeth in modern equids
(Bryant et al., 1996a), ended in the summer. Contrastingly, enamel mineralization for
UF221427 began in spring or early summer and was mostly completed by winter,
according to the high 68 O values from 20 to 5 mm from the tooth base that are within
error of one another. Although modern equid cheek teeth take a minimum of about 1.5
years to mineralize, it is possible for the teeth of 2erychippus primus to take less than a
year since the teeth are less high-crowned than modern equids. It should be noted that
each sample along the tooth bears a 6 O0 value that is time-averaged over a few months.
The other equid that was serially sampled, A. isonesus, reveals two distinct curves that
are also mirror images. UF221407 appears to have begun enamel mineralization towards
the end of summer or fall and continued through the entire winter and into the next
summer. UF217590 began enamel mineralization in early summer or spring, in the
Modern rhinocerotids are obligate drinkers (Clauss et al., 2005). Assuming that
Aphelops is also an obligate drinker, the 618O values of the enamel carbonate should
reflect any seasonal variation. UF 104227 displays considerable variation. Tooth
formation began during the 618O lows of winter. There are two summer peaks and three
winter troughs, indicating that the Aphelops cheek tooth formed over roughly two years.
The 618O variation of UF 1 16827 is not as pronounced as UF 104227, but a curve outside
the error ranges is discernible. Tooth mineralization for UF 104227 began in a summer
peak. The curve features two summer peaks and two winter lows, suggesting that the
Aphelops molar took slightly more than a year to mineralize. The lower 618O values of
UF 1 16827 indicates that the tooth mineralized during a period of cooler temperatures
than UF 104227. Overall, the 618O variation in both Aphelops specimens supports the
interpretation seasonality in northern Florida during the middle Miocene.
The variation of 68"O values for the serially sampled specimens ofM.~ primus, A .
isonesus, and Aphelops suggests that the WCF experienced significant seasonality. This
stable oxygen isotopic data supports the interpretations of periodic water-stress made
from the stable carbon isotopic data. The seasonality experienced by the WCF is typical
of the warming that took place in the middle Miocene.
The middle Miocene (early Barstovian) WCF represent an interesting transition in
the paleodiets of Floridian mammals. The mesodont equid Parahippus leonensis can be
found at the early Miocene (Hemingfordian) Thomas Farm locality, from about 19 to 18
Ma (Hulbert and MacFadden, 1991). The rate of wear for P. leonensis cheek teeth is
about half of wear rate of grazing equids, indicating a mixed diet of grass and browse
(Hulbert, 1984). This interpretation has been supported by mesowear analyses (Hoffman,
unpublished). This suggests that grasses, either C3 Of C4, eXiSted in central Florida as far
back as 19 Ma, but the mesodont equids at were still consuming significant amounts of
browse. Only hypsodont equids, like Acritohippus isonesus, were capable of exploiting
the more abrasive grasses. Thomas Farm also yields the brachydont equids Anchitherium
and Archaeohippus. A diminuitive presence of grasses would explain the lack of
hypsodont taxa at the Thomas Farm site. It was only after C3 graSses became more
abundant, by 15.8 Ma in northern Florida, that equids radiated into hypsodont lineages.
Mesowear analyses revealed grazing diets for all five of the hypsodont equids from
the last 10 million years. Coupled with previous isotopic data (Table 6-1), it is possible to
trace the existence of C3 graSses over the last 10 million years in Florida. All three horses
from the Love Site (Neohipparion trampasense,~rtrtrtrt~t~t~ Cormohipparion plicatile, and
Cormohipparion ingenuum) have 613C ValUeS indicative of a C3-dominated diet.
Furthermore, the most positive 613C Value Of these samples is -10.8%o, well below -8.7%o.
This indicates that the diets of these horses consisted purely of C3 plants. Since the
mesowear analyses indicate that these taxa were primarily grazers, they must have been
feeding on C3 graSses. The 613C ValUeS from enamel indicate a range of -26.7%o to
-24.9%o for the consumed plants. This suggests that the equids were eating water-stressed
C3 graSses in seasonal, open habitat. This interpretation compares favorably with other
studies. The population dynamics of N. trampasenser~rtrtrtrt~t~t~ from the Love Site reveal a high rate
of tooth wear, comparable to the modern zebra Equus burchelli, which indicates a highly
abrasive grass-dominated diet (Hulbert, 1982). The presence of discrete age classes at the
site suggests that the area was a wooded grassland savanna with seasonal rains that
controlled the migratory and birthing patterns (Hulbert, 1982). N. trampasenser~rtrtrtrt~t~t~ migrated
away from the Love site during the wet season to give birth, then returned during the dry
season (Hulbert, 1982). This interpretation is supported by the range of 613C ValUeS,
which suggest that the equids at the Love Site were consuming water-stressed grasses.
