Stable Isotope Diet Analysis of Teleoceras Fossiger from the High Plains of Kansas

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Master's Theses Graduate School

Spring 2017 Stable Isotope Diet Analysis Of Teleoceras Fossiger From The iH gh Plains Of Kansas Patrick J. Wilson Fort Hays State University, [email protected]

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Recommended Citation Wilson, Patrick J., "Stable Isotope Diet Analysis Of Teleoceras Fossiger From The iH gh Plains Of Kansas" (2017). Master's Theses. 20. https://scholars.fhsu.edu/theses/20

This Thesis is brought to you for free and open access by the Graduate School at FHSU Scholars Repository. It has been accepted for inclusion in Master's Theses by an authorized administrator of FHSU Scholars Repository. STABLE ISOTOPE DIET ANALYSIS OF TELEOCERAS FOSSIGER

FROM THE HIGH PLAINS OF KANSAS

being

A Thesis Presented to the Graduate Faculty

of the Fort Hays State University in

Partial Fulfillment of the Requirements for

the Degree of Master of Science

Patrick Wilson

B.S., Wichita State University

Date______Approved______Major Professor

Approved______Chair, Graduate Council

Graduate Committee Approval

The Graduate Committee of Patrick J. Wilson approves this thesis as meeting partial fulfillment of the requirements for the Degree of Master of Science.

Approved______Chairman, Graduate Committee

Approved______Committee Member

Date______

ABSTRACT

Stable isotope geochemistry provides a way of examining both modern and

ancient ecosystems. One important use of stable carbon isotopes is to determine the

amount of C3 versus C4 plant biomass in the diet of herbivorous mammals. C3 plants

have a more negative stable carbon isotope ratio (δ13C) than C4 plants because of the

different photosynthetic pathways each type uses. These different δ13C values are preserved in the hydroxyapatite of herbivores upon consumption of the different plant types. The purpose of this study it to use δ13C values from serial samples of tooth enamel

from six isolated permanent molars of Teleoceras fossiger, an extinct North American

rhinoceros from the Early Hemphillian, to determine what type of vegetation was

consumed by these animals and if the diet reflects the amount of C4 biomass present in

the ecosystem.

The δ13C values from the T. fossiger samples show a range between -11.2‰ and

-7.2‰ (all vs. VPDB, same below). These δ13C values indicate the diet of T. fossiger

contained little to no C4 biomass. Using an ecosystem model developed for the Late

Neogene of Nebraska, all of these data, except for two points, fall within an open canopy ecosystem (-12.1‰ to -7.7‰), an open grassland consisting of little to no C4 vegetation.

The two data points (-7.6‰ and -7.2‰) that do not fall within an open canopy ecosystem, fall within the mixed C3/C4 ecosystem (-7.7‰ to 2.1‰). Paleobotanical and paleosol carbonate data collected from the Minium Quarry, which is the quarry where the

sampled T. fossiger teeth were collected, shows approximately 20% C4 biomass in the i

ecosystem. The difference between the diet of T. fossiger and the percentage of C4 biomass in the ecosystem demonstrates that although there are C4 grasses present, these rhinoceroses did not incorporate a major C4 component into their diet.

ACKNOWLEDGEMENTS

I want to acknowledge Drs. Chunfu Zhang, Laura Wilson, Rob Channell, and

Reese Barrick for serving on my thesis committee for this project. I want to give special

thanks to Dr. Chunfu Zhang, my faculty advisor, for personally taking and analyzing the

samples at Florida State University’s Stable Isotope Laboratory at the National High

Magnetic Field Laboratory (NHMFL). I also want to thank Dr. Yang Wang and her

laboratory staff at NHMFL for assisting in sample analysis and for allowing the running

of the samples.

iii

TABLE OF CONTENTS

Page

ABSTRACT ...... i

ACKNOWLEDGEMENTS ...... iii

TABLE OF CONTENTS ...... iv

LIST OF FIGURES ...... vi

LIST OF TABLES ...... viii

LIST OF APPENDICES ...... ix

INTRODUCTION ...... 1

BACKGROUND ...... 9

Hydroxyapatite Properties and Deposition ...... 19

Minium Quarry and Bemis Local Fauna ...... 12

MATERIALS & METHODS ...... 16

Preparatory Methods ...... 17

Analytical Methods ...... 18

Carbon Isotope Values ...... 18

RESULTS ...... 20

Enamel δ13C Results ...... 20

Analytical Results ...... 20

DISCUSSION ...... 25

CONCLUSIONS...... 33 iv

REFERENCES ...... 35

APPENDICES ...... 51

LIST OF FIGURES

Figure Page

1 Model of C3 and C4 photosynthetic pathways showing the usage of Rubisco and

PEP enzymes (From Tipple and Pagani, 2007)...... 7

2 Histogram showing distribution of bulk C3 and C4 plant δ13C values (From

Tipple and Pagani, 2007)...... 8

3 Heat map of T. fossiger tooth showing the age of enamel formation with red being

the youngest and blue being the oldest ...... 11

4 Geologic Maps of Graham (upper left) and Ellis (lower right) counties [modified

from Prescott Jr (1955) and Kansas Geological Survey et al. (1988)]...... 14

5 Kansas County map indicating the location of the Minium Quarry and the Bemis

Local Fauna...... 15

6 85 serial δ13C values plotted against the distance from the bottom of the crown.

Red line denotes -7.7‰ upper limit of an open canopy ecosystem...... 22

7 Box plot of δ13C values for six specimens of Teleoceras fossiger showing two

statistical outliers (open circles) for VP-8745...... 23

8 Anterior view of FHSM VP-8745 showing alteration of the enamel and drill

tracks where samples were taken...... 30

9 Buccal view of FHSM VP-8745 showing alteration of the enamel and drill tracks

where samples were taken...... 30

vii

LIST OF TABLES

Table Page

1 Table 1. Plant Traces Recorded at Minium Quarry (From Thomasson, 1990) .....13

2 Kruskal-Wallis and KWMC Results ...... 24

viii

LIST OF APPENDICES

Appendix Page

I Data Table ...... 51

II Range Values ...... 53

INTRODUCTION

The genus Teleoceras was first named in 1894 by Hatcher and contains eight different species. The temporal range for Teleoceras is from the Early Hemingfordian to the latest Hemphillian. Teleoceras are one of the most wide-spread, easily recognizable, and distinctive North American fossil rhinoceroses. Distinguishing characteristics of

Teleoceras include the overall skull shape, distinctly robust, short limbs, and hypsodont teeth (Hatcher, 1894a, 1894b; Prothero, 2005). The hypsodont dentition of Teleoceras teeth suggests that these species were herbivores (Stebbins, 1981; Feranec and

MacFadden, 2000; Zazzo et al., 2000; Clementz and Koch, 2001; Strömberg, 2002;

Tafforeau et al., 2007; Fraser and Theodor, 2013). Teleoceras fossiger is known from localities in South Dakota, Nebraska, Kansas, Oklahoma, Texas, and Nevada and has a temporal range from the Early Hemphillian to the end of the Late Hemphillian (Prothero,

2005). This species is of interest because its hypsodont tooth morphology suggests individuals were eating vegetation present in the ecosystem during the spread of C4 plants.