The next site, the Upper Bone Valley Formation, features specimens ofl~annippus
aztecus that represent the 4.5 Ma level. The three 613C ValUeS for N. aztecus indicate a
diet composed of both C3 and C4 plant material (Table 6-1). Combined with the
mesowear grazer classification, N. aztecus can be interpreted as an opportunistic grazer,
feeding on both C3 and C4 graSses and possibly some browse. This level is dated after the
global shift in carbon biomass, when C4 graSses became dominant. However, the N.
aztecus specimens consist of an Ml, an M2, and an M1/2. First molars are completely
mineralized during the weaning process, suggesting that the isotopic compositions of
Mis are influenced by the mother's milk (Bryant et al., 1996a; Fricke and O'Neil, 1996;
Hoppe et al., 2004). Second molars are not completely mineralized until after the
weaning process ends, but they can still reflect the isotopic composition of the nursing
diet (Bryant et al., 1996a; Fricke and O'Neil, 1996; Hoppe et al., 2004). The M2 and
M1/2 613C ValUeS, therefore, might be skewed.
A study conducted on 6 sympatric horses from Upper Bone Valley Formation
concluded that, as recently as 5 million years ago, Cq tropical grasses in Florida coexisted
with C3 graSses. Using microwear, this study showed that Nannippus aztecus, along with
the sympatric late Hemphillian horse Pseudhipparion simpsoni, consumed both C3 and C4
grasses (MacFadden et al., 1999). This study substantiates the interpretation of
NNNNNNNNNNNNNNNNNNNNanipu aztecus as a C3 C4 grazer. Furthermore, older N. aztecus specimens from the 7
Ma Moss Acres Racetrack site (Table 6-1) have 613C ValUeS of -7.9%o and -5.3%o. These
values indicate a mixed diet of C3 and C4 plants. Mesowear analyses could not be
conducted on this population because the population size was too small (<20). However,
if this population behaved similarly to the younger Upper Bone Valley Nannippus
populations ("i.e.", they were primarily grazing), then this population was likely
composed of C3 C4 graZeTS.
Finally, the Leisey Shell Pit represents the 1.5 Ma level. The specimens of Equus
"~le/Jy/" have 613C ValUeS that are more negative than -1.3%o, indicating a C4-dominated
diet with a minor C3 COmponent. Modern Equus is predominantly a grazer, but is known
to eat other locally available plants (Berger, 1986). The isotopic and mesowear data
suggests that, like modern Equus, Equus "leidyi" was a dominant C4 grazer at 1.5 Ma.
Previous work (MacFadden et al., 1999) as well as the mesowear analyses of
various Florida equid populations have shown that C3 graSses were present in Florida
from ~10 to ~5 Ma. In light of the microwear and isotopic data retrieved from the WCF,
it appears that the record of C3 graSses in northern Florida and southern Georgia extends
back to at least 15 million years ago. As evidenced by the consumption C3 graSses by a
hypsodont taxon, it is possible that C3 graSses forced the adaptation of hypsodonty in
I I II
Pure C, IVixed Diet Pure C4
Q Acritohippus isonesus
-12 -11 -10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
8' C (V-PDB)
Figure 6-1. Plot of microwear index versus 613C ValUeS. The dashed line represents a
microwear index of 1.5, the boundary between a browser and a grazer
classification. Solid lines mark the boundaries between 'Pure C3 diet', 'Mixed
diet', 'Pure C4 diet" and transitional zones. 613C ValUeS below -8.7%o indicate
a pure C3 diet. Note that both Acritohippus isonesus and Neohipparion
trampasenser~rtrtrtrt~t~t~ have microwear indices above 1.5 and 813C ValUeS below -8.7%o,
suggesting diets composed purely of C3 graSSCS.
Table 6-1. Isotopic data for equids from 4 central Florida sites. Abbreviations: UF ID =
catalogue number in Florida Museum of Natural History collections.
(Modified woth permission from MacFadden and Cerling, 1996)
Taxon Site UF ID Material 613C (ooo
9.5 Ma Level, late Clarendonian, late Miocene
Love Bone Bed 32230 R. P3- 3
Love Bone Bed 32265 R. P2
Love Bone Bed 32265 R. P3
Love Bone Bed 32265 R. P4
Love Bone Bed 32265 R. M'
Love Bone Bed 60396 R. P2
Love Bone Bed 35979 R. P2
7.0 Ma level,
"middle" Hemphillian, late Miocene
69933 R. M3
None R. P2
4.5 Ma level, latest Hemphillian, early Pliocene
Upper Bone Valley None L. M
Upper Bone Valley 63633 L. M2
Upper Bone Valley 65796 R. M'
1.5 Ma level, early Irvingtonian, early Pleistocene
Equus "lleidyi"l Leisey 1A 80047 R. M3
Equus "leidvi Leisey 1A None R. P?