The expansion of C3 grasslands reached the Great Plains by the early Miocene

(Leopold and Denton, 1987; MacFadden and Cerling, 1996; Strömberg, 2002; Fox and

Koch, 2004; Janis, 2004; Kita, 2011; Fox et al., 2012; Fraser and Theodor, 2013; Kita et al., 2014). Fossil phytoliths and stable carbon isotopes from tooth enamel carbonates and soil carbonates from these early Miocene grasslands indicate that C4 biomass was present

(Thomasson, 1976, 1984, 1990, 1991, 2005; Thomasson et al., 1986; Strömberg, 2002;

Thomasson and Wester, 2003; Fox and Koch, 2004; Kita, 2011; Strömberg and

McInerney, 2011; Fox et al., 2012; Kita et al., 2014; Wang, 2016). Stable carbon isotopes

and fossil phytoliths indicate that the change from C3 dominated grasslands to C4

dominated grasslands continued through the early Pliocene (Cerling et al., 1993, 1997;

Strömberg, 2002; Fox and Koch, 2004; An et al., 2005; Behrensmeyer et al., 2007; Kita,

2011; Strömberg and McInerney, 2011; Fox et al., 2012; Kita et al., 2014). During the

Late Miocene, approximately 20 percent of plant biomass in the High Plains of Kansas

was composed of C4 plants (MacFadden, 1998; Fox and Koch, 2004; Strömberg and

McInerney, 2011), indicating that diets of herbivores, such as T. fossiger, probably would

not exclusively contain C4 plants. The change from the first occurrence of C4 plants to

C4 dominated ecosystems is poorly understood from 9 Ma to 2 Ma due to a lack of paleobotanical information (Strömberg and McInerney, 2011). However, this dearth of paleobotanical evidence does not account for the plant fossils of the Minium Quarry, which is an 8-9 Ma quarry located in northwestern Kansas. The T. fossiger teeth used in this study were recovered from the Minium Quarry and the Bemis Local Fauna, which is temporally and stratigraphically equivalent to the Minium Quarry.

Modern plants use three different types of photosynthesis. These photosynthetic pathways are C3, C4, and CAM (crassulacean acid metabolism). C3 and C4 pathways are named for the number of carbon atoms in the photosynthetic intermediates

(glyceraldehydes-3-phosphate or G3P for C3 and oxaloacetate for C4), and CAM plants are named for the family in which the CAM mechanism was first observed (Osmond,

1984; Keeley and Rundel, 2003; Rundel and Gibson, 2005; Osmond et al., 2012). In C3

plants, photosynthesis occurs in a single cell and uses an enzyme, called Rubisco or

RuBP (Ribulose-1, 5-biphosphate carboxylase/oxygenase), when fixing carbon for

energy production (Figure 1). Photosynthesis using RuBP exclusively causes higher fractionation – the separation of lighter isotopes from heavier isotopes – of the carbon isotopes than C4 plant photosynthesis (Koch, 1998; Tipple and Pagani, 2007). The range of δ13C values – a ratio of heavier isotopes to lighter isotopes – for fossil C3 plants is typically between -33.5‰ to -20.5‰ with a mean δ13C value of -25.5‰ (Figure 2)

(MacFadden and Cerling, 1996; Koch, 1998; Tipple and Pagani, 2007). Other factors

13 affect δ C values for C3 plants, such as temperature, light level, partial pressure of CO2

(which is the proportion of CO2 in the atmosphere), nutrient levels, and water availability.

Water stress (drought conditions), in particular, can cause δ13C values of C3 plants to

become more positive than under wetter conditions because plants close their stomata

during droughts to conserve water, which leads to internal production of CO2 and fixation

of carbon using O2 in a less efficient process called photorespiration (Koch, 1998; Tipple

and Pagani, 2007). Stomatal closing also occurs under high temperature and high-light

conditions to reduce water loss from the leaf (Ehleringer and Monson, 1993; Tipple and

Pagani, 2007). With C3 photosynthesis more efficient under lower temperatures, lower

light, and higher precipitation conditions, C3 plants are better adapted to temperate

climates than C4 plants.

C4 and CAM plants use the PEP (phosphoenolpyruvate) carboxylase enzyme to fix CO2 into a four carbon acid, but in CAM plants photosynthesis occurs in one cell instead of the two cells that C4 plants utilize (Ehleringer and Monson, 1993). The four- carbon acid is then transferred to an adjacent cell and converted back to CO2, which is

then used by the Rubisco enzyme to produce energy (Figure 1) (Tipple and Pagani,

2007). The use of the PEP enzyme in C4 and CAM plants causes less fractionation from

atmospheric carbon, which leads to more positive stable carbon isotope values than the

exclusive use of the RuBP enzyme in C3 photosynthesis. The more efficient CO2 fixation

makes C4 photosynthesis better adapted for low CO2 concentrations, hot temperatures,

high-light intensity, and dry environments. Fossil C4 plants have a range of δ13C values

between -13.5‰ and -9.5‰ with a mean δ13C value of -11.5‰ (Figure 2) (MacFadden and Cerling, 1996). Other factors that affect C4 plants tend to be abiotic. For instance, the distribution of C4 plants is associated with warm growing seasons. C4 plants can tolerate warmer temperatures, less precipitation, and lower partial pressures of CO2, resulting in

C4 plants showing less variability in their δ13C values than C3 plants (Koch, 1998).

The CAM pathway is the least common of the photosynthetic pathways, occurring

only in succulent plants, which are specially adapted to arid climates (Ehleringer and

Monson, 1993; Koch, 1998; Tipple and Pagani, 2007). In CAM plants, instead of a

spatial separation between PEP and RuBP enzymes there is a temporal separation and all

photosynthesis occurs in a single cell. During the evening, CAM plant stomata are open to allow CO2 to enter the cell and the PEP enzyme fixates carbon in a four carbon acid.

The four carbon acid is then decarboxylated during the day, when the stomata are closed

due to high temperatures and high-light intensity conditions, to release the CO2. The CO2 released from the four carbon acid is then used by the RuBP enzyme to produce energy.

The high concentration of CO2 in the cell when RuBP begins carbon fixation eliminates the photorespiration process, which occurs in C3 plants when the stomata are closed. The use of PEP and RuBP in a single cell produces an isotopic range, ~-33‰ to ~-11‰, in

CAM photosynthesis between C3 and C4 photosynthesis, which makes distinguishing

CAM plants from C3 and C4 plants in the fossil record nearly impossible (Ehleringer and

Monson, 1993; Koch, 1998; Tipple and Pagani, 2007). However, CAM plants are not typically part of ungulate diets (Ehleringer et al., 1997; Kita et al., 2014), so this type of

photosynthesis is not considered in this project.

Ecosystems have an effect on the δ13C value based on the type of plant biomass present within each ecosystem. Closed canopy forest ecosystems contain only C3 plants and are not affected by the factors that cause C3 plants to use photorespiration, such as rainforests, so δ13C values of closed canopy ecosystems are less than -26.2‰ (Kita et al.,

2014). Open canopy ecosystems contain C3 plants affected by photorespiration and/or a

minor percentage of C4 plants, such as woody scrubland, woodland-savannas, “dry” C3

grasslands, and “dry” forest. Thus, open canopy systems have δ13C values between

-26.2‰ and -21.8‰ (Kita et al., 2014). Mixed ecosystems contain C3 and C4 plant

material, and have δ13C values between -21.8‰ and -12.0‰ (Kita et al., 2014). Pure C4

grasslands have δ13C values greater than -12.0‰ (Kita et al., 2014).

C3 plants have a more negative δ13C value (greater proportion of 12C) than C4

plants, thus animals that eat a diet rich in C3 plants will have δ13C values which are more

negative than animals with a diet rich in C4 plants. Fractionation from biological

processes within animals, such as metabolic rates, increase the δ13C by 14.1‰ from the

original plant material (e.g., DeNiro and Epstein, 1978; Koch et al., 1992, 1995a, 1995b,

1997, 1998; Koch, 1998; Kohn and Cerling 2002; Tipple and Pagani, 2007). As an

example, fossil organisms eating a pure C3 diet, with the plant δ13C represented by

-25.5‰, will preserve a δ13C isotopic signature of -11.4‰ in the carbonate molecule of

its hydroxyapatite (Ca10[PO4,CO3]6[OH,CO3]2), the inorganic component of bone, dentine, and enamel. However, animals eating a pure C4 diet, with plant δ13C represented

by -11.5‰, will preserve an isotopic signature of 2.6‰ (e.g., DeNiro and Epstein, 1978;

Koch et al., 1992, 1995a, 1995b, 1997, 1998; Koch, 1998; Kohn and Cerling 2002;

Tipple and Pagani, 2007; Kita et al., 2014).

The fractionation between plants and animals means the isotopic signal preserved

in the hydroxyapatite will be more positive than the isotopic signal of the plants. This

leads to values of the different ecosystems increasing from their baseline. For closed

13 canopy forest systems the δ Cenamel value will be less than -12.1‰ (Kita et al., 2014). For

13 open canopy systems, such as the grassland of the Great Plains, the δ Cenamel values fall

between -12.1‰ and -7.7‰ (Kita et al., 2014). In mixed ecosystems, where the

percentage of C4 biomass contributes a significant amount to the overall ecosystem, the

13 δ Cenamel values range between -7.7‰ and 2.1‰ (Kita et al., 2014). Ecosystems which

13 have δ Cenamel values greater than 2.1‰ are biomes with pure C4 grasses (Kita et al.,

2014).

The purpose of this study is to analyze the diet of the Early Hemphillian

rhinoceros, Teleoceras fossiger, using isotope samples collected from six isolated

permanent molars to determine what type of vegetation was consumed by these animals

and if the diet reflects the amount of C4 biomass present in the ecosystem. This study

helps establish the spread of C4 plant dominance of C4 plants between 8 and 9 Ma and

fills in a gap in the literature regarding the diet of T. fossiger from the High Plains of

Kansas during the Late Miocene.

C3 plant

Mesophyll cell ATP

2 G3P},NADPH ,.e Calvin- .,.,,~,~ 0 Benson ,.,.0, cycle ATP co, = co,-::; co, RuBP ATP 02 -= 02 -: :;. 02 RuBP ~ NADPH Photo- G3P resplrato):y cycle co,

PG ATP G3P NADPH

C4 plant

Mesophyll cell Bundle sheath cell

C3Acids1\ J C3Acidsl ATP L 2G3P NADPH "'1''e I Calvin- .,.,,~' _ Benson ,ef>'o co,= co,-:.T,:_CO,-:;_Hco3r PEP c, photosynthetic cycle} o, = o, -:.•·; o, Y cycle C3 Acids ATP RuBP PEP·C I Jubisco Acidsj C4 Acids t CO2

Figure 1. Model of C3 and C4 photosynthetic pathways showing the usage of Rubisco and PEP enzymes (From Tipple and Pagani, 2007).

180 ~------~ 160 C4 plants 140

>. 120 u I I r:: 100 I Q) I ::::, C3 plants c- 80

it 60

0 '-=-'l:..Cl.L..L...L.J-'-'-L..I...L..L...I.U_L.JL..L.= =-=LU...J...L.J-L.u..J....::...___,J__ __, -40 -35 -30 -25 -20 -15 -10 -5 0

Figure 2. Histogram showing distribution of bulk C3 and C4 plant δ13C values (From Tipple and Pagani, 2007).

BACKGROUND

Fractionation is caused by differences in the masses of atoms’ nuclei. The

difference in mass results in atoms reacting at different rates during thermodynamic and kinetic processes. The delta notation (δ) is a measure of small differences in the isotope ratios expressed in parts per thousand or per mil (‰). When standardizing δ13C values,

the formula δX = [(Rsample/Rstandard)-1]∗1000 is used, where X is the isotope in question and R is the ratio of the heavier isotope over the lighter isotope. For instance, δ13C =

13 12 13 12 ([( C/ C)sample/( C/ C)standard]-1)*1000. This formula calculates delta values by

comparing the ratios of the isotopes (R) to a known standard. For analysis of stable carbon isotopes, the typical standard is the Vienna PeeDee Belemnite (VPDB).

Hydroxyapatite Properties and Deposition

Apatite is a calcium phosphate mineral with the general composition of

Ca5(PO4)3(OH,F,Cl). When the last group is the hydroxide group (OH), the mineral is

called hydroxyapatite. Occasionally, the composition of the hydroxyapatite is altered by

replacing the phosphate and the hydroxide groups with a carbonate group

(Ca10[PO4,CO3]6[OH,CO3]2) (Koch, 1998). Apatite plays an important role in the structure of hard tissues within the vertebrate body.

The hard tissues of the vertebrate body include bone, dentine, and enamel. These tissues contain different amounts of organic materials. Bone tissue can have approximately 30% organic material by dry weight (LeGeros, 1981; Schoeninger and

DeNiro, 1982; Tieszen et al., 1983; Simkiss and Wilbur, 1989; Lee-Thorp and van der

Merwe, 1991; Koch et al., 1997; Kohn and Cerling, 2002), making it highly porous. This

porosity leads to complications when analyzing the tissue for stable carbon isotopes because it has higher rates of alteration of isotopic composition from diagenesis

(LeGeros, 1981; Schoeninger and DeNiro, 1982; Tieszen et al., 1983; Simkiss and

Wilbur, 1989; Lee-Thorp and van der Merwe, 1991; Koch et al., 1997; Kohn and Cerling,

2002). Dentine has approximately the same percentage of organic material by dry weight

as bone tissue (LeGeros, 1981; Schoeninger and DeNiro, 1982; Tieszen et al., 1983;

Simkiss and Wilbur, 1989; Lee-Thorp and van der Merwe, 1991; Koch et al., 1997; Kohn

and Cerling, 2002). However, due to the arrangement of the organic materials in the

dentine, it is less porous than bone. Enamel is the least porous of all the hard tissues

because it contains less than 5% organic material (LeGeros, 1981; Schoeninger and

DeNiro, 1982; Tieszen et al., 1983; Simkiss and Wilbur, 1989; Lee-Thorp and van der

Merwe, 1991; Koch et al., 1997; Kohn and Cerling, 2002). The low porosity makes

enamel resistant to problems caused by diagenesis and is thus an excellent tissue for

stable isotope analysis. Tooth enamel is also an ideal tissue for isotopic analysis because

enamel can be prepared for either phosphate or carbonate analysis. The carbonate

component can be used to examine the amount of C3 versus C4 plant biomass in the diet

of large herbivores.