Stable carbon isotope analyses of the Willacoochee Creek Fauna, the oldest
isotopically sampled taxa in Florida, provide no evidence for the presence of C4 graSSCS
in the diets of ungulates in northern Florida and southern Georgia at from 15.3 to 15.9
Ma. This suggests that C4 graSses were not present in the area at important time in
mammal evolution and, since they are not a significant dietary component, it is unlikely
that they were responsible for hypsodonty adaptations. Furthermore, the microwear of the
most hypsodont taxon from the WCF, Acritohippus isonesus, indicates a grazing diet.
Combined with the C3-COnSuming classification, based on 6 13C ValUeS from bioapatite,
the microwear suggests that Acritohippus isonesus ate C3 graSses. This substantiates the
presence of C3 graSses in the middle Miocene of northern Florida and supports Fox and
Koch's (2003) claim that C3 graSses were responsible for the hypsodonty adaptations.
Stable carbon and oxygen isotope values also indicate that the Willacoochee Creek Fauna
ate many water-stressed plants and lived in an open, arid, and seasonal environment, such
as open-country plains
Mesowear analyses and previously published isotope data reveal that the C3
grasses were the primary dietary component in some Floridian horses at ~9.5 Ma. Even
after the global carbon biomass shift at ~7 Ma, C3 graSses persisted in Florida and were
significant dietary components until at least ~4.5 Ma. By ~1.5 Ma, it appears that
abundance of C3 graSses finally subsided in Florida, where the dominant grazer (Equus
"le/Jyr )i~ was consuming mostly C4 graSs. This long reliance off of C3 graSses suggests
that C3 graSses were a driving mechanism for the appearance of hypsodonty in ungulates.
The combinations of mesowear and stable isotope analyses and microwear and stable
isotope analyses have proven to be valuable tools in assessing the origins of hypsodonty.
Future work will involve applying these combined methods to the northern Great Plains,
where there is a rich record of ungulate fossils.
UF ID Taxon Locality
62251 Neohipparion trampasense Love Site
25637 Neohipparion trampasense Love Site
27992 Neohipparion trampasense Love Site
32273 Neohipparion trampasense Love Site
32258 Neohipparion trampasense Love Site
27991 Neohipparion trampasense Love Site
32253 Neohipparion trampasense Love Site
53428 Neohipparion trampasense Love Site
53427 Neohipparion trampasense Love Site
32256 Neohipparion trampasense Love Site
32272 Neohipparion trampasense Love Site
32252 Neohipparion trampasense Love Site
53429 Neohipparion trampasense Love Site
53230 Neohipparion trampasense Love Site
53243 Neohipparion trampasense Love Site
53256 Neohipparion trampasense Love Site
53269 Neohipparion trampasense Love Site
53244 Neohipparion trampasense Love Site
53257 Neohipparion trampasense Love Site
53232 Neohipparion trampasense Love Site
53271 Neohipparion trampasense Love Site
53246 Neohipparion trampasense Love Site
53272 Neohipparion trampasense Love Site
53234 Neohipparion trampasense Love Site
53247 Neohipparion trampasense Love Site
53273 Neohipparion trampasense Love Site
53235 Neohipparion trampasense Love Site
53248 Neohipparion trampasense Love Site
53236 Neohipparion trampasense Love Site
53249 Neohipparion trampasense Love Site
53275 Neohipparion trampasense Love Site
53237 Neohipparion trampasense Love Site
53250 Neohipparion trampasense Love Site
53276 Neohipparion trampasense Love Site
53225 Neohipparion trampasense Love Site
53251 Neohipparion trampasense Love Site
MESOWEAR VALUES LISTED BY SAMPLE
Table A-1. Abbreviations: UF ID = catalogue number in Florida Museum of Natural
History collections, CS = Cusp Shape, OR = Occlusal Relief, BVF = Bone
Valley Formation, Leisey = Leisey Shell Pit, B = Blunt, R = Round, S =
Sharp, H = High, L = Low.
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
Equus "leidyi "
SERIAL STABLE ISOTOPE VALUES
Table B-1. Abbreviations: ELC
Farm Mine, and LC2
Englehard La Camelia Mine, MGF = Milwhite Gunn
La Camelia 2 Mine.
Table B-1. Continued
813 o/oo) 6180 (%o)
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