Enamel is deposited throughout the growth of the tooth (Boyde and Fortelius,

1986; Hillson, 1986; Lowenstam and Weiner, 1989; Simkiss and Wilbur, 1989; Passey

11 and Cerling, 2002, 2004). Once the enamel is laid down, it is chemically and physically invariant (Boyde and Fortelius, 1986; Hillson, 1986; Lowenstam and Weiner, 1989;

Simkiss and Wilbur, 1989; Kohn and Cerling, 2002). Unlike bone tissue, enamel does not remodel after it is deposited, but preserves the original isotopic composition of the tooth

(Koch, 1998; Kohn and Cerling, 2002).

When enamel is deposited, it forms in

layers that move outward from the enamel-

dentine junction and toward the tooth root

(Hillson, 1986; Lowenstam and Weiner, 1989;

Fricke and O’Neil, 1996; Passey and Cerling,

2002; Tafforeau et al., 2007). First a protein-rich

layer deposited on the enamel-dentine junction.

Then the hydroxyapatite crystals accumulate on

the protein layer. The formation of the Figure 3. Heat map of T. fossiger tooth showing the age of enamel hydroxyapatite crystals happens over a period of formation with red being the youngest and blue being the oldest time, ranging from days to months, causing an amount of time averaging in each tooth (Passey and Cerling, 2002; Taffoeau et al., 2007).

In the black rhinoceroses (Diceros bicornis), m1 molars take 34 months to form; m2 molars take 45 months to form; and m3 molars take 48 months to form (Goddard, 1970).

The process of enamel deposition leads to enamel at the top of the tooth being older than 12

the enamel deposited at the bottom of the tooth crown, as seen in Figure 3 with the red

being younger in age than blue.

Minium Quarry and Bemis Local Fauna

The Minium Quarry is an attritional deposit in the Ogallala Formation (Darnell,

2000; Ludvigson et al., 2009), and is located in a Late Miocene fluvial deposit located

~7 km northwest of Morland, Kansas in Graham County (Figures 4 & 5). Various fossil

taxa have been recovered from the Minium Quarry, dating the deposit at 8-9 Ma (Darnell,

2000). According to Thomasson and Wester (2003), these fossils include ~30 different

species of vertebrates and ~30 different species of flowering plants. The plants from the

Minium Quarry are an important factor to consider for this project because they represent

a sample of the flora present during the Early Hemphillian. The Minium Quarry flora

provides the paleobotanical record between 8-9 Ma, and shows if any C4 biomass is present. The paleobotanical data could help verify the diet analysis of herbivores living in

the area.

The flora of the Minium quarry indicates that some shallow, permanent water was

nearby and that there were grasses with some trees (Thomasson, 1990; Thomasson and

Wester, 2003). The grass seeds and leaf fragments show C4 plants and C3 trees, forbs,

and grasses already present in the ecosystem (Table 1). The C4 plants present include numerous genera which have been shown to prefer warm climates and near aquatic ecosystems, such as chloridoid leaf fragments and fossil sedge seeds (Thomasson, 1990).

There is also a species of Midravalva, which is a pond weed, indicating the presence of a

13 permanent body of water. This creates a marginal-aquatic setting with a warm temperate to subtropical grassland (Thomasson, 1991).

Table 1. Plant Traces Recorded at Minium Quarry (From Thomasson, 1990)

Plant Name C3 or C4 Type of Plarit Climat e Indication Ce/tis wilfistonii C3 Tree Biorbia Jossi!ia C3 Farb Chen opo clium spp. C3 Farb Cryptantha aruiculata C3 Farb Prolappula spp. C3 Farb Ar:ch aeoleersia nebraskensis C3 Grass W arm, nea r aquat ic Berriochloa amphom fis C3 Grass W.arm Berriochloa conica C3 Grass w .arm Berriochloa clartonii C3 Grass w .arm Berrioch/oa maxima C3 Grass Warm Berrioch /oa minuta C3 Grass w .arm Berriochloa varieg ata C3 Grass Warm Nassef/a poh!ii Grass w .arm Panicum elegans C4 Grass Arnri dinoid le.a t fragm ents C3 Grass Nea r aquaUc Ba mbl!l soid le.af fragme11 t s C3 Grass W arm, nea r aquat i,c Chloridoid leaf fragments C4 Grass w .arm Carex graceii C4 Se dge Nea r aquat i,c Cyperocarpus pu/cherrim a C4 Se dge Nea r aquat i·c Eleofimbris svensonii C4 Se dge Nea r aquaUc Cyp eraceae leaf fragments C4 Se dge Miclravalva spp. C3 Pond W eed Aquat ic

Figure 4. Geologic Maps of Graham (upper left) and Ellis (lower right) counties [modified from Prescott Jr (1955) and Kansas Geological Survey et al. (1988)]. 15

Figure 5. Kansas County map indicating the location of the Minium Quarry and the Bemis Local Fauna.

MATERIALS & METHODS

Six T. fossiger adult molars housed at Fort Hays State University’s Sternberg

Museum of Natural History (FHSM) were selected for this project. Specimens FHSM

VP-8968, FHSM VP-8294, FHSM VP-8290, FHSM VP-8913, and FHSM VP-8745 came from the Minium Quarry in Graham County, Kansas; FHSM VP-5496 came from the Bemis Local Fauna (LF) in Ellis County, Kansas (contact FHSM for detailed locality data). FHSM VP-8968, 8290, 8913, and 8745 are all identified as lower right molars.

FHSM VP-8294 and 5496 are identified as lower left molars. These teeth were chosen because of their isolated nature, type of tooth, and specimen access. The isolated nature of the teeth was necessary for better handling while drilling, and isolated teeth were the only specimens allowed to be sampled. The type of tooth was the most important factor when selecting teeth, because premolars and incisors might be deciduous teeth, which show the isotopic signature from the mother’s milk rather than the vegetation in the diet of the individual (LeGeros, 1981; Boyde and Fortelius, 1986; Hillman‐Smith et al.,

1986; Hillson, 1986; Passey and Cerling, 2002). Molars show the isotopic signal from the vegetation in the diet of the individual because they begin developing after weaning occurs in rhinoceroses (Goddard, 1970; LeGeros, 1981; Boyde and Fortelius, 1986;

Hillman‐Smith et al., 1986; Hillson, 1986; Passey and Cerling, 2002). Molars were also chosen following previous stable isotope methods using tooth enamel (Quade et al., 1992;

MacFadden, 1998; Wang and Deng, 2005; Zhang et al., 2009, 2012; Wang, 2016).

Preparatory Methods

The preparation method used in this study follows Zhang et al. (2009; 2012) and

focused on isolating the structural carbonate in the tooth enamel. Using a variable speed

rotary tool, the teeth were drilled perpendicular to the growth axis of the enamel to create a fine powder. The powder was then collected and placed in microcentrifuge vials, and reacted with 5% sodium hypochlorite (NaOCl) for at least 12 hours to remove any

remaining organic material. Then the enamel powder was centrifuged and rinsed three

times with deionized water to remove any remaining NaOCl. Once the powder was

rinsed, it was reacted with 1 M acetic acid. Again, this mixture was allowed to soak for

12 hours. The purpose of the acetic acid bath was to remove the non-structural carbonate

from the enamel. To stop the reaction, the enamel samples were centrifuged and rinsed

three times with deionized water to remove any remaining acetic acid.

The powder was sent to Florida State University (FSU) for carbonate isotope analysis. Sample analysis took place at FSU’s Stable Isotope Laboratory at the National

High Magnetic Field Laboratory, which is equipped with a continuous flow Finnigan

MAT Delta Plus XP stable isotope ratio mass spectrometer. After freeze drying, the

enamel samples were weighed into individual vials. This was also done with a universal carbonate standard and three sets of in-house laboratory carbonate standards. Then the vials were injected with 100% phosphoric acid on the sampling block held at 25.0 ±

0.1°C, transferred to a water bath maintained at 25.0 ± 0.5°C, and allowed to react for at least 72 hours to produce carbon dioxide gas from the enamel powder, universal

carbonate standard (NBS-19), and in-house carbonate standards (MERK, Roy-Cc, and

YW-Cc). This carbon dioxide gas was passed through the mass spectrometer to measure

stable carbon isotope ratios.

Analytical Methods

Isotope values produced by the mass spectrometer were standardized to Vienna

PeeDee Belemnite (VPDB) to produce delta values (δ). Data were analyzed to look for statistical outliers and visualized using a box plot created from the statistical program R

x64 version 3.2.2. R was also used to compare the central tendency of the stable isotope

values among teeth via a one-way analysis of variance (ANOVA) or a Kruskal-Wallis test. An ANOVA was used to determine if the means of the individual T. fossiger teeth differed significantly from the mean of all the sampled teeth. To assess the ANOVA’s assumptions of normal distribution and homogeneity variances, Shapiro-Wilks and

Levene’s tests were used. When assumptions for an ANOVA were not met, a Kruskal-

Wallis test was used to compare the medians instead. If the ANOVA or Kruskal-Wallis showed a significant difference, a post-hoc Tukey’s honest significant difference (HSD) or Kruskal-Wallis Multiple Comparison (KWMC) test was conducted. All statistics for

this project use a significance level of 0.05.

Carbon Isotope Values

The null hypothesis for this study is the diet of T. fossiger only contained C3 plants. To determine if there is a presence of C4 plant biomass in the diet of T. fossiger,

13 δ Cenamel values were evaluated using a model developed by Kita et al. (2014) based on

19 extant plants and normalized to parameters for the late Neogene of Nebraska. This model accounts for diet-enamel enrichment, changes in the atmospheric CO2 composition due to the use of fossil fuels, effects of latitude, and effects of altitude. The model indicates that

13 ecosystems will have different δ Cenamel values based on the type of vegetation present and separates the ecosystems into four categories: closed canopy forest (δ13C ≤ -12.1‰), open canopy forest (δ13C between -12.1‰ and -7.7‰), mixed C3/C4 grasslands (δ13C between -7.7‰ and 2.1‰), and pure C4 grasslands (δ13C ≥ 2.1‰) (Kita et al., 2014).

Eighty-five tooth enamel δ13C values were compared to these four ranges to determine if

C4 biomass played a role in the diet of T. fossiger in Kansas during the Early

Hemphillian.

Following this model, tooth enamel δ13C values below -7.7‰ suggests a pure C3 diet, while δ13C values between -7.7‰ and 2.1‰ suggests as a mixed diet. However, water-stressed (drought-stricken) C3 vegetation can cause tooth enamel of mammals to

13 have δ Cenamel values above -7.7‰ (Kita et al., 2014). The effect of water-stress conditions were considered when making interpretations.

RESULTS

Enamel δ13C Results

All δ13C values from the 85 samples drilled from the six teeth are less the -7‰

and greater than -12.1‰ (Figure 6) (Appendix I). Of these samples, there are only two

values between -7.0‰ and -7.7‰; they both belong to FHSM VP-8745. The rest of the

sample δ13C values fall between -7.7‰ and -11.2‰. Of the 85 samples, 71 of the samples

have δ13C values that are less than -9.0‰. The maximum δ13C value is -7.2‰ and

belongs to FHSM VP-8745. The minimum δ13C value is -11.1‰ and belongs to FHSM

VP-8290. FHSM VP-8745 has the largest range of δ13C values of 2.3‰ with two statistical outliers (-7.2‰ and -7.6‰) at the nearest two drill tracks to the occlusal surface (Figure 7), which represent the youngest enamel in ontogenetic age (Appendix

II).

Analytical Results

All teeth, except for FHSM VP-8745, which fails normality of the δ13C values

(W = 0.83306, p = 0.010), pass a Shapiro-Wilk normality test for δ13C values. The δ13C

values of the six sampled teeth fail Levene’s test for homogeneity of variance

(F = 3.2311, df = 5, p-value = 0.010), so a non-parametric Kruskal-Wallis test is run

instead of an ANOVA (Table 2). The Kruskal-Wallis test shows that there is a significant

difference in the median δ13C values of the six teeth (X2 = 68.23, df = 5, p-value =

2.393e-13). A post-hoc KWMC test determines the significant differences are between

FHSM VP-8968 and VP-8290, FHSM VP-8294 and VP-8290, FHSM VP-8294 and

VP-5496, FHSM VP-8294 and VP-8745, FHSM VP-8290 and VP-5496, FHSM VP-8290 and VP-8745, FHSM VP-8913 and VP-5496, and FHSM VP-8913 and VP-8745 (Table

2). Statistical outliers remain in the data set for all analyses because of their possible ecological impact and minimal statistical impact.

FHSM V P-6496 FHSM VP-8290 22

_.,,,,·-·-·...... -•-...... --.. .-· ,.,.• ..._. -- . --· ~• ·----· -• -·-·-·-· -·-· __ ,..,. -· -• ~- ----.

15 20 25 30 35 40 45 50 25 30 35 40 45 50 55

Distance from Bottomr.J. Crown (rrm) Distance fro mBottom r.J. Crown (ll'Til

FHSM VP-8294 FHSM VP-8745

8Cl. 8Cl. > > "/ "/

'\' '\' - .,....• __....• / •-• ...... -·-·.,.,..--· -· . -. -·

15 20 25 30 35 40 45 15 20 25 30 35 40 45 50

Distance fro m Bottom of Crow, (nm) Ois1ance fromBottomr.J.Crow,(fl'lTV

FHSMV P-8913 FHSM VP-8968

It'•-. - • ,..,. • .,,,•.,....,... -· -· - • -·-..,_. ---•.• ·-·-----·-·--~· ----.,,.-,--·---. "·

15 20 25 30 35 40 45 15 20 25 30 35

Distance from Bottom cl Cro'Nrl (rrm) Distance from Bottom ct Crooo (nm

Figure 6. 85 serial δ13C values plotted against the distance from the bottom of the crown. Red line denotes -7.7‰ upper limit of an open canopy ecosystem. 23

co > '<' g .; i, -n().,

VP-5496 VP-8290 VP-8294 VP-8745 VP-8913 VP-8968

Specirren

Figure 7. Box plot of δ13C values for six specimens of Teleoceras fossiger showing two statistical outliers (open circles) for VP-8745.

Table 2. Kruskal-Wallis and KWMC Results

Kruskal-Wa llis Test chi-squared = 68.23 df =5 p-va lue = 2.393e-13

Kruskal-Wa llis Mult ip le Comparison Test (KWMC) Specimens Examined Observed Diffe rence Crit ical Diffe rence Significant Diffe rence VP-8968 - VP-8294 14.38333 29.57536 NO VP-8968 - VP-8290 43.85000 29.57536 YES VP-8968 - VP-8913 23 .68333 29.57536 NO VP-8968 - VP-5496 18.98333 29.57536 NO VP-8968 - VP-8745 12.78333 29.57536 NO VP-8294 - VP-8290 49.46667 26.45301 YES VP-8294 - VP-8913 9.30000 26.45301 NO VP-8294 - VP-5496 33.36670 26.45301 YES VP-8294 - VP-8745 27.16670 26.45301 YES VP-8290 - VP-8913 20.16670 26.45301 NO VP-8290 - VP-5496 62.83333 26.45301 YES VP-8290 - VP-8745 56.63333 26.45301 YES VP-8913 - VP-5496 42.66667 26.45301 YES VP-8913 - VP-8745 36.46667 26.45301 YES VP-5496 - VP-8745 6.20000 26.45301 NO

DISCUSSION

Grasses, such as Panicum elegans and chloridoid leaf fragments, from the

Minium Quarry demonstrate the presence of C4 plants in the ecosystem (Thomasson,

1976, 1984, 1990, 1991, 2005; Waller and Lewis, 1979; Thomasson et al., 1986;

Thomasson and Wester, 2003), but forb seeds and tree seeds and bambusoid leaf fragments found in the quarry demonstrate the presence of C3 plants (Table 1). Overall, only six of the 16 grass and sedge species found at the Minium Quarry can be identified as using C4 photosynthesis (Thomasson, 1976, 1984, 1990, 1991, 2005; Waller and

Lewis, 1979; Thomasson et al., 1986; Thomasson and Wester, 2003). From seeds and leaf fragments found at the Minium Quarry, seven plant fossils are identified as being near aquatic or aquatic indicators (Table 1). Of these seven fossils, four have been identified as using C3 photosynthesis. The other three fossils are near aquatic sedges using C4 photosynthesis. All of these plant fossils support the conclusion of a C3 dominated ecosystem with a small percentage of C4 plants available (MacFadden, 1998;

Fox and Koch, 2004; Strömberg and McInerney, 2011).

Soil carbonates can preserve the carbon isotope signal of the relative abundance of C3 and C4 plants within an ecosystem (Koch, 1998; Fox and Koch, 2004; Passey et al., 2009, 2010; Fox et al., 2012). The soil carbonates collected from the Minium Quarry have δ13C values of -6.4‰, -7.0‰, and -7.5‰ (Fox and Koch, 2004). These values fall within the mixed C3/C4 ecosystem of Kita et al. (2014), further supporting the conclusion of C4 being present but not dominating the ecosystem.

The conclusion of a C3 dominated ecosystem is also supported by the enamel

δ13C values in this study. The approximately 20% C4 biomass present in the ecosystem

(MacFadden, 1998; Fox and Koch, 2004; Strömberg and McInerney, 2011) indicates that

δ13C values above 2.1‰ are unlikely. The percentage of C4 plants also indicate that a

13 pure C3 regime without seasonal variations (δ Cenamel values ≤ -12.1‰) is not likely to occur. The enamel δ13C values from the six T. fossiger specimens sampled all show an

13 open canopy ecosystem (δ Cenamel values between -12.1‰ and -7.7‰) except for the two outlier values belonging to FHSM VP-8745 (-7.6‰ and -7.2‰), which show a mixed

13 C3/C4 ecosystem (δ Cenamel values between -7.7‰ and 2.1‰). These model results were expected for the diet of T. fossiger, and indicate that T. fossiger had a diet with little to no

C4 plant material.

The two data points identified as statistical outliers (-7.6‰ and -7.2‰), which fall into the mixed C3/C4 category of the model, indicate that one of the individuals of T. fossiger in this study either included a small portion of C4 biomass into the diet, deposited enamel during a period of drought, or the data points are a result of error. The data points are the closest to the occlusal surface. If they are caused by an increased amount of C4 biomass in the diet, then it means the individual transitioned from incorporating C4 in the diet at a younger age to eating a pure C3 diet as the individual grew older. This could be a significant interpretation. None of the other individuals in this study are interpreted as incorporating C4 biomass in their diets. If this individual is the geologically youngest specimen, it could indicate this species of rhinoceros was starting

27 to eat C4 plant material as it became more abundant. However, without knowing the exact stratigraphic position in this attritional deposit it is not possible to determine if the individual with these two data points is the geologically youngest specimen.

Another possibility is a period of drought occurring during the time of enamel deposition. A drought would decrease the amount of precipitation and cause C3 plants to

13 increase their δ C values by reusing the CO2. If an animal were to eat these C3 plants during this time, they would record an increased δ13C value reflecting the plant material.

Determining the length of the drought is difficult without knowing exactly how long it took the tooth to form. A rhinoceros molar takes between three to four years to completely form and erupt depending on which molar it is in the jaw (Goddard, 1970).

The two data points are from enamel formed early in the tooth development and could represent one year to two years of tooth growth. This would mean that the drought causing these two data points could have lasted between one to two years. A drought lasting this duration is not uncommon in the modern climate of Kansas.

These data points also could have been caused by a slightly elevated reading from the mass spectrometer creating laboratory error or from a human error during the process of drilling, preparation, or treatment prior to being run by the mass spectrometer.

However, the raw data produced by the mass spectrometer does not show any abnormalities in the results. This indicates that these two values are authentic signals from the animal’s diet.

Although there is a 20% presence of C4 grasses recorded in the Minium Quarry community at the time of deposition (MacFadden, 1998; Fox and Koch, 2004; Strömberg and McInerney, 2011), the amount of C4 biomass consumed by T. fossiger is interpreted as being zero, including the two points falling in the mixed C3/C4 biome. This indicates a discrepancy between plant material available in the ecosystem and plant biomass in the diet of T. fossiger. The difference between the diet and the ecosystem could result from the ecological and foraging behavior Teleoceras is thought to have filled. Based on the stout, barrel-chested skeletal morphology and a few artists’ reconstructions, Teleoceras is

thought to fill a semi-aquatic niche, similar to modern hippopotamuses (Webb, 1983;

Prothero and Schoch, 1989; Prothero, 1993; MacFadden, 1998). This would mean that T. fossiger was constrained to near aquatic environments but travel away from the water source to forage. The plant material present at the Minium Quarry support these organisms moving away from the water source to feed. The ratio of C3 to C4 plants near the water source is three to four, as indicated by the near aquatic indicator plants, while

the C3 to C4 ratio is seven to two (Table 1) (Thomasson, 1976, 1984, 1990, 1991, 2005;

Waller and Lewis, 1979; Thomasson et al., 986; Thomasson and Wester, 2003). If T.

fossiger was truly eating a pure C3 diet, it would make sense that they would travel away

from the permanent water source to feed because the diversity of C3 vegetation is lower

next to the water than it is away from the water source.

The discrepancy between what is available in the ecosystem and the diet of T.

fossiger could also be caused by selective eating. The nutritional value of C3 plants is

29 greater than the nutritional value of C4 plants (Wilson et al., 1983; Barbehenn and

Bernays, 1992; Barbehenn, 1993; Van Soest, 1994; Barbehenn et al., 2004). Sedges are a

C4 plant and make up most of the plant types found in the near aquatic environment of

Minium. In general, C4 plants are known to be more fibrous than C3 plants (Wilson et al., 1983; Barbehenn and Bernays, 1992; Barbehenn, 1993; Van Soest, 1994; Barbehenn et al., 2004). This means that it is possible T. fossiger was moving away from the water source to avoid eating the sedges present in the near aquatic environment. A selective diet would support a pure C3 diet and the idea of T. fossiger migrating away from the water source to forage for food like modern hippopotamuses.

Diagenesis, alteration of fossil material after burial, poses complications to isotopic studies (e.g. Sullivan and Krueger, 1981; Nelson et al., 1986; Lee-Thorp and Van

Der Merwe, 1987, 1991; Wang and Cerling, 1994; Koch et al., 1997; Kohn and Cerling,

2002; Schoeninger et al., 2003). If diagenetic alteration occurs, the isotopic signal of the hydroxyapatite will change to reflect the composition of the altering solution. Recent diagenetic processes would decrease the δ13C in fossil specimens to reflect modern values. However, groundwater exposure is necessary for any diagenetic alteration to occur. Enamel’s low porosity does not allow fluids, such as ground water, to penetrate.

Along with extremely low porosity, enamel has the largest hydroxyapatite crystals of any hard tissue (LeGeros, 1981; Schoeninger and DeNiro, 1982; Tieszen et al., 1983; Simkiss and Wilbur, 1989; Lee-Thorp and van der Merwe, 1991; Koch et al., 1997; Kohn and

Cerling, 2002), which affects the diagenetic susceptibility with smaller crystals being

more susceptible to diagenesis than larger crystals. It is because of the large crystal size

and low porosity that enamel is considered to be resistant to diagenesis and is not tested

for diagenetic alteration (e.g. Sullivan and Krueger, 1981; Nelson et al., 1986; Lee-Thorp

and Van Der Merwe, 1987, 1991; Wang and Cerling, 1994; Koch et al., 1997; Kohn and

Cerling, 2002; Schoeninger et al., 2003).

For this project, the major criteria for tooth selection was the type of tooth and

whether the tooth was an isolated element. FHSM VP-8745 was one of the teeth selected

because it is an isolated, permanent molar. However, FHSM VP-8745 also shows some erosion of the enamel surface, which is likely caused by acid dissolution (Figures 8 & 9).

It is unlikely that the dissolution process would alter the original isotopic composition of the hydroxyapatite crystals because the structural carbonate of tooth enamel is considered to be resistant to diagenesis (e.g. Sullivan and Krueger, 1981; Nelson et al., 1986; Wang and Cerling, 1994; Koch et al., 1997; Kohn and Cerling, 2002; Schoeninger et al., 2003).

Figure 8 (above). Anterior view of FHSM VP-8745 showing alteration of the enamel and drill tracks where samples were taken. Figure 9 (right). Buccal view of FHSM VP-8745 showing alteration of the enamel and drill tracks where samples were taken.

The post hoc KWMC test showed that significant differences exist among three

different groupings in the teeth. The first grouping consists of FHSM VP-8968, FHSM

VP-8294, and FHSM VP-8913. The second grouping consists of FHSM VP-8968, FHSM

VP-5496, and FHSM VP-8745. The third grouping contains only one specimen, FHSM

VP-8290. There are a number of factors which could be causing these groupings

including differences in individual crown lengths, differences in vegetation due to

seasonal precipitation changes, and differences in vegetation due to yearly climate

changes.

The crown length affects where samples are taken on the tooth, in respect to the bottom of the tooth crown, and the total length of the crown is different for each molar.

This could cause the grouping in the data because samples from similar distances from the bottom of the crown could have similar δ13C values if the individuals were living at

the same time. FHSM VP-8290 has the longest crown with a crown length of 59.1 mm.

FHSM VP-5496 and FHSM VP- 8745 have the second longest crowns with total crown

length of 56.1 mm and 57.9 mm, respectively. FHSM VP-8294 and FHSM VP-8913 have

crown lengths of 49.97 mm and 48.58 mm, respectively. FHSM VP-8968 is an

ambiguous result because even though FHSM VP-8968 has the shortest crown length,

with a total crown length of 40.8 mm, it groups with the second longest crown length

group and the group with the third longest crown length.

The effect of seasonal changes in vegetation on this data set is difficult to

determine because of the time resolution of the Minium Quarry and the time resolution of

32 each tooth. Minium Quarry represents approximately a million years of time and the enamel of each tooth is deposited over several years, depending on the amount of time required to deposit the tooth enamel. This means each individual and each tooth could represent different years, which is beneficial of broad climatic trend but is not a small enough scale to examine any seasonal variations within the same year. Even though climatic changes between years can be analyzed, conclusions must include possible overlap of data and/or the possible thousands of years between sampled individuals.

Overall, the lack of time resolution on these teeth could be showing enough variation in years to cause the different groupings in the teeth.

CONCLUSIONS

δ13C values from 85 serial samples from six T. fossiger molars were used to

reconstruct the diet and examine the abundance of C4 plant biomass between 8 and 9 Ma.

These values were placed in a model for late Neogene Nebraska developed by Kita et al.

13 (2014), which separates ecosystems into four categories: closed canopy (δ Cenamel ≤ -

13 13 12.1‰), open canopy (-12.1‰ < δ Cenamel < -7.7‰), mixed C3/C4 (-7.7‰ < δ Cenamel <

13 13 2.1‰), and pure C4 (δ Cenamel ≥ 2.1‰). Eighty-three of the δ Cenamel values collected in this study fit within the open canopy ecosystem. Two samples fit within the mixed C3/C4

13 ecosystem of the model. However, the δ Cenamel values for the two samples (-7.6‰ and -

7.2‰) are close to the open canopy ecosystem. These samples are the first two drill

tracks of FHSM VP-8745, with the rest of the values fitting the open canopy ecosystem.

It is possible that these two values could represent a shift in the diet of this individual

from incorporating some C4 biomass in the diet to eating a pure C3 diet, a period of

drought during the time of the enamel deposition, or an error from laboratory or

pretreatment processing (this last possibility is unlikely based on the raw mass

spectrometer data).

A Kruskal-Wallis test showed a significant difference in the medians of the six

teeth (X2 = 68.23, df = 5, p << 0.05). A post-hoc KWMC determined where the significant difference was among the teeth, and indicated that there were three groupings in the teeth. Although the reason for the significant difference in the medians cannot be

determined exactly, it is possible the variation results from where the enamel was

sampled on each crown or the climatic changes between different years.

Overall, T. fossiger shows an open canopy ecosystem (sensu Kita et al., 2014) and

little to no C4 biomass was incorporated into the diet. The paleobotanical (Thomasson,

1976, 1984, 1990, 1991, 2005; Waller and Lewis, 1979; Thomasson et al., 1986;

Strömberg, 2002; Thomasson and Wester, 2003; Strömberg and McInerney, 2011), soil carbonate (Fox and Koch, 2004; Fox et al., 2012), and the diet of T. fossiger support the presence, but not the dominance, of C4 biomass in the ecosystem between 8 and 9 Ma.

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Appendix I

Data Table Distance Specimen Sample from Bottom Statistics ID δ13C δ18O ID Number of Crown (mm) 1 T.fossiger 1 36.51 -9.61 -1.02 2 T.fossiger 1 33.27 -9.13 -0.62 3 T.fossiger 1 30.47 -9.57 -0.91 4 T.fossiger 1 27.44 -9.15 -1.26 VP-8968 5 T.fossiger 1 25.26 -9.22 -1.75 6 T.fossiger 1 22.58 -9.16 -1.45 7 T.fossiger 1 19.91 -9.34 -0.61 8 T.fossiger 1 16.88 -9.51 -0.44 9 T.fossiger 1 14.52 -9.31 -0.49 10 T.fossiger 1 12.16 -9.12 -1.05 1 T.fossiger 2 47.73 -9.85 -2.14 2 T.fossiger 2 45.47 -10.03 -1.64 3 T.fossiger 2 43.21 -10.27 -1.77 4 T.fossiger 2 40.78 -9.75 -1.62 5 T.fossiger 2 38.50 -9.61 -1.50 6 T.fossiger 2 36.25 -9.41 -1.65 7 T.fossiger 2 34.36 -9.70 -1.94 VP-8294 8 T.fossiger 2 32.26 -9.50 -2.40

9 T.fossiger 2 29.12 -9.27 -2.75 10 T.fossiger 2 26.76 -9.11 -2.99 11 T.fossiger 2 23.01 -9.27 -3.69 12 T.fossiger 2 20.32 -9.56 -2.37 13 T.fossiger 2 18.59 -9.73 -2.73 14 T.fossiger 2 15.76 -9.90 -2.87 15 T.fossiger 2 12.97 -9.46 -2.53 1 T.fossiger 3 56.77 -11.12 -2.80 2 T.fossiger 3 53.98 -10.94 -2.99 3 T.fossiger 3 52.02 -10.98 -3.07 VP-8290 4 T.fossiger 3 49.90 -10.94 -2.56 5 T.fossiger 3 47.73 -10.78 -2.16 6 T.fossiger 3 45.47 -11.09 -1.44 7 T.fossiger 3 43.03 -11.16 -1.07 8 T.fossiger 3 41.23 -11.10 -0.79

9 T.fossiger 3 38.71 -11.08 -0.92 10 T.fossiger 3 36.56 -11.06 -1.35 11 T.fossiger 3 34.34 -11.13 -1.37 12 T.fossiger 3 31.78 -11.11 -1.04 13 T.fossiger 3 28.68 -10.97 -1.15 14 T.fossiger 3 26.14 -10.95 -1.92 15 T.fossiger 3 23.28 -10.85 -2.24 1 T.fossiger 4 46.37 -10.63 -2.03 2 T.fossiger 4 44.02 -10.15 -2.38 3 T.fossiger 4 42.21 -10.10 -2.46 4 T.fossiger 4 39.12 -9.90 -2.84 5 T.fossiger 4 36.31 -9.60 -3.11 6 T.fossiger 4 34.03 -9.53 -3.89 7 T.fossiger 4 31.71 -9.45 -3.93 VP-8913 8 T.fossiger 4 29.25 -9.47 -4.08

9 T.fossiger 4 26.71 -9.57 -4.32 10 T.fossiger 4 24.02 -9.70 -4.28 11 T.fossiger 4 21.78 -10.08 -3.99 12 T.fossiger 4 19.57 -10.19 -3.81 13 T.fossiger 4 17.25 -10.30 -3.93 14 T.fossiger 4 14.84 -10.18 -3.37 15 T.fossiger 4 12.78 -9.80 -2.84 1 T.fossiger 5 51.77 -8.95 -2.19 2 T.fossiger 5 48.50 -9.06 -2.64 3 T.fossiger 5 45.13 -9.32 -3.28 4 T.fossiger 5 42.18 -9.19 -3.65 5 T.fossiger 5 39.74 -8.91 -2.33 6 T.fossiger 5 37.10 -9.13 -1.93 7 T.fossiger 5 34.47 -9.22 -2.27 VP-5496 8 T.fossiger 5 31.59 -9.00 -1.86

9 T.fossiger 5 29.17 -8.70 -1.56 10 T.fossiger 5 26.71 -8.58 -2.56 11 T.fossiger 5 23.77 -8.61 -3.46 12 T.fossiger 5 21.24 -8.24 -3.37 13 T.fossiger 5 18.76 -8.19 -3.91 14 T.fossiger 5 15.84 -8.17 -3.63 15 T.fossiger 5 13.16 -8.59 -2.41 1 T.fossiger 6 54.43 -7.24 -4.00 2 T.fossiger 6 40.68 -7.65 -4.11 3 T.fossiger 6 47.84 -8.20 -3.34 VP-8745 4 T.fossiger 6 45.31 -8.69 -2.91

5 T.fossiger 6 42.69 -9.15 -2.04 6 T.fossiger 6 39.85 -9.19 -2.05 7 T.fossiger 6 37.04 -9.05 -1.45

8 T.fossiger 6 34.84 -8.86 -1.81 9 T.fossiger 6 31.50 -8.80 -2.41 10 T.fossiger 6 29.20 -9.01 -2.77 11 T.fossiger 6 26.47 -9.20 -3.06 12 T.fossiger 6 24.00 -9.37 -2.81 13 T.fossiger 6 21.61 -9.56 -2.47 14 T.fossiger 6 18.88 -9.35 -2.19 15 T.fossiger 6 15.96 -9.35 -1.91

Appendix II

Range Values

Specimen ID δ13C Range δ18O Range

VP-8968 0.48 1.31 VP-8294 1.16 1.49 VP-8290 0.39 2.27 VP-8913 1.18 2.30 VP-5496 1.15 2.10 VP-8745 2.32 2.66